Part Second

Part Second

SUGARS AND STARCHES.

=44. Introduction.=—Carbohydrates, of which sugars and starches are the chief representatives, form the great mass of the results of vegetable metabolism. The first functions of the chlorophyll cells of the young plant are the condensation of carbon dioxid and water. The simplest form of the condensation is formaldehyd, CH₂O. There is no convincing evidence, however, that this is the product resulting from the functional activity of the chlorophyll cells. The first evidence of the condensation is found in more complex molecules; viz., those having six atoms of carbon. It is not the purpose of this work to discuss the physiology of this process, but the interested student can easily find access to the literature of the subject.[23] When a sample of a vegetable nature reaches the analyst he finds by far the largest part of its substance composed of these products of condensation of the carbon dioxid and water. The sugars, starches, pentosans, lignoses, and celluloses all have this common origin. Of many air-dried plants these bodies form more than eighty per cent.

In green plants the sugars exist chiefly in the sap. In plants cut green and quickly dried by artificial means the sugars are found in a solid state. They also exist in the solid state naturally in certain sacchariferous seeds. Many sugar-bearing plants when allowed to dry spontaneously lose all or the greater part of their sugar by fermentation. This is true of sugar cane, sorghum, maize stalks, and the like. The starches are found deposited chiefly in tubers, roots or seeds. In the potato the starch is in the tuber, in cassava the tuber holding the starch is also a root, in maize, rice and other cereals the starch is in the seeds. The wood-fibers; viz., pentosans, lignose, cellulose, etc., form the framework and support of the plant structure. Of all these carbohydrate bodies the most important as foods are the sugars and starches, but a certain degree of digestibility cannot be denied to other carbohydrate bodies with the possible exception of pure cellulose. In the following paragraphs the general principles of determining the sugars and starches will be given and afterwards the special processes of extracting these bodies from vegetable substances preparatory to quantitive determination.

=45. Nomenclature.=—In speaking of sugars it has been thought best to retain for the present the old nomenclature in order to avoid confusion. The terms dextrose, levulose, sucrose, etc., will therefore be given their commonly accepted significations.

A more scientific nomenclature has recently been proposed by Fischer, in which glucose is used as the equivalent of dextrose and fructose as the proper name for levulose. All sugars are further classified by Fischer into groups according to the number of carbon atoms found in the molecule. We have thus trioses, tetroses, pentoses, hexoses, etc. Such a sugar as sucrose is called hexobiose by reason of the fact that it appears to be formed of two molecules of hexose sugars. For a similar reason raffinose would belong to the hexotriose group.[24]

Again, the two great classes of sugars as determined by the structure of the molecule are termed aldoses and ketoses according to their relationship to the aldehyd or ketone bodies.

Since sugars may be optically twinned, that is composed of equal molecules of right and left-handed polarizing matter it may happen that apparently the same body may deflect the plane of polarization to the right, to the left, or show perfect neutrality.

Natural sugars, as a rule, are optically active, but synthetic sugars being optically twinned are apt to be neutral to polarized light.

To designate the original optical properties of the body therefore the symbols d, l, and i, meaning dextrogyratory, levogyratory, and inactive, respectively, are prefixed to the name. Thus we may have d, l, or i glucose, d, l, or i fructose, and so on.

The sugars that are of interest here belong altogether to the pentose and hexose groups; viz., C₅H₁₀O₅ and C₆H₁₂O₆, respectively. Of the hexobioses, sucrose, maltose, and lactose are the most important, and of the hexotrioses, raffinose. In this manual, unless otherwise stated, the term dextrose corresponds to d glucose, and levulose to d fructose. In this connection, however, it should be noted that the levulose of nature, or that which is formed by the hydrolysis of inulin or sucrose is not identical in its optical properties with the l fructose of Fischer.

=46. Preparation of Pure Sugar.=—In using the polariscope or in testing solutions for the chemical analysis of samples, the analyst will be required to keep always on hand some pure sugar. Several methods of preparing pure sugar have been proposed. The finest granulated sugar of commerce is almost pure. In securing samples for examination those should be selected which have had a minimum treatment with bluing in manufacture. The best quality of granulated sugar when pulverized, washed with ninety-five per cent and then with absolute alcohol and dried over sulfuric acid at a temperature not exceeding 50° will be found nearly pure. Such a sugar will, as a rule, not contain more than one-tenth per cent of impurities, and can be safely used for all analytical purposes. It is assumed in the above that the granulated sugar is made from sugar cane.

Granulated beet sugars may contain raffinose and so may show a polarization in excess of 100. This sugar may be purified by dissolving seventy parts by weight in thirty parts of water. The sugar is precipitated by adding slowly an equal volume of ninety-six per cent alcohol with constant stirring, the temperature of the mixture being kept at 60°. While still warm the supernatant liquor is decanted and the precipitated sugar washed by decantation several times with strong warm alcohol. The sugar, on a filter, is finally washed with absolute alcohol and dried in a thin layer over sulfuric acid at from 35° to 40°. By this process any raffinose which the sugar may have contained is completely removed by the warm alcohol. Since beet sugar is gradually coming into use in this country it is safer to follow the above method with all samples.[25] In former times it was customary to prepare pure sugar from the whitest crystals of rock candy. These crystals are powdered, dissolved in water, filtered, precipitated with alcohol, washed and dried in the manner described above.

=47. Classification of Methods.=—In the quantitive determination of pure sugar the various processes employed may all be grouped into three classes. In the first class are included all those which deduce the percentage of sugar present from the specific gravity of its aqueous solution. The accuracy of this process depends on the purity of the material, the proper control of the temperature, and the reliability of the instruments employed. The results are obtained either directly from the scale of the instruments employed or are calculated from the arbitrary or specific gravity numbers observed. It is evident that any impurity in the solution would serve to introduce an error of a magnitude depending on the percentage of impurity and the deviation of the density from that of sugar. The different classes of sugars, having different densities in solution, give also different readings on the instruments employed. It is evident, therefore, that a series of tables of percentages corresponding to the specific gravities of the solutions of different sugars would be necessary for exact work. Practically, however, the sugar which is most abundant, viz., sucrose, may be taken as a representative of the others and for rapid control work the densimetric method is highly useful.

In the second class of methods are grouped all those processes which depend upon the property of sugar solutions to rotate the plane of polarized light. Natural sugars all have this property and if their solutions be found neutral to polarized light it is because they contain sugars of opposite polarizing powers of equal intensity. Some sugars turn the polarized plane to the right and others to the left, and the degree of rotation in each case depends, at equal temperatures and densities of the solutions, on the percentages of sugars present. In order that the optical examination of a sugar may give correct results the solution must be of a known density and free of other bodies capable of affecting the plane of polarized light. In the following paragraphs an attempt will be made to give in sufficient detail the methods of practice of these different processes in so far as they are of interest to the agricultural analyst. The number of variations, however, in these processes is so great as to make the attempt to fully discuss them here impracticable. The searcher for additional details should consult the standard works on sugar analysis.[26]

In the third class of methods are included those which are of a chemical nature based either on the reducing power which sugar solutions exercise on certain metallic salts, upon the formation of certain crystalline and insoluble compounds with other bodies or upon fermentation. Under proper conditions solutions of sugar reduce solutions of certain metallic salts, throwing out either the metal itself or a low oxid thereof. In alkaline solutions of mercury and copper, sugars exercise a reducing action, throwing out in the one case metallic mercury and in the other cuprous oxid. With phenylhydrazin, sugars form definite crystalline compounds, quite insoluble, which can be collected, dried and weighed. There is a large number of other chemical reactions with sugars such as their union with the earthy bases, color reactions with alkalies, oxidation products with acids, and so on, which are of great use qualitively and in technological processes, but these are of little value in quantitive determinations.

THE DETERMINATION OF THE PERCENTAGE OF SUGAR BY THE DENSITY OF ITS SOLUTION.

=48. Principles of the Method.=—This method of analysis is applied almost exclusively to the examination of one kind of sugar, viz., the common sugar of commerce. This sugar is derived chiefly from sugar cane and sugar beets and is known chemically as sucrose or saccharose. The method is accurate only when applied to solutions of pure sucrose which contain no other bodies. It is evident however, that other bodies in solution can be determined by the same process, so that the principle of the method is broadly applicable to the analyses of any body whatever in a liquid state or in solution. Gases, liquids and solids, in solution, can all be determined by densimetric methods.

Broadly stated the principle of the method consists in determining the specific gravity of the liquid or solution, and thereafter taking the percentage of the body in solution from the corresponding specific gravity in a table. These tables are carefully prepared by gravimetric determinations of the bodies in solution of known densities, varying by small amounts and calculation of the percentages for the intervening increments or decrements of density. This tabulation is accomplished at definite temperatures and the process of analysis secured thereby is rapid and accurate, with pure or nearly pure solutions.

=49. Determination of Density.=—While not strictly correct from a physical point of view, the terms density and specific gravity are here used synonymously and refer to a direct comparison of the weights of equal volumes of pure water and of the solution in question, at the temperature named. When not otherwise stated, the temperature of the solution is assumed to be 15°.5.

The simplest method of determining the density of a solution is to get the weight of a definite volume thereof. This is conveniently accomplished by the use of a pyknometer. A pyknometer is any vessel capable of holding a definite volume of a liquid in a form suited to weighing. It may be a simple flask with a narrow neck distinctly marked, or a flask with a ground perforated stopper, which, when inserted, secures always the same volume of liquid contents. A very common form of pyknometer is one in which the central stopper carries a thermometer and the constancy of volume is secured by a side tubulure of very small or even capillary dimensions, which is closed by a ground glass cap.

The apparatus may not even be of flask form, but assume a quite different shape as in Sprengel’s tube. Pyknometers are often made to hold an even number of cubic centimeters, but the only advantage of this is in the ease of calculation which it secures. As a rule, it will be found necessary to calibrate even these, and then the apparent advantage will be easily lost. A flask which is graduated to hold fifty cubic centimeters, may, in a few years, change its volume at least slightly, due to molecular changes in the glass. Some of the different forms of pyknometers are shown in the accompanying figures.

In use the pyknometer should be filled with pure water of the desired temperature and weighed. From the total weight the tare of the flask and stopper, weighed clean and dry, is to be deducted. The remainder is the weight of the volume of water of the temperature noted, which the pyknometer holds. The weight of the solution under examination is taken in the same way and at the same temperature, and thus a direct comparison between the two liquids is secured.

Example.—Let the weight of the pyknometer be 15.2985 grams. and its weight with pure water at 15°.5 be 26.9327 ” Then the weight of water is 11.6342 ” The weight filled with the sugar solution is 28.3263 ” Then the weight of the sugar solution is 13.0278 ”

The specific gravity of the sugar solution is therefore, 13.0278 ÷ 11.6342 = 1.1198.

For strictly accurate results the weight must be corrected for the volume of air displaced, or in other words, be reduced to weights in vacuo. This however is unnecessary for the ordinary operations of agricultural analysis.

If the volume of the pyknometer be desired, it can be calculated from the weight of pure water which it holds, one cubic centimeter of pure water weighing one gram at 4°.

The weights of one cubic centimeter of water at each degree of temperature from 1° to 40°, are given in the following table:

TABLE SHOWING WEIGHTS OF ONE CUBIC CENTIMETER OF PURE WATER AT TEMPERATURES VARYING FROM 1° TO 40°.

Weight, Weight, Temperature. Gram. Temperature. Gram.

0° 0.999871 21° 0.998047 1° 0.999928 22° 0.997826 2° 0.999969 23° 0.997601 3° 0.999991 24° 0.997367 4° 1.000000 25° 0.997120 5° 0.999990 26° 0.996866 6° 0.999970 27° 0.996603 7° 0.999933 28° 0.998331 8° 0.999886 29° 0.995051 9° 0.999824 30° 0.995765 10° 0.999747 31° 0.995401 11° 0.999655 32° 0.995087 12° 0.999549 33° 0.994765 13° 0.999430 34° 0.994436 14° 0.999299 35° 0.994098 15° 0.999160 36° 0.993720 16° 0.999002 37° 0.993370 17° 0.998841 38° 0.993030 18° 0.998654 39° 0.992680 19° 0.998460 40° 0.992330 20° 0.998259

From the table and the weight of water found, the volume of the pyknometer is easily calculated.

Example.—Let the weight of water found be 11.72892 grams, and the temperature 20°. Then the volume of the flask is equal to 11.72892 ÷ 0.998259, viz., 11.95 cubic centimeters.

=50. Use of Pyknometer at High Temperatures.=—It is often found desirable to determine the density of a liquid at temperatures above that of the laboratory, e. g., at the boiling-point of water. This is easily accomplished by following the directions given below:

Weight of Flask.—Use a small pyknometer of from twenty-five to thirty cubic centimeters capacity. The stopper should be beveled to a fine edge on top and the lower end should be slightly concave to avoid any trapping of air. The flask is to be thoroughly washed with hot water, alcohol and ether, and then dried for some time at 100°. After cooling in a desiccator the weight of the flask and stopper is accurately determined.[27]

Weight of Water.—The flask in an appropriate holder, Fig. 28, conveniently made of galvanized iron, is filled with freshly boiled and hot distilled water and placed in a bath of pure, very hot distilled water, in such a way that it is entirely surrounded by the liquid with the exception of the top.

The water of the bath is kept in brisk ebullition for thirty minutes, any evaporation from the flask being replaced by the addition of boiling distilled water. The stopper should be kept for a few minutes before use in hot distilled water and is then inserted, the flask removed, wiped dry, and, after it is nearly cooled to room temperature, placed in the balance and weighed when balance temperature is reached. A convenient size of holder will enable the analyst to use eight or ten flasks at once. The temperature at which water boils in each locality may also be determined; but unless at very high altitudes, or on days of unusual barometric disturbance the variations will not be great, and will not appreciably affect the results.

=51. Alternate Method of Estimating the Weight of Water in Flasks.=—Formulas for calculating the volume V, in cubic centimeters, of a glass vessel from the weight P of water at the temperature t contained therein, and the volume Vʹ at any other temperature t’ are given by Landolt and Börnstein.[28] They are as follows:

p V = P --- d

p Vʹ = P ---- [1 + γ(tʹ- t)] d

p = weight (in brass weights) of one cubic centimeter H₂O in vacuo. This is so nearly one gram that it will not affect the result in the fifth place of decimals and may therefore be disregarded. Hence the formula stands:

1 Vʹ = P ---- [1 + γ(tʹ-t)]; in which d

d = density of water at temperature t.

γ = 0.000025, the cubical expansion coefficient of glass.

From this volume the weight of the water may be readily obtained by referring to tables 13, 14 and 15_a_ in Landolt and Börnstein’s book.

=52. Example Showing Determination of Specific Gravity of a Fat.=—The flask is emptied of its water, rinsed with alcohol and ether, and dried again for a few minutes at 100°. It is then filled with the dry, hot, fresh-filtered fat, which should be entirely free from air bubbles.

The stoppered flask is then replaced in the water-bath, kept for thirty minutes at the temperature of boiling water, removed, and treated as above. The weight of fat having been determined, the specific gravity is obtained by dividing it by the weight of water previously found.

Example. Grams. Weight of flask, dry 10.0197 Weight of flask, plus water 37.3412 Weight of water 27.3215 Weight of flask, plus fat 34.6111 Weight of fat 24.5914

Specific gravity = 24.5914 ÷ 27.3215 = 0.90008.

The weight of the flask dry and empty and the weight of water at 99° to 100° contained therein may be used constantly if great care be taken in handling and cleaning the apparatus.

Example. Grams. Weight of flask, dry and empty 10.0028 Weight of flask after three weeks’ use 10.0030

=53. Determination of Density by the Hydrostatic Balance.=—While the pyknometer is useful in control work and in fixing standards of comparison, it is not used extensively in practical work. Quicker methods of determination are desired in such work, and these are found in the use of other forms of apparatus. A convenient method of operation consists in determining the weight of a sinker, whose exact weights in air and in pure water of a definite temperature, have been previously determined. The instrument devised by Mohr and modified by Westphal, is based upon that principle, and is extensively used in practical work. The construction of this apparatus and also that of the pyknometers and areometers is shown in the illustrations, figures 29 and 30.

The weight of the sinker is so adjusted that the index of the balance arm marks zero when the sinker is wholly immersed in pure water at the standard temperature. The density of a solution of sugar at the same temperature, is then determined by placing the rider-weights on the divided arm of the balance, until the index again marks zero. The density can then be read directly from the position of the weights in the arm of the balance or calculated therefrom.

=54. The Areometric Method.=—The most rapid method of determining the density of a solution and the one in most common use, is based on the distance to which a heavy bulb with a slender graduated stem will sink therein. An instrument of this kind is called an areometer. Many forms of this instrument are employed but they all depend on the same principle and differ only in the manner of graduation. The one of widest application has the stem graduated in such a manner as to give directly the specific gravity of the solution in which it is placed.

Others are made with a special graduation giving directly the percentage of solid matter in the solution. These instruments can be used only for the special purposes for which they are constructed. Other forms are provided with an arbitrary graduation, the numbers of which by appropriate tables can be converted into expressions of specific gravity or of per cents of dissolved matters. It is not practicable to give here, a discussion of the principles of the construction of areometers.[29] The two which are commonly used, are the baumé hydrometer and the balling or brix spindle.

In the baumé instrument the zero of the scale is fixed at the point marked by the surface of distilled water at 15°, and the point to which it sinks in pure monohydrated sulfuric acid at the same temperature is marked 66, corresponding to a specific gravity of 1.8427.

The specific gravity corresponding to any degree of the scale, may be calculated in the absence of a table giving it, by the following formula

144.3 P = -----------. 144.3 - d

In this formula P is the density and d the degree of the scale.[30] In former times the baumé instruments were graduated with a solution of common salt and a different formula was employed for calculating specific gravity, but these older instruments are no longer in common use.

The following table shows the specific gravities of solutions corresponding to baumé degrees from 1° to 75° consecutively[31]:

Degree Specific Degree Specific Degree Specific Degree Specific baumé gravity baumé gravity baumé gravity baumé gravity 0 1.0000 19 1.1516 38 1.3574 57 1.6527 1 1.0069 20 1.1608 39 1.3703 58 1.6719 2 1.0140 21 1.1702 40 1.3834 59 1.6915 3 1.0212 22 1.1798 41 1.3968 60 1.7115 4 1.0285 23 1.1895 42 1.4104 61 1.7321 5 1.0358 24 1.1994 43 1.4244 62 1.7531 6 1.0433 25 1.2095 44 1.4386 63 1.7748 7 1.0509 26 1.2197 45 1.4530 64 1.7968 8 1.0586 27 1.2301 46 1.4678 65 1.8194 9 1.0665 28 1.2407 47 1.4829 66 1.8427 10 1.0744 29 1.2514 48 1.4983 67 1.8665 11 1.0825 30 1.2624 49 1.5140 68 1.8909 12 1.0906 31 1.2735 50 1.5301 69 1.9161 13 1.0989 32 1.2849 51 1.5465 70 1.9418 14 1.1074 33 1.2964 52 1.5632 71 1.9683 15 1.1159 34 1.3081 53 1.5802 72 1.9955 16 1.1246 35 1.3201 54 1.5978 73 2.0235 17 1.1335 36 1.3323 55 1.6157 74 2.0523 18 1.1424 37 1.3447 56 1.6340 75 2.0819

=55. Correction for Temperature.=—The baumé hydrometer should be used at the temperature for which it is graduated, usually 15°. In this country the mean temperature of our working rooms is above 15°. The liquid in the hydrometer flask should therefore be cooled to a trifle below 15°, or kept in a bath exactly at 15° while the observation is made. When this is not convenient, the observation may be made at any temperature, and the reading corrected as follows: When the temperature is above 15° multiply the difference between the observed temperature and fifteen, by 0.0471 and add the product to the observed reading of the baumé hydrometer; when the temperature on the other hand, is below fifteen, the corresponding product is subtracted.[32]

=56. The Balling or Brix Hydrometer.=—The object of the balling or brix instrument is to give in direct percentages the solid matter in solution. It is evident that for this purpose the instrument must be graduated for a particular kind of material, since ten per cent of sugar in solution, might have a very different specific gravity from a similar quantity of another body. Instruments of this kind graduated for pure sugar, find a large use in technical sugar analysis. To attain a greater accuracy and avoid an instrument with too long a stem, the brix hydrometers are made in sets. A convenient arrangement is to have a set of three graduated as follows; one from 0° to 30°, one from 25° to 50°, and one from 45° to 85°. When the percentage of solid matter dissolved is over seventy the readings of the scale are not very reliable.

=57. Correction for Temperature.=—The brix as the baumé scale is graduated at a fixed temperature. This temperature is usually 17°.5. The following table shows the corrections to be applied to the scale reading when made at any other temperature:[33]

PER CENT OF SUGAR IN SOLUTION.

0. 5. 10. 15. 20. 25. 30. 35. 40. 50. 60. 70. 75. Temp. To be subtracted from the degree read. 0° 0.17 0.30 0.41 0.52 0.62 0.72 0.82 0.92 0.98 1.11 1.22 1.25 1.29 5° 0.23 0.30 0.37 0.44 0.52 0.59 0.65 0.72 0.75 0.80 0.88 0.91 0.94 10° 0.20 0.26 0.29 0.33 0.36 0.39 0.42 0.45 0.48 0.50 0.54 0.58 0.61 11° 0.18 0.23 0.26 0.28 0.31 0.34 0.36 0.39 0.41 0.43 0.47 0.50 0.53 12° 0.16 0.20 0.22 0.24 0.26 0.29 0.31 0.33 0.34 0.36 0.40 0.42 0.46 13° 0.14 0.18 0.19 0.21 0.22 0.24 0.26 0.27 0.28 0.29 0.33 0.35 0.39 14° 0.12 0.15 0.16 0.17 0.18 0.19 0.21 0.22 0.22 0.23 0.26 0.28 0.32 15° 0.09 0.11 0.12 0.14 0.14 0.15 0.16 0.16 0.17 0.17 0.19 0.21 0.25 16° 0.06 0.07 0.08 0.09 0.10 0.10 0.11 0.12 0.12 0.12 0.14 0.16 0.18 17° 0.02 0.02 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.04 0.05 0.05 0.06

To be added to the degree read. 18° 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 19° 0.06 0.08 0.08 0.09 0.09 0.10 0.10 0.10 0.10 0.10 0.10 0.08 0.06 20° 0.11 0.14 0.15 0.17 0.17 0.18 0.18 0.18 0.19 0.19 0.18 0.15 0.11 21° 0.16 0.20 0.22 0.24 0.24 0.25 0.25 0.25 0.26 0.26 0.25 0.22 0.18 22° 0.21 0.26 0.28 0.31 0.31 0.32 0.32 0.32 0.33 0.34 0.32 0.29 0.25 23° 0.27 0.32 0.35 0.37 0.38 0.39 0.39 0.39 0.40 0.42 0.39 0.36 0.33 24° 0.32 0.38 0.41 0.43 0.44 0.46 0.46 0.47 0.47 0.50 0.46 0.43 0.40 25° 0.37 0.44 0.47 0.49 0.51 0.53 0.54 0.55 0.55 0.58 0.54 0.51 0.48 26° 0.43 0.50 0.54 0.56 0.58 0.60 0.61 0.62 0.62 0.66 0.62 0.58 0.55 27° 0.49 0.57 0.61 0.63 0.65 0.68 0.68 0.69 0.70 0.74 0.70 0.65 0.62 28° 0.56 0.64 0.68 0.70 0.72 0.76 0.76 0.78 0.78 0.82 0.78 0.72 0.70 29° 0.63 0.71 0.75 0.78 0.79 0.84 0.84 0.86 0.86 0.90 0.88 0.80 0.78 30° 0.70 0.78 0.82 0.87 0.87 0.92 0.92 0.94 0.94 0.98 0.94 0.88 0.86 35° 1.10 1.17 1.22 1.24 1.30 1.32 1.33 1.35 1.36 1.39 1.34 1.27 1.25 40° 1.50 1.61 1.67 1.71 1.73 1.79 1.79 1.80 1.82 1.83 1.78 1.69 1.65 50° ---- 2.65 2.71 2.74 2.78 2.80 2.80 2.80 2.80 2.79 2.70 2.56 2.51 60° ---- 3.87 3.88 3.88 3.88 3.88 3.88 3.88 3.90 3.82 3.70 3.43 3.41 70° ---- ---- 5.18 5.20 5.14 5.13 5.10 5.08 5.06 4.90 4.72 4.47 4.35 80° ---- ---- 6.62 6.59 6.54 6.16 6.38 6.30 6.26 6.06 5.82 5.50 5.33

According to observations of Gerlach, the correction for temperature varies with the concentration of the solution and the range of temperature as shown in the table.

=58. Comparison of Brix and Baumé Degrees.=—The following table shows the degree baumé and the specific gravity of a sugar solution for each degree brix (per cent of sugar in solution) from zero to ninety-five:[34]

Degree Degree Specific Degree Degree Specific brix. baumé. gravity. brix. baumé. gravity. 1.0 0.6 1.00388 37.0 20.7 1.16413 2.0 1.1 1.00779 38.0 21.2 1.16920 3.0 1.7 1.01173 39.0 21.8 1.17430 4.0 2.3 1.01570 40.0 22.3 1.17943 5.0 2.8 1.01970 41.0 22.9 1.18460 6.0 3.4 1.02373 42.0 23.4 1.18981 7.0 4.0 1.02779 43.0 24.0 1.19505 8.0 4.5 1.03187 44.0 24.5 1.20033 9.0 5.1 1.03599 45.0 25.0 1.20565 10.0 5.7 1.04014 46.0 25.6 1.21100 11.0 6.2 1.04431 47.0 26.1 1.21639 12.0 6.8 1.04852 48.0 26.6 1.22182 13.0 7.4 1.05276 49.0 27.2 1.22128 14.0 7.9 1.05703 50.0 27.7 1.23278 15.0 8.5 1.06133 51.0 28.2 1.23832 16.0 9.0 1.06566 52.0 28.8 1.24390 17.0 9.6 1.07002 53.0 29.3 1.24951 18.0 10.1 1.07441 54.0 29.8 1.25517 19.0 10.7 1.07884 55.0 30.4 1.26086 20.0 11.3 1.08329 56.0 30.9 1.26658 21.0 11.8 1.08778 57.0 31.4 1.27235 22.0 12.4 1.09231 58.0 31.9 1.27816 23.0 13.0 1.09686 59.0 32.5 1.28400 24.0 13.5 1.10145 60.0 33.0 1.28989 25.0 14.1 1.10607 61.0 33.5 1.29581 26.0 14.6 1.11072 62.0 34.0 1.30177 27.0 15.2 1.11541 63.0 34.5 1.30177 28.0 15.7 1.12013 64.0 35.1 1.31381 29.0 16.3 1.12488 65.0 35.6 1.31989 30.0 16.8 1.12967 66.0 36.1 1.32601 31.0 17.4 1.13449 67.0 36.6 1.33217 32.0 18.0 1.13934 68.0 37.1 1.33836 33.0 18.5 1.14423 69.0 37.6 1.34460 34.0 19.1 1.14915 70.0 38.1 1.35088 35.0 19.6 1.15411 71.0 38.6 1.35720 36.0 20.1 1.15911 72.0 39.1 1.36355 73.0 39.6 1.36995 85.0 45.5 1.44986 74.0 40.1 1.37639 86.0 46.0 1.45678 75.0 40.6 1.38287 87.0 46.5 1.46374 76.0 41.1 1.38939 88.0 47.0 1.47074 77.0 41.6 1.39595 89.0 47.5 1.47778 78.0 42.1 1.40254 90.0 49.9 1.48486 79.0 42.6 1.40918 91.0 48.4 1.49199 80.0 43.1 1.41586 92.0 48.9 1.49915 81.0 43.6 1.42258 93.0 49.3 1.50635 82.0 44.1 1.42934 94.0 49.8 1.51359 83.0 44.6 1.43614 95.0 50.3 1.52087 84.0 45.1 1.44298

=59. Error Due to Impurities.=—The fact that equal per cents of solid bodies in solution affect the specific gravity in different degrees has already been noted. The specific gravities of the solutions of the common sugars, however, are so nearly the same for equal per cents of solid matter in solution as to render the use of a brix hydrometer quite general for technical purpose. For the mineral salts which often occur in sugar solutions the case is quite different. A twenty per cent solution of cane sugar at 17°.5 has a specific gravity 1.08329 and of dextrose 1.08310, practically identical. But a solution of calcium acetate of similar strength has a specific gravity of 1.0874; of sodium sulfate 1.0807, and of potassium nitrate 1.1359. This latter number would correspond to a sugar content of nearly twenty-seven per cent. The brix scale can, therefore, be regarded as giving only approximately the percentage of solid matter in sugar solutions and, while useful in technical work, should never be relied upon for exact analytical data.

THE DETERMINATION OF SUGAR WITH POLARIZED LIGHT.

=60. Optical Properties of Natural Sugars.=—The solutions of all natural sugars have the property of deflecting the plane of polarized light and the degree of deflection corresponds to the quantity of sugar in solution. By measuring the amplitude of the rotation produced the percentage of sugar in the solution can be determined. In order to secure accuracy in the determinations it is necessary that only one kind of sugar be present, or, if more than one, that the quantities of all but one be determined by other means, and the disturbances produced thereby in the total rotation be properly arranged. In point of fact the process in practice is applied chiefly to cane and milk sugars, both of which occur in nature in an approximately pure state. The process is also useful in determining cane sugar when mixed with other kinds, by reason of the fact that this sugar after hydrolysis by treatment with a weak acid for a long or a strong acid for a short time, definitely changes its rotating power. Since, by the same treatment, the rotating power of other sugars which may be present is only slightly altered, the total disturbance produced is approximately due to the inversion of the cane sugar.

Dextrose and maltose arising from the hydrolysis of starch may also be determined with a fair degree of accuracy by their deportment with polarized light. When a solution of natural sugars shows negative results when examined with polarized light, it is due to an admixture of two or more sugars of opposite polarizing powers in such proportions as to produce neutrality. This condition often occurs in the examination of honeys or in submitting artificial sugars to polarimetric observations. In the latter case the neutrality is caused by the tendency manifested by artificially produced sugars to form twin compounds of optically opposite qualities.

The instrument used for measuring the degree of deflection produced in a plane of polarized light is called a polariscope, polarimeter, or optical saccharimeter. For a theoretical discussion of the principles of polarization and the application of these principles in the construction of polariscopes, the reader is referred to the standard works on optics and the construction of optical instruments.[35] For the purposes of this work a description of the instruments commonly employed and the methods of using them will be sufficient.

=61. Polarized Light.=—When a ray of light has been repeatedly reflected from bright surfaces or when it passes through certain crystalline bodies it acquires peculiar properties and is said to be polarized.

Polarization is therefore a term applied to a phenomenon of light, in which the vibrations of the ether are supposed to be restricted to a particular form of an ellipse whose axes remain fixed in direction. If the ellipse become a straight line it is called plane polarization. This well-known phenomenon is most easily produced by a nicol prism, consisting of a cut crystal of calcium carbonate (Iceland spar). This rhombohedral crystal, the natural ends of which form angles of 71° and 109°, respectively, with the opposite edges of its principal section, is prepared as follows:

The ends of the crystals are ground until the angles just mentioned become 68° and 112°. The crystal is then divided diagonally at right angles with the planes of the ends and with the principal section, and after the new surfaces are polished they are joined again by canada balsam. The principal section of this prism passes through the shorter diagonal of the two rhombic ends. If now a ray of light fall on one of the ends of this prism, parallel with the edge of its longer side, it suffers double refraction, and each ray is plane polarized, the one at right angles with the other. That part of the entering ray of light which is most refracted is called the ordinary and the other the extraordinary ray. The refractive index of the film of balsam being intermediate between those of the rays, permits the total reflection of the ordinary ray, which, passing to the blackened sides of the prism, is absorbed. The extraordinary ray passes the film of balsam without deviation and emerges from the prism in a direction parallel with the incident ray, having, however, only half of its luminous intensity.

Two such prisms, properly mounted, furnish the essential parts of a polarizing apparatus. They are called the polarizer and the analyzer, respectively.

If now the plane of vibration in each prism be regarded as coincident with its principal section, the following phenomena are observed: If the prisms are so placed that the principal sections lie in the prolongation of the same plane, then the extraordinary polarized ray from the polarizer passes into the analyzer, which practically may be regarded in this position as a continuation of the same prism. It happens, therefore, that the extraordinary polarized ray passes through the analyzer exactly as it did through the polarizer, and is not reflected by the film of balsam, but emerges from the analyzer in seemingly the same condition as from the polarizer. If now the analyzer be rotated 180°, bringing the principal section again in the same plane, the same phenomenon is observed. But if the rotation be in either direction only 90°, then the polarized ray from the first prism, incident on the second, deports itself exactly as the ordinary ray, and on meeting the film of balsam is totally reflected. The field of vision, therefore, is perfectly dark.

In all other inclinations of the planes of the principal sections of the two prisms the ray incident in the analyzer is separated into two, an ordinary and extraordinary, varying in luminous intensity in proportion to the square of the cosine of the angle of the two planes.

Thus, by gradually turning the analyzer, the field of vision passes slowly from maximum luminosity to complete obscurity. The expression crossed nicols refers to the latter condition of the field of vision.

=62. Description of the Prism.=—In a nicol made as described above, Fig. 31, suppose a ray of light parallel with the longer side of the prism be incident to the end a b at m. By the double refracting power of the spar the ray is divided into two, which traverse the first half of the prism. The two rays are polarized at right angles to one another. The less refracted ray when it strikes the film of Canada balsam passes through it without interference. The more refracted ray strikes the balsam at o at such an angle as to be totally reflected and made to pass out of the prism in the direction o r. If the prism be blackened at the surface the ray will be entirely absorbed. The other ray passes on through the other half of the prism and emerges in the direction of qs. It is evident that the emergent light from a nicol has only half the illuminating power possessed by the immergent rays.

The polarized plane of light from the nicol just described may be regarded as passing also into a second nicol of essentially the same construction as the first.

This second nicol, called the analyzer, is so constructed as to revolve freely about its longitudinal axis, and is attached to a graduated circle in such a way that the degree of rotation can be accurately read. If the planes of polarization of the two nicols are coincident when prolonged, the ray of light passing from the first nicol will pass through the second practically unchanged in character or intensity. If, however, the analyzing nicol be turned until the plane of polarization is at right angles to that of the polarizer the immergent ray will suffer refraction in such a manner as to be totally reflected when reaching the film of balsam and will be thus entirely lost. In making a complete revolution of the analyzer, therefore, two positions of maximum intensity of light and two of darkness will be observed. In intermediate positions the ray immergent to the analyzer will be separated as in the first instance into two rays g p varying intensities, one of which will be always totally reflected.

In Fig. 32 is given a more detailed illustration of the action of the rays of light. The film of balsam is represented as enlarged and of the thickness bb. Draw the perpendiculars represented by the dotted lines _n_₁ _n_ʹ₁, _n_₂ _n_ʹ₂, _n_₃ _n_ʹ₃ and _n_₄ _n_ʹ₄. In passing into the prism at m both refracted rays are bent towards the normal _m n_ʹ₁. The degree of deflection depends on the refractive index of the two rays 1.52 and 1.66 respectively. The refractive index of the extraordinary ray in calcspar being 1.52, and in Canada balsam 1.54, it suffers but little disturbance in passing from one to the other. On the other hand the balsam, being considerably less refractive for the ordinary ray than the calcspar, causes that ray to diverge outwards from the normal _o n_ʹ₂, and to such a degree as to suffer total reflection. The critical angle, that is the angle at which a ray issuing from a more refractive into a less refractive medium, emerges just parallel to the bounding surfaces, depends on the relative index of refraction. In the case under consideration the ratio for balsam and spar is 1.54/1.66 = 0.928 = sin 68°. Therefore the limiting value of m o _n_₃ so that m o may just emerge in the direction od is 68°. If now mo were parallel to o d the angle m o n, would be just 68°, being opposite b a d. which has been ground to 68° in the construction of the prism. But in passing into the prism, m o is refracted so that the angle _m o n_₃ is greater than b a d. It is therefore always certain that by grinding b a d to 68° the ordinary ray m o will be with certainty entirely thrown out in every case. In respect of the analyzing nicol the following additional observations will be found useful. In all uniaxial crystals there are two directions at right angles to each other, one of greatest and one of least resistance to the propagation of luminous vibrations. These planes are in the direction of the principal axis and at right angles thereto. Only light vibrating in these two directions can be transmitted through calcspar; and all incident light propagated by vibrations in a plane at any other angle to the principal section is resolved into two such component rays. But the velocities of transmission in the two directions are unequal, that is, the refractive index of the spar for the two rays is different. If the analyzing nicol be so adjusted as to receive the emergent light from the polarizer when the corresponding planes of the two prisms are coincident when extended, the emergent extraordinary ray falling into a plane of the same resistance as that it had just left is propagated through the second nicol with the same velocity that it passed the first one. It is therefore similarly refracted. If, however, the two prisms be so arranged that corresponding planes cross then the extraordinary ray falls into a plane which it traverses with greater velocity than it had before and is accordingly refracted and takes the course which ends in total reflection at the film of balsam. No light therefore can pass through the prism in that position. If any other substance, as for instance a solution of sugar, capable of rotating a plane of polarized light, be interposed between the two nicols the effect produced is the same as if the analyzer had been turned to a corresponding degree. When the analyzer is turned to that degree the corresponding planes again coincide and the light passes. This is the principle on which the construction of all polarizing instruments is based.[36]

=63. The Polariscope.=—A polariscope for the examination of solutions of sugar consists essentially of a prism for polarizing the light, called a nicol, a tube of definite length for holding the sugar solution, a second nicol made movable on its axis for adjustment to the degree of rotation and a graduated arc for measuring it. Instead of having the second nicol movable, many instruments have an adjusting wedge of quartz of opposite polarizing power to the sugar, by means of which the displacement produced on the polarized plane is corrected. A graduated scale and vernier serve to measure the movement of the wedges and give in certain conditions the desired reading of the percentage of sugar present. Among the multitude of instruments which have been devised for analytical purposes, only three will be found in common use, and the scope of this volume will not allow space for a description of a greater number. For a practical discussion of the principles of polarization and their application to optical saccharimetry, the reader may conveniently refer to the excellent manuals of Sidersky, Tucker, Landolt, and Wiechmann.[37]

=64. Kinds of Polariscopes.=—The simplest form of a polarizing apparatus consists of two nicol prisms, one of which, viz., the analyzer, is capable of rotation about its long axis. The prolongation of this axis is continuous with that of the other prism, viz., the polarizer. The two prisms are sufficiently removed from each other to allow of the interposition of the polarizing body whose rotatory power is to be measured.

For purposes of description three kinds of polarimeters may be mentioned.

1. Instruments in which the deviation of the plane of polarization is measured by turning the analyzer about its axis.

Instruments of this kind conform to the simple type first mentioned, and are coeteris paribus the best. The Laurent, Wild, Landolt-Lippich, etc., belong to this class.

2. Instruments in which both nicols are fixed and the direction of the plane of polarized light corrected by the interposition of a wedge of a solid polarizing body.

Belonging to this class are the apparatus of Soleil, Duboscq, Scheibler, and the compensating apparatus of Schmidt and Haensch.

3. Apparatus in which the analyzer is set at a constant angle with the polarizer, and the compensation secured by varying the length or concentration of the interposed polarizing liquid.

The apparatus of Trannin belongs to this class.

=65. Appearance of Field of Vision.=—Polarimeters are also classified in respect of the appearance of the field of vision.

1. Tint Instruments.—The field of vision in these instruments in every position of the nicols, except that on which the plane of vibration of the polarized light is coincident with the three principal sections, is composed of two semi-disks of different colors.

2. Shadow Instruments.—The field of vision in this class of polarimeters in all except neutral positions, is composed of two semi-disks, one dark and one yellow. As the neutral position is approximated the two disks gradually assume a light yellow color, and when neutrality is reached they appear to be equally colored.

The Laurent, Schmidt and Haensch shadow and Landolt-Lippich instruments, are of this class.

3. Striated Instruments.—In this class the field of vision is striated. The lines may be tinted as in Wild’s polaristrobometer or black, as in the Duboscq and Trannin instruments. The neutral position is indicated either by the disappearance of the striae (Wild) or by the phenomenon of their becoming continuous. (Duboscq, Trannin.)

=66. Character of Light Used.=—Polariscopes may be further divided into two classes, based on the kind of light employed.

1. Instruments which Use Ordinary White Light.—(Oil lamp, etc.) Scheibler, Schmidt and Haensch.

2. Instruments Employing Monochromatic Light.—(Sodium flame, etc.) Laurent, Landolt-Lippich, etc.

=67. Interchangeable Instruments.=—Some of the instruments in common use are arranged to be used either with ordinary lamp or gas light, or with a monochromatic flame. Laurent’s polarimeter is one of this kind. The compensating instruments also may have the field of vision arranged for tints or shadows. Theoretically the best instrument would be one in which the light is purely monochromatic, the field of vision a shadow, and the compensation secured by the rotation of the second nicol.

The accuracy of an instrument depends, however, on the skill and care with which it is constructed and used. With quartz wedges properly ground and mounted, and with ordinary white light, polariscopes may be obtained which give readings as accurate as can be desired.

Since many persons are more or less affected with color-blindness, the shadow are to be preferred to the tint fields of vision.

For practical use in sugar analysis the white light is much more convenient than the monochromatic light.

For purposes of general investigation the polarimeters built on the model of the laurent are to be preferred to all others. Such instruments are not only provided with a scale which shows the percentage of sucrose in a solution, but also with a scale and vernier by means of which the angular rotation which the plane of vibration has suffered, can be accurately measured in more than one-quarter of the circle.

DESCRIPTION OF POLARIZING INSTRUMENTS.

=68. Rotation Instruments.=—This instrument has already been described as one in which the extent of deviation in the plane of polarized light caused by the intervention of an optically active substance is measured by rotating one of the nicols about its axis and measuring the degree of this rotation by a vernier on a graduated arc.

In Germany these instruments are called polaristrobometers, and in France polarimètrés. In England and this country the term polariscope or polarimeter is applied without discrimination to all kinds of optical saccharimeters.

The polariscope of Mitscherlich was one of the earliest in use. It has now been entirely superseded by more modern and accurate instruments.

=69. The Laurent Instrument.=—A polariscope adapted by Laurent to the use of monochromatic yellow light is almost exclusively used in France and to a considerable extent in this country. In case a worker is confined to the use of a single instrument, the one just mentioned is to be recommended as the best suited to general work. It has the second nicol, called the analyzer, movable and the degree of rotation produced is secured in angular terms directly on a divided circle. The scale is graduated both in angular measurements and in per cents of sugar for a definite degree of concentration of the solution and length of observation tube. The normal solution in the laurent instrument contains 16.19 grams of pure sugar in 100 true cubic centimeters, and the length of the observation tube is 200 millimeters. Both the angular rotation and the direct percentage of sugar can be read at the same time. Great accuracy can be secured by making the readings in each of the four quadrants. The light is rendered yellow monochromatic by bringing into the flames of a double bunsen, spoons made of platinum wire, which carry fragments of fused sodium chlorid.

=70. The Laurent Burner.=—The theory of the illumination of the laurent burner is illustrated by the accompanying Fig. 33. The lamp consists essentially of two bunsens, surmounted by a chimney.[38] A curved spoon made of platinum gauze serves to hold the fused particles of sodium chlorid which are used to produce the yellow light. The spoon is shown at G, held by the arm F, fastened by the key P. The interior intense flame B B is surrounded by an exterior less highly colored flame A A. It is important that the optical axis of the polariscope be directed accurately upon the disk B, which is the most intense part of the illumination. The point of the spoon carrying the salt should be coincident with the prolongation of the lamp TT, so that it just strikes the edge of the blue flame. Care should be taken not to press the spoons into the interior of the flame as by so doing the intensity of the illumination is very much diminished. Great care must be observed in the position of the spoon G, and the platinum arm F being flexible, the operator with a little patience, will be enabled to properly place the spoon by bending it. Moreover, if the spoon be pressed too far into the flame, the melted particles of salt collecting in the bottom of G may drop into the lamp and occlude the orifices through which the gas enters. The light of the yellow flame produced by the lamp may be further purified by passing through a prism filled with a solution of potassium dichromate, or better, a homogeneous disk cut from a crystal of that salt.

Since the flame produced by the above device is not perfectly constant, being more intense at the moment of introducing a fresh portion of the fused salt, the author has used a lamp designed to furnish an absolutely constant flame.[39] This device which is shown in Fig. 34, is based on the principle of adding constantly a fresh portion of the salt to the flame. The flame is thus kept perfectly uniform in its intensity.

The lamp consists essentially of two wheels with platinum gauze perimeters and platinum wire spokes, driven by a clock-work D, and mounted by the supports AAʹ as shown in the figure. The sodium salt, chlorid or bromid, in dilute solution, is placed in the porcelain crucibles F, supported by BBʹ as indicated in the figure, to such a depth that the rims of the platinum wheels dip beneath the surface as they revolve. The salt is volatilized by the lamp E. By means of the crossed bands the wheels are made to revolve in opposite directions as indicated by the arrows. The solution of the salt which is taken up by the platinum net-work of the rim of the wheel, thus has time to become perfectly dry before it enters the flame and the sputtering which a moist salt would produce is avoided. At every instant, by this arrangement, a minute fresh portion of salt is introduced into the flame with the result of making a perfectly uniform light which can be used for hours without any perceptible variation. The mechanism of the apparatus is so simple that no further description is necessary. The polariscope should be so directed toward the flame as to bring into the field of vision its most luminous part. The platinum wheels are adjustable and should be so arranged as to produce between them an unbroken yellow flame. The wheels are eight centimeters in diameter and are driven at a rate to make one revolution in from six to ten minutes.

=71. Construction of Laurent’s Apparatus.=—The shadow polariscope invented by Laurent is constructed as follows: The polarizer is a special nicol which is not fixed in its position, but is so arranged as to be turned through a small arc about its axis. By this device, the quantity of light passing through it can be regulated, and the apparatus is thus useful with colored solutions which are not easily cleared by any of the common bleaching agents. The greater the quantity of light admitted, however, the less delicate is the reading of the shadow produced. The plane of polarized light emergent from this prism, falls on a disk of glass half covered by a thin lamina of quartz which thus divides the field of vision into halves. It is this semi-disk of quartz which is the distinguishing feature of the apparatus.[40] The polarized light thus passes without hindrance the half field of vision which is covered by the glass only, but can not pass the quartz plate unless its axis is set in a certain way. The field of vision may be thus half dark, or both halves may be equally illuminated or equally dark according to the position of the nicol analyzer which is freely movable about its axis and carries a vernier and reading glass over a graduated circle. The field of vision in the laurent may have any of the following forms.[41] Let the polarizer be first so adjusted that the plane of polarization of the transmitted pencil of light is parallel to the axis of the plate lying in the direction A B. The two halves of the field of vision will then appear equally illuminated in every position of the analyzer. But if the polarizing nicol be inclined to AB at an angle a, the plane of polarization of the rays passing through the quartz plate will undergo deviation through an equal angle in the opposite direction.

It happens from this, that when in the uncovered half of the field, the plane of polarization has the direction AC, in the other half it will have the direction ACʹ. When the analyzer is rotated, if its plane of polarization lie in the direction cc, the rays polarized parallel to AC will be completely extinguished and the corresponding half of the field will be dark. The opposite happens when the plane of polarization lies in the direction of cʹcʹ. When one-half of the field is thus obscured, the other suffers only a partial diminution in the intensity of its illumination. When the middle position bb is reached in the rotation of the analyzer, the illumination of the two halves is uniform, and this is the point at which the zero of the scale is reached. The slightest rotation of the analyzer to the right or left of this neutral point will cause a shadow to appear on one of the halves of the field, which by an oscillatory movement of the analyzer, seems to leap from side to side. The smaller the angle a or BAC, the more delicate will be the shading and the more accurate the observation. This angle being adjustable by the mechanism already described, should be made as small as will permit the admission of the quantity of light requisite for accurate observation.

The various pieces composing the polariscope are arranged in the following positions, beginning on the right of Fig. 36, and passing to the left, where the observer is seated.[42]

1. The lamp VV, TT, AA, or the wheel burner:

2. The lens B for condensing the rays and rendering them parallel:

3. The tube I, blackened inside to carry the lens:

4. A thin lamina E, cut from a crystal of potassium bichromate, serving to render the sodium light more monochromatic: When the saccharine liquids under examination are colored the crystal of bichromate is removed before the observation is made.

5. The polarizer R, which is rotatable through a small angle by the lever K:

6. The lever JK for rotating the tube containing the polarizer: This is operated by the rod X extending to the left.

7. Diaphragm D, half covered with a lamina of quartz.

8. Trough L for holding the observation tube: In the large instrument shown in the figure, it is more than half a meter in length and arranged to hold an observation tube 500 millimeters long.

9. Disk C, carrying divided circle and arbitrary sugar scale:

10. Mirror M, to throw the light of the lamp on the vernier of the scale:

11. Reading glass N, carried on the same radius as the mirror and used to magnify and read the scale:

12. Device F, to regulate the zero of the instrument:

13. Tube H, carrying a nicol analyzer and ocular O for defining the field of vision: This tube is rotated by the radial arm G, carrying the mirror and reading glass.

=72 Manipulation.=—The lamp having been adjusted, the instrument, in a dark room, is so directed that the most luminous spot of the flame is in the line of vision. An observation tube filled with water is placed in the trough and the zero of the vernier is placed accurately on the zero of the scale. The even tint of the field of vision is then secured by adjusting the apparatus by the device number 12.

=73 The Soleil-Ventzke Polariscope.=—A form of polariscope giving a colored field of vision was in use in this country almost exclusively until within ten years, and is still largely employed. There are many forms of tint instruments, but the one almost exclusively used here is that mentioned. A full description of their construction and manipulation is given by Tucker.[43] By the introduction of a third rotating nicol in front of the lens next to the lamp, the sensitive tint at which the reading is made can be kept at a maximum delicacy. These instruments are capable of rendering very reliable service, especially in the hands of those who have a delicate perception of color. They are inferior, however, to the shadow instruments in delicacy, and are more trying to the eye of the observer. The shadow instruments therefore, especially those making use of an ordinary kerosene lamp, have practically driven the tint polariscopes out of use.

The general arrangement of a tint instrument as modified by Scheibler is shown in Fig. 37.

Beginning on the right of the figures, its optical parts are as follows: A is a nicol which, with the quartz plate B, forms the apparatus for producing the light rose neutral tint. The proper degree of rotation of these two parts is secured by means of the button L attached to the rod carrying the ratchet wheel as shown. The polarizing nicol is at C, and D is a quartz disk, one-half of which is right-handed and the other left-handed. At G is another quartz plate belonging to the analyzing part of the apparatus. This, together with the fixed quartz wedge F, and the movable quartz wedge E, constitute the compensating apparatus of the instrument whereby the deviation produced in the plane of polarized light by the solution in the tube is restored.

Next to the compensating apparatus is the analyzing nicol which in this instrument is fixed in a certain place, viz., the zero of the scale. The analyzer and the telescope for observing the field of vision are carried in the tube HJ. The movable quartz wedge has a scale which is read with a telescope K, provided with a mirror inclined at an angle of 45°, just over the scale and serving to illuminate it. The quartz wedges are also provided with a movement by which the zero point of the scale can be adjusted. A kerosene lamp with two flat wicks is the best source of illumination and the instrument should be used in a dark room and the light of the lamp, save that which passes through the polariscope, be suppressed by a shade. The sensitive or transition tint is produced by that position of the regulating apparatus which gives a field of view of such a nature that a given small movement of the quartz compensating wedge gives the greatest contrast in color between the halves of the field of vision. For most eyes a faint rose-purple tint, as nearly colorless as possible, possesses this quality. A slight movement of the quartz wedge by means of the screw head M will, with this tint, produce on one side a faint green and on the other a pink color, which are in strong contrast. The neutral point is reached by so adjusting the quartz wedge as to give to both halves of the field the same faint rose-purple tint.

=74. The Shadow Polariscope for Lamp Light.=—This form of instrument is now in general use for saccharimetric purposes. It possesses on the one hand, the advantages of those instruments using monochromatic light, and on the other, the ease of manipulation possessed by the tint polariscopes. It differs from the tint instrument in dispensing with the nicol and quartz plate used to regulate the sensitive tint, and in having its polarizing nicol peculiarly constructed in harmony with the optical principles of the jellet-corny prism. The more improved forms of the apparatus have a double quartz wedge compensation. The two wedges are of opposite optical properties, and serve to make the observations more accurate by mutual correction. The optical arrangement of the different parts of such a polariscope is shown in the following figure.

The lenses for concentrating the rays of light and rendering them parallel are contained in the tube N. At O is placed the modified polarizing nicol. The two compensating quartz wedges are moved by the milled screw-heads EG. The rest of the optical apparatus is arranged as described under the tint polariscope. For practical purposes, only one of the wedges is employed, but for all accurate work the readings should be made with both wedges and thus every possible source of error eliminated.

=75. The Triple Shadow Instrument.=—When properly made, all the instruments which have been mentioned, are capable of giving accurate results if used in harmony with the directions given. In the use of polariscopes having colored fields of vision a delicate sense of distinguishing between related tints is necessary to good work. Color-blind observers could not successfully use such apparatus. In the shadow instruments it is only necessary to distinguish between the halves of a field of vision unequally illuminated and to reduce this inequality to zero. A neutral field is thus secured of one intensity of illumination and of only one color, usually yellow. Such a field of vision permits of the easy discrimination between the intensity of the coloration of its two halves, and is consequently not trying to the eye of the observer, and allows of great accuracy of discrimination. This field of vision has lately been still further improved by dividing it into three parts instead of two. An instrument of this kind, Fig. 39, in use in this laboratory, permits a delicacy of reading not possessed by any other instrument used for sugar analysis, and approaching that of the standard Landolt-Lippich apparatus, used by us for research work and for determining the rotation of quartz plates and testing the accuracy of other polariscopes.

The triple shadow is secured by interposing in front of the polarizing nicol two small nicols as indicated in Fig. 40. The end views in different positions of the polarizer are shown in the lower part of the diagrams.

Instead of the comparison of the intensity of the illumination being made on the halves of the field of vision it is made by comparing the segments of the halves with a central band, which also changes in intensity synchronously with the two segments, but in an opposite direction.

THE ANALYTICAL PROCESS.

=76. General Principles.=—Having described the instruments chiefly employed in the optical examination of sugar solutions, the next step is to apply them to the analytical work. A common set of directions for use will be found applicable to all instruments with such modifications only as are required by peculiarities of construction. With the best made instruments it is always advisable to have some method of controlling the accuracy of the observation. The simplest way of doing this is to test the apparatus by standard quartz plates. These plates are made from right-handed polarizing quartz crystal ground into plates of definite thickness and accurately tested by standard instruments. Theoretically such quartz plates deflect the plane of polarized light in a degree proportionate to their thickness, but practically some small deviations from the rule are found. With a source of light of the same tint, and at a constant temperature, such plates become a safe test for the accuracy of the graduation of polariscopes. They are more convenient for use than pure sugar solutions of known strength which are the final standards in all disputed cases. These quartz plates are conveniently mounted in tubes of the same size as those holding the sugar solution, and thus fit accurately into the trough of the polariscope, the optical axis of which passes through their center. The quartz plate when used for setting the scale of a polariscope should be placed always in the same position. In some plates slight differences of reading may be noticed on rotating the tubes holding them. Theoretically, such differences should not exist, but in practice they are sometimes found. The temperature of observation should also be noted, and if not that at which the value of the plate was fixed a proper correction should be made.

=77. Setting the Polariscope.=—While mention has been made of several forms of apparatus in the preceding paragraphs, those in common use are limited to a very small number. In this country quite a number of color instruments may still be found, together with a few laurents, and a constantly increasing number of shadow instruments for use with lamp light. The following description of setting the polariscope is especially adapted to the last named instrument, but the principles of adjustment are equally applicable to all.

The scale of the instrument is first so adjusted by means of the adjusting screws provided with each instrument, as to bring the zero of the vernier and that of the scale exactly together. The telescope or ocular is then adjusted until the sharp line separating the halves of the field of vision is brought into focus. This being accomplished an observation tube filled with pure water is placed in the apparatus and the telescope again adjusted to bring the dividing line of the field into focus. The beginner especially, should repeatedly study this adjustment and be impressed with the fact that only in a sharply defined field are practical observations of any worth. The importance of having all the lenses perfect and all the cover glasses without a flaw may be fully appreciated when it is remembered that the polarized ray, already deprived of half its original luminous power, must pass through several centimeters of crystallized calcium carbonate, and half a dozen disks of glass and quartz, and as many lenses before reaching the eye of the observer. Only with the greatest care and neatness is it possible to secure the required degree of illumination. The zero point having been well studied and accurately adjusted, the scale of the instrument may be tried with a series of quartz plates of known polarizing power at the temperature of the observation. In the apparatus with double quartz wedge compensation, it will be noticed that the marks on one scale are black and on the other red. The black is the working and the red the control scale. To operate this instrument, the red scale is placed exactly at the zero point. The black scale is also placed at zero, and if the field of vision is not neutral, it is made so by the micrometer screw with which the black scale is provided. In a right-handed solution, the red scale is left at zero and the black one moved to the right until neutrality in the field of vision is reached and the reading is taken. The observation tube containing the sugar solution is taken out and the red scale moved until the field of vision is again neutral and the reading of the red scale taken. The two readings should agree. Any failure in the agreement shows some fault either in adjusting the apparatus or in its construction, or some error in manipulation.

The double compensating shadow instruments are more readily tested for accuracy in all parts of the scale than those of any other construction. The two compensating wedges are cut with the greatest care, one from a left-handed and the other from a right-handed perfectly homogeneous quartz crystal. Since faults in these wedges are due either to lack of parallelism of surface, or of perpendicularity to the optical axis of the crystal, and since these faults of crystallization or construction must be in a very limited degree common they would not coincide once in many thousand times in the two wedges. This is easily shown by the theory of probabilities. If, therefore, the two readings made at any point, should not agree, it must be due either to a fault in one of the wedges, or to a fault in reading or a lack of adjustment, as has been mentioned. In such cases the readings should be retaken and the errors are usually easily discovered.

=78. Control Observation Tube.=—Instead of using quartz plates of known values for testing the accuracy of the scale, an observation tube may be used, the length of which can be varied at the pleasure of the observer.

The construction of a tube of this kind is shown in Fig. 40. The tube B is movable telescopically in A by means of the ratchet wheel shown. It is closed at D water-tight by a glass disk. The tube B fits as accurately into A as is possible to permit of free movement, and any liquid which may infilter between its outer surface and the inner surface of A is prevented from gaining exit by the washer C, which fits both tubes water-tight. The ratchet which moves B in A carries a millimeter scale and vernier N whereby the exact thickness of the liquid solution between the surfaces of the glass disks D and E can be always determined.

By this device the length of liquid under observation can be accurately read to a tenth of a millimeter. The cover glass E is held in position by any one of the devices in common use for this purpose in the case in question, by a bayonet fastening. The funnel T, communicating directly with the interior of A, serves to hold the solution, there being always enough of it to fill the tube when D is removed to the maximum distance from C, which is usually a little more than 200 millimeters.

Let the control tube be adjusted to 200 millimeters and filled with a solution of pure sugar, which reads 100 per cent or degrees in a 200 millimeter tube. Since the degree of rotation is, other things being equal, proportional to the length of the column of polarizing solution, it follows that if the tube B be moved inward until the distance between D and C is 100 millimeters, the scale should read 50° or per cent. By adjusting the length of the distance between B and C it is easily seen that every part of the scale can be accurately tested.

The tube should be filled by removing the funnel and closing the orifice with a screw cap which comes with the apparatus. The cap E is then removed and the tube filled in the ordinary manner. This precaution is practiced to avoid carrying air bubbles into the tube when filled directly through the funnel. With a little care, however, this danger may be avoided, or should air bubbles enter they can be easily removed by inclining the tube.

In case the solution used be not strictly pure it may still be employed for testing the scale. Suppose, for instance, that a solution made up in the usual way, has been made from a sample containing only 99.4 per cent of sugar. Then in order to have this solution read 100° on the scale the tube should be set at 201.2 millimeters, according to the formula

200 × 100 --------- = 201.2. 99.4

By a similar calculation the position of the tube for reading any desired degree on the scale can be determined. The importance of controlling all parts of the scale in compensating instruments is emphasized by the fact that a variation of only 0.016 millimeter in the thickness of the compensating wedge will cause a change of one degree in the reading of the instrument.

=79. Setting the Polariscope with Quartz Plates.=—Pure sugar is not always at the command of the analyst, and it is more convenient practically to adjust the instrument by means of quartz plates, the sugar values of which have been previously tested for the character of the light used. Assuming the homogeneity of a plate of quartz, the degree of deflection which it imparts to a plane of polarized light depends on the quality of the light, the thickness of the plate, and the temperature.

In respect of the quality of light, red polarized rays are least, and violet most deflected. The degree of rotation produced with any ray, at a given temperature, is directly proportional to the thickness of the plate. Temperature affects the rotating power of a quartz plate in a degree highly significant from a scientific point of view and not wholly negligible for practical purposes. The rotating power of a quartz plate increases with the temperature and the variation may be determined by the formula given below:[44]

The formula is applicable for temperatures between 0° and 100°. Its values are expressed in degrees of angular measure which can be converted into degrees of the sugar scale by appropriate factors:

_Formula.—a_ᵗ = a°(1 + 0.000146_t_);

in which a° = polarization in angular degrees at 0°, t the temperature of observation and _a_ᵗ the rotation desired.

Example.—A quartz plate which has an angular rotation of 33° at 0° will have a rotation at 20° of 33°.09834.

_a_ᵗ = 33(1 + 0.000146 × 20) = 33.09834.

Since in instruments using the ventzke scale one degree of the sugar scale is equal to 0.3467 degree angular measure, the sugar value of the quartz plate mentioned is equal to 95.47 percent; 33.09834 ÷ 0.3467 = 95.47.

The sugar value of this plate at 0° is 95.18 per cent; 33 ÷ 0.3467 = 95.18.

=80. Tables for Correcting Quartz Plates.=—Instead of calculating the variation in quartz plates for each temperature of observation, it is recommended by the Bureau of Internal Revenue of the Treasury, to use control quartz plates the values of which at any given temperature, are found on a card which accompanies each one.[45] The variations given, are from temperatures between 10° and 35°. Three control plates are provided with each instrument used by the Bureau, for polarimetric work in the custom houses, or in ascertaining bounties to be paid on the production of domestic sugars. For example, the case of a sugar which polarizes 80°.5 may be cited. One of the control plates nearest to this number, is found to have at the temperature of observation, a polarization of 91°.4, the reading being made in each case at 25°. On consulting the card which accompanies the control plate, it is seen that its value at the temperature mentioned, is 91°.7. The reading of the instrument is therefore too low by three-tenths of a degree, and this quantity should be added to the observed polarization, making it 80°.8. In this method of correcting the reading for temperature, it is assumed that the compensating wedges of the instrument, are free of error at the points of observation. The plates used for the purpose above, are all standardized in the office of weights and measures of the Coast and Geodetic Survey, before delivery to the analysts.

=81. Applicability of Quartz Plates.=—Quartz plates which are correctly set for one instrument or kind of light, should be equally accurate for the sugar scales of all instruments, using the same sugar factor. In other words a quartz plate which reads 99° on a scheibler color polariscope, should give the same reading on the sugar scale of a shadow compensating or a monochromatic direct reading apparatus using 26.048 grams of sugar.

The most useful quartz plates for sugar analysis, are those which give the readings at points between 80° and 96°, which cover the limits of ordinary commercial sugars. For molasses the plates should read from 45° to 55°. For sugar juices of the cane and beet, the most convenient graduation would be from 10° to 20°, but plates of this value would be too thin for practical work and are not in use. When quartz plates are to be used for control purposes, they should be purchased from reliable manufacturers, or better, tested directly against pure sugar solutions by the observer.

In practice we have found quartz plates as a rule, true to their markings.

=82. The Sugar Flask.=—Sugar solutions are prepared for polarization in flasks graduated to hold fifty or one hundred cubic centimeters. For scientific work a flask is marked to hold 100 grams of distilled water at 4°. The weights are all to be reduced to a vacuum standard. One flask having been marked in this way, others may be compared directly therewith by means of pure mercury. For this purpose the flasks must be perfectly dry and the mercury pure, leaving no stain on the sides of the flask. The glass must also be strong enough to undergo no change in shape from the weight of mercury used.

For sugar work the true 100 gram flask is not usually employed, but one graduated by weighing at 17°.5. These flasks are graduated by first weighing them perfectly dry, filling with distilled water and again weighing fifty and fifty-five, or 100 and 110 grams of water at the temperature named. Since the volume of water at 17°.5 is greater than at 4° the sugar flask in ordinary use has a greater volume by about 0.25 cubic centimeter than the true flask. The observer should always secure a statement from the dealer in respect of the volume of the flask used in testing the scale of the polariscope purchased. In the graduation of a flask in true cubic centimeters, when brass weights are used it will be necessary to correct the weight of each gram of water by adding to it one milligram, which is almost exactly the weight of the volume of air displaced by one gram of water in the circumstances named. If the flask be first counterbalanced and it be desired to mark it at 100 cubic centimeters the sum of the weights placed in the opposite pan should be 100 - 0.100 = 99.900 grams. While this is not a rigidly exact correction it will be sufficient for all practical purposes. A liter of dry air weighs 1.29366 grams; and 100 cubic centimeters of water would therefore displace 0.129 gram of air. But the brass weights also displace a volume of air which when deducted reduces the correction to be made for the water to nearly the one named. For convenience in inverting sugar solutions the flasks used in practical work are graduated at fifty and fifty-five and 100 and 110 cubic centimeters respectively.

=83. Preparing Sugar Solutions for Polarization.=—If sugar samples were always pure the percentage of sugar in a given solution could be directly determined by immediate polarization. Such cases, however, are rarely met in practice. In the majority of cases the sample is not only to be brought into solution but is also to be decolorized and rendered limpid by some one of the methods to be described. A perfectly limpid liquid is of the highest importance to secure correct observations. With a cloudy solution the field of vision is obscured, the dividing line of the two halves, or the double line in the triple field, becomes blurred or invisible and the intensity of illumination is diminished. A colored liquid which is bright is far more easy to polarize than a colorless liquid which is turbid. In fact, it is only rarely in sugar work that samples will be found which require any special decolorizing treatment other than that which is received in applying the reagents which serve to make the solutions limpid. In the following paragraphs the approved methods of clarifying sugar solutions preparatory to observation in the polariscope will be described.

=84. Alumina Cream.=—The hydrate of alumina, commonly known as alumina cream, is always to be preferred as a clarifying agent in all cases where it can be successfully applied.[46] It is a substance that acts wholly in a mechanical way and therefore leaves the sugars in solution unchanged, carrying out only suspended matters. In the preparation of this reagent a solution of alum is treated with ammonia in slight excess, the aluminum hydroxid produced washed on a filter or by decantation until neutral in reaction. The hydroxid is suspended in pure water in proportions to produce a creamy liquid. Although apparently very bulky, the actual space occupied by the amount of dry hydroxid added in a few cubic centimeters is so small as to produce no disturbing effect of importance on the volume of the sugar solution. The cream thus prepared is shaken just before using and from one to five cubic centimeters of it, according to the degree of turbidity of the saccharine solution, are added before the volume in the flask is completed to the mark. After filling the flask to the mark the ball of the thumb is placed over the mouth and the contents well shaken and allowed to stand for a few moments before filtering.

The alumina cream is well suited to use with solutions of commercial sugars of not too low a grade and of most honeys and high grade sirups. It is usually not powerful enough to clarify beet and cane juices, molasses and massecuites.

=85. Basic Lead Acetate.=—A solution of basic lead acetate is an invaluable aid to the sugar analyst in the preparation of samples for polarimetric observation. It acts as a clarifying agent by throwing out of solution certain organic compounds and by uniting with the organic acids in solution forms an additional quantity of precipitate, and these precipitates act also mechanically in removing suspended matters from solution. The action of this reagent is therefore much more vigorous than that of alumina cream. Coloring matters are often precipitated and removed by treatment with lead acetate. It happens therefore that there are few samples of saccharine bodies whose solutions cannot be sufficiently clarified by lead acetate to permit of polarimetric observation.

The reagent is most frequently employed of the following strength:[47] Boil for half an hour in one and a half liters of water 464 grams of lead acetate and 264 grams of litharge with frequent stirring. When cool, dilute with water to two liters, allow to stand until clear, and decant the solution. The specific gravity of this solution is about 1.267.

In a solution of basic lead acetate of unknown strength the percentage of lead acetate may be determined from its specific gravity by the following table:[48]

PERCENTAGE OF LEAD ACETATE CORRESPONDING TO DIFFERENT SPECIFIC GRAVITIES AT 15°.

Percentage of Percentage of Specific gravity. lead acetate. Specific gravity. lead acetate. 1.0127 2 1.2040 28 1.0255 4 1.2211 30 1.0386 6 1.2395 32 1.0520 8 1.2579 34 1.0654 10 1.2768 36 1.0796 12 1.2966 38 1.0939 14 1.3163 40 1.1084 16 1.3376 42 1.1234 18 1.3588 44 1.1384 20 1.3810 46 1.1544 22 1.4041 48 1.1704 24 1.4271 50 1.1869 26

=86. Errors Due to use of Lead Solutions.=—In the use of lead solutions there is danger of errors intruding into the results of the work. These errors are due to various sources. Lead subacetate solution, when used with low grade products, or sugar juices, or sirups from beets and canes, precipitates albuminous matters and also the organic acids present. The bulk occupied by these combined precipitates is often of considerable magnitude, so that on completing the volume in the flask the actual sugar solution present is less than indicated. The resulting condensation tends to give too high a polarimetric reading. With purer samples this error is of no consequence, but especially with low grade sirups and molasses it is a disturbing factor, which must be considered.

One of the best methods of correcting it has been proposed by Scheibler.[49] To 100 cubic centimeters of a solution of the sample, ten of lead solution are added, and after shaking and filtering the polarimetric reading is taken. Another quantity of 100 cubic centimeters of the solution with ten of lead is diluted to 220 cubic centimeters, shaken, filtered, and polarized. Double the second reading, subtract it from the first, multiply the difference by 2.2, and deduct the product from the first reading. The remainder is the correct polarization.

The process just described is for the usual work with beet juices and sirups. For cane juices measured by the graduated pipette, hereafter to be described, and for weighed samples of molasses and massecuites, the following method of calculation is pursued.[50] To the sample dissolved in water, add a measured portion of the lead subacetate solution, make its volume 100 cubic centimeters and observe the polarimetric reading. Prepare a second solution in the same way and make the volume double that of the first and again take the polarimetric reading. Multiply the second reading by two, subtract the product from the first reading and multiply the remainder by two, and subtract the product from the first reading.

Example.—First polarization 30.0 Second polarization 14.9 Then 30 - (2 × 14.9 = 29.8) = 0.2 0.2 × 2 = 0.4 and 30 - 0.4 = 29.6

The corrected reading therefore shows that the sample contained 29.6 per cent of sugar.

=87. Error Due to Action of Lead Subacetate on Levulose.=—In the use of lead subacetate solution not only is there danger of error due to the causes just described, but also to a more serious one, arising from the chemical interaction of the clarifying agent and levulose.[51]

Lead subacetate forms a chemical union with levulose and the resulting compound has a different rotatory power from the left-handed sugar in an uncombined state. By adding a sufficient quantity of subacetate solution, the left-handed rotation of levulose may be greatly diminished if not entirely destroyed. In this case the dextrose, which with levulose forms inverted sugar, serves to increase the apparent right rotation due to the sucrose in solution. The reading of the scale is therefore higher than would be given by the sucrose alone. If the lead subacetate could be added in just the proportion to make the invert sugar neutral to polarized light, its use would render the analysis more accurate; but such a case could only arise accidentally. To correct the error, after clarification, the compound of levulose and lead may be decomposed by the addition of acetic acid according to the method of Spencer. In this case the true content of sucrose can only be obtained by the method of inversion proposed by Clerget, which will be described in another paragraph.

=88. Clarification with Mercuric Compounds.=—Where the disturbing bodies in a solution are chiefly of an albuminoid nature, one of the best methods of securing clarification is by the use of a solution containing an acid mercuric compound.[52] In the case of milk this method is to be preferred to all others. Albuminoid bodies themselves, have the property of deflecting the plane of polarization, as a rule, to the left, and therefore, should be completely removed from solutions containing right-handed sugars such as lactose. For this purpose the mercuric compound is more efficient than any other. It is prepared and used as follows.[53] Dissolve mercury in double its weight of strong nitric acid and dilute the solution with an equal volume of water. One cubic centimeter of this solution is sufficient to clarify fifty times its volume of milk.

=89. Decolorization by Means of Bone-Black.=—Where the means already described fail to make a solution sufficiently colorless to permit of the passage of a ray of polarized light, recourse should be had to a decolorizing agent. The most efficient of these is bone-black. For laboratory work it is finely ground and should be dry if added to an already measured solution. When moist it should be added to the flask before the volume is completed, and a correction made for the volume of the dry char employed. Bone-black has the power of absorbing a certain quantity of sugar, and for this reason as little of it should be employed as is sufficient to secure the end in view. If not more than one gram of the char be used for 100 cubic centimeters of solution, the error is not important commercially. The error may be avoided by placing the char on the filter and rejecting the first half of the filtered solution. The char becomes saturated with the first portion of the solution, and does not absorb any sugar from the second. This method, however, does not secure so complete a decolorization as is effected by adding the black directly to the solution and allowing to stand for some time with frequent shaking.

=90. Remarks on Analytical Process.=—Since large weights of sugar are taken for polarization, a balance which will weigh accurately to one milligram may be used. In commercial work the weighing is made in a counterpoised dish with a prominent lip, by means of which the sample can be directed into the mouth of the flask after partial solution. Where the air in the working room is still, an uncovered balance is most convenient. With a little practice the analyst will be able to dissolve and transfer the sample from the dish to the flask without danger of loss. The source of light used in polarizing should be in another room, and admitted by a circular opening in the partition. In a close polarizing room, which results from the darkening of the windows, the temperature will rapidly rise if a lamp be present, endangering notably the accuracy of the work, and also interfering with the comfort of the observer. The greatest neatness must be practiced in all stages of the work, and especially the trough of the polariscope must be kept from injury which may arise from the leaking of the observation tubes. Dust and dirt of all kinds must be carefully excluded from the lenses, prisms, wedges and plates of the instrument.

=91. Determination of Sucrose by Inversion.=—In the foregoing paragraphs directions have been given for the estimation of sugar (sucrose) by its optical properties. It has been assumed so far, that no other disturbing bodies have been present, save those which could be removed by the clarifying agents described. The case is different when two or more sugars are present, each of which has a specific relation to polarized light. In such cases some method must be used for the optical determination of sucrose, which is independent of the influence of the other polarizing bodies, or else recourse must be had to other methods of analysis. The conversion of the sucrose present into invert sugar by the action of an acid or a ferment, affords an opportunity for the estimation of sucrose in mixed sugars, by purely optical methods. This process rests upon the principle that by the action of a dilute acid for a short time, or of a ferment for a long time, the sucrose is completely changed, while other sugars present are not sensibly affected. Neither of these assumptions is rigidly correct but each is practically applicable.

The sucrose by this process of hydrolysis is converted into an equal mixture of levulose and dextrose. The former, at room temperatures, has the higher specific rotating power, and the deflection of the plane of polarization in a solution of inverted sugar is therefore to the left. The levorotatory power of invert sugar varies with the temperature, and this arises from the optical properties of the levulose. The influence of temperature on the rotating power of other sugars, is not imperceptible in all cases, but in practice is negligible.

This method of analysis is invaluable in control work in factories, in the customs and in agricultural laboratories. Since the rotating power of levulose diminishes as the temperature rises, an accurate thermometric observation must accompany each polarimetric reading. At about 88° the rotatory powers of dextrose and levulose are equal, and a solution of pure invert sugar examined at that degree, is found to be neutral to polarized light.

=92. Clerget’s Method of Inversion.=—The classical method of Clerget for the determination of cane sugar by double polarization before and after inversion, was first described in a memoir presented to the Society of Encouragement for National Industry on the 14th of October, 1846. The following description of the original method is taken from a reprint of the proceedings of that Society, dated Nov. 1846:

Clerget points out first the observation of Mitscherlich regarding the influence of temperature on the rotatory power of invert sugar, and calls attention to the detailed experiments he has made which resulted in the determination of the laws of the variation. From these studies he was able to construct a table of corrections, applicable in the analysis of all saccharine substances in which the cane sugar is polarized before and after inversion. The basis of the law rests upon the observation that a solution of pure sugar, polarizing 100° on the sugar scale, before inversion, will polarize 44° to the left after inversion at a temperature of zero. The quantity of sugar operated upon by Clerget amounted to 16.471 grams in 100 cubic centimeters of liquid. On the instrument employed by him this quantity of sugar in 100 cubic centimeters gave a reading of 100° to the right on the sugar scale when contained in a tube twenty centimeters in length. The process of inversion carried on by Clerget is as follows:

The sugar solution is placed in a flask, marked on the neck at 100 and 110 cubic centimeters; or if smaller quantities are used, in a flask marked on the neck at fifty and fifty-five cubic centimeters. The flask is filled with the sugar solution to the first mark and then a sufficient quantity of strong hydrochloric acid added to bring the volume of the liquid to the second mark. The mouth of the flask is then closed with the thumb and its contents thoroughly mixed by shaking. A thermometer is placed in the flask which is set in a water-bath in such a way that the water comes just above the level of the liquid in the neck of the flask. The water is heated in such a manner as to bring the temperature of the contents of the flask, as determined by the thermometer, exactly to 68° and at such a rate as to require fifteen minutes to reach this result. At the end of fifteen minutes the temperature having reached 68° the flask is removed and placed at once in another water-bath at the temperature of the room, to which temperature the contents of the flask are cooled as rapidly as possible. To make the polarimetric observation a tube twenty-two centimeters in length is filled with the inverted sugar solution by means of a tubulure in its center, which serves not only the purpose of filling the tube but also afterwards to carry the thermometer, by means of which the temperature of observation can be taken. If the sugar solution be turbid, or contain any lead chlorid due to the previous use of basic lead acetate in clarification, it should be filtered before being introduced into the observation tube. This tube being one-tenth longer than the original compensates for the dilution caused by the addition of the hydrochloric acid in inversion.

When reading, the bulb of the thermometer should be withdrawn far enough to permit the free passage of the ray of light and the exact temperature of the solution noted.

The above outline of Clerget’s method of inversion is given in order that the analyst may compare it with any of the variations which he may find in other works. The chief points to which attention is called, are, first, the fact that only a little over sixteen grams of sugar are used for ten cubic centimeters of strong hydrochloric acid, and second, that the time of heating is exactly fifteen minutes, during which time the contents of the flask should be raised from room temperature to exactly 68°.

From the above it is seen that the process of Clerget, as originally described, can be applied directly to all instruments, using approximately sixteen grams of sugar in 100 cubic centimeters. Experience has also shown that even when larger quantities of sugar are employed, as for instance, approximately twenty-six grams, the inversion is effected with practical completeness in the same circumstances. It is advised, therefore, that in all analytical processes, in which cane sugar is to be determined by the process of inversion with an acid, the original directions of Clerget be followed as strictly as possible. Experience has shown that no one of the variations proposed for Clerget’s original method has any practical advantage and the analyst is especially cautioned against those methods of inversion in which the temperature is continued at 68° for fifteen minutes or in which it is allowed to go above that degree.

=93. Influence of Strength of Solution and Time of Heating on the Inversion of Sucrose.=—As has been intimated, the strength of a sugar solution and the time of heating with hydrochloric acid are factors that must be considered in determining a formula for the calculation of sucrose by inversion. The Clerget formula holds good only for the conditions specified and these conditions must be rigidly adhered to in order to secure the proper results. This matter has been thoroughly studied by Bornträger, who also gives a nearly complete bibliography of the subject.[54] As a result of his investigations it seems well established that the original Clerget formula is practically correct for the conditions indicated, Bornträger modifying it only by substituting in the formula 143.66 for 144. This is so nearly the same as the Clerget factor that it is not advisable to substitute it therefor. If, however, the inverted sugar solution be diluted to double its volume before polarization the factor proposed by Landolt, viz., 142.4, gives more nearly accurate results. If the hydrochloric acid be neutralized before polarization by an alkaline body, the character of the salt which is formed also influences, to a greater or less extent, the specific rotatory power of the solution. Hydrochloric acid itself also influences the rotation to a certain degree.[55]

=94. Calculation of Results.=—The percentage of sucrose in a solution which has been polarized before and after inversion is calculated by an appropriate formula from the data obtained or is taken directly from tables. These tables are too long to insert here, and in point of fact the calculation can be made from the formula almost as quickly as the result can be taken from a table.

Two factors are commonly used in the calculations, one based on the supposition that a sugar solution polarizing 100° to the right will, after inversion, give a reading of 44° to the left, at zero temperature. In the second formula in common use the polarization to the left in the circumstances mentioned above is assumed to be 42.4, a number reached by Landolt after a long series of experiments.[56] The principle of the calculation of the percentage of sucrose is based upon the original observation of Clerget to the effect that the algebraic difference of the two readings, divided by 144, less half of the temperature, will give the percentage of sucrose desired. The formula by which this is obtained is

a - b S = -----------. K - t ---- 2

In this formula a is the polarization on the sugar scale before inversion, b the polarization after inversion, K the constant representing the algebraic difference of the two polarizations of pure sugar at 0° and t the temperature of the observation. To K may be assigned the values 144 or 142.4, the one in more common use. In case the polarization, after inversion, is to the left, which is more commonly the case, the sum of the two readings is taken for a - (-b) = a + b; when both polarizations are to the right or left the difference is taken. S is the percentage of sucrose desired.

_Example.—_Let the polarization before inversion be +95 and after inversion -26 and the temperature 20°

95 + 26 Then S= -------- = 121 ÷ 134 = 90.6. 144 - 10

Substituting the value 142.4 for K, the result of the calculation is 91.4.

In high grade sugars, therefore, the difference in the results secured by taking the two values of K amounts to about 1 per cent of sucrose.

For a further discussion of the theory and practice of inversion the reader is referred to the articles of Herles, Herzfeld, and Wohl.[57]

=95. Method Of Lindet.=—Courtonne recommends the method of Lindet for securing the inversion instead of the method of Clerget.[58] Modified by Courtonne, the method is as follows:

Make two or three times the normal weight of sugar dissolved in water to a volume of 200 or 300 cubic centimeters, as the case may be. After thoroughly mixing proceed as follows:

First, to Obtain the Polarization Direct.—Place fifty or 100 cubic centimeters of the prepared solution in a flask marked at fifty and fifty-five or at 100 and 110 cubic centimeters, add a sufficient quantity of lead acetate to secure a complete clarification, make the volume to fifty-five or 110 cubic centimeters, shake thoroughly, filter, and polarize in a 220 millimeter tube.

Second, to Obtain the Rotation after Inversion.—Place twenty cubic centimeters of the original solution, in a flask marked at fifty cubic centimeters, containing five grams of powdered zinc. The flask should be placed in boiling water. Add, little by little so as to avoid a too rapid evolution of hydrogen, ten cubic centimeters of hydrochloric acid made of equal parts of the strongest acid and water. After the operation is terminated, cool to the temperature of the room, make the volume to fifty cubic centimeters, polarize, and determine the rotation. The volume occupied by the zinc which is not dissolved, will be about one-half cubic centimeter, hence the deviation should be multiplied by the factor 2.475 in order to get the true deviation which would have been produced by the pure liquor. We have then:

A = the deviation direct. B = the deviation after inversion. C = the algebraic difference of the deviations.

The amount of sucrose, therefore, would be calculated by the formula of Creydt,[59]

C - 0.493_A_ X = --------------; 0.827

for raffinose the formula would be

A - S Y = ---------, 1.57

in which S is the deviation due to the sucrose present. The solutions inverted in the manner described are absolutely colorless. There is no need of employing bone-black to secure the saccharimetric reading nor does it present any uncertainty. It is thought by Courtonne that this method will soon take the place of the method of Clerget on account of the advantages above mentioned. The method will be somewhat improved by adopting the following suggestions:

1. Instead of allowing any arbitrary number for the volume of the undissolved zinc, decant the liquid, after inversion, into another flask and wash repeatedly with hot water until all trace of sugar is removed from the flask in which the inversion took place.

2. Instead of polarizing in a 200 millimeter tube make the observation in a 500 millimeter tube, which will permit of the reading being made without any correction whatever.

=96. Inversion by Means of Invertase.=—Instead of using acids for the inversion of cane sugar the hydrolysis can be easily effected by means of a ferment derived from yeast. A complete history of the literature and characteristics of this ferment, together with a study of its properties and the various methods of preparing it, has been given by O’Sullivan and Tompson.[60] In the preparation of invertase, the method found most effective is the following:

The yeast is allowed to liquify for at least a month in a fairly warm room without stirring. At the end of this time the surface is removed and any supernatant liquid poured away. The lower sedimentary part is thrown on a quick-acting filter and allowed to drain for two days. To the filtrate, alcohol of specific gravity 0.87 is gradually added to the extent of one and a half times its volume, with continued and vigorous stirring. The process of adding the alcohol and stirring should require about half an hour, after which the mixture is allowed to stand for twenty-four hours to allow the precipitated invertase to settle. The supernatant liquid is poured away and the precipitate washed several times on successive days by decantation with alcohol of 0.92 specific gravity. When the washings become nearly colorless the precipitate is thrown on a filter, allowed to drain, and immediately removed and mixed with a large bulk of alcohol of 0.92 specific gravity. The precipitate is again collected, mixed thoroughly with its own bulk of water, and some alcohol of 0.97 specific gravity, allowed to stand for a few hours and thrown on a filter. The filtrate contains the invertase.

=97. Determination of Activity of Invertase.=—The activity of a solution of invertase, prepared as above, is measured by the number of minutes required for it to reduce to zero the optical power of a solution of 100 times its weight of cane sugar at a temperature of 15°.5. In order to facilitate the action of the invertase, a trace of sulfuric acid is added to the solution. The manipulation is as follows:

Fifty grams of sucrose are dissolved in water and made to a volume of nearly a quarter of a liter and placed in a bath maintained at 15°.5. Half a gram of the invertase is added, the time noted, the solution immediately made up to a quarter of a liter and well shaken. The contents of the flask are poured rapidly into five beakers; the actual quantity in each beaker is not necessarily the same. To each of these beakers, in succession, are added the following amounts of decinormal sulfuric acid, viz., one-tenth, three-tenths, six-tenths, one, and one and four-tenths cubic centimeters. After an hour a small quantity of the solution is taken from beaker No. 3 and the reaction of the invertase stopped by adding a few drops of strong potassium hydroxid and the time of adding this reagent noted. This solution is then read in the polariscope and the percentage of sugar inverted is calculated from the formula C₁₂H₂₂O₁₁ + H₂O = C₆H₁₂O₆ + C₆H₁₂O₆.

The calculation of the amount of cane sugar inverted is based on the formula, (38.4 - d) ÷ 0.518 = p. In this formula d equals the divisions of the sugar scale read on the polariscope; p the percentage of cane sugar inverted; 38.4 the reading on the sugar scale of the original sugar solution and 51.8 the total number of divisions of the cane sugar scale that the polariscope reading would fall through if all the sugar were inverted. The observation tubes used in the polarization are only 100 millimeters in length. After stopping the action of the invertase with potassium hydroxid the solution is allowed to stand for some time before polarization inasmuch as the dextrose formed appears to assume the state of birotation and some time is required for it to reach its normal rotatory power. If the invertase be used in the alcoholic solution a sufficient quantity should be added to be equivalent to 0.01 of the sucrose present. The time which the contents of beaker No. 3 will take to reach optical activity is calculated in a manner described by O’Sullivan and Tompson, but too long to be inserted here.[61] The five beakers mentioned above are examined in succession and the amount of sulfuric acid best suited to the maximum inversion thus determined. This quantity is then used in subsequent hydrolyses with the given sample of invertase.

The action of invertase on sucrose is very rapid at the first and becomes very much slower towards the end. At a temperature of 15°.5 it is advisable to let the solution stand for forty-eight hours in order to be sure that complete inversion has taken place. For this reason the method by inversion by means of invertase is one of no great practical importance, but it may often be useful to the analyst when the employment of an acid is inadmissible.

=98. Inversion by Yeast.=—Owing to the difficulty of preparing invertase, O’Sullivan and Thompson[62] propose to use yeast as the hydrolytic agent, as first suggested by Kjeldahl. It is shown that in the use of yeast it is not necessary to employ thymol or any other antiseptic. The method of procedure is as follows: The cane sugar solution of usual strength should not be alkaline, but, if possible, should be exactly neutral. If there be any ferment suspected, the temperature should be momentarily raised to 80° to destroy its activity. The polariscopic reading of the solution is then taken at 15°.5 and the amount of copper reduced by the solution should also be determined.

Fifty cubic centimeters of the solution are poured into a beaker and raised to a temperature of 55° in a constant temperature bath. Some brewers yeast amounting to about one-tenth of the total amount of sugar to be inverted, pressed in a towel, is thrown into the hot solution and the whole stirred until mixture is complete. The solution is left for four hours in the water-bath, at the end of this time it is cooled to 15°.5, a little freshly precipitated aluminum hydroxid added, and the volume made to 100 cubic centimeters. A portion of this solution is filtered and its polariscopic reading observed. The solution is then left till the next day, when another polariscopic reading is taken in order to prove that inversion is complete. The copper reducing power is also determined. The method of calculating the results is the same as when invertase is used. The following formulas are employed.

a = the number of divisions indicated by the polariscopic reading for a 200 millimeter tube:

aʹ = the same number after inversion:

m = the number of the divisions of the polariscopic scale which 200 millimeters of the sugar solution containing one gram of cane sugar per 100, alter at 15°.5 on being inverted: In the case of the ventzke polarimeter scale, one gram of cane sugar in 100 cubic centimeters, indicates +3.84 divisions and after inversion it gives -1.34 div. In experiments of this kind, therefore, m = 5.18.

P = the weight of cane sugar present in 100 cubic centimeters of the original solution:

The formula employed then is

a - 2_a_ʹ P = -----------. m

For the copper reduction data the following are used:

G = the weight of 100 cubic centimeters of the original solution:

Gʹ; = the same for the inverted solution: Allowance must be made here both for the dilution and for the 5 per cent increase of the inverted sugar, but the latter number is so small that it need not be calculated accurately.

w = the weight of the original solution used for the estimation:

wʹ = the same factor for the inverted solution:

k = the weight of cupric oxid reduced by w:

kʹ = the same factor for wʹ:

p = the weight of cane sugar present in 100 cubic centimeters of the original solution: The formula to be employed then is

Gʹ kʹ G k p = 0.4308(2 ------ - -----). wʹ w

This method has been applied to the estimation of cane sugar in molasses, apple juices and other substances. It is recommended by the authors as a simple and accurate means of estimating sucrose in all solutions containing it. The methods of making the copper reductions will be given hereafter.

=99. Application of the Process.=—In practice the process of inversion is used chiefly in the analysis of molasses and low grade massecuites. In approximately pure sugars the direct polarization is sufficiently accurate for all practical purposes. In molasses resulting from the manufacture of beet sugar are often found considerable quantities of raffinose, and the inversion process has been adapted to that character of samples. In molasses, in sugar cane factories, the disturbing factors are chiefly invert sugars and gums. The processes used for molasses will be given in another paragraph. In certain determinations of lactose the process of inversion is also practiced, but in this case the lactose is converted into dextrose and galactose, and the factors of calculation are altogether different. The process has also been adapted by McElroy and Bigelow to the determination of sucrose in presence of lactose, and this method will be described further on. In general the process of inversion is applicable to the determination of sucrose in all mixtures of other optically active bodies, which are not affected by the methods of inversion employed.

=100. Determination of Sucrose and Raffinose.=—Raffinose is a sugar which often occurs in beets, and is found chiefly in the molasses after the chief part of the sucrose has been removed by crystallization. It is also found in many seeds, notably in those of the cotton plant. In a pure solution of sucrose and raffinose, both sugars may be determined by the inversion method of Creydt.[63] The inversion is effected by means of hydrochloric acid in the manner described by Clerget. The following formulas are calculated for a temperature of observation of 20°, and the readings should be made as near that temperature as possible.

C - 0.493_A_ (1) S = -------------- 0.827

A - S 6 (2) R = --------- = 1.017_A_ - ------. 1.57 1.298

In these formulas S and R are the respective per cents of sucrose and raffinose desired, A the polarization in sugar degrees before inversion, B the polarization after inversion read at 20°, and C is the algebraic difference between A and B. It must be understood that these formulas are applicable only to a solution containing no other optically active substances, save sucrose and raffinose.

=101. Specific Rotatory Power.=—In order to compare among themselves the rotations produced on a plane of polarized light by different optically active bodies in solution, it is convenient to refer them all to an assumed standard. The degree of rotation which the body would show in this condition, is found by calculation, since, in reality, the conditions assumed are never found in practice. In the case of sugars and other optically active bodies, the standard of comparison is called the specific rotatory power. This factor in any given case, is the angular rotation which would be produced by any given substance in a pure anhydrous state if it were one decimeter in length and of a specific gravity equal to water. These are conditions which evidently do not exist in the case of sugars, since crystalline sugar particles have no polarizing power, and it would be impossible to pass a ray of light through an amorphous sugar column of the length specified. The specific rotatory power is therefore to be regarded as a purely theoretical factor, calculated from the actual data obtained by the examination of the solution of any given substance. If the length of the observation tube in decimeters be represented by l, the percentage of the polarizing body in 100 grams by p, and the specific gravity of the solution by d, and the observed angle of rotation by a, then the factor is calculated from the formula:

a. 100 [a]_{Dj} = ---------------. p. d. l.

The symbols Dj refer to the character of light employed, D indicating the monochromatic sodium flame, and j the transition tint from white light.

If the weight of the polarizing body c be given or known for 100 cubic centimeters of the solution the formula becomes

a. 100 [a]_{Dj} = ----------. c. l.

The latter formula is the one easier of application since it is only necessary in applying it to dissolve a given weight of the active body in an appropriate solvent and to complete the volume of the solution exactly to 100 cubic centimeters. It is therefore unnecessary in this case to determine the specific gravity.

=102. Formulas for Calculating Specific Rotatory Power.=—In order to determine the specific rotatory power (gyrodynat[64]) of a given substance it is necessary to know the specific gravity and percentage composition or concentration of its solution, and to examine it with monochromatic polarized light in an instrument by which the angular rotation can be measured. The gyrodynat of any body changes with its degree of concentration, in some cases with the temperature, and always with the color of the light. With the red rays the gyrodynat is least and itprogressively increases as the violet end of the spectrum is approached. In practice the yellow ray of the spectrum has been found most convenient for use, and in the case of sugars the gyrodynat is always expressed either in terms of this ray or if made with color compensating instruments in terms of the sensitive or transition tint. In the one case the symbol used is (a)_{D} and in the other (a)_{j}. From this statement it follows that (a)_{D} is always numerically less than (a)_{j}. Unless otherwise specified the gyrodynat of a body is to be considered as determined by yellow monochromatic light, and therefore corresponds to _a__{D}.[65]

=103. Variations in Specific Rotatory Power.=—The gyrodynat of any optically active body varies with the nature of the solvent, the strength of the solution, and the temperature.[66]

Since water is the only solvent of importance in determining the gyrodynat of sugars it will not be necessary here to discuss the influence of the nature of the solvent. In respect of the strength of the solution it has been established that in the case of cane sugar the gyrodynat decreases while with dextrose it increases with the degree of concentration. The influence of temperature on the gyrodynat of common sugars is not of great importance save in the case of levulose, where it is the most important factor, the gyrodynat rapidly increasing as the temperature falls. It is of course understood that the above remarks do not apply to the increase or decrease in the volume of a solution at changed temperatures. This influence of temperature is universally proportional to the change of volume in all cases, and this volumetric change is completely eliminated when the polarizations are made at the temperatures at which the solutions are completed to standard volumes.

=104. Gyrodynatic Data for Common Sugars.=—In the case of cane sugar the gyrodynat for twenty-five grams of sugar in 100 grams of solution at 20° is [a]_{D} = 66°.37. This is about the degree of concentration of the solutions employed in the shadow lamplight polariscopes. For seventeen grams of sugar in 100 grams of solution the number is [a]_{D} = 66°.49. This is approximately the degree of concentration for the laurent instrument.

For any degree of concentration according to Tollens the gyrodynat may be computed by the following formula: [a]_{D} = 66°.386 + 0.015035_p_ - 0.0003986_p_², in which p is the number of grams of sugar in 100 grams of the solution.[67] In the table constructed by Schmitt the data obtained are as follows:

In 100 parts by weight Specific Rotation a of solution. gravity Concentration for 100 mm. Sugar p. Water q. at 20° C. d. c = pd. at 20° C. [a]_{D}. 64.9775 35.0225 1.31650 85.5432 56°.134 65°.620 54.9643 45.0357 1.25732 69.1076 45°.533 65°.919 39.9777 60.0223 1.17664 47.0392 31°.174 66°.272 25.0019 74.9981 1.10367 27.5938 18°.335 66°.441 16.9926 83.0074 1.06777 18.1442 12°.064 66°.488 9.9997 90.0003 1.03820 10.3817 6°.912 66°.574 4.9975 95.0025 1.01787 5.0868 3°.388 66°.609 1.9986 98.0014 1.00607 2.0107 1°.343 66°.802

=105. Bi-Rotation.=—Some sugars in fresh solution show a gyrodynat much higher than the normal, sometimes lower. The former phenomenon is called bi- the latter semi-rotation. Dextrose shows birotation in a marked degree, also maltose and lactose. After standing for a few hours, or immediately on boiling, solutions of these sugars assume their normal state of rotation. The addition of a small quantity of ammonia also causes the birotation to disappear.[68] This phenomenon is doubtless due to a certain molecular taxis, which remains after solution is apparently complete. The groups of molecules thus held in place have a certain rotatory power of their own and this is superadded to that of the normal solution. After a time, under the stress of the action of the solvent, these groups are broken up and the solution then assumes its normal condition.

=106. Gyrodynat of Dextrose.=—The gyrodynat of dextrose, as has already been mentioned, increases with the degree of concentration, thus showing a property directly opposite that of sucrose.

The general formula for the anhydrous sugar is [a]_{D} = 52.°718 + 0.017087_p_ + 0.0004271_p_². In this formula p represents the grams of dextrose in 100 grams of the solution. In a ten per cent solution the gyrodynat of dextrose is therefore nearly exactly [a]_{D}20° = 53°. As calculated by Tollens the gyrodynats corresponding to several degrees of concentration are shown in the following table:

p = grams in 100 [a]_{D}20° calculated for grams of solution. anhydrous dextrose. 7.6819 52°.89 9.2994 52°.94 9.3712 52°.94 10.0614 52°.96 10.6279 52°.98 12.9508 53°.05 18.6211 53°.25 31.6139 53°.83 40.7432 54°.34 43.9883 54°.54 53.0231 55°.17 82.6111 57°.80

=107. Gyrodynats of Other Sugars.=—Of the other sugars it will be sufficient to mention only levulose, maltose, lactose, and raffinose. For complete tables of gyrodynatic powers the standard books on carbohydrates may be consulted.[69]

The gyrodynat of levulose is not definitely established. At 14° the number is nearly expressed by [a]_{D}14° = -93°.7.

Invert sugar, which should consist of exactly equal molecules of dextrose and levulose, has a gyrodynat expressed by the formula [a]_{D}0° = -27°.9, with a concentration equivalent to 17.21 grams of sugar in 100 cubic centimeters. The gyrodynat decreases with increase of temperature, according to the formula [a]_{D}t° = -(27°.9 - 0.32_t_°). According to this formula the solution is neutral to polarized light at 87°.2, and this corresponds closely to the data of experiment.

Maltose, in a ten per cent solution at 20°, shows a gyrodynat of [a]_{D}20° = 138°.3.

The general formula for other degrees of concentration is [a]_{D} = 140°.375 - 0.01837_p_ - 0.095_t_, in which p represents the number of grams in 100 grams of the solution and t the temperature of observation.

In the case of lactose [a]_{D} = 52°.53, and this number does not appear to be greatly influenced by the degree of concentration; but is somewhat diminished by a rising temperature.

The gyrodynat of raffinose in a ten per cent solution is [a]_{D} = 104°.5.

CHEMICAL METHODS OF ESTIMATING SUGARS.

=108. General Principles.=—The methods for the chemical estimation of sugars in common use depend on the reducing actions exerted on certain metallic salts, whereby the metal itself or some oxid thereof, is obtained. The reaction is either volumetric or the resulting oxid or metal may be weighed. The common method is, therefore, resolved into two distinct processes, and each of these is carried out in several ways. Not all sugars have the faculty of exerting a reducing action on highly oxidized metallic salts and the most common of them all, viz., sucrose is practically without action. This sugar, however, by simple hydrolysis, becomes reducing, but the two components into which it is resolved by hydrolytic action do not reduce metallic salts in the same proportion. Moreover, in all cases the reducing power of a sugar solution is largely dependent on its degree of concentration, and this factor must always be taken into consideration. Salts of copper and mercury are most usually selected to measure the reducing power of a sugar and in point of fact copper salts are almost universally used. Copper sulfate and carbonate are the salts usually employed, and of these the sulfate far more frequently, but after conversion into tartrate. Practically, therefore, the study of the reducing action of sugar as an analytical method will be confined almost exclusively to the determination of its action on copper tartrate.

Direct gravimetric methods are also practiced to a limited extent in the determination of sugars as in the use of the formation of sucrates of the alkaline earths and of the combinations which certain sugars form with phenylhydrazin. Within a few years this last named reaction has assumed a marked degree of importance as an analytical method. The most practical treatment of this section, therefore, for the limited space which can be given it, will be the study of the reducing action of sugars, both from a volumetric and gravimetric point of view, followed by a description of the best approved methods of the direct precipitation of sugars by such reagents as barium hydroxid and phenylhydrazin.

VOLUMETRIC METHODS.

=109. Classification.=—Among the volumetric methods will be given those which are in common use or such as have been approved by the practice of analysts. Since the use of mercuric salts is now practiced to a limited extent, only a brief study of that process will be attempted. With the copper methods a somewhat extended description will be given of those depending on the use of copper sulfate, and a briefer account of the copper carbonate process.

In the copper sulfate method two distinct divisions must be noted, viz., first an indirect process depending first upon the reduction of the copper to a suboxid, the subsequent action of this body on iron salts, measured finally by titration with potassium permanganate; and second, a direct process determined either by the disappearance of the blue color from the copper solution, or by the absence of copper from a drop of the solution withdrawn and tested with potassium ferrocyanid. This last mentioned reaction is one which is found in common use. The volumetric methods are not, as a rule, as accurate as the gravimetric, depending on weighing the resultant metal, but they are far more rapid and well suited to technical control determinations.

=110. Reduction of Mercuric Salts.=—The method of determining sugar by its action on mercuric salts, is due to Knapp.[70] The method is based on the observation that dextrose and other allied sugars, will reduce an alkaline solution of mercuric cyanid, and that the mercury will appear in a metallic state.

The mercuric liquor is prepared by adding to a solution of ten grams of mercuric cyanid, 100 cubic centimeters of a solution of caustic soda of 1.145 specific gravity, and making the volume to one liter with water. The solution of sugar to be titrated, should be as nearly as possible of one per cent strength.

To 100 cubic centimeters of the boiling solution, the sugar solution is added in small portions from a burette and in such a way as to keep the whole mass in gentle ebullition.

To determine when all the mercuric salt has been decomposed, a drop of the clear boiling liquid is removed and brought into contact with a drop of stannous chlorid solution on a white surface. A brownish black coloration or precipitate will indicate that the mercury is not all precipitated. Fresh portions of the sugar must then be added, until no further indication of the presence of mercury is noted. The approximate quantity of sugar solution required to precipitate the mercury having thus been determined, the process is repeated by adding rapidly, nearly the quantity of sugar solution required, and then only a few drops at a time, until the reduction is complete.

One hundred cubic centimeters of the mercuric cyanid solution prepared as directed above, will be completely reduced by

202 milligrams of dextrose, 200 ” ” invert sugar, 198 ” ” levulose, 308 ” ” maltose, 311 ” ” lactose.

By reason of the unpleasant odor of the boiling mercuric cyanid when in presence of a reducing agent, the process should be conducted in a well ventilated fume chamber. With a little practice the process is capable of rapid execution, and gives reasonably accurate results.

=111. Sachsse’s Solution.=—The solution of mercuric salts proposed by Sachsse, is made by dissolving eighteen grams of mercuric iodid in twenty-five cubic centimeters of an aqueous solution of potassium iodid. To this solution are added 200 cubic centimeters of potash lye, containing eighty grams of caustic potash. After mixing the solution, the volume is completed to one liter. The sugar solutions used to reduce this mixture, should be more dilute than those employed with the mercuric cyanid, and should not be over one-half per cent in strength. The end of the reduction is determined as already described. After a preliminary trial, nearly all the sugar necessary to complete reduction, should be added at once, and the end of the reduction then determined by the addition of successive small quantities. One hundred cubic centimeters of the mercuric iodid solution prepared as directed above, require the following quantities of sugar to effect a complete reduction:

325 milligrams of dextrose, 269 ” ” invert sugar, 213 ” ” levulose, 491 ” ” maltose, 387 ” ” lactose.

By reason of the great difference between the reducing power of dextrose and levulose in this solution, it has been used in combination with the copper reduction method, to be described, to determine the relative proportion of dextrose and levulose in a mixture.[71]

It is now known that copper solutions require slightly different quantities of dextrose, levulose, or invert sugar to effect complete reduction, but the variations are not great and in the calculation above mentioned, it may be assumed that these differences do not exist.

Instead of using stannous chlorid as an indicator, the end of the reaction may be determined as follows: A disk of filtering paper is placed over a small beaker containing some ammonium sulfid. A drop of the clear hot solution is placed on this disk, and if salts of mercury be still present a dark stain will be produced; or a drop of the ammonium sulfid may be brought near the moist spot formed by the drop of mercury salt. An alkaline solution of zinc oxid may also be used.

The methods depending on the use of mercuric salts have, of late, been supplanted by better processes, and space will not be given here to their further discussion.

=112. The Volumetric Copper Methods.=—The general principle on which these methods depend, is found in the fact that certain sugars, notably, dextrose, (glucose), levulose, (fructose), maltose and lactose, have the property of reducing an alkaline solution of copper to a lower state of combination, in which the copper is separated as cuprous oxid. The end of the reaction is either determined by the disappearance of the blue color of the solution, or by the reaction produced by a drop of the hot filtered solution, when placed in contact with a drop of potassium ferrocyanid acidified with acetic.

The copper salt which is found to give the most delicate and reliable reaction, is the tartrate. The number of volumetric processes proposed and which are in use, is very great, and an attempt even to enumerate all of these can not be made in this volume. A few of the most reliable and best attested methods will be given, representing if possible, the best practice in this and other countries. The rate of reduction of the copper salt to suboxid, is influenced by the rate of mixing with the sugar solutions, the temperature, the composition of the copper solution and the strength of the sugar solution.

The degree of reduction is also modified by the rate at which the sugar solution is added, and by the degree and duration of heating, and all these variables together, make the volumetric methods somewhat difficult and their data, to a certain extent, discordant. By reason, however, of the ease with which they are applied and the speed of their execution, they are invaluable for approximately correct work and for use in technical control.

=113. Historical.=—It is not the purpose in this paragraph to trace the development of the copper reduction method for the determination of reducing sugars, but only to refer to the beginning of the exact analytical application of it.

Peligot, as early as 1844, made a report to the Society for the Encouragement of National Industry on methods proposed by Barreswil and Fromherz for the quantitive estimation of sugar by means of copper solution.[72] These methods were based on the property of certain sugars to reduce alkaline copper solution to a state of cuprous oxid first announced by Trommer.[73] This was followed by a paper by Falck on the quantitive determination of sugar in urine.[74]

In 1848 the methods, which have been proposed, were critically examined by Fehling, and from the date of his paper the determination of sugar by the copper method may be regarded as resting on a scientific basis.[75]

Since the date mentioned the principal improvements in the process have been in changing the composition of the copper solution in order to render it more stable, which has been accomplished by varying the proportions of copper sulfate, alkali and tartaric acid. For the better keeping of the solution the method of preserving the copper sulfate and the alkaline tartrates in separate flasks and only mixing them at the time of use has been found very efficacious.[76] For testing for the end of the reaction by means of an acetic acid solution of potassium ferrocyanid the filtering tube suggested by the author, the use of which will be described further on, has proved quite useful. Pavy has suggested that by the addition of ammonia to the copper solution the precipitated suboxid may be kept in solution and the end of the reaction thus easily distinguished by the disappearance of the blue color.[77] Allen has improved on this method by covering the hot mixture with a layer of paraffin oil whereby any oxidation of the suboxid is prevented.[78]

The introduction and development of the gravimetric process depending on securing the reduced copper oxid in a metallic state as developed by Allihn, Soxhlet, and others, completes the resumé of this brief sketch of the rise and development of the process.

=114. Action of Alkaline Copper Solution on Dextrose.=—The action to which dextrose and other reducing sugars are subjected in the presence of a hot alkaline copper solution is two-fold in its nature. In the first place there is an oxidation of the sugar which is transformed into tartronic, formic and oxalic acids, the two latter in very small quantities. At the same time another part of the sugar is attacked directly by the alkali and changed to complex products among which have been detected lactic, oxyphenic and oxalic acids, also two bodies isomeric with dioxyphenolpropionic acid. When the sugar is in large excess melassic and glucic acids have also been detected. The glucic acid may be regarded as being formed by simple dehydration but becomes at once resolved into pyrocatechin and gluconic acid according to the reaction C₁₂H₁₈O₉ = C₆H₆O₂ + C₅H₁₂O₇. The gluconic acid also is decomposed and gives birth to lactic and glyceric acids according to the formula C₆H₁₂O₇ = C₃H₆O₃ + C₃H₆O₄. The glyceric acid also in the presence of a base is changed into lactic and oxalic acids. Between lactic acid and pyrocatechin, existing in a free state, there is produced a double reciprocal etherification in virtue of which there arise two ethers isomeric with hydrocaffeic acid, C₉H₁₀O₄. One of these bodies is an acid and corresponds to the constitution

CH₃ / O ── CH / \ C₆H₄ CO₂H (2) \ OH (2)

and the other is of an alcoholic nature corresponding to the formula

CO₂ ── CHOH ── CH₃ (1) / C₆H₄ \ OH₂ (2)

Of all these products only oxyphenic and lactic acids and their ethers and oxalic acid remain unchanged and they can be isolated. All the others are transformed in an acid state and they can only be detected by operating in the presence of metallic oxids capable of precipitating them at the time of their formation.[79]

=115. Fehling’s Solution.=—The copper solution which has been most used in the determination of reducing sugars is the one proposed by Fehling as a working modification of the original reagent used by Trommer.[80]

Following is the formula for the preparation of the fehling solution:

Pure crystallized copper sulfate CuSO₄.5H₂O, 34.64 grams: Potassium tartrate, 150.00 ” Sodium hydroxid, 90.00 ”

The copper sulfate is dissolved in water and the potassium tartrate in the aqueous solution of the sodium hydroxid which should have a volume of about 700 cubic centimeters. The two solutions are mixed and the volume completed to a liter. Each cubic centimeter of this solution will be reduced by five milligrams of dextrose, equivalent to four and a half milligrams of sucrose.

The reaction which takes place is represented by the following molecular proportions:

C₆H₁₂O₆ = 10CuSO₄.5H₂O Dextrose. Copper sulfate. 180 2494

Fehling’s solution is delicate in its reactions but does not keep well, depositing cuprous oxid on standing especially in a warm place exposed to light. The fehling liquor was soon modified in its constitution by substituting 173 grams of the double sodium and potassium tartrate for the neutral potassium tartrate first used, and, in fact, the original fehling reagent contained forty grams of copper sulfate instead of the quantity mentioned above. Other proportions of the ingredients are also given by many authors as fehling solution.

=116. Comparison of Copper Solutions for Oxidizing Sugars.=—For the convenience of analysts there is given below a tabular comparison of the different forms of fehling liquor which have been proposed for oxidizing sugars. The table is based on a similar one prepared by Tollens and Rodewald, amended and completed by Horton.[81] The solutions are arranged alphabetically according to authors’ names:

1. Allihn:

34.6 grams copper sulfate, solution made up to half a liter; 173 grams potassium-sodium tartrate; 125 grams potassium hydroxid (equivalent to 89.2 grams sodium hydroxid) solution made up to half a liter.

2. A. H. Allen:

34.64 grams copper sulfate, solution made up to 500 cubic centimeters; 180 grams potassium-sodium tartrate; 70 grams sodium hydroxid (not less than 97° NaOH), solution made up to half a liter.

3. Bödeker:

34.65 grams copper sulfate; 173 grams potassium-sodium tartrate; 480 cubic centimeters sodium hydroxid solution, 1.14 specific gravity; 67.3 grams sodium hydroxid; fill to one liter; 0.180 gram grape sugar reduces according to Bödeker, 36.1 cubic centimeters of the copper solution = 0.397 gram copper oxid. The same quantity of milk sugar reduces, however, only twenty-seven cubic centimeters copper solution = 0.298 copper oxid.

4. Boussingault:

40 grams copper sulfate; 160 grams potassium tartrate; 130 grams sodium hydroxid.

5. Dietzsch:

34.65 grams copper sulfate; 150 grams potassium-sodium tartrate; 250 grams sodium hydroxid solution, 1.20 specific gravity; 150 grams glycerol.

6. Fleischer:

69.278 grams copper sulfate dissolved in about half a liter of water, add to this 200 grams tartaric acid; fill to one liter with concentrated sodium hydroxid solution; twenty cubic centimeters copper solution = forty cubic centimeters sugar solution, that contain in every cubic centimeter five milligrams grape sugar.

7. Fehling:

40 grams copper sulfate; 160 grams di-potassium tartrate = 600-700 cubic centimeters sodium hydroxid solution, 1.12 specific gravity, or from 54.6 to 63.7 grams sodium hydroxid, fill to 1154.4 cubic centimeters.

8. Gorup-Besanez:

34.65 grams copper sulfate; 173 grams potassium-sodium tartrate; 480 cubic centimeters sodium hydroxid solution, 1.14 specific gravity; equal 67.3 sodium hydroxid. Fill to one liter.

9. Grimaux:

40 grams copper sulfate; 160 grams potassium-sodium tartrate; 600-700 cubic centimeters sodium hydroxid solution, 1.20 specific gravity, equal to 92.5-107.9 grams sodium hydroxid. Fill to 1154.4 cubic centimeters. Ten cubic centimeters of this solution are completely decolorized by 0.050 gram glucose.

10. Holdefleis:

34.632 grams copper sulfate in one liter of water; 125 grams potassium hydroxid, equivalent to 89.2 grams sodium hydroxid; 173 grams potassium-sodium tartrate. Fill to one liter.

11. Hoppe-Seyler:

34.65 grams copper sulfate; 173 grams potassium-sodium tartrate; 600-700 cubic centimeters sodium hydroxid solution, 1.12 specific gravity; equal to 63.0-73.5 grams potassium hydroxid. Fill to one liter. One cubic centimeter is reduced by exactly 0.005 gram grape sugar.

12. Krocker:

6.28 grams copper sulfate; 34.6 grams potassium-sodium tartrate; 100 cubic centimeters sodium hydroxid solution, 1.14 specific gravity. Fill to 200 cubic centimeters. In 100 cubic centimeters of this solution is contained 0.314 gram copper sulfate, which is reduced by 0.050 grape sugar.

13. Liebermann:

4 grams copper sulfate; 20 grams potassium-sodium tartrate; 70 grams sodium hydroxid solution, 1.12 specific gravity. Fill to 115.5 cubic centimeters.

14. Löwe:

15 grams copper sulfate; 60 grams glycerol; 80 cubic centimeters sodium hydroxid, 1.34 specific gravity; 160 cubic centimeters water. Fill to half a liter.

15. Mohr:

34.64 copper sulfate; 150 grams di-potassium tartrate; 600-700 cubic centimeters sodium hydroxid solution, 1.12 specific gravity, equal to 70.5-82.3 grams sodium hydroxid. Fill to one liter.

16. Märcker:

35 grams copper sulfate, solution made up to one liter: 175 grams potassium-sodium tartrate; 125 grams potassium hydroxid, equivalent to 89.2 grams sodium hydroxid, solution made up to one liter.

17. Maumenè:

375 grams copper sulfate; 188 grams potassium-sodium tartrate; 166 grams potassium hydroxid. Fill to nine liters.

18. Monier:

40 grams copper sulfate; 3 grams stannic chlorid; 80 grams cream of tartar; 130 grams sodium hydroxid. Fill to one liter.

19. Neubauer and Vogel:

34.639 grams copper sulfate; 173 grams potassium-sodium tartrate; 500-600 grams sodium hydroxid solution, 1.12 specific gravity. Fill to one liter.

20. Pasteur:

40 grams copper sulfate; 105 grams tartaric acid; 80 grams potassium hydroxid; 130 grams sodium hydroxid.

21. Possoz:

40 grams copper sulfate; 300 grams potassium-sodium tartrate; 29 grams sodium hydroxid; 159 grams sodium bicarbonate, allow to stand six months before use. Fill to one liter. One cubic centimeter equals 0.0577 gram dextrose. One cubic centimeter equals 0.0548 gram cane sugar.

22. Rüth:

34.64 grams copper sulfate; 143 grams potassium-sodium tartrate; 600-700 cubic centimeters sodium hydroxid solution, 1.12 specific gravity. Fill to one liter.

23. Rodewald and Tollens:

34.639 grams copper sulfate, solution made up to half a liter; 173 grams potassium-sodium tartrate; 60 grams sodium hydroxid, solution made up to half a liter.

24. Schorlemmer:

34.64 grams copper sulfate; 200 grams potassium-sodium tartrate; 600-700 cubic centimeters sodium hydroxid solution, 1.20 specific gravity. Fill to one liter.

25. Soxhlet:

34.639 grams copper sulfate, solution made up to half a liter. 173 grams potassium-sodium tartrate; 51.6 grams sodium hydroxid, solution made up to half a liter.

26. Soldaini:

3.464 grams copper sulfate; 297 grams potassium bicarbonate. Fill to one liter.

27. Violette:

34.64 grams copper sulfate; 187.0 potassium-sodium tartrate; 78.0 sodium hydroxid made up to one liter. Ten cubic centimeters equal 0.050 gram dextrose. Ten cubic centimeters equal 0.0475 gram cane sugar.

=117. Volumetric Method used in this Laboratory.=—The alkaline copper solution preferred in this laboratory has the composition proposed by Violette. The copper sulfate and alkaline tartrate solutions are kept in separate vessels and mixed in proper proportions immediately before use, and diluted with about three volumes of water. The reduction is accomplished in a long test tube at least twenty-five centimeters in length, and from thirty-five to forty millimeters in diameter.

The sugar solution employed should contain approximately one per cent of reducing sugar. If it should have a greater content it should be reduced with water to approximately the one named. If it have a less content, it should be evaporated in a vacuum at a low temperature until it reaches the strength mentioned above. A preliminary test will indicate almost the exact quantity of the sugar solution to be added to secure a complete reduction of the copper. This having been determined the whole quantity should be added at once to the boiling copper solution, the test tube held in the open flame of a lamp giving a large circular flame and the contents of the tube kept in brisk ebullition for just two minutes. The lamp is withdrawn and the precipitated suboxid allowed to settle. If a distinct blue color remain an additional quantity of the sugar solution is added and again boiled for two minutes. When the blue coloration is no longer distinct, the presence or absence of copper is determined by aspirating a drop or two of the hot solution with the apparatus described below. This clear filtered liquor is then brought into contact with a drop of potassium ferrocyanid solution acidulated with acetic. The production of a brown precipitate or color indicates that some copper is still present, in which case an additional quantity of the sugar solution is added and the operation continued as described above until after the last addition of sugar solution no coloration is produced.

=118. The Filtering Tube.=—The filtering tube used in the above operation is made of a long piece of narrow glass tubing with thick walls. The length of the tube should be from forty to forty-five centimeters. One end of the tube being softened in the flame is pressed against a block of wood so as to form a flange. Over this flange is tied a piece of fine linen.[82]

Instead of using a linen diaphragm the tube is greatly improved, as suggested by Knorr, by sealing into the end of the tube while hot a perforated platinum disk. Before using, the tube is dipped into a vessel containing some suspended asbestos felt and by aspiration a thin felt of asbestos is formed over the outer surface of the platinum disk. By inverting the tube the water which has entered during aspiration is removed. The tube thus prepared is dipped into the boiling solution in the test tube above described and aspiration continued until a drop of the liquor has entered the tube. It is then removed from the boiling solution, the asbestos felt wiped off with a clean towel, and the drop of liquor in the tube blown through the openings in the platinum disk and brought into contact with a drop of potassium ferrocyanid in the usual way. In this way a drop of the liquor is secured without any danger of a reoxidation of the copper which may sometimes take place on cooling.

The careful analyst by working in this way with the volumetric method is able to secure highly accurate results. The apparatus used is shown in the accompanying illustration.

=119. Suppression of the Error Caused by the Action of the Alkali on Reducing Sugars.=—Three methods are proposed by Gaud for correcting or suppressing the error due to the action of the alkali upon reducing sugars. In the first place, the common method followed may be employed, depending upon the use of an alkaline copper solution of known composition and the employment of a reducing sugar solution of a strength varying between one-half and one per cent. The error which is introduced into such a reaction is a constant one and the solution having been tested once for all against pure sugar is capable of giving fairly accurate results.

In the second place, a table may be constructed in which the error is determined for sugar solutions for varying strengths, viz., from one-tenth of one per cent to ten per cent. If y represent the error and x the exact percentage of reducing sugar present then the correction may be made by the following formula;

y = -0.00004801_x_ + 0.02876359_x_².

In order to use this formula in practice the percentage of reducing sugar obtained by the actual analysis must be introduced and may be represented by θ. The formula for correction then becomes 0.02876_x_² - 1.000048_x_ + θ = 0; whence the value of x is easily computed.

In the third place, the error may be eliminated by substituting for an alkali which acts upon the glucose one which does not, viz., ammonia. At the temperature of boiling water ammonia does not have any decomposing effect upon reducing sugars. It is important, however, that the reduction take place in an inert atmosphere in order to avoid the oxidation of the dissolved cuprous oxid and the temperature need not be carried beyond 80°. The end of the reaction can be easily distinguished in this case by the disappearance of the blue color. When one reaction is finished the copper may be completely reoxidized by conducting through it a current of air or oxygen for half an hour, when an additional quantity of ammonia may be added to supply any that may have evaporated, and a new reduction accomplished with exactly the same quantity of copper as was used in the first. The solution used by Gaud contains 36.65 grams of crystallized copper sulfate dissolved in water and the volume completed to one liter with ordinary aqueous ammonia.[83]

=120. Permanganate Process for the Estimation of Reducing Sugars.=—Dextrose, invert sugar, and other reducing sugars can also be determined with a fair degree of accuracy by an indirect volumetric process, in which a standard solution of potassium permanganate is used as the final reagent.[84] The principle of the process is based upon the observation that two molecules of dextrose reduce from an alkaline cupric tartrate solution five molecules of cuprous oxid. The five molecules of cuprous oxid thus precipitated when added to an acid solution of ferric sulfate, will change five molecules of the ferric sulfate to ten molecules of ferrous sulfate. The reaction is illustrated by the following equation:

{ 5Cu₂O } + { 5Fe₂(SO₄)₃ } + { 5H₂SO₄ } = { 10CuSO₄ } {715 parts} { 2000 parts } {490 parts} {1595 parts}

+ { 10FeSO₄ } + { 5H₂O } {1520 parts} {90 parts}

The ten molecules of ferrous sulfate formed as indicated in the above reaction, are reoxidized to ferric sulfate by a set solution of potassium permanganate. This reaction is illustrated by the equation given below:

{ 10FeSO₄ } + { K₂Mn₂O₈ } + { 5H₂SO₄ } = { 5Fe₂(SO₄)₂ } {1520 parts} {316.2 parts} {784 parts} { 2000 parts }

+ { 2MnSO₄ } + { K₂SO₄ } + { 8H₂O } {302 parts} {174.2 parts} {144 parts}.

By the study of the above equations it is seen that two molecules of dextrose or other similar reducing sugar, are equivalent to one molecule of potassium permanganate, as is shown by the following equations:

{ 2C₆H₁₂O₆ } = { 5Cu₂O } = { 10FeSO₄ } = { K₂Mn₂O₈ } { 360 parts } {715 parts} {1520 parts} {316.2 parts}

It is thus seen that 316.2 parts by weight of potassium permanganate are equivalent to 360 parts by weight of dextrose; or one part of permanganate corresponds to 1.1385 parts by weight of dextrose. If, therefore, the amount of permanganate required in the above reaction to restore the iron to the ferric condition, be multiplied by the factor mentioned above, the quotient will represent in weight the amount of dextrose which enters into the reaction. The standard solution of potassium permanganate should contain 4.392 grams of the salt in a liter. One cubic centimeter of this solution is equivalent to five milligrams of dextrose.

=121. Manipulation.=—The saccharine solution whose strength is to be determined should contain approximately about one per cent of sugar. Of this solution ten cubic centimeters are placed in a porcelain dish together with a considerable excess of fehling solution. When no sucrose is present, the mixture may be heated to the temperature of boiling water and kept at that temperature for a few minutes until all the reducing sugar is oxidized. There should be enough of the copper solution used to maintain a strong blue coloration at the end of the reaction. A greater uniformity of results will be secured by using in all cases a considerable excess of the copper solution. When sucrose or other non-reducing sugars are present, the temperature of the reaction should not be allowed to exceed 80° and the heating may be continued somewhat longer. At this temperature the copper solution is absolutely without action on sucrose. The precipitated suboxid is allowed to settle, the supernatant liquid poured off through a filter and the suboxid washed thoroughly a number of times by decantation with hot water, the washings being poured through the filter. This process of washing is greatly facilitated by decanting the supernatant liquid from the porcelain dish first into a beaker and from this into a third beaker and so on until no suboxid is carried off. Finally the wash water is poured through a filter-paper bringing as little as possible of the suboxid onto the paper. The suboxid on the filter-paper and in the beakers is next dissolved in a solution of ferric sulfate made strongly acid with sulfuric; or in a sulfuric acid solution of ammonia ferric sulfate which is more easily obtained free from impurities than the ferric sulfate. When all is dissolved from the beakers the solution is poured upon the suboxid which still remains in the porcelain dish. When the solution is complete it is washed into a half liter flask and all the vessels which contain the suboxid are also thoroughly washed and the wash waters added to the same flask. The whole is rendered strongly acid with sulfuric and made up to a volume of half a liter.

The process carried out as directed, when tested against pure sugar, gives good results, not varying from the actual content of the sugar by more than one-tenth per cent below or three-tenths above the true content. The distinct pink coloration imparted to the solution by the permanganate solution as soon as the iron is all oxidized to the ferric state marks sharply the end of the reaction. In this respect this process is very much to be preferred to the usual volumetric processes depending upon the coloration produced with potassium ferrocyanid by a copper salt for distinguishing the end of the reaction. It is less convenient than the ordinary volumetric process by reason of the somewhat tedious method of washing the precipitated cuprous oxid. When a large number of analyses is to be made, however, the whole can be washed with no more expenditure of time than is required for a single sample. One analyst can, in this way, easily attend to fifty or a hundred determinations at a time.

In the application of the permanganate method to the analysis of the juices of sugar cane and sorghum it is directed to take 100 cubic centimeters of the expressed juice and clarify by the addition of twenty-five cubic centimeters of basic lead acetate, diluted with water, containing enough of the lead acetate, however, to produce a complete clarification. It is not necessary to remove the excess of lead from the filtrate before the determination. Ten cubic centimeters of the filtrate correspond to eight cubic centimeters of the original juice. For percentage calculation the specific gravity of the original juice must be known. Before the addition of the alkaline copper solution, from fifty to seventy-five cubic centimeters of water should be added to the clarified sugar juice and the amount of fehling solution used in each case should be from fifty to seventy-five cubic centimeters. The heating at 75° should be continued for half an hour in order to insure complete reduction and oxidation of the sugar. The sucrose can also be estimated in the same juices by inverting five cubic centimeters of the clarified juice with five cubic centimeters of dilute hydrochloric acid, by heating for an hour at a temperature not above 90°. Before adding the acid for inversion, about 100 cubic centimeters of water should be poured over the five cubic centimeters of sugar solution. The washing of the suboxid and the estimation of the amount reduced are accomplished in the manner above described.

This method has been extensively used in this laboratory and with very satisfactory results. The only practical objection which can be urged to it is in the time required for filtering. This fault is easily remedied by adopting the method of filtering through asbestos felt described in the next paragraph.

For the sake of uniformity, however, the copper solution should be boiled for a few minutes before the addition of the sugar in order to expel all oxygen, the sugar solutions should be made with recently boiled water and the precipitation of the suboxid should be accomplished by heating for just thirty minutes at 75°. At the end of this time an equal volume of cold, recently boiled, water should be added and the filtration at once accomplished.

=122. Modified Permanganate Method.=—The permanganate method as used by Ewell, in this laboratory, is conducted as follows: After the precipitate is obtained, according to the directions given in the methods described, it is thoroughly washed with hot, recently boiled water, on a gooch. The asbestos, with as much of the precipitate as possible, is transferred to the beaker in which the precipitation was made, beaten up with from twenty-five to thirty cubic centimeters of hot, recently boiled water, and from fifty to seventy-five cubic centimeters of a saturated solution of ferric sulfate in twenty-five per cent sulfuric acid are added to the beaker and then poured through the crucible to dissolve the cuprous oxid remaining therein. If the precipitate be first beaten up with water as directed, so that no large lumps of it remain, there is no difficulty in dissolving the oxid in the ferric salt; while if any lumps of the oxid be allowed to remain there is great difficulty. After the solution is obtained, it is titrated with a solution of potassium permanganate of such a strength that each cubic centimeter is equivalent to 0.01 gram of copper.

In triplicate determinations made by this method the precipitates obtained required after solution in the ferric salt, 28.7, 28.9, 28.6 cubic centimeters of potassium permanganate solution, respectively. For the quantities taken this was equivalent to an average percentage of reducing sugars of 4.19. The percentage obtained by the gravimetric method was 4.26.

The method seems to be sufficiently accurate for all ordinary purposes and is extremely rapid.

The permanganate solution used should be standardized by means of metallic iron, but in ordinary work it is also recommended to standardize by check determinations of reducing sugars in the same sample by the gravimetric method.

=123. Determination of Reducing Sugar by the Specific Gravity of the Cuprous Oxid.=—Gaud proposes to determine the percentage of reducing sugar from the specific gravity of the cuprous oxid. The manipulation is carried out as follows:

In a porcelain dish are placed fifty cubic centimeters of the alkaline copper solution and an equal quantity of water and the mixture maintained in ebullition for two or three minutes. The dish is then placed on a boiling water-bath and twenty-five cubic centimeters of a reducing sugar of approximately one per cent strength added at once. The reduction is thus secured at a temperature below 100°, which is an important consideration in securing the minimum decomposing effect of the alkali upon the sugar. The dish is kept upon the water-bath for about ten minutes when the reduction is complete and the supernatant liquor should still be intensely blue. The precipitate is washed by decantation with boiling water, taking care to avoid the loss of any of the cuprous oxid. The washing is continued until the wash waters are neutral to phenolphthalein. The cuprous oxid is then washed into a pyknometer of from twenty to twenty-five cubic centimeters capacity, the exact content of which has been previously determined at zero. It is filled with boiling water, the stopper inserted, and after cooling the flask is weighed. Let P be the weight of the pyknometer plus the liquid and the precipitate, the total volume of which is equal to the capacity of the flask at the temperature at which it was filled, that is _V_ₜ = _V_₀ [1 + 3β(t-_t_₀)].

This formula is essentially that given in paragraph =51=, for calculating the volume of a pyknometer at any temperature, substituting for 3β, γ the cubical expansion of glass, viz., 0.000025.

The specific gravity of the dry cuprous oxid is Δ = 5.881 and let the specific gravity of water at the temperature of filling, which can be taken from any of the tables of the density of water, be d. The total weight p of the precipitated suboxid may then be calculated by the following formula:

P-_V_ₜ_d_ P = -----------. 1 - d ---- Δ

The density of water at 99°, which is about the mean temperature of boiling water for laboratories in general, is 0.95934, and this may be taken as the weight of one cubic centimeter for purposes of calculation in the formula above.

In order to obtain exact results, it is important that the weight P be reduced to a vacuum. The weight of cuprous oxid not varying proportionally to the weight of reducing sugar, it is necessary to prepare a table showing the principal numerical values of the two, in order to be able to calculate easily all the possible values, either directly from the table or by appropriate interpolations. Following are the chief values which are necessary for the calculation:

Milligrams Milligrams Milligrams Milligrams cuprous oxid. dextrose. cuprous oxid. dextrose.

10 5.413 100 46.221 20 9.761 200 91.047 30 14.197 300 138.842 50 23.036 400 188.928

It is claimed by the author that the above method is both simple and rapid and can be applied with an error of not more than one-thousandth if the corrections for temperature and pressure be rigorously applied.[85]

=124. The Copper Carbonate Process.=—While the copper solutions which have been mentioned in previous paragraphs have only a slight action on sucrose and dextrin yet on prolonged boiling even these bodies show a reducing effect due probably to a preliminary change in the sugar molecules whereby products analogous to dextrose or invert sugar are formed. In order to secure a reagent, to which the sugar not reducing alkaline copper solutions might be more resistant Soldaini has proposed to employ a liquor containing the copper as carbonate instead of as tartrate.[86] This solution is prepared by adding to a solution of forty grams of copper sulfate one of equal strength of sodium carbonate. The resulting copper carbonate and hydroxid are collected on a filter, washed with cold water, and dried. The reaction which takes place is represented by the following formula:

2CuSO₄ + 2Na₂CO₃ + H₂O = CuCO₃ + CuO₂H₂ + 2Na₂SO₄ + CO₂.

The dry precipitate obtained, which will weigh about fifteen grams, is placed in a large flask with about 420 grams of potassium bicarbonate and 1400 cubic centimeters of water. The contents of the flask are heated on a steam-bath for several hours with occasional stirring until the evolution of carbon dioxid has ceased. During this time the liquid is kept at the same volume by the addition of water, or by attaching a reflux condenser to the flask. The potassium and copper compounds at the end of this time will be found dissolved and the resulting liquor will have a deep blue color. After filtration the solution is boiled for a few minutes and cooled to room temperature. The volume is then completed to two liters. A more direct method of preparing the solution, and one quite as effective, consists in adding the solution of the copper sulfate directly to the hot solution of potassium bicarbonate and heating and shaking the mixture until the copper carbonate formed is dissolved. After filtering the volume is made as above. The proportions of reagents employed are placed by Preuss at 15.8 grams of crystallized copper sulfate and 594 grams of potassium bicarbonate.[87] The soldaini reagent is extremely sensitive and is capable of detecting as little as half a milligram of invert sugar. The presence of sucrose makes the reagent more delicate, and it is especially useful in determining the invert sugar arising during the progress of manufacture by the action of heat and melassigenic bodies on sucrose.

=125. The Analytical Process.=—As in the case of fehling solution a great many methods of conducting the analysis with the soldaini reagent have been proposed. The general principle of all these processes is the one already described for the alkaline copper tartrate solution, viz., the addition of the reducing sugar solution to the boiling reagent, and the determination of the end of the reaction by the disappearance of the copper.[88]

Practically, however, these methods have had no general application, and the use of the soldaini reagent has been confined chiefly to the determination of invert sugar in presence of a large excess of sucrose. For this purpose the sugar solution is not added until the blue color of the reagent has been destroyed, but on the other hand, the reagent has been used in excess, and the cuprous oxid formed collected and weighed as metallic copper. The weight of the metallic copper found, multiplied by the factor 0.3546, gives the weight of invert sugar in the volume of the sugar solution used. According to Preuss, the factor is not a constant one, but varies with the quantity of invert sugar present, as is seen in the formula y = 2.2868 + 3.3_x_ + 0.0041_x_², in which x = the invert sugar, and y the metallic copper.[89]

=126. Tenth Normal Copper Carbonate Solution.=—In the study of some of the solutions of copper carbonate, proposed for practical work, Ettore Soldaini was impressed with the difficulty of dissolving so large a quantity of carbonate in the solvent employed.[90] The solution recommended by Bodenbender and Scheller,[91] in which forty grams of the crystallized copper sulfate were used, failed to disclose an equivalent amount of copper in the reagent ready for use. For this reason a tenth-normal copper solution is prepared by Soldaini containing the equivalent of 3.464 grams of copper sulfate in one liter. The reagent is easily prepared by adding slowly the dissolved or finely powdered copper salt to a solution of 297 grams of potassium bicarbonate, and after complete solution of the copper carbonate formed, completing the volume to one liter. With this reagent as little as one-quarter of a milligram of reducing sugar can be easily detected. For the quantitive estimation of sugar a solution of the above strength is to be preferred to the other forms of the soldaini reagent by reason of the ease of direct comparison with standard fehling solutions.

The analytical process is conducted with the tenth-normal solution, prepared by Soldaini and described above, as follows: Place 100 cubic centimeters of the reagent in each of several porcelain dishes heat to boiling, and add little by little the sugar solution to one dish until the blue color has disappeared. Having thus determined nearly the exact quantity of sugar solution required for the copper in 100 cubic centimeters of the reagent the whole of the sugar solution is added at once, varying slightly the amounts added to each dish. The boiling is continued for fifteen minutes, and the contents of the dishes poured on filters. That filtrate which contains neither copper nor sugar represents the exact quantity of sugar solution which contained fifty milligrams of dextrose.

=127. Relation of Reducing Sugar to Quantity of Copper Suboxid Obtained.=—The relation of the quantity of copper reduced to the amount of sugar oxidized by the copper carbonate solution has been determined by Ost, and the utility of the process thereby increased.[92] The solution used should have the following composition: 23.5 grams of crystallized copper sulfate, 250 grams of potassium carbonate, and 100 grams of potassium bicarbonate in one liter. Without an indicator the end reaction is distinctly marked by the passage of the blue color into a colorless solution. Ost affirms that this solution is preferable to any form of fehling liquor because it can be kept indefinitely unchanged; it attacks sucrose far less strongly, and an equal quantity of sugar precipitates nearly double the quantity of copper. The boiling requires a longer time, as a rule ten minutes, but this is a matter of no importance, when the other advantages are taken into consideration. The relations of the different sugars to the quantity of copper precipitated are given in the table in the next paragraph.

=128. Factor for Different Sugars.=—For pure dextrose the relation between sugar and copper reduced has been determined by Ost, and the data are given in the table below. The data were obtained by adding to fifty cubic centimeters of the copper solution twenty-five cubic centimeters of sugar solutions of varying strength and collecting, washing, and reducing the cuprous oxid obtained in a current of hydrogen in a glass tube by the method described further on.

The boiling in all cases was continued, just ten minutes, although a slight variation from the standard time did not produce so great a difference as with fehling reagent. In the case of dextrose, when fifty milligrams were used with fifty cubic centimeters of the solution, the milligrams of copper obtained after six, ten and twenty minutes’ boiling were 164.6, 165.5, and 166.9 respectively.[93]

The data differ considerably from those obtained by Herzfeld, but in his experiments the boiling was continued only for five minutes, and this is not long enough to secure the proper reduction of the copper.[94]

TABLE SHOWING THE QUANTITY OF COPPER REDUCED BY DIFFERENT SUGARS.

Copper. Invert Sugar. Dextrose. Levulose. Galactose. Arabinose. Milli- Milli- Milli- Milli- Milli- Milli- grams. grams. grams. grams. grams. grams. 50 15.2 15.6 14.7 17.4 17.0 55 16.6 17.0 16.1 19.1 18.6 60 18.0 18.5 17.5 20.8 20.3 65 19.4 19.9 18.9 22.5 21.9 70 20.8 21.4 20.3 24.2 23.5 75 22.3 22.9 21.7 25.9 25.1 80 23.7 24.4 23.0 27.7 26.7 85 25.2 25.8 24.3 29.3 28.3 90 26.6 27.3 25.7 31.1 29.9 95 28.1 28.8 27.1 32.8 31.5 100 29.5 30.3 28.5 34.5 33.1 105 31.0 31.8 29.9 36.2 34.7 110 32.4 33.3 31.2 38.0 36.3 115 33.9 34.8 32.6 39.7 37.9 120 35.3 36.3 34.0 41.4 39.5 125 36.8 37.8 35.4 43.1 41.1 130 38.2 39.3 36.8 44.8 42.8 135 39.7 40.8 38.2 46.5 44.4 140 41.1 42.3 39.6 48.3 46.0 145 42.6 43.8 41.0 50.0 47.6 150 44.0 45.3 42.5 51.8 49.3 155 45.5 46.8 43.9 53.6 50.9 160 47.0 48.3 45.3 55.4 52.6 165 48.5 49.8 46.7 57.2 54.3 170 50.0 51.4 48.1 59.0 55.9 175 51.5 52.9 49.5 60.8 57.5 180 53.0 54.5 51.0 62.7 59.2 185 54.5 56.0 52.5 64.5 60.9 190 56.0 57.6 54.0 66.4 62.7 195 57.5 59.2 55.5 68.3 64.4 200 59.1 60.8 57.0 70.3 66.2 205 60.7 62.4 58.6 72.3 68.0 210 62.4 64.1 60.2 74.3 69.8 215 64.1 65.8 61.8 76.3 71.6 220 65.8 67.5 63.5 78.3 73.5 225 67.5 69.2 65.2 80.3 75.4 230 69.3 70.9 66.9 82.4 77.3 235 71.1 72.7 68.7 84.5 79.3 240 72.9 74.5 70.6 86.6 81.3 245 74.8 76.4 72.5 88.9 83.4 250 76.7 78.4 74.4 91.2 85.5 255 78.6 80.5 76.5 93.5 87.6 260 80.5 82.8 78.8 95.9 89.8 265 82.5 85.1 81.1 98.3 92.2 270 84.7 87.5 83.5 100.7 94.6 275 87.1 89.9 85.9 103.3 97.1 280 89.7 92.4 88.6 106.1 99.6 285 92.3 94.9 91.3 109.0 102.3 290 95.1 97.6 94.2 112.0 105.1 295 98.0 100.4 97.2 115.1 107.9 298 100.0 102.5 99.0 117.0 109.5

VOLUMETRIC METHODS BASED UPON THE USE OF AN AMMONIACAL COPPER SOLUTION.

=129. Pavy’s Process.=—The well-known solubility of cuprous oxid in ammonia led Pavy to adopt a copper reagent containing ammonia in the volumetric determination of reducing sugars.[95] In Pavy’s process an alkaline copper solution is employed made up in the usual way, to which a sufficient quantity of ammonia is added to hold in solution all the copper when precipitated as cuprous oxid. The solution used by Pavy has the following composition: One liter contains

Crystallized copper sulfate 34.65 grams Potassium-sodium tartrate 173.00 ” Caustic potash 160.00 ”

For use 120 cubic centimeters of the above reagent are mixed with 300 of ammonia of specific gravity 0.88, and the volume completed to one liter with distilled water. Twenty cubic centimeters of this reagent are equivalent to ten milligrams of dextrose or invert sugar when added in a one per cent solution.

In the use of ammoniacal copper solution, care must be taken that all the liquids employed be entirely free of oxygen and that the contents of the flask in which the reduction takes place be in some way excluded from contact with the air. Pavy secured this by conducting the reduction in a flask closed with a stopper carrying two holes; one of these served for the introduction of the burette carrying the sugar solution and the other carried a tube dipping into a water seal by means of a slit rubber tube, which would permit of the exit of the vapors of steam and ammonia, but prevent the regurgitation of the water into the flask.

The complete decoloration of the copper solution marks the end of the reaction. The usual precautions in regard to the length of the time of boiling must be observed.

It is easy to see that in the Pavy process the quantity of ammonia in the solution is rapidly diminished during the boiling and this has led to the suggestion of other methods to exclude the air. Among these have been recommended the introduction of a current of hydrogen or carbon dioxid. One of the best methods of procedure is that proposed by Allen, who recommends covering the copper solution by a layer of paraffin oil (kerosene).[96]

=130. Process Of Peska.=—Peska has also independently made use of Allen’s method of covering the solutions with a layer of paraffin oil and finds it reliable.[97] The copper reagent employed by him has the following composition:

Crystallized copper sulfate 6.927 grams Ammonia, twenty-five per cent strength 160.00 cc.

The copper sulfate is dissolved in water, the ammonia added, and the volume completed to half a liter with distilled water. A second solution containing half a liter is made by dissolving 34.5 grams of potassium-sodium tartrate and ten grams of sodium hydroxid and completing the cool solution to half a liter with distilled water. In all cases the water used in making up the above solutions must be freshly boiled to exclude the air.

For the titration, fifty cubic centimeters of each of the above solutions are taken, mixed and covered with a layer of paraffin oil half a centimeter in depth. The reduction is not accomplished at a boiling temperature, but at from 80° to 85°. The manipulation is conducted as follows:

The mixed solutions are placed in a beaker, covered with oil, and heated to 80°. The temperature is measured by a thermometer which also serves as a stirring rod. The sugar solution is run down the sides of the beaker from a burette of such a shape as to be protected from the heat. After each addition of the sugar solution the mixture is carefully stirred, keeping the temperature at from 80° to 85°. The first titration is made to determine approximately the quantity of sugar solution necessary to decolorize the copper. This done, the actual titration is accomplished by adding at once the total amount of sugar solution necessary to decolorize, less about one cubic centimeter. Any sugar solution adhering to the side of the beaker is washed down by distilled water, the contents of the beaker well stirred, and the temperature kept at 85° for two minutes. The rest of the sugar solution is then added in quantities of one-tenth of a cubic centimeter until the decoloration is completed. The total time of the final titration should not exceed five minutes. The sugar solution should be as nearly as possible of one per cent strength. If a lower degree of strength be employed a larger quantity of the sugar is necessary to reduce a given quantity of copper.

In the case of dextrose, when a one per cent solution is used, eight and two-tenths cubic centimeters, corresponding to 80.2 milligrams of dextrose, are required to reduce 100 cubic centimeters of the mixed reagent. On the other hand, when the sugar solution is diluted to one-tenth of a per cent strength 82.1 milligrams are required.

With invert sugar slightly larger quantities are necessary, the reducing power being as 94.9 to 100 as compared with dextrose. With a one per cent strength of invert sugar it is found that eighty-four milligrams are required to reduce 100 cubic centimeters of the mixed reagent and when the strength of the invert sugar is reduced to one-tenth per cent 87.03 milligrams are required.

=131. Method Of Allein and Gaud.=—Allein and Gaud have proposed a further modification of the ammonia process which consists essentially in the suppression of rochelle salt and fixed caustic alkali and the entire substitution therefor of ammonia. Ammonia acts with much less vigor upon sugars than the caustic alkalies, and it is therefore claimed that the decomposition of the sugar due to the alkali is reduced to a minimum when ammonia is employed.[98] The copper solution is made as follows:

Dissolve 8.7916 grams of electrolytic copper in ninety-three grams of concentrated sulfuric acid diluted with an equal volume of water. Complete the resulting solution to one liter with concentrated ammonia. Ten cubic centimeters of this solution are equal to fifty grams of dextrose.

It is recommended that the reduction be accomplished in an atmosphere of hydrogen, but it is apparent that the use of kerosene is permissible in this case, and on account of its greater simplicity it is to be recommended as the best means of excluding the oxygen. The reduction is accomplished at a temperature of about 80°.

It is also proposed to reoxidize the copper by substituting a current of air for the hydrogen at the end of the reaction, and thus use the same copper a number of times. The danger of loss of ammonia, and the difficulty of determining when the oxidation is complete, render this regeneration of the reagent undesirable.

=132. Method of Gerrard.=—The method of Gerrard does not depend upon the use of ammonia, but the principle involved is the same, viz., the holding of the separated cuprous oxid in solution and the determination of the end of the reaction by the disappearance of the blue color. As first proposed by Gerrard, the copper sulfate solution is made of double the strength usually employed and to each 100 cubic centimeters thereof, before use, three and three-tenths grams of potassium cyanid are added. This is sufficient to hold the precipitated cuprous oxid in solution.[99]

The original method of Gerrard is found difficult of execution and the author, in conjunction with Allen, has lately modified it and reduced it to a practical working basis.[100]

In the new method the ordinary fehling solution is employed and it is prepared for use in the following way: Ten cubic centimeters of the fehling solution, or half that quantity of each of the component parts kept in separate bottles, are placed in a porcelain dish with forty cubic centimeters of water and brought to the boiling-point. To the boiling liquid is added, from a pipette, a five per cent solution of potassium cyanid until the blue color just disappears, or only a very faint tint of blue remains, avoiding any excess of the cyanid. A second portion of the fehling solution equal to that first employed is added, and to the boiling mixture the solution of sugar is added, from a burette, until the blue color disappears. The contents of the dish should be kept boiling during the addition of the sugar solution. The volume used will contain fifty milligrams of dextrose. The sugar solution should be of such a strength as to contain no more than half a per cent of reducing sugar.

The principle of the preparation of the solution may be stated as follows: If to a solution of copper sulfate, potassium be added until the blue color disappears, a double cyanid of copper and potassium cyanid is formed according to the following reaction: CuSO₄ + 4KCN = Cu(CN)₂.2KCN + K₂SO₄. This double cyanid is a salt of considerable stability. It is not decomposed by alkalies, hydrogen or ammonium, sulfid. With mineral acids it gives a whitish, curdy precipitate. With fehling solution the same double cyanid is formed as that described above. If, however, fehling solution be present in excess of the amount necessary to form the double cyanid of copper, this excess can be used in the oxidation of reducing sugar and the colorless condition of the solution will be restored as soon as the excess of the fehling is destroyed. The double cyanid holds in solution the cuprous oxid formed and thus complete decoloration is secured.

=133. Sidersky’s Modification of Soldaini’s Process.=—In all cases where the sugar solutions are not too highly colored, Sidersky finds that the method of reduction in a large test tube, as practiced by Violette, is applicable with the copper carbonate solution.[101] For more exact work it is preferred to determine the quantity of copper reduced by an indirect volumetric method. The sugar solution, properly clarified and the lead removed if subacetates have been used, is made of such a volume as to contain less than one per cent of reducing sugars. In a flask or large test tube are placed 100 cubic centimeters of the copper solution, which is boiled for a short time and the sugar solution added, little by little, from a pipette, at such a rate as not to stop the ebullition. The boiling is continued for five minutes after the last addition of the sugar. The vessel is taken from the flame and 100 cubic centimeters of cold water added, the whole brought on an asbestos felt and the cuprous oxid washed with hot water until the alkaline reaction has disappeared. The residual cuprous oxid is dissolved in a measured quantity of set sulfuric acid, semi- or fifth-normal, a few particles of potassium chlorate added, and the mixture boiled to convert any cuprous into cupric sulfate. The reaction is represented by the following formula:

3Cu₂O + 6H₂SO₄ + KClO₃ = 6CuSO₄ + KCl + 6H₂O.

The residual sulfuric acid is titrated with a set alkali in excess, ammonia being preferred.

The solution of ammonia is made by diluting 200 cubic centimeters of commercial aqua ammonia with 800 of water. Its strength is determined by adding a little copper sulfate solution as indicator and then the set solution of sulfuric acid until the blue color disappears. The copper sulfate secured from the cuprous sulfate as described above is cooled, and a quantity of the ammonia, equal to twenty-five cubic centimeters of the set sulfuric acid, added. The excess of the ammonia is then determined by titration with the sulfuric acid, the disappearance of the blue color being the indication of the end of the reaction. The number of cubic centimeters of the set sulfuric acid required to saturate the ammonia represents the equivalent of cuprous oxid originally present. One cubic centimeter of normal sulfuric acid is equivalent to 0.0317 gram of metallic copper.

To determine the weight of invert sugar oxidized, multiply the weight of copper, calculated as above described, by the factor 0.3546.[102] For a general application of this method of analysis the relative quantities of copper reduced by different quantities of sugar must be taken into consideration.

While, as has already been stated, the copper carbonate process has heretofore been applied chiefly to the detection of invert sugar, it has merits which justify the expectation that it may some time supplant the fehling liquor both for volumetric and gravimetric work. Large volumes of the reagent can be prepared at once and without danger of subsequent change. The action of the reagent on the hexobioses and trioses is far less vigorous than that of the alkaline copper tartrate, and the end reactions for volumetric work are, at least, as easily determined in the one case as the other.

=134. Method Depending on Titration of Excess of Copper.=—Instead of measuring the quantity of copper reduced, either by its disappearance or by reducing the cuprous oxid to a metallic state, Politis has proposed a method of analysis depending on the titration of the residual copper.[103] The reagents employed are:

(1) A copper solution containing 24.95 grams of crystallized copper sulfate, 140 grams of sodium and potassium tartrate, and twenty-five grams of sodium hydroxid in one liter:

(2) A solution of sodium thiosulfate containing 24.8 grams of the salt in one liter:

(3) A solution of potassium iodid containing 12.7 grams of iodin in one liter.

The reaction is represented by the formula

2CuCl₂ + 4KI = Cu₂I₂ + 4KCl + I₂.

The analytical process is carried out as follows: In a 100 cubic centimeter flask are boiled fifty cubic centimeters of the copper solution, ten cubic centimeters of about one-tenth per cent reducing sugar solution are added, the boiling continued for five minutes, the flask filled to the mark with boiling water and its contents filtered. Fifty cubic centimeters of the hot filtrate are cooled, slightly acidified, potassium iodid solution added in slight excess; and the iodin set free determined by titration with sodium thiosulfate. The quantity of iodin obtained corresponds to the unreduced copper remaining after treatment with the reducing sugar. The number of cubic centimeters of thiosulfate used subtracted from twenty-five will give the number of cubic centimeters of the copper solution which would be reduced by five cubic centimeters of the sugar solution used.

Example.—In the proportions given above it was found that eleven cubic centimeters of thiosulfate were required to saturate the iodin set free. Then 25 - 11 = 14 cubic centimeters of copper solution reduced by five cubic centimeters of the sugar solution. Since one cubic centimeter of the copper solution is reduced by 0.0036 gram of dextrose the total dextrose in the five cubic centimeters = 0.0036 × 5 = 0.0180 gram.

The above method does not seem to have any practical advantage over those based on noting the disappearance of the copper and is given only to illustrate the principle of the process. While the titration of the iodin by sodium thiosulfate is easily accomplished in the absence of organic matter, it becomes difficult, as shown by Ewell, when organic matters are present, as they always are in the oxidation of a sugar solution. Ewell has therefore proposed to determine the residual copper by a standard solution of potassium cyanid, but the method has not yet been developed.[104]

GRAVIMETRIC COPPER METHODS.

=135. General Principles.=—In the preceding pages the principles of the volumetric methods of sugar analysis by means of alkaline copper solution have been set forth. They depend either on the total decomposition of the copper solution employed by the reducing sugar, or else on the collection and titration of the cuprous oxid formed in the reaction. In the gravimetric methods the general principle of the process rests upon the collection of the cuprous oxid formed and its reduction to metallic copper, the weight of which serves as a starting point in the calculations of the weight of reducing sugar, which has been oxidized in the solution.

The factors which affect the weight of copper obtained are essentially those which influence the results in the volumetric method. The composition of the copper solution, the temperature at which the reduction is accomplished, the time of heating, the strength of the sugar solution and the details of the manipulation, all affect more or less the quantity of copper obtained. As in the volumetric method also, the kind of reducing sugar must be taken in consideration, dextrose, levulose, invert sugar, maltose and other sugars having each a definite factor for reduction in given conditions. It follows, therefore, that only those results are of value which are obtained under definite conditions, rigidly controlled.

=136. Gravimetric Methods of the Department of Agriculture Laboratory.=—The process used in this laboratory is based essentially on the methods of Maercker, Behrend, Morgen, Meissl, Hiller and Allihn.[105] Where dextrose alone is present, the table of factors proposed by Allihn is used and also the copper solution corresponding thereto.

For pure invert sugar, the tables and solutions of Meissl are used. For invert sugar in the presence of sucrose, the table and process proposed by Hiller are used.

The reduction of the copper solution and the electrolytic deposition of the copper are accomplished as follows:

The copper and alkali solutions are kept in separate bottles. After mixing the equivalent volume of the two solutions in a beaker, heat is applied and the mixture boiled. To the boiling liquid the proper volume of the cold sugar solution is added. This must always be less than the amount required for complete reduction. The solution is again brought into ebullition and kept boiling exactly two minutes. A two-minute sand glass is conveniently used to determine the time of boiling. At the end of this time an equal volume of freshly boiled cold water is added, and the supernatant liquor at once passed through a gooch under pressure. The residual cuprous oxid is covered with boiling water and washed by decantation until the wash water is no longer alkaline. It is more convenient to wash in such a way that, at the end, the greater part of the cuprous oxid is in the gooch. The felt and cuprous oxid are then returned to the beaker in which the reduction is made. The gooch is moistened with nitric acid to dissolve any adhering oxid and then is washed into the beaker. Enough nitric acid is added to bring all the oxid into solution, an excess being avoided, and a small amount of water added. The mixture is again passed under pressure through a gooch having a thin felt, to remove the asbestos and the filtrate collected in a flask of about 150 cubic centimeters capacity. The washing is continued until the gooch is free of copper, when the volume of the filtrate should be about 100 cubic centimeters. The liquid is transferred to a platinum dish holding about 175 cubic centimeters and the flask washed with about twenty-five cubic centimeters of water. From three to five cubic centimeters of strong sulfuric acid are added and the copper deposited by an electric current.

=137. Precipitating the Copper.=—When no more nitric acid is used than indicated in the previous paragraph, it will not be necessary to remove it by evaporation. The platinum dishes containing the solutions of the cuprous oxid are arranged as shown in the figure for the precipitation of the copper by the electric current. Each of the supporting stands has its base covered with sheet-copper, on which the platinum dishes rest. The uprights are made of heavy glass rods and carry the supports for the platinum cylinders which dip into the copper solutions. The current used is from the city service and is brought in through the lamp shown at the right of the figure. This current has a voltage of about 120. After passing the lamp it is conducted through the regulator shown at the right, a glass tube closed below by a stopper carrying a piece of platinum foil, and above by one holding a glass tube, in the lower end of which is sealed a piece of sheet platinum connected, through the glass tube, with the lamp. The regulating tube contains dilute sulfuric acid. The strength of current desired is secured by adjusting the movable pole. A battery of this kind easily secures the precipitation of sixteen samples at once, but only twelve are shown in the figure. The practice here is to start the operation at the time of leaving the laboratory in the afternoon. The next morning the deposition of the copper will be found complete. The wiring of the apparatus is shown in the figure. The wire from the regulator is connected with the base of the first stand, and thence passes through the horizontal support to the base of the second, and so on. The return to the lamp is accomplished by means of the upper wire. This plan of arranging the apparatus has been used for two years, and with perfect satisfaction.

Where a street current is not available, the following directions may be followed: Use four gravity cells, such as are employed in telegraphic work, for generating the current. This will be strong enough for one sample and by working longer for two. Connect the platinum dish with the zinc pole of the battery. The current is allowed to pass until all the copper is deposited. Where a larger number of samples is to be treated at once, the size of the battery must be correspondingly increased.

=138. Method Used at the Halle Station.=—The method used at the Halle station is the same as that originally described by Maercker for dextrose.[106] The copper solution employed is the same as in the allihn method, viz., 34.64 grams of copper sulfate in 500 cubic centimeters, and 173 grams of rochelle salt and 125 grams of potassium hydroxid in the same quantity of water. In a porcelain dish are placed thirty cubic centimeters of copper solution and an equal quantity of the alkali, sixty cubic centimeters of water added and the mixture boiled. To the solution, in lively ebullition, are added twenty-five cubic centimeters of the dextrose solution to be examined which must not contain more than one per cent of sugar. The mixture is again boiled and the separated cuprous oxid immediately poured into the filter and washed with hot water, until the disappearance of an alkaline reaction. For filtering, a glass tube is employed, provided with a platinum disk, and resembling in every respect similar tubes used for the extraction of substances with ether and alcohol. The arrangement of the filtering apparatus is shown in Fig. 44. In the Halle method it is recommended that the tubes be prepared by introducing a platinum cone in place of the platinum disk and filling it with asbestos felt, pressing the felt tightly against the sides of the glass tube and making the asbestos fully one centimeter in thickness. This is a much less convenient method of working than the one described above. After filtration and washing, the cuprous oxid is washed with ether and alcohol and dried for an hour at 110°, and finally reduced to metallic copper in a stream of pure dry hydrogen, heat being applied by means of a small flame. The apparatus for the reduction of the cuprous oxid is shown in Fig. 45. The metallic copper, after cooling and weighing, is dissolved in nitric acid, the tube washed with water, ether and alcohol, and again dried, when it is ready for use a second time. The percentage of dextrose is calculated from the milligrams of copper found by Allihn’s table.

=139. Tables for Use in the Gravimetric Determination of Reducing Sugars.=—The value of a table for computing the percentage of a reducing sugar present in a solution, is based on the accuracy with which the directions for the determination are followed. The solution must be of the proper strength and made in the way directed. The degree of dilution prescribed must be scrupulously preserved and the methods of boiling during reduction and washing the reduced copper, followed. The quantity of copper obtained by the use of different alkaline copper solutions and of sugar solutions of a strength different from that allowed by the fixed limits, is not a safe factor for computation. It must be understood, therefore, that in the use of the tables the directions which are given are to be followed in every particular.

=140. Allihn’s Gravimetric Method for the Determination of Dextrose.=—Reagents:

I. 34.639 grams of CuSO₄.5H₂O, dissolved in water and diluted to half a liter:

II. 173 grams of rochelle salts } dissolved in water and diluted 125 grams of KOH, } to half a liter.

Manipulation: Place thirty cubic centimeters of the copper solution (I), thirty cubic centimeters of the alkaline tartrate solution (II), and sixty cubic centimeters of water in a beaker and heat to boiling. Add twenty-five cubic centimeters of the solution of the material to be examined, which must be so prepared as not to contain more than one per cent of dextrose, and boil for two minutes. Filter immediately after adding an equal volume of recently boiled cold water and obtain the weight of copper by one of the gravimetric methods given. The corresponding weight of dextrose is found by the following table:

ALLIHN’S TABLE FOR THE DETERMINATION OF DEXTROSE.

(A) = Milligrams of copper. (B) = Milligrams of dextrose.

(A) (B) (A) (B) (A) (B) (A) (B) (A) (B) 10 6.1 46 23.9 82 41.8 118 60.1 154 78.6 11 6.6 47 24.4 83 42.3 119 60.6 155 79.1 12 7.1 48 24.9 84 42.8 120 61.1 156 79.6 13 7.6 49 25.4 85 43.4 121 61.6 157 80.1 14 8.1 50 25.9 86 43.9 122 62.1 158 80.7 15 8.6 51 26.4 87 44.4 123 62.6 159 81.2 16 9.0 52 26.9 88 44.9 124 63.1 160 81.7 17 9.5 53 27.4 89 45.4 125 63.7 161 82.2 18 10.0 54 27.9 90 45.9 126 64.2 162 82.7 19 10.5 55 28.4 91 46.4 127 64.7 163 83.3 20 11.0 56 28.8 92 46.9 128 65.2 164 83.8 21 11.5 57 29.3 93 47.4 129 65.7 165 84.3 22 12.0 58 29.8 94 47.9 130 66.2 166 84.8 23 12.5 59 30.3 95 48.4 131 66.7 167 85.3 24 13.0 60 30.8 96 48.9 132 67.2 168 85.9 25 13.5 61 31.3 97 49.4 133 67.7 169 86.4 26 14.0 62 31.8 98 49.9 134 68.2 170 86.9 27 14.5 63 32.3 99 50.4 135 68.8 171 87.4 28 15.0 64 32.8 100 50.9 136 69.3 172 87.9 29 15.5 65 33.3 101 51.4 137 69.8 173 88.5 30 16.0 66 33.8 102 51.9 138 70.3 174 89.0 31 16.5 67 34.3 103 52.4 139 70.8 175 89.5 32 17.0 68 34.8 104 52.9 140 71.3 176 90.0 33 17.5 69 35.3 105 53.5 141 71.8 177 90.5 34 18.0 70 35.8 106 54.0 142 72.3 178 91.1 35 18.5 71 36.3 107 54.5 143 72.9 179 91.6 36 18.9 72 36.8 108 55.0 144 73.4 180 92.1 37 19.4 73 37.3 109 55.5 145 73.9 181 92.6 38 19.9 74 37.8 110 56.0 146 74.4 182 93.1 39 20.4 75 38.3 111 56.5 147 74.9 183 93.7 40 20.9 76 38.8 112 57.0 148 75.5 184 94.2 41 21.4 77 39.3 113 57.5 149 76.0 185 94.7 42 21.9 78 39.8 114 58.0 150 76.5 186 95.2 43 22.4 79 40.3 115 58.6 151 77.0 187 95.7 44 22.9 80 40.8 116 59.1 152 77.5 188 96.3 45 23.4 81 41.3 117 59.6 153 78.1 189 96.8

(A) (B) (A) (B) (A) (B) (A) (B) (A) (B) 190 97.3 233 120.1 276 143.3 319 167.0 362 191.1 191 97.8 234 120.7 277 143.9 320 167.5 363 191.7 192 98.4 235 121.2 278 144.4 321 168.1 364 192.3 193 98.9 236 121.7 279 145.0 322 168.6 365 192.9 194 99.4 237 122.3 280 145.5 323 169.2 366 193.4 195 100.0 238 122.8 281 146.1 324 169.7 367 194.0 196 100.5 239 123.4 282 146.6 325 170.3 368 194.6 197 101.0 240 123.9 283 147.2 326 170.9 369 195.1 198 101.5 241 124.4 284 147.7 327 171.4 370 195.7 199 102.0 242 125.0 285 148.3 328 172.0 371 196.3 200 102.6 243 125.5 286 148.8 329 172.5 372 196.8 201 103.1 244 126.0 287 149.5 330 173.1 373 197.4 202 103.7 245 126.6 288 149.4 331 173.7 374 198.0 203 104.2 246 127.1 289 150.9 332 174.2 375 198.6 204 104.7 247 127.6 290 151.0 333 174.8 376 199.1 205 105.3 248 128.1 291 151.6 334 175.3 377 199.7 206 105.8 249 128.7 292 152.1 335 175.9 378 200.3 207 106.3 250 129.2 293 152.7 336 176.5 379 200.8 208 106.8 251 129.7 294 153.2 337 177.0 380 201.4 209 107.4 252 130.3 295 153.8 338 177.6 381 202.0 210 107.9 253 130.8 296 154.3 339 178.1 382 202.5 211 108.4 254 131.4 297 154.9 340 178.7 383 203.1 212 109.0 255 131.9 298 155.4 341 179.3 384 203.7 213 109.5 256 132.4 299 156.0 342 179.8 385 204.3 214 110.0 257 133.0 300 156.5 343 180.4 386 204.8 215 110.6 258 133.5 301 157.1 344 180.9 387 205.4 216 111.1 259 134.1 302 157.6 345 181.5 388 206.0 217 111.6 260 134.6 303 158.2 346 182.1 389 206.5 218 112.1 261 135.1 304 158.7 347 182.6 390 207.1 219 112.7 262 135.7 305 159.3 348 183.2 391 207.7 220 113.2 263 136.2 306 159.8 349 183.7 392 208.3 221 113.7 264 136.8 307 160.4 350 184.3 393 208.8 222 114.3 265 137.3 308 160.9 351 184.9 394 209.4 223 114.8 266 137.8 309 161.5 352 185.4 395 210.0 224 115.3 267 138.4 310 162.0 353 186.0 396 210.6 225 115.9 268 138.9 311 162.6 354 186.6 397 211.2 226 116.4 269 139.5 312 163.1 355 187.2 398 211.7 227 116.9 270 140.0 313 163.7 356 187.7 399 212.3 228 117.4 271 140.6 314 164.2 357 188.3 400 212.9 229 118.0 272 141.1 315 164.8 358 188.9 401 213.5 230 118.5 273 141.7 316 165.3 359 189.4 402 214.1 231 119.0 274 142.2 317 165.9 360 190.0 403 214.6 232 119.6 275 142.8 318 166.4 361 190.6 404 215.2

(A) (B) (A) (B) (A) (B) (A) (B) (A) (B) 405 215.8 417 222.8 429 229.8 441 236.9 453 244.0 406 216.4 418 223.3 430 230.4 442 237.5 454 244.6 407 217.0 419 223.9 431 231.0 443 238.1 455 245.2 408 217.5 420 224.5 432 231.6 444 238.7 456 245.7 409 218.1 421 225.1 433 232.2 445 239.3 457 246.3 410 218.7 422 225.7 434 232.8 446 239.8 458 246.9 411 219.3 423 226.3 435 233.4 447 240.4 459 247.5 412 219.9 424 226.9 436 233.9 448 241.0 460 248.1 413 220.4 425 227.5 437 234.5 449 241.6 461 248.7 414 221.0 426 228.0 438 235.1 450 242.2 462 249.3 415 221.6 427 228.6 439 235.7 451 242.8 463 249.9 416 222.2 428 229.2 440 236.3 452 243.4

=141. Meissl’s Table for Invert Sugar.=—Invert sugar is usually the product of the hydrolysis of sucrose. The following table is to be used when the hydrolysis is complete, i. e., when no sucrose is left in the solution. The solution of copper sulfate and of the alkaline tartrate are made up as follows: 34.64 grams of copper sulfate in half a liter, and 173 grams of rochelle salt and 51.6 grams sodium hydroxid in the same volume. The quantity of sugar solution used must not contain more than 245 nor less than ninety milligrams of invert sugar.

In the determination twenty-five cubic centimeters of the copper solution and an equal volume of the alkaline tartrate are mixed and boiled, the proper amount of sugar solution added to secure a quantity of invertose within the limits named, the volume completed to 100 cubic centimeters with boiling water, and the mixture kept in lively ebullition for two minutes. An equal volume of recently boiled cold water is added and the cuprous oxid at once separated by filtration on asbestos under pressure, and washed free of alkali with boiling water. The metallic copper is secured by one of the methods already described.

TABLE FOR INVERT SUGAR BY MEISSL AND WIEN.[107]

(A) = Milligrams of copper. (B) = Milligrams of invert sugar.

(A) (B) (A) (B) (A) (B) (A) (B) 90 46.9 133 69.7 176 93.0 219 117.0 91 47.4 134 70.3 177 93.5 220 117.5 92 47.9 135 70.8 178 94.1 221 118.1 93 48.4 136 71.3 179 94.6 222 118.7 94 48.9 137 71.9 180 95.2 223 119.2 95 49.5 138 72.4 181 95.7 224 119.8 96 50.0 139 72.9 182 96.2 225 120.4 97 50.5 140 73.5 183 96.8 226 120.9 98 51.1 141 74.0 184 97.3 227 121.5 99 51.6 142 74.5 185 97.8 228 122.1 100 52.1 143 75.1 186 98.4 229 122.6 101 52.7 144 75.6 187 99.0 230 123.2 102 53.2 145 76.1 188 99.5 231 123.8 103 53.7 146 76.7 189 100.1 232 124.3 104 54.3 147 77.2 190 100.6 233 124.9 105 54.8 148 77.8 191 101.2 234 125.5 106 55.3 149 78.3 192 101.7 235 126.0 107 55.9 150 78.9 193 102.3 236 126.6 108 56.4 151 79.4 194 102.9 237 127.2 109 56.9 152 80.0 195 103.4 238 127.8 110 57.5 153 80.5 196 104.0 239 128.3 111 58.0 154 81.0 197 104.6 240 128.9 112 58.5 155 81.6 198 105.1 241 129.5 113 59.1 156 82.1 199 105.7 242 130.0 114 59.6 157 82.7 200 106.3 243 130.6 115 60.1 158 83.2 201 106.8 244 131.2 116 60.7 159 83.8 202 107.4 245 131.8 117 61.2 160 84.3 203 107.9 246 132.3 118 61.7 161 84.8 204 108.5 247 132.9 119 62.3 162 85.4 205 109.1 248 133.5 120 62.8 163 85.9 206 109.6 249 134.1 121 63.3 164 86.5 207 110.2 250 134.6 122 63.9 165 87.0 208 110.8 251 135.2 123 64.4 166 87.6 209 111.3 252 135.8 124 64.9 167 88.1 210 111.9 253 136.3 125 65.5 168 88.6 211 112.5 254 136.9 126 66.0 169 89.2 212 113.0 255 137.5 127 66.5 170 89.7 213 113.6 256 138.1 128 67.1 171 90.3 214 114.2 257 138.6 129 67.6 172 90.8 215 114.7 258 139.2 130 68.1 173 91.4 216 115.3 259 139.8 131 68.7 174 91.9 217 115.8 260 140.4 132 69.2 175 92.4 218 116.4 261 140.9

(A) (B) (A) (B) (A) (B) (A) (B) 262 141.5 305 166.8 348 192.6 391 219.3 263 142.1 306 167.3 349 193.2 392 219.9 264 142.7 307 167.9 350 193.8 393 220.5 265 143.2 308 168.5 351 194.4 394 221.2 266 143.8 309 169.1 352 195.0 395 221.8 267 144.4 310 169.7 353 195.6 396 222.4 268 144.9 311 170.3 354 196.2 397 223.1 269 145.5 312 170.9 355 196.8 398 223.7 270 146.1 313 171.5 356 197.4 399 224.3 271 146.7 314 172.1 357 198.0 400 224.9 272 147.2 315 172.7 358 198.6 401 225.7 273 147.8 316 173.3 359 199.2 402 226.4 274 148.4 317 173.9 360 199.8 403 227.1 275 149.0 318 174.5 361 200.4 404 227.8 276 149.5 319 175.1 362 201.1 405 228.6 277 150.1 320 175.6 363 201.7 406 229.3 278 150.7 321 176.2 364 202.3 407 230.0 279 151.3 322 176.8 365 203.0 408 230.7 280 151.9 323 177.4 366 203.6 409 231.4 281 152.5 324 178.0 367 204.2 410 232.1 282 153.1 325 178.6 368 204.8 411 232.8 283 153.7 326 179.2 369 205.5 412 233.5 284 154.3 327 178.8 370 206.1 413 234.3 285 154.9 328 180.4 371 206.7 414 235.0 286 155.5 329 181.0 372 207.3 415 235.7 287 156.1 330 181.6 373 208.0 416 236.4 288 156.7 331 182.2 374 208.6 417 237.1 289 157.2 332 182.8 375 209.2 418 237.8 290 157.8 333 183.5 376 209.9 419 238.5 291 158.4 334 184.1 377 210.5 420 239.2 292 159.0 335 184.7 378 211.1 421 239.9 293 159.6 336 185.4 379 211.7 422 240.6 294 160.2 337 186.0 380 212.4 423 241.3 295 160.8 338 186.6 381 213.0 424 242.0 296 161.4 339 187.2 382 213.6 425 242.7 297 162.0 340 187.8 383 214.3 426 243.4 298 162.6 341 188.4 384 214.9 427 244.1 299 163.2 342 189.0 385 215.5 428 244.9 300 163.8 343 189.6 386 216.1 429 245.6 301 164.4 344 190.2 387 216.8 430 246.3 302 165.0 345 190.8 388 217.4 303 165.6 346 191.4 389 218.0 304 166.2 347 192.0 390 218.7

=142. Table for the Determination of Invert Sugar (Reducing Sugars) in the Presence of Sucrose.=—The method adopted by the Association of Official Agricultural Chemists is essentially that proposed by Meissl and Hiller.[108] Prepare a solution of the material to be examined in such a manner that it contains twenty grams of the mixed sugars in one hundred cubic centimeters, after clarification and the removal of the excess of lead. Prepare a series of solutions in large test tubes by adding one, two, three, four, five etc. cubic centimeters of this solution to each tube successively. Add five cubic centimeters of the mixed copper reagent to each, heat to boiling, boil two minutes and filter. Note the volume of sugar solution which gives the filtrate lightest in tint, but still distinctly blue. Place twenty times this volume of the sugar solution in a 100 cubic centimeter flask, dilute to the mark, and mix well. Use fifty cubic centimeters of the solution for the determination, which is conducted as already described, until the weight of copper is obtained. For the calculation of the results use the following formulas and table of factors of Meissl and Hiller:[109]

Let Cu = the weight of the copper obtained; P = the polarization of the sample; W = the weight of the sample in the fifty cubic centimeters of the solution used for determination; F = the factor obtained from the table for conversion of copper to invert sugar; Cu ---- = approximate absolute weight of invert sugar = Z; 2

100 Z × ----- = approximate per cent of invert sugar = y; W

100P ------- = R, relative number for sucrose; P + y

100 - R = I, relative number for invert sugar;

Cu ---- = per cent of invert sugar. W

Z indicates the vertical column, and the ratio of R to I, the horizontal column of the table, which are to be used for the purpose of finding the factor (F) for calculating copper to invert sugar.

Example:—The polarization of a sugar is 86.4, and 3.256 grams of it (W) are equivalent to 0.290 gram of copper. Then:

Cu 0.290 ---- = ----- = 0.145 = Z 2 2

100 100 Z × ----- = 0.145 × ------ = 4.45 = y W 3.256

100P 8640 -------- = ------------ = 95.1 = R P + y 86.4 + 4.45

100 - R = 100 - 95.1 = 4.9 = I

R : I = 95.1 : 4.9

By consulting the table it will be seen that the vertical column headed I = 150 is nearest to Z, 145, the horizontal column headed 95: 5 is nearest to the ratio of R to I, 95.1: 4.9. Where these columns meet we find the factor 51.2, which enters into the final calculation:

CuF .290 × 51.2 ----- = ------------- = 4.56 the true per cent of invert sugar. W 3.256

MEISSL AND HILLER’S FACTORS FOR THE DETERMINATION OF MORE THAN ONE PER CENT OF INVERT SUGAR.

Ratio of Approximate absolute weight of invert sugar = Z. sucrose I = I = I = I = I = I = I = to invert 200 175 150 125 100 75 50 sugar = mg. mg. mg. mg. mg. mg. mg. R : I. 0 : 100 56.4 55.4 54.5 53.8 53.2 53.0 53.0 10 : 90 56.3 55.3 54.4 53.8 53.2 52.9 52.9 20 : 80 56.2 55.2 54.3 53.7 53.2 52.7 52.7 30 : 70 56.1 55.1 54.2 53.7 53.2 52.6 52.6 40 : 60 55.9 55.0 54.1 53.6 53.1 52.5 52.4 50 : 50 55.7 54.9 54.0 53.5 53.1 52.3 52.2 60 : 40 55.6 54.7 53.8 53.2 52.8 52.1 51.9 70 : 30 55.5 54.5 53.5 52.9 52.5 51.9 51.6 80 : 20 55.4 54.3 53.3 52.7 52.2 51.7 51.3 90 : 10 54.6 53.6 53.1 52.6 52.1 51.6 51.2 91 : 9 54.1 53.6 52.6 52.1 51.6 51.2 50.7 92 : 8 53.6 53.1 52.1 51.6 51.2 50.7 50.3 93 : 7 53.6 53.1 52.1 51.2 50.7 50.3 49.8 94 : 6 53.1 52.6 51.6 50.7 50.3 49.8 48.9 95 : 5 52.6 52.1 51.2 50.3 49.4 48.9 48.5 96 : 4 52.1 51.2 50.7 49.8 48.9 47.7 46.9 97 : 3 50.7 50.3 49.8 48.9 47.7 46.2 45.1 98 : 2 49.9 48.9 48.5 47.3 45.8 43.3 40.0 99 : 1 47.7 47.3 46.5 45.1 43.3 41.2 38.1

=143. Table for the Estimation of Milk Sugar.=—The solutions to be used for this table are the same as those employed in the preceding table for the estimation of invert sugar. The milk sugar is supposed to be in a pure form in solution before beginning the analysis. The method to be employed for milk will be given in the part devoted to dairy products.

In the conduct of the work twenty-five cubic centimeters of the copper solution are mixed with an equal quantity of the alkaline tartrate mixture, and from twenty to one hundred cubic centimeters of the sugar solution added, according to its concentration. This solution should not contain less than seventy nor more than 306 milligrams of lactose. The volume is completed to 150 cubic centimeters with boiling water and kept in lively ebullition for six minutes. The rest of the operation is conducted in the manner already described. From the weight of copper obtained the quantity of milk sugar is determined by inspecting the table. It is recommended to use such a weight of milk sugar as will give about 200 milligrams of copper.

TABLE FOR DETERMINING MILK SUGAR.

(A) = Milligrams of copper. (B) = Milligrams of milk sugar.

(A) (B) (A) (B) (A) (B) (A) (B) 100 71.6 120 86.4 140 101.3 160 116.4 101 72.4 121 87.2 141 102.0 161 117.1 102 73.1 122 87.9 142 102.8 162 117.9 103 73.8 123 88.7 143 103.5 163 118.6 104 74.6 124 89.4 144 104.3 164 119.4 105 75.3 125 90.1 145 105.1 165 120.2 106 76.1 126 90.9 146 105.8 166 120.9 107 76.8 127 91.6 147 106.6 167 121.7 108 77.6 128 92.4 148 107.3 168 122.4 109 78.3 129 93.1 149 108.1 169 123.2 110 79.0 130 93.8 150 108.8 170 123.9 111 79.8 131 94.6 151 109.6 171 124.7 112 80.5 132 95.3 152 110.3 172 125.5 113 81.3 133 96.1 153 111.1 173 126.2 114 82.0 134 96.9 154 111.9 174 127.0 115 82.7 135 97.6 155 112.6 175 127.8 116 83.5 136 98.3 156 113.4 176 128.5 117 84.2 137 99.1 157 114.1 177 129.3 118 85.0 138 99.8 158 114.9 178 130.1 119 85.7 139 100.5 159 115.6 179 130.8

(A) (B) (A) (B) (A) (B) (A) (B) 180 131.6 223 164.2 266 197.2 309 231.4 181 132.4 224 164.9 267 198.0 310 232.2 182 133.1 225 165.7 268 198.8 311 232.9 183 133.9 226 166.4 269 199.5 312 233.7 184 134.7 227 167.2 270 200.3 313 234.5 185 135.4 228 167.9 271 201.1 314 235.3 186 136.2 229 168.6 272 201.9 315 236.1 187 137.0 230 169.4 273 202.7 316 236.8 188 137.7 231 170.1 274 203.5 317 237.6 189 138.5 232 170.9 275 204.3 318 238.4 190 139.3 233 171.6 276 205.1 319 239.2 191 140.0 234 172.4 277 205.9 320 240.0 192 140.8 235 173.1 278 206.7 321 240.7 193 141.6 236 173.9 279 207.5 322 241.5 194 142.3 237 174.6 280 208.3 323 242.3 195 143.1 238 175.4 281 209.1 324 243.1 196 143.9 239 176.2 282 209.9 325 243.9 197 144.6 240 176.9 283 210.7 326 244.6 198 145.4 241 177.7 284 211.5 327 245.4 199 146.2 242 178.5 285 212.3 328 246.2 200 146.9 243 179.3 286 213.1 329 247.0 201 147.7 244 180.1 287 213.9 330 247.7 202 148.5 245 180.8 288 214.7 331 248.5 203 149.2 246 181.6 289 215.5 332 249.2 204 150.0 247 182.4 290 216.3 333 250.0 205 150.7 248 183.2 291 217.1 334 250.8 206 151.5 249 184.0 292 217.9 335 251.6 207 152.2 250 184.8 293 218.7 336 252.5 208 153.0 251 185.5 294 219.5 337 253.3 209 153.7 252 186.3 295 220.3 338 254.1 210 154.5 253 187.1 296 221.1 339 254.9 211 155.2 254 187.9 297 221.9 340 255.7 212 156.0 255 188.7 298 222.7 341 256.5 213 156.7 256 189.4 299 223.5 342 257.4 214 157.5 257 190.2 300 224.4 343 258.2 215 158.2 258 191.0 301 225.2 344 259.0 216 159.0 259 191.8 302 225.9 345 259.8 217 159.7 260 192.5 303 226.7 346 260.6 218 160.4 261 193.3 304 227.5 347 261.4 219 161.2 262 194.1 305 228.3 348 262.3 220 161.9 263 194.9 306 229.1 349 263.1 221 162.7 264 195.7 307 229.8 350 263.9 222 163.4 265 196.4 308 230.6 351 264.7

(A) (B) (A) (B) (A) (B) (A) (B) 352 265.5 365 276.2 377 286.5 389 296.8 353 266.3 366 277.1 378 287.4 390 297.7 354 267.2 367 277.9 379 288.2 391 298.5 355 268.0 368 278.8 380 289.1 392 299.4 356 268.8 369 279.6 381 289.9 393 300.3 357 269.6 370 280.5 382 290.8 394 301.1 358 270.4 371 281.4 383 291.7 395 302.0 359 271.2 372 282.2 384 292.5 396 302.8 360 272.1 373 283.1 385 293.4 397 303.7 361 272.9 374 283.9 386 294.2 398 304.6 362 273.7 375 284.8 387 295.1 399 305.4 363 274.5 376 285.7 388 296.0 400 306.3 364 275.3

=144. Table for the Determination of Maltose.=—The copper and alkaline solutions employed for the oxidation of maltose are the same as those used for invert and milk sugars.

In the manipulation twenty-five cubic centimeters each of the copper and alkali solutions are mixed and boiled and an equal volume of the maltose solution added, which should not contain more than one per cent of the sugar. The boiling is continued for four minutes, an equal volume of cold recently boiled water added, the cuprous oxid separated by filtration and the metallic copper obtained in the manner already described. The weight of maltose oxidized is then ascertained from the table.

Example. Weight of impure maltose taken, ten grams to a liter: Quantity used, twenty-five cubic centimeters: Weight of copper obtained 268 milligrams: Weight of maltose oxidized 237 milligrams: Weight of impure maltose taken 250 milligrams: Percentage of maltose in sample 94.8.

TABLE FOR MALTOSE.

(A) = Milligrams of copper. (B) = Milligrams of maltose.

(A) (B) (A) (B) (A) (B) (A) (B) 30 25.3 35 29.6 40 33.9 45 38.3 31 26.1 36 30.5 41 34.8 46 39.1 32 27.0 37 31.3 42 35.7 47 40.0 33 27.9 38 32.2 43 36.5 48 40.9 34 28.7 39 33.1 44 37.4 49 41.8

(A) (B) (A) (B) (A) (B) (A) (B) 50 42.6 94 81.2 138 120.6 182 160.1 51 43.5 95 82.1 139 121.5 183 160.9 52 44.4 96 83.0 140 122.4 184 161.8 53 45.2 97 83.9 141 123.3 185 162.7 54 46.1 98 84.8 142 124.2 186 163.6 55 47.0 99 85.7 143 125.1 187 164.5 56 47.8 100 86.6 144 126.0 188 165.4 57 48.7 101 87.5 145 126.9 189 166.3 58 49.6 102 88.4 146 127.8 190 167.2 59 50.4 103 89.2 147 128.7 191 168.1 60 51.3 104 90.1 148 129.6 192 169.0 61 52.2 105 91.0 149 130.5 193 169.8 62 53.1 106 91.9 150 131.4 194 170.7 63 53.9 107 92.8 151 132.3 195 171.6 64 54.8 108 93.7 152 133.2 196 172.5 65 55.7 109 94.6 153 134.1 197 173.4 66 56.6 110 95.5 154 135.0 198 174.3 67 57.4 111 96.4 155 135.9 199 175.2 68 58.3 112 97.3 156 136.8 200 176.1 69 59.2 113 98.1 157 137.7 201 177.0 70 60.1 114 99.0 158 138.6 202 177.9 71 61.0 115 99.9 159 139.5 203 178.7 72 61.8 116 100.8 160 140.4 204 179.6 73 62.7 117 101.7 161 141.3 205 180.5 74 63.6 118 102.6 162 142.2 206 181.4 75 64.5 119 103.5 163 143.1 207 182.3 76 65.4 120 104.4 164 144.0 208 183.2 77 66.2 121 105.3 165 144.9 209 184.1 78 67.1 122 106.2 166 145.8 210 185.0 79 68.0 123 107.1 167 146.7 211 185.9 80 68.9 124 108.0 168 147.6 212 186.8 81 69.7 125 108.9 169 148.5 213 187.7 82 70.6 126 109.8 170 149.4 214 188.6 83 71.5 127 110.7 171 150.3 215 189.5 84 72.4 128 111.6 172 151.2 216 190.4 85 73.2 129 112.5 173 152.0 217 191.2 86 74.1 130 113.4 174 152.9 218 192.1 87 75.0 131 114.3 175 153.8 219 193.0 88 75.9 132 115.2 176 154.7 220 193.9 89 76.8 133 116.1 177 155.6 221 194.8 90 77.7 134 117.0 178 156.5 222 195.7 91 78.6 135 117.9 179 157.4 223 196.6 92 79.5 136 118.8 180 158.3 224 197.5 93 80.3 137 119.7 181 159.2 225 198.4

(A) (B) (A) (B) (A) (B) (A) (B) 226 199.3 245 216.3 264 233.4 283 250.4 227 200.2 246 217.2 265 234.3 284 251.3 228 201.1 247 218.1 266 235.2 285 252.2 229 202.0 248 219.0 267 236.1 286 253.1 230 202.9 249 219.9 268 237.0 287 254.0 231 203.8 250 220.8 269 237.9 288 254.9 232 204.7 251 221.7 270 238.8 289 255.8 233 205.6 252 222.6 271 239.7 290 256.6 234 206.5 253 223.5 272 240.6 291 257.5 235 207.4 254 224.4 273 241.5 292 258.4 236 208.3 255 225.3 274 242.4 293 259.3 237 209.1 256 226.2 275 243.3 294 260.2 238 210.0 257 227.1 276 244.2 295 261.1 239 210.9 258 228.0 277 245.1 296 262.0 240 211.8 259 228.9 278 246.0 297 262.8 241 212.7 260 229.8 279 246.9 298 263.7 242 213.6 261 230.7 280 247.8 299 264.6 243 214.5 262 231.6 281 248.7 300 265.5 244 215.4 263 232.5 282 249.6

=145. Preparation of Levulose.=—It is not often that levulose, unmixed with other reducing sugars, is brought to the attention of the analyst. It probably does not exist in the unmixed state in any agricultural product. The easiest method of preparing it is by the hydrolysis of inulin. A nearly pure levulose has also lately been placed on the market under the name of diabetin. It is prepared from invert sugar.

Inulin is prepared from dahlia bulbs by boiling the pulp with water and a trace of calcium carbonate. The extract is concentrated to a sirup and subjected to a freezing temperature to promote the crystallization of the inulin. The separated product is subjected to the above operations several times until it is pure and colorless. It is then washed with alcohol and ether and is reduced to a fine powder. Before the repeated treatment with water it is advisable to clarify the solution with lead subacetate. The lead is afterwards removed by hydrogen sulfid and the resultant acetic acid neutralized with calcium carbonate.

By the action of hot dilute acids inulin is rapidly converted into levulose.

Levulose may also be prepared from invert sugar, but in this case it is difficult to free it from traces of dextrose. The most successful method consists in forming a lime compound with the invert sugar and separating the lime levulosate and dextrosate by their difference in solubility. The levulose salt is much less soluble than the corresponding compound of dextrose. In the manufacture of levulose from beet molasses, the latter is dissolved in six times its weight of water and inverted with a quantity of hydrochloric acid, proportioned to the quantity of ash present in the sample. After inversion the mixture is cooled to zero and the levulose precipitated by adding fine-ground lime. The dextrose and coloring matters in these conditions are not thrown down. The precipitated lime levulosate is separated by filtration and washed with ice-cold water. The lime salt is afterwards beaten to a cream with water and decomposed by carbon dioxid. The levulose, after filtration, is concentrated to the crystallizing point.[110]

=146. Estimation of Levulose.=—Levulose, when free of any admixture with other reducing sugars, may be determined by the copper method with the use of the subjoined table, prepared by Lehmann.[111] The copper solution is the same as that used for invert sugar, viz., 69.278 grams of pure copper sulfate in one liter. The alkali solution is prepared by dissolving 346 grams of rochelle salt and 250 grams of sodium hydroxid in water and completing the volume to one liter.

Manipulation.—Twenty-five cubic centimeters of each solution are mixed with fifty of water and boiled. To the boiling mixture twenty-five cubic centimeters of the levulose solution are added, which must not contain more than one per cent of the sugar. The boiling is then continued for fifteen minutes, and the cuprous oxid collected, washed and reduced to the metallic state in the usual way. The quantity of levulose is then determined by inspection from the table given below. Other methods of determining levulose in mixtures will be given further on.

TABLE FOR THE ESTIMATION OF LEVULOSE.

(A) = Milligrams of copper. (B) = Milligrams of levulose.

(A) (B) (A) (B) (A) (B) (A) (B) 20 7.15 62 31.66 104 56.85 146 82.81 21 7.78 63 32.25 105 57.46 147 83.43 22 8.41 64 32.84 106 58.07 148 84.06 23 9.04 65 33.43 107 58.68 149 84.68 24 9.67 66 34.02 108 59.30 150 85.31 25 10.30 67 34.62 109 59.91 151 85.93 26 10.81 68 35.21 110 60.52 152 86.55 27 11.33 69 35.81 111 61.13 153 87.16 28 11.84 70 36.40 112 61.74 154 87.88 29 12.36 71 37.00 113 62.36 155 88.40 30 12.87 72 37.59 114 62.97 156 89.05 31 13.46 73 38.19 115 63.58 157 89.69 32 14.05 74 38.78 116 64.21 158 90.34 33 14.64 75 39.38 117 64.84 159 90.98 34 15.23 76 39.98 118 65.46 160 91.63 35 15.82 77 40.58 119 66.09 161 92.26 36 16.40 78 41.17 120 66.72 162 92.90 37 16.99 79 41.77 121 67.32 163 93.53 38 17.57 80 42.37 122 67.92 164 94.17 39 18.16 81 42.97 123 68.53 165 94.80 40 18.74 82 43.57 124 69.13 166 95.44 41 19.32 83 44.16 125 69.73 167 96.08 42 19.91 84 44.76 126 70.35 168 96.77 43 20.49 85 45.36 127 70.96 169 97.33 44 21.08 86 45.96 128 71.58 170 97.99 45 21.66 87 46.57 129 72.19 171 98.63 46 22.25 88 47.17 130 72.81 172 99.27 47 22.83 89 47.78 131 73.43 173 99.90 48 23.42 90 48.38 132 74.05 174 100.54 49 24.00 91 48.98 133 74.67 175 101.18 50 24.59 92 49.58 134 75.29 176 101.82 51 25.18 93 50.18 135 75.91 177 102.46 52 25.76 94 50.78 136 76.53 178 103.11 53 26.35 95 51.38 137 77.15 179 103.75 54 26.93 96 51.98 138 77.77 180 104.39 55 27.52 97 52.58 139 78.39 181 105.04 56 28.11 98 53.19 140 79.01 182 105.68 57 28.70 99 53.79 141 79.64 183 106.33 58 29.30 100 54.39 142 80.28 184 106.97 59 29.89 101 55.00 143 80.91 185 107.62 60 30.48 102 55.62 144 81.55 186 108.27 61 31.07 103 56.23 145 82.18 187 108.92

(A) (B) (A) (B) (A) (B) (A) (B) 188 109.56 232 138.57 276 168.68 320 199.97 189 110.21 233 139.25 277 169.37 321 200.71 190 110.86 234 139.18 278 170.06 322 201.44 191 111.50 235 140.59 279 170.75 323 202.18 192 112.14 236 141.27 280 171.44 324 202.91 193 112.78 237 141.94 281 172.14 325 203.65 194 113.42 238 142.62 282 172.85 326 204.39 195 114.06 239 143.29 283 173.55 327 205.13 196 114.72 240 143.97 284 174.26 328 205.88 197 115.38 241 144.65 285 174.96 329 206.62 198 116.04 242 145.32 286 175.67 330 207.36 199 116.70 243 146.00 287 176.39 331 208.10 200 117.36 244 146.67 288 177.10 332 208.83 201 118.02 245 147.35 289 177.82 333 209.57 202 118.68 246 148.03 290 178.53 334 210.30 203 119.33 247 148.71 291 179.24 335 211.04 204 119.99 248 149.40 292 179.95 336 211.78 205 120.65 249 150.08 293 180.65 337 212.52 206 121.30 250 150.76 294 181.63 338 213.25 207 121.96 251 151.44 295 182.07 339 213.99 208 122.61 252 152.12 296 182.78 340 214.73 209 123.27 253 152.81 297 183.49 341 215.48 210 123.92 254 153.49 298 184.21 342 216.23 211 124.58 255 154.17 299 184.92 343 216.97 212 125.24 256 154.91 300 185.63 344 217.72 213 125.90 257 155.65 301 186.35 345 218.47 214 126.56 258 156.40 302 187.06 346 219.21 215 127.22 259 157.14 303 187.78 347 219.97 216 127.85 260 157.88 304 188.49 348 220.71 217 128.48 261 158.49 305 189.21 349 221.46 218 129.10 262 159.09 306 189.93 350 222.21 219 129.73 263 159.70 307 190.65 351 222.96 220 130.36 264 160.30 308 191.37 352 223.72 221 131.07 265 160.91 309 192.09 353 224.47 222 131.77 266 161.63 310 192.81 354 225.23 223 132.48 267 162.35 311 193.53 355 225.98 224 133.18 268 163.07 312 194.25 356 226.74 225 133.89 269 163.79 313 194.97 357 227.49 226 134.56 270 164.51 314 195.69 358 228.25 227 135.23 271 165.21 315 196.41 359 229.00 228 135.89 272 165.90 316 197.12 360 229.76 229 136.89 273 166.60 317 197.83 361 230.52 230 137.23 274 167.29 318 198.55 362 231.28 231 137.90 275 167.99 319 199.26 363 232.05

(A) (B) (A) (B) (A) (B) (A) (B) 364 232.81 370 237.39 376 241.87 382 246.25 365 233.57 371 238.16 377 242.51 383 247.17 366 234.33 372 238.93 378 243.15 384 248.08 367 235.10 373 239.69 379 243.79 385 248.99 368 235.86 374 240.46 380 244.43 369 236.63 375 241.23 381 245.34

=147. Precipitation of Sugars with Phenylhydrazin=.—The combination of phenylhydrazin with aldehyds and ketones was first studied by Fischer, and the near relationship of these bodies to sugar soon led to the investigation of the compounds formed thereby with this reagent.[112] Reducing sugars form with phenylhydrazin insoluble crystalline bodies, to which the name osazones has been given. The reaction which takes place is a double one and is represented by the following formulas:

Dextrose. Phenylhydrazin. Dextrose-phenylhydrazone.

C₆H₁₂O₆ + C₆H₅NH.NH₂ = C₆H₁₂O₅.N.NHC₆H₅ + H₂O and C₆H₁₂O₅.N.NHC₆H₅ + C₆H₅NH.NH₂ = Phenyldextrosazone. C₆H₁₀O₄(N.NHC₆H₅)₂ + 2H₂O.

The dextrosazone is commonly called glucosazone. The osazones formed with the commonly occurring reducing sugars are crystalline, stable, insoluble bodies which can be easily separated from any attending impurities and identified by their melting points. Glucosazone melts at 205°, lactosazone at 200° and maltosazone at 206°.

The osazones are precipitated in the following way: The reducing sugar, in about ten per cent solution, is treated with an excess of the acetate of phenylhydrazin in acetic acid and warmed to from 75° to 85°. In a short time the separation is complete and the yellow precipitate formed is washed, dried and weighed. The sugar can be recovered from the osazone by decomposing it with strong hydrochloric acid by means of which the phenylhydrazin is displaced and a body, osone, is formed, which by treatment with zinc dust and acetic acid, is reduced to the original sugar. The reactions which take place are represented by the following equations:[113]

Glucososone. C₆H₁₀O₄(N.NH.C₆H₅)₂ + 2H₂O = C₆H₁₀O₆ + 2C₆H₅N₂H₂

Dextrose (Glucose). C₆H₁₀O₆ + H₂ = C₆H₁₂O₆.

For the complete precipitation of dextrose as osazone Lintner and Kröber show that the solution of dextrose should not contain more than one gram in 100 cubic centimeters. Twenty cubic centimeters containing 0.2 gram dextrose should be used for the precipitation.[114] To this solution should be added one gram of phenylhydrazin and one gram of fifty per cent acetic acid. The solution is then to be warmed for about two hours and the precipitate washed with from sixty to eighty cubic centimeters of hot water and dried for three hours at 105°. One part of the osazone is equivalent to one part of dextrose when maltose and dextrin are absent. When these are present the proportion is one part of osazone to 1.04 of dextrose. Where levulose is precipitated instead of dextrose 1.43 parts of the osazone are equal to one part of the sugar.

Sucrose is scarcely at all precipitated as osazone until inverted.

After inversion and precipitation as above, 1.33 parts osazone are equal to one part of sucrose.

The reaction with phenylhydrazin has not been much used for quantitive estimations of sugars, but it has been found especially useful in identifying and separating reducing sugars. It is altogether probable, however, that in the near future phenylhydrazin will become a common reagent for sugar work.

Maquenne has studied the action of phenylhydrazin on sugars and considers that this reaction offers the only known means of precipitating these bodies from solutions where they are found mixed with other substances.[115] The osazones, which are thus obtained, are usually very slightly soluble in the ordinary reagents, for which reason it is easy to obtain them pure when there is at the disposition of the analyst a sufficient quantity of the material. But if the sugar to be studied is rare and if it contain, moreover, several distinct reducing bodies, the task is more delicate. It is easy then to confound several osazones which have almost identical points of fusion; for example, glucosazone with galactosazone. Finally, it becomes impossible by the employment of phenylhydrazin to distinguish glucose, dextrose or mannose from levulose alone or mixed with its isomers. Indeed, these three sugars give, with the acetate of phenylhydrazin the same phenylglucosazone which melts at about 205°. It is noticed that the weights of osazones which are precipitated when different sugars are heated for the same time with the same quantity of the phenylhydrazin, vary within extremely wide limits. It is constant for each kind of sugar if the conditions under which the precipitation is made are rigorously the same. There is then, in the weight of the osazones produced, a new characteristic of particular value. The following numbers have been obtained by heating for one hour at 100°, one gram of sugar with 100 cubic centimeters of water and five cubic centimeters of a solution containing forty grams of phenylhydrazin and forty grams of acetic acid per hundred. After cooling the liquid, the osazones are received upon a weighed filter, washed with 100 cubic centimeters of water, dried at 110° and weighed. The weights of osazones obtained are given in the following table:

Weight of the osazones. Character of the sugar. gram.

Sorbine, crystallized 0.82 Levulose ” 0.70 Xylose ” 0.40 Glucose, anhydrous 0.32 Arabinose, crystallized 0.27 Galactose ” 0.23 Rhamnose ” 0.15 Lactose ” 0.11 Maltose ” 0.11

With solutions twice as dilute as those above, the relative conditions are still more sensible, and the different sugars arrange themselves in the same order, with the exception of levulose, which shows a slight advantage over sorbine and acquires the first rank. From the above determinations, it is shown that levulose and sorbine give vastly greater quantities of osazones, under given conditions, than the other reducing sugars. It would be easy, therefore, to distinguish them by this reaction and to recognize their presence also even in very complex mixtures, where the polarimetric examination alone would furnish only uncertain indications.

It is remarkable that these two sugars are the only ones among the isomers or the homologues of dextrose, actually known, which possess the functions of an acetone. They are not, however, easily confounded, since the glucosazone forms beautiful needles which are ordinarily visible to the naked eye, while the sorbinosazone is still oily and when heated never gives perfectly distinct crystals.

This method also enables us to distinguish between dextrose and galactose, of which the osazone is well crystallized and melts at almost the same temperature as the phenylglucosazone. Finally, it is observed that the reducing sugars give less of osazones than the sugars which are not capable of hydrolysis, and consequently differ in their inversion products. It is specially noticed in this study of the polyglucoses (bioses, trioses), that this new method of employing the phenylhydrazin appears very advantageous. It is sufficient to compare the weights of the osazones to that which is given under the same conditions by a known glucose, in order to have a very certain verification of the probabilities of the result of the chemical or optical examination of the mixture which is under study. All the polyglucoses which have been examined from this point of view give very decided results. The numbers which follow have reference to one gram of sugar completely inverted by dilute sulfuric acid, dissolved in 100 cubic centimeters of water, and treated with two grams of phenylhydrazin, the same quantity of acetic acid, and five grams of crystallized sodium acetate. All these solutions have been compared with the artificial mixtures and corresponding glucoses, with the same quantities of the same reagents. The following are the results of the examination:

Weight of the osazone. Character of the sugar. gram.

1 {Saccharose, ordinary 0.71 {Glucose and levulose (.526 g each) 0.73

2 {Maltose 0.55 {Glucose (1.052 g) 0.58

3 {Raffinose, crystallized 0.48 {Levulose, glucose and galactose (.333 g each) 0.53

4 {Lactose, crystallized 0.38 {Glucose and galactose (.500 g each) 0.39

It is noticed that the agreement for each saccharose is as satisfactory as possible. Numbers obtained with the products of inversion are always a little low by reason of the destructive action of sulfuric acid, and in particular, upon levulose. This is, moreover, quite sensible when the product has to be heated for a long time with sulfuric acid in order to secure a complete inversion. It is evident from the data cited from the papers of Fischer, Maquenne, and others, that the determination of sugars by this method is not a very difficult analytical process and may, in the near future, become of great practical importance.

=148. Molecular Weights of Carbohydrates.=—In the examination of carbohydrates the determination of the molecular weights is often of the highest analytical value.

The uncertainty in respect of the true molecular weights of the carbohydrates is gradually disappearing by reason of the insight into the composition of these bodies, which recently discovered physical relations have permitted.

Raoult, many years ago,[116] proposed a method of determining molecular weights which is particularly applicable to carbohydrates soluble in water.

The principle of Raoult’s discovery may be stated as follows: The depression of the freezing point of a liquid, caused by the presence of a dissolved liquid or solid, is proportionate to the absolute amount of substance dissolved and inversely proportionate to its molecular weight.

The following formulas may be used in computing results:

C = observed depression of freezing point:

P = weight of anhydrous substance in 100 grams:

C --- = A = depression produced by one gram substance in 100 grams: P

K = depression produced by dissolving in 100 cubic centimeters a number of grams of the substance corresponding to its molecular weight:

M = molecular weight:

C Then we have, K = ---- × M. P

K is a quantity varying with the nature of the solvent but with the same solvent remaining sensibly constant for numerous groups of compounds.

The value of

C A ---- P

can be determined by experiment. The molecular weight can therefore be calculated from the formula

K M = ----. A

With organic compounds in water the value of K is almost constant.

Brown and Morris[117] report results of their work in extending Raoult’s investigations of the molecular weight of the carbohydrates. The process is carried on as follows:

A solution of the carbohydrate is prepared containing a known weight of the substance in 100 cubic centimeters of water. About 120 cubic centimeters of the solution are introduced into a thin beaker of about 400 capacity. This beaker is closed with a stopper with three holes. Through one of these a glass rod for stirring the solution is inserted. The second perforation carries a delicate thermometer graduated to 0°.05. The temperature is read with a telescope. The beaker is placed in a mixture of ice and brine at a temperature from 2° to 3° below the freezing point of the solution. The solution is cooled until its temperature is from 0°.5 to 1° below the point of congelation. Through the third aperture in the stopper a small lump of ice taken from a frozen portion of the same solution, is dropped, causing at once the freezing process to begin. The liquid is briskly stirred and as the congelation goes on the temperature rises and finally becomes constant. The reading is then taken. The depression in the freezing point, controlled by the strength of the solution, should never be more than from 1° to 2°.

The molecular weights may also be determined by the boiling points of their solutions as indicated by the author,[118] Beckmann,[119] Hite, Orndorff and Cameron.[120]

The method applied to some of the more important carbohydrates gave the following results:

DEXTROSE.

Calculated for C₆H₁₂O₆. Found. M = 180 M = 180.2

SUCROSE.

Calculated for C₁₂H₂₂O₁₁. Found. M = 342 M = 337.5

INVERTOSE (DEXTROSE AND LEVULOSE).

Calculated for C₆H₁₂O₆. Found. M = 180 M = 174.3

MALTOSE.

Calculated for C₁₂H₂₂O₁₁. Found. M = 342 M = 322

LACTOSE.

Calculated for C₁₂H₂₂O₁₁. Found. M = 342 M = 345

ARABINOSE.

Calculated for C₅H₁₀O₅. Found. M = 150 M = 150.3

RAFFINOSE. Calculated for C₁₈H₃₂O₁₆.5H₂O. Found. M = 594 M = 528

=149. Birotation.=—As is well known, dextrose exhibits in fresh solutions the phenomenon of birotation. The authors supposed that this phenomenon might have some relation to the size of the molecule. They, therefore, determined the molecular volume of freshly dissolved dextrose by the method of Raoult and found M = 180. The high rotatory power of recently dissolved dextrose is therefore not due to any variation in the size of its molecule.

The mathematical theory of birotation is given by Müller as follows.[121] In proportion as the unstable modification A is transformed into the stable modification B, the rotation will vary. Let ρ = the specific rotatory power of B and _a_ρ = that of A, both in the anhydrous state. Let now p grams of the substance be dissolved in V cubic centimeters of solvent and observed in a tube l decimeters in length. The time from making the solution is represented by θ. The angle of rotation α is read at the time θ. Let x = the mass of A, and y = that of B, and the equation is derived.

_a_ρ_xl_ ρ_yl_ α = --------- + ------: V V

But x + y = p

ρ_l_ whence α = [(a - 1)x + p] ----. V

If now there be introduced into the calculation the final angle of rotation αₙ, which can be determined with great exactness; we have

_p_ρ_l_ (a - 1)x αₙ = ------- and consequently α = αₙ[1 + ------------], V p

(a - 1)x α whence ------------- = --- - 1. p αₙ

This equation gives the quantity x of the unstable matter which is transformed into the stable modification in the time θ.

It must be admitted that the quantity dx which is changed during the infinitely small time _d_θ is proportional to the mass x which still exists at the moment θ, whence dx = -Cʹ_xd_θ where Cʹ represents a constant positive factor. From this is derived the equation

dx ----- = -Cʹ_d_θ. x

Integrating and calling x the quantity of matter changed to the stable form at the moment θ, corresponding to a rotation α₀, we have

1 _x_₀ Cʹ = ------- log. nap. ----, and taking into consideration θ - θ₀ x

the equation given above, and substituting common for superior logarithms we get

1 α₀ - αₙ C = --------- log. ---------. θ - θ₀ α - αₙ

Experience has shown that such a constant C really exists, and its value can be easily calculated from the data of Parcus and Tollens.[122] The mean value of C from these data is 0.0301 for arabinose; 0.0201 for xylose; 0.0393 for rhamnose; 0.0202 for fucose; 0.00927 for galactose; 0.00405 for lactose; 0.00524 for maltose, and for dextrose, 0.00348 at 11° to 13° and 0.00398 from 13° to 15°. The constant C as is well known, increases as the temperature is raised.

The constant C, at a given temperature, measures the progress of the phenomenon of the change from the unstable to the stable state. It will be noticed that among the sugars possessing multirotation properties the pentoses possess a much higher speed of transformation than the others.

=150. Estimation of Pentose Sugars and Pentosans as Furfurol.=—The production of furfurol by distilling carbohydrates with an acid has already been mentioned. Tollens and his associates have shown that with pentose sugars, and carbohydrate bodies yielding them, the production of furfurol is quantitive.

The production and estimation of furfurol have been systematically studied by Krug, to whose paper the reader is referred for the complete literature of the subject.[123] The essential principles of the operation are based on the conversion of the pentoses into furfurol by distilling with a strong acid, and the subsequent precipitation and estimation of the furfurol formed in the first part of the reaction.

The best method of conducting the distillation is as follows:

Five grams of the pentose substance are placed in a flask of about a quarter liter capacity, with 100 cubic centimeters of hydrochloric acid of 1.06 specific gravity. The arrangement of the apparatus is shown in Fig. 46. The flame of the lamp is so regulated as to secure about two cubic centimeters of distillate per minute.

The distillate is received in a graduated cylinder and as soon as thirty cubic centimeters are collected, an equal quantity of hydrochloric acid, of the strength noted, is added to the distilling flask, allowing it to flow in slowly so as not to stop the ebullition. The process is continued until a drop of the distillate gives no sensible reaction for furfurol when tested with anilin acetate. The test is applied as follows: Place a drop of the distillate on a piece of filter paper moistened with anilin acetate. The presence of furfurol will be disclosed by the production of a brilliant red color. Usually about three hours are consumed in the distillation, during which time a little less than 400 cubic centimeters of distillate is obtained. The distillate is neutralized with solid sodium carbonate and, in order to have always the same quantity of common salt present, 10.2 grams of sodium chlorid are added for each fifty cubic centimeters of water necessary to make the total volume to half a liter.[124]

The reactions with pentosans probably consist in first splitting up of the molecule into a pentose and the subsequent conversion of the latter into furfurol according to the following equations:

(C₅H₈O₄)ₙ + (H₂O)ₙ = (C₅H₁₀O₅)ₙ Pentosan. Water. Pentose.

and

(C₅H₁₀O₅)ₙ = (C₅H₄O₂)ₙ + (3H₂O)ₙ. Pentose. Furfurol. Water.

=151. Determination of Furfurol.=—The quantity of furfurol obtained by the process mentioned above may be determined in several ways.

As Furfuramid.—When ammonia is added to a saturated solution of furfurol, furfuramid, (C₅H₄O)₃N₂, is formed. In order to secure the precipitate it is necessary that the furfurol be highly concentrated and this can only be accomplished by a tedious fractional distillation. This method, therefore, has little practical value.

As Furfurolhydrazone.—Furfurol is precipitated almost quantitively, even from dilute solutions, by phenylhydrazin. The reaction is represented by the equation:

C₆H₈N₂ + C₅H₄O₂ = C₁₁H₁₀N₂O + H₂O. Phenylhydrazin. Furfurol. Furfurolhydrazone. Water.

=152. Volumetric Methods.=—Tollens and Günther have proposed a volumetric method which is carried out as follows:[125] The distillation is accomplished in the manner described. The distillate is placed in a large beaker, neutralized with sodium carbonate and acidified with a few drops of acetic. Phenylhydrazin solution of known strength is run in until a drop of the liquid, after thorough mixing, shows no reaction for furfurol with anilin acetate. The reagent is prepared by dissolving five grams of pure phenylhydrazin and three of glacial acetic acid in distilled water, and diluting to 100 cubic centimeters. The solution is set by dissolving from two-tenths to three-tenths gram of pure furfurol in half a liter of water and titrating with the phenylhydrazin as indicated above. The quantity of the pentose used has a great influence on the result.

With nearly a gram of arabinose about fifty per cent of furfurol were obtained while when nearly five grams were used only about forty-six per cent of furfurol were found. With xylose a similar variation was found, the percentage of furfurol, decreasing as the quantity of pentose increased. The method, therefore, gives only approximately accurate results.

=153. Method of Stone.=—Another volumetric method proposed by Stone is based on the detection of an excess of phenylhydrazin by its reducing action on the fehling reagent.[126] A standard solution of phenylhydrazin is prepared by dissolving one gram of the hydrochlorate and three grams of sodium acetate in water and completing the volume of the liquor to 100 cubic centimeters. This solution contains 1.494 milligrams of phenylhydrazin in each cubic centimeter, theoretically equivalent to 1.328 milligrams of furfurol. The reagent is set by titrating against a known weight of furfurol. Pure furfurol may be prepared by treating the crude article with sulfuric acid and potassium dichromate, and subjecting the product to fractional distillation. The distillate is treated with ammonia and the furfuramid formed is purified by recrystallizing from alcohol and drying over sulfuric acid. One gram of this furfuramid is dissolved in dilute acetic acid and the volume completed to one liter with water.[127] The phenylhydrazin solution being unstable, is to be prepared at the time of use.

The titration is conducted as follows: Twenty-five cubic centimeters of the distillate obtained from a pentose body, by the method described above, are diluted with an equal volume of water, a certain quantity of the phenylhydrazin solution added to the mixture from a burette and the whole heated quickly to boiling. The flask is rapidly cooled and a portion of its contents poured on a filter. The filtrate should have a pale yellow color and be perfectly clear. If it become turbid on standing, it should be refiltered. Two cubic centimeters of the clear filtrate are boiled with double the quantity of the fehling reagent. If phenylhydrazin be present, the color of the mixture will change from blue to green. By repeating the work, with varying quantities of phenylhydrazin, a point will soon be reached showing the end of the reaction in a manner entirely analogous to that observed in volumetric sugar analysis.

In practice the volumetric methods have given place to the more exact gravimetric methods described below.

=154. Gravimetric Methods.=—The distillation is carried on and the volume of the distillate completed to half a liter as described above. Chalmot and Tollens then proceed as follows:[128] Ten cubic centimeters of a solution of phenylhydrazin acetate, containing in 100 cubic centimeters twelve grams of the phenylhydrazin and seven and a half grams of glacial acetic acid dissolved and filtered, are added to the distillate and the mixture stirred with an appropriate mechanism for half an hour. The furfurolhydrazone at the end of this time will have separated as small reddish-brown crystals. The mixture is then thrown onto an asbestos filter and the liquid separated with suction. The suction should be very gradually applied so as not to clog the felt. The precipitate adhering to the beaker is washed into the filter with 100 cubic centimeters of water. The precipitate is dried at about 60° and weighed. As a check the hydrazone may be dissolved in hot alcohol, the filter well washed, dried and again weighed. To obtain the weight of furfurol the weight of hydrazone found is multiplied by 0.516 and 0.025 added to compensate for the amount which was held in solution or removed by washing. Less than one per cent of pentose can not be determined by this method since that amount is equalled by the known losses during the manipulation.

Factor.—To convert the furfurol found into pentoses, the following factors are used:

Per cent furfurol Multiply for obtained from Multiply for Multiply for penta-glucoses five grams of arabinose by. xylose by. by. pentoses.

2.5 per cent or less 1.90 1.70 1.67 5.0 ” ” ” more 2.04 1.90 1.92

=155. Method Of Krug.=—In conducting the determination of furfurol, according to the method of Chalmont and Tollens just noticed, Krug observed that the filtrate, after standing for some time, yielded a second precipitate of furfurol hydrazone. Great difficulty was also experienced in collecting the precipitate upon the filter on account of the persistency with which it stuck to the sides of the vessel in which the precipitation took place.[129] In order to avoid these two objections, Krug modified the method as described below and this modified method is now exclusively used in this laboratory.

After the precipitation of the furfurol hydrazone, it is stirred vigorously, by means of an appropriate mechanical stirrer, for at least half an hour and then allowed to rest for twenty-four hours. On filtering after that length of time the filtrate remains perfectly clear and no further precipitation takes place. After the filtration is complete and the beaker and filtering tube well washed, no attempt is made to detach the part of the filtrate adhering to the beaker but the whole of the precipitate, both that upon the filter and that adhering to the sides of the beaker, is dissolved in strong alcohol, from thirty to forty cubic centimeters being used. The alcoholic solution is collected in a small weighed flask, the alcohol evaporated at a gentle heat and the last traces of water removed by heating to 60° and blowing a current of dry air through the flask. After weighing the precipitate of furfurol hydrazone, obtained as above, the calculation of the weight of pentose bodies is accomplished by means of the usual factors.

=156. Precipitation of Furfurol with Pyrogalol.=—Furfurol is thrown out of solution in combination with certain phenol bodies by heating together in an acid solution. Hotter has proposed a method for the determination of furfurol based on the above fact.[130] The furfurol is obtained by distillation in the manner already described and hydrochloric acid is added if necessary to secure twelve per cent of that body in a given volume. The furfurol is thrown out of an aliquot portion by heating with an excess of pyrogalol in closed tubes for about two hours at 110°. The reaction takes place in two stages, represented by the following equations:

C₅H₄O₂ + C₆H₆O₃ = C₁₁H₁₀O₅

and

2C₁₁H₁₀O₅ = C₂₂H₁₈O₉ + H₂O.

The aliquot part of the distillate used should not contain more than one-tenth of a gram of furfurol. The precipitate formed in this way is collected on an asbestos felt, dried at 103° and weighed. The weight obtained divided by 1.974 gives the corresponding amount of furfurol. There is some difficulty experienced in loosening the precipitate from the sides of the tubes in which the heating takes place, but this defect can be overcome by heating in covered beakers in an autoclave.

=157. Precipitation with Phloroglucin.=—Instead of using pyrogalol for the precipitating reagent phloroglucin may be employed. The method of procedure proposed by Councler for this purpose is given below.[131] The furfurol is prepared by distillation in the usual way. The volume of the distillate obtained is completed to half a liter with twelve per cent hydrochloric acid, and an aliquot portion, varying in volume with the percentage of furfurol is withdrawn for precipitation. This portion is placed in a glass-stoppered flask with about twice the quantity of finely powdered phloroglucin necessary to combine with the furfurol present. The contents of the flask are well shaken and allowed to stand fifteen hours. The precipitate is collected on an asbestos filter, washed free of chlorin, dried at the temperature of boiling water and weighed.

The theoretical quantity of precipitate corresponding to one part of furfurol, viz., 2.22 parts, is never obtained since the precipitate is not wholly insoluble in water. The actual proportions between the precipitate and the original furfurol vary with the amount of precipitate obtained.

When the weight of the precipitate is 200 milligrams and over, 2.12 parts correspond to one part of furfurol. When the weight of the precipitate varies from fifty to 100 milligrams, the ratio is as 2.05:1 and when only about twenty-five milligrams of precipitate are obtained the ratio is as 1.98:1.

The quantity of pentose bodies corresponding to the furfurol is calculated from the factors given by Tollens in a preceding paragraph.

The reaction which takes place with furfurol and phloroglucin is simply a condensation of the reagents with the separation of water. It is very nearly represented by the following formula:

2C₅H₄O₂ + C₆H₆O₃ = C₁₆H₁₂O₆ + H₂O. Furfurol. Phloroglucin Condensation Water. product.

It has been shown by Welbel and Zeisel,[132] that in the presence of twelve per cent of hydrochloric acid phloroglucin itself is condensed into dark insoluble compounds. When three molecules of furfurol and two molecules of phloroglucin are present, the bodies are both condensed and separated by continued action. When from one and a quarter to three parts of phloroglucin by weight are used to one part of furfurol, the weight of the precipitate obtained under constant conditions may serve sufficiently well for the calculation of the furfurol. The precipitates contain chlorin, which they give up even in the cold, to water. For these reasons the analytical data obtained by the method of Councler, given above, are apt to be misleading. It is probable also that similar conditions may to a certain extent prevail in the separation of furfurol with phenylhydrazin, and further investigation in this direction is desirable. For the present the very best method that can be recommended for the estimation of pentoses and pentosans is the conversion thereof into furfurol and the separation of the compound with phenylhydrazin acetate.

=158. Estimation of Sugars by Fermentation.=—When a solution of a hexose sugar is subjected to the action of certain ferments a decomposition of the molecule takes place with the production of carbon dioxid and various alcohols and organic acids. Under the action of the ferment of yeast Saccharomyces cerevisiae the sugar yields theoretically only carbon dioxid and ethyl alcohol, as represented by the equation:

C₆H₁₂O₆ = 2C₂H₆O + 2CO₂.

The theoretical quantities of alcohol and carbon dioxid obtained according to this equation are 51.11 percent of alcohol and 48.89 per cent of carbon dioxid.

When the yeast ferment acts on cane sugar the latter first suffers inversion, and the molecules of dextrose and levulose produced are subsequently converted into alcohol and carbon dioxid as represented below:

C₁₂H₂₂O₁₁ + H₂O = 2C₆H₁₂O₆

2C₆H₁₂O₆ = 4C₂H₆O + 4CO₂.

Cane sugar, plus the water of hydrolysis, will yield theoretically 53.8 per cent of alcohol and 51.5 per cent of carbon dioxid.

In practice the theoretical proportions of alcohol and carbon dioxid are not obtained because of the difficulty of excluding other fermentative action, resulting in the formation especially of succinic acid and glycerol. Moreover, a part of the sugar is consumed by the yeast cells to secure their proper growth and development. In all only about ninety-five per cent of the sugar can be safely assumed as entering into the production of alcohol. About 48.5 per cent of alcohol are all that may be expected of the weight of dextrose or invert sugar used. Only sugars containing three molecules of carbon or some multiple thereof are fermentable. Thus the trioses, hexoses, nonoses, etc., are susceptible of fermentation, while the tetroses, pentoses, etc., are not.

=159. Estimating Alcohol.=—In the determination of sugar by fermentation, a rather dilute solution not exceeding ten per cent should be used. A quantity of pure yeast, equivalent to four or five per cent of the sugar used, is added, and the contents of the vessel, after being well shaken, exposed to a temperature of from 25° to 30° until the fermentation has ceased, which will be usually in from twenty-four to thirty-six hours. The alcohol is then determined in the residue by the methods given hereafter.

The weight of the alcohol obtained multiplied by 100 and divided by 48.5, will give the weight of the hexose reducing sugar which has been fermented. Ninety-five parts of sucrose will give 100 parts of invert sugar.

Example.—Let the weight of alcohol obtained be 0.625 gram. Then 0.625 × 100 ÷ 48.5 = 1.289 grams, the weight of the hexose, which has been fermented; 1.289 grams of dextrose or levulose correspond to 1.225 of sucrose.

=160. Estimating Carbon Dioxid.=—The sugar may also be determined by estimating the amount of carbon dioxid produced during the fermentation. For this purpose the mixture of sugar solution and yeast, prepared as above mentioned, is placed in a flask whose stopper carries two tubes, one of which introduces air free of carbon dioxid into the contents of the flask, and the other conducts the evolved carbon dioxid into the absorption bulbs. In passing to the absorption bulbs the carbon dioxid is freed of moisture by passing through another set of bulbs filled with strong sulfuric acid. During the fermentation, the carbon dioxid is forced through the bulbs by the pressure produced, or better, a slow current of air is aspirated through the whole apparatus. The aspiration is continued after the fermentation, has ceased, until all the carbon dioxid is expelled. Towards the end, the contents of the flask may be heated to near the boiling-point. The increase of the weight of the potash bulbs will give the weight of carbon dioxid obtained. A hexose reducing sugar will yield about 46.5 per cent of its weight of carbon dioxid. The calculation is made as suggested for the alcohol process.

The fermentation process has little practical value save in determining sucrose in presence of lactose, as will be described in another place.

=161. Precipitation of Sugars in Combination with the Earthy Bases.=—The sugars combine in varying proportions with the oxids and hydroxids of calcium, strontium and barium. Sucrose especially, furnishes definite crystalline aggregates with these bases in such a way as to form the groundwork of several technical processes in the separation of that substance from its normally and abnormally associated compounds. These processes have little use as analytical methods, but are of great value, as mentioned, from a technical point of view.

=162. Barium Saccharate.=—This compound is formed by mixing the aqueous solutions of barium hydroxid and sugar. The saccharate separates in bright crystalline plates or needles from the warm solution, as C₁₂H₂₂O₁₁BaO. One part of this precipitate is soluble in about forty-five parts of water, both at 15° and 100°.

=163. Strontium Saccharates.=—Both the mono- and distrontium saccharates are known, viz., C₁₂H₂₂O₁₁SrO + 5H₂O and C₁₂H₂₂O₁₁2SrO.

The monosalt may be easily secured by adding a few of its crystals to a mixture of sugar and strontium hydroxid solutions.

The disaccharate is precipitated as a granular substance when from two to three molecules of strontium hydroxid are added to a boiling sugar solution. The reaction is extensively used in separating the sugar from beet molasses.

=164. Calcium Saccharates.=—Three calcium saccharates are known in which one molecule of sugar is combined with one, two and three molecules of lime respectively.

The monosaccharate is obtained by mixing the sugar and lime in the proper proportion and precipitating by adding alcohol.

The precipitate is partly granular and partly jelly-like, and is soluble in cold water. The dicalcium compound is obtained in the same way and has similar properties. Both, on boiling, with water, form the trisaccharate and free sugar.

The tricalcium saccharate is the most important of these compounds, and may be obtained directly by mixing freshly burned and finely ground lime (CaO) with a very cold dilute solution of sugar.

The compound crystallizes with three or four molecules of water. When precipitated as described above, however, it has a granular, nearly amorphous structure, and the process is frequently used in the separation of sugar from beet molasses.

In the laboratory but little success has been had in using even the barium hydroxid as a chemical reagent, and therefore the reactions mentioned above are of little value for analytical purposes. In separating sugar from vegetable fibers and seeds, however, the treatment with strontium hydroxid is especially valuable the sugar being subsequently recovered in a free state by breaking up the saccharate with carbon dioxid. The technical use of these reactions also is of great importance in the beet sugar industry.

=165. Qualitive Tests for the Different Sugars.=—The analyst will often be aided in examining an unknown substance by the application of qualitive tests, which will disclose to him the nature of the saccharine bodies with which he has to deal.

=166. Optical Test for Sucrose.=—The simplest test for the presence of sucrose is made with the polariscope. A small quantity of the sample under examination is dissolved in water, clarified by any of the usual methods, best with alumina cream, and polarized. A portion of the liquor is diluted with one-tenth its volume of strong hydrochloric acid and heated to just 68°, consuming about fifteen minutes time in the operation. The mixture is quickly cooled and again polarized in a tube one-tenth longer than before used; or the same tube may be used and the observed reading of the scale increased by one-tenth. If sucrose be present the second reading will be much lower than the first, or may even be to the left.

=167. Cobaltous Nitrate Test for Sucrose.=—Sucrose in solution may be distinguished from other sugars by the amethyst violet color which it imparts to a solution of cobaltous nitrate. This reaction was first described by Reich, in 1856, but has only lately been worked out in detail. The test is applied as follows:

To about fifteen cubic centimeters of the sugar solution add five cubic centimeters of a five per cent solution of cobaltous nitrate. After thoroughly mixing the two solutions, add two cubic centimeters of a fifty per cent solution of sodium hydroxid. Pure sucrose gives by this treatment an amethyst violet color, which is permanent. Pure dextrose gives a turquoise blue color which soon passes into a light green. When the two sugars are mixed the coloration produced by the sucrose is the predominant one, and one part of sucrose in nine parts of dextrose can be distinguished. If the sucrose be mixed with impurities such as gum arabic or dextrin, they should be precipitated by alcohol or basic lead acetate, before the application of the test. Dextrin may be thrown out by treatment of the solution with barium hydroxid and ammoniacal lead acetate. The reaction may also be applied to the detection of cane sugar in wines, after they are thoroughly decolorized by means of lead acetate and bone-black. The presence of added sucrose to milk, either in the fresh or condensed state, may also be detected after the disturbing matters are thrown out with lead acetate. The presence of sucrose in honey may also be detected by this process. The reaction has been tried in this laboratory with very satisfactory results. The amethyst violet coloration with sucrose is practically permanent. On boiling the color is made slightly bluish, but is restored to the original tint on cooling. Dextrose gives at first a fine blue color which in the course of two hours passes into a pale green. A slight flocculent precipitate is noticed in the tube containing the dextrose. Maltose and lactose act very much as dextrose, but in the end do not give so fine a green color. If the solutions containing dextrose, lactose and maltose be boiled, the original color is destroyed and a yellow-green color takes its place. The reaction is one which promises to be of considerable practical value to analysts, as it may be applied for the qualitive detection of sucrose in seeds and other vegetable products.[133]

=168. The Dextrose Group.=—In case the carbohydrate in question shows a right-handed rotation and the absence of sucrose is established by the polariscopic observation described above, the presence of the dextrose group may be determined by the following test.[134]

Five grams of the carbohydrate are oxidized by boiling with from twenty to thirty cubic centimeters of nitric acid of 1.15 specific gravity, and then at gentle heat evaporated to dryness with stirring. If much mucic acid be present, as will be the case if the original matter contained lactose some water is added and the mixture well stirred, and again evaporated to dryness to expel all nitric acid. The residue should be of a brown color. The mass is again mixed with a little water and the acid reaction neutralized by rubbing with fine-ground potassium carbonate. The carbonate should be added in slight excess and acetic acid added to the alkaline mixture, which is concentrated by evaporation and allowed to stand a few days. At the end of this time potassium saccharate has formed and is separated from the mother liquid by pouring on a porous porcelain plate. The residue is collected, dissolved in a little water and again allowed to crystallize, when it is collected on a porous plate, as before, and washed by means of an atomizer with a little aqueous spray until it is pure white and free of any oxalic acid. The residual acid potassium saccharate may be weighed after drying and then converted into the silver salt. The potash salt for this purpose is dissolved in water, neutralized with ammonia and precipitated with a solution of silver nitrate. The precipitate is well stirred, collected on a gooch and washed and dried in a dark place. It contains 50.94 percent of silver. All sugars which contain the dextrose group yield silver saccharate when treated as above described. Inulin, sorbose, arabinose and galactose yield no saccharic acid under this treatment, and thus it is shown that they contain no dextrose group. Milk sugar, maltose, the dextrins, raffinose and sucrose yield saccharic acid when treated as above and therefore all contain the dextrose group.

=169. Levulose.=—The levulose group of sugars, wherever it occurs, when oxidized with nitric acid, gives rise to tartaric, racemic, glycolic and oxalic acids, which are not characteristic, being produced also by the oxidation of other carbohydrates. A more distinguishing test is afforded by the color reactions produced with resorcin.[135] The reagent is prepared by dissolving half a gram of resorcin in thirty cubic centimeters each of water and strong hydrochloric acid. To the sugar solution under examination an equal volume of strong hydrochloric acid is added, and then a few drops of the reagent. The mixture is gently warmed, and in the presence of levulose develops a fire-red color. Dextrose, lactose, mannose and the pentoses do not give the coloration, but it is produced by sorbose in a striking degree, and also by sucrose and raffinose since these sugars contain the levulose group.

=170. Galactose.=—The galactose which arises from the hydrolysis of milk sugar is readily recognized by the mucic acid which it gives on oxidation with nitric acid.[136] The analytical work is conducted as follows: The body containing galactose or galactan is placed in a beaker with about sixty cubic centimeters of nitric acid of 1.15 specific gravity for each five grams of the sample used. The beaker is placed on a steam-bath and heated, with frequent stirring, until two-thirds of the nitric acid have been evaporated. The residual mixture is allowed to stand over night and the following morning is treated with ten cubic centimeters of water, allowed to stand for twenty-four hours, filtered through a gooch, and the collected matter washed with twenty-five cubic centimeters of water, dried at 100° and weighed. The mucic acid collected in this way will amount to about thirty-seven per cent of the milk sugar or seventy-five per cent of the galactose oxidized. Raffinose yields under similar treatment, about twenty-three per cent of mucic acid, which proves that the galactose group is contained in that sugar. Raffinose, therefore, is composed of one molecule each of dextrose, levulose, and galactose.

=171. Invert Sugar.=—The presence of a trace of invert sugar accompanying sucrose can be determined by Soldaini’s solution, paragraph =124=, or by boiling with methyl blue.[137] Methyl blue is the hydrochlorate of an ammonium base, which, under the influence of a reducing agent, loses two atoms of hydrogen and becomes a colorless compound. The test for invert sugar is made as follows: The reagent is prepared by dissolving one gram of methyl blue in water. If the sugar solution is not clear, twenty grams of the sugar are dissolved in water clarified by lead subacetate, the volume completed to 100 cubic centimeters, and the solid matters separated by filtration. The filtrate is made slightly alkaline with sodium carbonate to remove the lead. A few drops of soda lye solution are then added and the mixture thrown on a filter. To twenty-five cubic centimeters of the filtrate a drop of the methyl blue solution is added, and a portion of the liquor heated in a test tube over the naked flame. If, after boiling for one minute, the coloration disappear, the sample contains at least 0.01 per cent of invert sugar; if the solution remain blue it contains none at all or less than 0.01 per cent. The test may also be made with the dilute copper carbonate solution of Ost described further on.

=172. Compounds with Phenylhydrazin.=—Many sugars may also be qualitively distinguished by the character of their compounds with phenylhydrazin. In general, it may be said that those sugars which reduce fehling solution form definite crystalline compounds with the reagent named. If a moderately dilute hot solution of a reducing sugar be brought into contact with phenylhydrazin acetate, a crystalline osazone is separated. The reaction takes place between one molecule of the sugar and two molecules of the hydrazin compound, according to the following formula:

C₆H₁₂O₆ + 2C₆H₅N₂H₃ = C₁₈H₂₂N₄O₄ + 2H₂O + H₂.

The hydrogen does not escape but combines in the nascent state with the excess of phenylhydrazin to form anilin and ammonia.

The precipitation is accomplished as follows:

One part by weight of the sugar, two parts of phenylhydrazin hydrochlorate, and three parts of sodium acetate are dissolved in twenty parts of water and gradually heated on the water-bath.

The osazone slowly separates in a crystalline form and it is freed from the mother liquor by filtration, and purified by solution in alcohol and recrystallization. The crystals are composed of yellow needles, which are difficultly soluble in water and more easily in hot alcohol. The crystals are not decomposed by a dilute acid but are destroyed by the action of strong acids.

Dextrosazone.—The crystals melt at from 204° to 205°, reduce fehling liquor, and dissolved in glacial acetic acid are slightly left rotating.

Levulosazone.—This body has the same properties as the dextrose compound.

Maltosazone.—This substance melts at 206° with decomposition. It is left rotating. Its structure is represented by the formula C₂₄H₃₂N₄O₉.

Galactosazone.—This substance, C₁₈H₂₂N₄O₄, has the same centesimal composition as the corresponding bodies produced from dextrose and levulose. It is distinguished from these compounds, however, by its low melting point, viz., 193°.

The above comprise all the phenylosazones which are important from the present point of view. Sucrose, by inversion, furnishes a mixture of dextros- and levulosazones when treated with phenylhydrazin, while starch and dextrin yield the dextros- or maltosazone when hydrolyzed. Lactose yields a mixture of dextros- and galactosazones when hydrolyzed and treated as above described.

The reactions with phenylhydrazin are approximately quantitive and it is possible that methods of exact determination may be based on them in the near future.[138]

=173. Other Qualitive Tests for Sugars.=—The analyst may sometimes desire a more extended test of qualitive reactions than those given above. The changes of color noticed on heating with alkalies may often be of advantage in discriminating between different sugars. The formation of definite compounds with the earthy and other mineral bases may also be used for qualitive determinations. One of the most delicate qualitive tests is found in the production of furfurol and this will be described in the following paragraphs.

=174. Detection of Sugars and Other Carbohydrates by Means of Furfurol.=—The production of furfurol (furfuraldehyd) as noted in paragraph =150=, is also used quantitively for the determination of pentose sugars and pentosans.

Furfurol was first obtained from bran (furfur), whence its name, by treating this substance with sulfuric acid, diluted with three volumes of water, and subjecting the mixture to distillation. Its percentage composition is represented by the symbol C₅H₄O₂, and its characteristics as an aldehyd by the molecular structure C₄H₃O,C-HO.

Carbohydrates in general, when treated as described above for bran, yield furfurol, but only in a moderate quantity, with the exception of the pentoses.

Mylius has shown[139] that Pettenkofer’s reaction for choleic acid is due to the furfurol arising from the cane sugar employed, which, with the gall acid, produces the beautiful red-blue colors characteristic of the reaction.

Von Udránszky[140] describes methods for detecting traces of carbohydrates by the furfurol reaction, which admit of extreme delicacy. The solution of furfurol in water, at first proposed by Mylius, is to be used and it should not contain more than two and two-tenths per cent, while a solution containing five-tenths per cent furfurol is found to be most convenient. The furfurol, before using, should be purified by distillation, and, as a rule, only a single drop of the solution used for the color reaction.

The furfurol reaction proposed by Schiff[141] appears to be well suited for the detection of carbohydrates. It is made as follows:

Xylidin is mixed with an equal volume of glacial acetic acid and the solution treated with some alcohol. Strips of filter paper are then dipped in the solution and dried. When these strips of prepared paper are brought in contact with the most minute portion of furfurol, furoxylidin is formed, C₄H₃OCH(C₈H₈NH₂)₂, producing a beautiful red color. In practice, a small portion of the substance, supposed to contain a carbohydrate, is placed in a test tube and heated with a slight excess of concentrated sulfuric acid. The prepared paper is then placed over the mouth of the test tube so as to be brought into contact with the escaping vapors of furfurol.

The furfurol reaction with α-naphthol for some purposes, especially the detection of sugar in urine, is more delicate than the one just described. This reaction was first described by Molisch[142], who, however, did not understand its real nature.

The process is carried on as follows: The dilute solution should contain not to exceed from 0.05 to one-tenth per cent of carbohydrates. If stronger, it should be diluted. Place one drop of the liquid in a test tube with two drops of fifteen per cent alcoholic solution of α-naphthol, add carefully one-half cubic centimeter of concentrated sulfuric acid, allowing it to flow under the mixture. The appearance of a violet ring over a greenish fringe indicates the presence of a carbohydrate. If the substance under examination contain more than a trace of nitrogenous matter, this must be removed before the tests above described are applied.

If the liquids be mixed by shaking when the violet ring is seen, a carmine tint with a trace of blue is produced. If this be examined with a spectroscope, a small absorption band will be found between D and E, and from F outward the whole spectrum will be observed. One drop of dextrose solution containing 0.05 per cent of sugar gives a distinct reaction by this process. It can be used, therefore, to detect the presence of as little as 0.028 milligram of grape sugar. This test has been found exceedingly delicate in this laboratory, and sufficiently satisfactory without the spectroscopic adjunct.

The furfurol reaction is useful in detecting the presence of minute traces of carbohydrates but is of little value in discriminating between the different classes of these bodies.

It is not practical here to go into greater detail in the description of qualitive reactions. The analyst, desiring further information, should consult the standard works on sugar chemistry.[143]

=175. Detection of Sugars by Bacterial Action.=—Many forms of bacteria manifest a selective action towards sugars and this property may in the future become the basis of a qualitive and even quantitive test for sugars and other carbohydrates. Our present knowledge of the subject is due almost exclusively to the researches of Smith, conducted at the Department of Agriculture.[144] Dextrose is the sugar first and most vigorously attacked by bacterial action, and by proper precautions the whole of the dextrose may be removed from mixtures with sucrose and lactose.

The development of other forms of micro-organisms which will have the faculty of attacking other and special forms of carbohydrates is to be looked for with confident assurance of success.

DETERMINATION OF STARCH.

=176. Constitution of Starch.=—The molecule of starch is without doubt formed by the condensation of a large number of hexose bodies. On account of its great insolubility its molecular weight has not been determined with any degree of accuracy. Its formula may be expressed either as (C₆H₁₀O₅)ₙ or (C₁₂H₂₀O₁₀)ₙ. It is insoluble in cold water and other common solvents and does not pass into solution in any reagent without undergoing a change of structure. In hot water it forms a paste and when heated under pressure with water it undergoes a partial change and becomes soluble. Heated with acids or subjected to the action of certain ferments it suffers hydrolysis and is transformed into dextrin, maltose and dextrose. In analytical work an attempt is usually made to transform the starch entirely into dextrose, the quantity of which is then determined by some of the processes already given. All starches possess the property of giving an intensely blue color with iodin and this reaction serves to detect the most minute quantity of the material.

Starch grains derived from different sources are distinguished by differences in size and appearance. In most cases a careful examination of the starch particles will reveal their origin.[145] The greatest part of the cereal grains is composed of starch, the percentage ranging from sixty to eighty. Rice has the greatest percentage of starch in its composition of any substance. Certain root crops are also rich in starch, such as the potato, artichoke and cassava. Starch appears as one of the first products of vegetable metabolism, according to some authorities, preceding the formation of sugars. By reason of its greater complexity, however, it is more probable that the production of simple sugars precedes the formation of the more complex molecule. Starch granules are probably used as a food by the plant in the building of more complex structures and the excess of this food is stored in the seeds and in tubers.

=177. Separation of Starch Particles.=—Advantage is taken of the insolubility of the starch particles to secure their separation from the other vegetable structures with which they are associated. The substances containing starch are reduced to a pulp as fine as possible, and this pulp being placed in a fine cloth the starch particles are washed through the cloth with water. The milky filtrate carrying the starch is collected in an appropriate holder and, after some time, the particles subside. They may then be collected and dried. While this process is the one used commercially in the manufacture of starch, it can only give approximate data respecting the actual quantity of starch in a given weight of the sample. It is not quite possible by this method to get all the starch separated from the rest of the vegetable matter, and particles of foreign substances, such as cellulose and albuminoid matters, may pass through the filter cloth and be found with the deposited granules. It follows from this that the quantitive determination of the starch in a given sample by any direct method is only approximately exact.

=178. Methods of Separation.=—Hot acids cannot be safely employed to dissolve starch from its natural concomitants because other carbohydrate bodies become soluble under similar conditions. In such cases the natural sugars which are present should be removed by cold water and the starch dissolved from the residue by a diastatic ferment. Instead of this the sugars may be determined in a separate portion of the pulped material and the starch, together with the sugars, determined, and the quantity of sugar found deducted from the final result.

In these cases the final determinations are made on the sugars, after inverting the sucrose, and proceeding as directed for invert sugars in paragraph =141=. The starch, after separation with diastase, is converted into dextrose by one of the methods to be given and the resulting dextrose determined by one of the approved methods.

=179. Separation with Diastase.=—Diastase or malt extract at a temperature of about 65° rapidly renders starch soluble. Cereals, potato meal and other starch-holding bodies are dried, first at a low temperature, and extracted with ether or petroleum to remove fat. The material is then rubbed up with water, boiled, cooled to 65°, and treated with malt extract (diastase) prepared as given below. One kilogram of ground green malt is mixed with one liter of glycerol and an equal quantity of water, and allowed to stand, with frequent shaking, for eight days. After that time the mixture is filtered, first through a small filter press and afterwards through paper. In case no filter press is at hand the mixture may be pressed in a bag and the liquor obtained, filtered. Malt extract obtained in this way will keep its diastatic properties for a long time. In its use, blank determinations must be made of the dextrose produced by treating equal portions of it with hydrochloric acid. For three grams of starchy material twenty-five cubic centimeters of the malt solution should be used and the mixture kept at 65° for two hours.[146]

=180. Method in Use at the Halle Station.=—The method of separating starch from cereals, potatoes and other starch-holding materials, employed at the Halle station, is essentially the same as already described.[147]

The malt extract used is prepared immediately beforehand, inasmuch as no preservative is added to it. It can be quickly prepared by digesting, for a short time at not above 50°, 100 grams of finely ground dried malt with one liter of water and separating the extract by filtration. This extract will keep only a few hours.

The material in which the starch is to be determined is dried and extracted with ether. From two to four grams of the extracted material, according to the amount of starch which it contains, are boiled for half an hour with 100 cubic centimeters of water, cooled to 65°, treated with ten cubic centimeters of malt extract and kept at the temperature named for half an hour. It is then again boiled for fifteen minutes, cooled to the temperature mentioned and again treated with malt extract as above. Two treatments with malt extract are usually sufficient to bring all the starch into solution. Finally it is again boiled and the volume completed to 250 cubic centimeters and thrown upon a filter. Two hundred cubic centimeters of the filtrate are converted into dextrose by boiling with hydrochloric acid, and the rest of the analysis is conducted in the usual manner. The dextrose value of the quantity of malt extract used must be determined upon a separate portion thereof, and the quantity of dextrose found deducted from the total amount obtained in the analysis.

=181. Separation by Hydrolysis with Water at High Temperatures.=—Instead of dissolving the starch with diastase, it may be brought into solution by heating with water under pressure. The former method employed of heating in sealed flasks has been entirely superceded by heating in an autoclave. The materials are best held in metal beakers furnished with a cover which prevents loss from boiling if the pressure should be removed too rapidly after the completion of the operation. The autoclave is a strong metal vessel capable of resisting the pressure of several atmospheres. It is furnished with a pressure gauge C and a safety valve D, as shown in the figure. The top is securely screwed on by means of a wrench, shown at the right hand side. In the figure a portion of the case is represented cut away to show the arrangement of the metal beakers inside.

In the method of Reinke, as practiced at the Halle station, and in this laboratory, about three grams of the starchy substance are placed in each of the metal beakers with twenty-five cubic centimeters of a one per cent lactic acid solution and thirty cubic centimeters of water. The contents of the beaker are thoroughly mixed and they are then heated for two and a half hours in the autoclave, at a pressure of three and a half atmospheres. The addition of the lactic acid is for the purpose of protecting any sugar which may be present from decomposition at the high pressure and temperature employed. After the completion of the heating, the autoclave is allowed to cool, the cover is removed and the beakers taken out and their contents washed with hot water into quarter liter flasks. After cooling, the volume is completed with cold water, and after standing for half an hour, with frequent shaking, the contents of the flasks are filtered and 200 cubic centimeters of the filtrate in each case converted into dextrose with hydrochloric acid in the usual way. In order to obtain agreeing results, it is highly necessary that the substance before treatment should be ground to a fine powder. The addition of the lactic acid, as practiced in the reinke method, tends to give somewhat high results, due probably to the hydrolytic action of the acid on the fiber present. When starchy bodies are heated in the autoclave for the determination of their starch by polarimetric methods, or for ordinary determinations, the use of lactic acid should be omitted.

Example.—The following data indicate the methods of calculation to be followed in the determination of the percentage of starch in the material by diastatic hydrolysis: Three grams of a barley were inverted by diastase, as directed above, the volume of the solution made a quarter of a liter, filtered, 200 cubic centimeters of the filtrate converted into dextrose by hydrochloric acid, the volume completed to half a liter with water and fifty cubic centimeters thereof oxidized by the alkaline copper solution in the usual way. The amount of copper obtained was 331 milligrams, corresponding to 174 milligrams of dextrose. The amount of malt extract used in hydrolyzing the barley mentioned above, was ten cubic centimeters. The diastatic solution inverted with hydrochloric acid and treated as indicated above, yielded 191 milligrams of copper, corresponding to ninety-eight milligrams of dextrose in ten cubic centimeters of the malt extract. The quantity of malt extract represented in the final determination of copper, however, was only one and six-tenths cubic centimeters. We then have:

Total dextrose 174 milligrams Dextrose in one and six-tenths cubic centimeters malt extract 16 milligrams Dextrose corresponding to 240 milligrams of barley 158 milligrams

Calculated on the proportion that dextrose is to starch, as ten is to nine, this is equivalent to 142 milligrams of starch. The percentage of starch in the original substance, therefore, was equivalent to 142 multiplied by 100, divided by 240, viz., 59.17.

=182. Principles of the Methods of Determination.=—In the approximately pure state in which starch exists in the trade, it may be determined by conversion into dextrose and estimating the latter by one of the methods given. It is probable that there is no known method by which starch can be entirely converted into dextrose, and all the methods of hydrolysis, when used for quantitive purposes, must be standardized, not by the theoretical quantity of dextrose which a given weight of pure starch should yield, but by the actual quantity obtained. Starch is not largely converted into dextrose by any of the diastatic ferments which produce principally maltose and dextrins. Recourse must therefore be had to strong acids. In practice, hydrochloric is the one usually employed. By the action of a hot mineral acid, not only is starch converted into dextrose, but also the dextrose found is subjected to changes. In such cases an opposing action seems to be exerted by the hydrolytic agent, a part of the dextrose formed suffering a partial condensation, and thus assuming a state of higher molecular weight, approaching the constitution of the dextrins. Another part of the dextrose may also suffer oxidation and thus disappear entirely in respect of the further steps in starch analysis.

In such cases, the best the analyst can do is to conduct the hydrolysis in as nearly as possible constant conditions, and to assume that the percentage of dextrose present at a given time bears a constant ratio to the quantity of starch hydrolyzed. In reality almost all the starch appears finally as dextrose, and by proceeding on the assumption noted above a fairly satisfactory accounting may be made of the remainder.

Starch being insoluble, it cannot be determined directly by its rotatory power. When heated for a few hours in contact with water at a high pressure, starch becomes soluble, and in this state has a fairly constant gyrodynat, viz., [a]_{D} = 197°.

Starch is also rendered soluble by rubbing it in a mortar for about ten minutes with an excess of strong hydrochloric acid, and in this way a quick approximate idea may be obtained of the percentage present. Starch prepared in this manner, however, has a strong reducing power on metallic salts, showing that a part of it has already, even in so short a time, assumed the state of maltose or dextrose. The gyrodynat of pure anhydrous starch in such conditions varies from [a]_{D} = 197° to [a]_{D} = 194°. Starch is also rendered soluble by boiling with salicylic acid, whereby a solution is obtained having a gyrodynat of [a]_{D} = 200°(circa). The methods of procedure for the analysis of starch will be set forth in detail in the following paragraphs.

=183. Estimation of Water.=—In prepared or commercial starches the water may be determined by heating in a partial vacuum. The temperature at first should be low, not exceeding 60°. After drying for an hour at that heat the temperature may be gradually increased. The last traces of water come off from starch with difficulty, and the final temperature may be carried a little beyond 100° without danger of decomposition.

Ost recommends the use of an atmosphere of hydrogen or illuminating gas.[148] One and a half grams of the finely powdered sample are placed in the drying tube described in paragraph =23=, and heated in a stream of dry hydrogen. The temperature at first is kept at about 60° for several hours and is then gradually increased to 120°. Ost states that even at 150° the sample preserves its pure white color, but so high a temperature is not necessary. Maercker, at the Halle station, makes use of the same process, but employs illuminating gas instead of hydrogen. The importance of beginning the desiccation at a low temperature arises from the fact that at a higher temperature, before the greater part of the water is driven off, the starch will suffer a partial fusion and form a paste which is very difficult to dry. The dried sample must be kept in a stoppered vessel to prevent the absorption of hygroscopic moisture.

=184. Estimation of Ash.=—When the drying is accomplished in a flat platinum dish, the same sample may serve for incineration. Otherwise the incineration may be accomplished in another portion of the sample by following directions already given.[149]

=185. Nitrogen.=—Even very pure samples of starch may contain a little nitrogen which is most conveniently determined by moist combustion.[150]

As a rule, in commercial starches of good quality, the quantity of pure starch may be considered to be the remainder after subtracting the sum of the weights of water, ash and nitrogen multiplied by 6.25, from the original weight of the sample taken.

Example:—

Per cent of moisture found 12.85 ” ” ” ash found 0.08 ” ” ” nitrogen × 6.25 0.27 ----- Sum 13.20 Per cent of pure starch in sample 86.80

Samples of starch usually contain also traces of fat and fiber, and these when present in weighable quantities, should be determined and proper deductions made.

=186. Hydrolysis with Acids.=—The acids commonly chosen for hydrolyzing starch are sulfuric and hydrochloric. The former has the advantage of being more easily removed from the finished product but the latter performs the work with less damage to the sugars formed. For commercial purposes sulfuric and for analytical practice hydrochloric acids are commonly employed.

The best process for analytical purposes is the one proposed by Sachsse.[151] In this method the starch is heated with the hydrolyzing mixture in the proportion of three grams to 200 cubic centimeters of water and twenty of hydrochloric acid of 1.125 specific gravity, containing five and six-tenths grams of the pure gas. The heating is continued for three hours on a steam-bath. Maercker recommends, instead of the above procedure, heating for two hours at gentle ebullition in an oil-bath. In this method three grams of the starch are reduced to paste with 200 cubic centimeters of water, and then boiled for two hours with fifteen cubic centimeters of hydrochloric acid of 1.125 specific gravity. The erlenmeyers in which the hydrolysis takes place are heated in an oil-bath and are provided with reflux condensers made of long glass tubes on which some bulbs have been blown, as shown in the accompanying figure. In all cases after hydrolysis the solution is neutralized, made to a standard volume and an aliquot part, after filtration, diluted to contain an amount of dextrose suited to the use of the table by Allihn for calculating the percentage of sugar. In diluting the solution preparatory to the estimation of dextrose, it is well to remember that nine parts of starch will furnish theoretically ten parts of dextrose. Since three grams of the sample are used, containing approximately eighty-five per cent of starch, the quantity of dextrose present is a little less than three grams. The solution should therefore contain not less than 300 cubic centimeters.

=187. Factor for Calculating Starch from the Dextrose Obtained.=—If all the starch could be converted into dextrose without loss, the quantity of it could be easily calculated theoretically on the supposition that the formula of starch is (C₆H₁₀O₅)ₙ. The factor by this assumption is, starch = dextrose × 0.90. If the starch have the formula assigned to it by Nägeli, viz., C₃₆H₆₂O₃₁ the formula becomes, starch = dextrose × 0.918.

Ost prefers to work by Sachsse’s method and to use the factor 0.925 to convert the dextrose into starch.[152]

In view of all the facts in the case it appears that the analyst will reach nearly correct results by converting the starch into dextrose by heating for three hours at 100° with hydrochloric acid or for two hours at gentle ebullition as directed above, determining the resultant dextrose and multiplying the weight thereof by 0.92.

=188. Polarization of Starch.=—Starch may be prepared for polarization by dissolving it in cold hydrochloric acid. The process as carried out by Effront is as follows.[153] Five grams of starch are rubbed with twenty cubic centimeters of cold concentrated hydrochloric acid for nearly ten minutes or until the solution is quite clear. The volume is completed to 200 cubic centimeters with water and the solution polarized. By this process there is always produced a notable quantity of reducing sugars, and for this reason it must be admitted that a portion of the starch has suffered complete hydrolysis. Ost therefore recommends the use of an acid of 1.17 specific gravity, and the gyrodynat of the soluble starch thus produced is found to vary from [a]_{D} = 196°.3 to 196°.7. When acid of 1.20 specific gravity is employed the gyrodynat falls to [a]_{D} = 194.2.[154] For approximately correct work the solution with the weaker hydrochloric acid and subsequent polarization is to be recommended as the most rapid method for starch determination.

It will be of interest to add the observation that the gyrodynat of maltose has lately been redetermined by Ost, who finds it to be [a]_{D}²⁰ ° = 137°.04 ± 0.19.[155]

=189. Solutions of Starch at High Pressure.=—Starch may also be brought into a condition suited to polarization by dissolving in water at a high temperature and pressure. The solution is accomplished in an autoclave as described in =181=.

From two to three grams of starch are used and from eighty to ninety cubic centimeters of water. The starch is first reduced to a pasty state by heating with the water and, when evenly distributed throughout the flask, is rendered soluble by heating from three to five hours in an autoclave at from two to three atmospheres. The material is entirely without action on an alkaline copper solution. After heating, the volume of the solution is completed to 100 cubic centimeters and it is then polarized. The gyrodynat of starch dissolved in this way varies from [a]_{D} = 196°.5 to 197°.[156]

Starch is prepared by Baudry for polarization by boiling with salicylic acid.[157] The gyrodynat of starch dissolved in this way is [a]_{D} = 200°.25.

=190. Polarization after Solution in Dilute Nitric Acid.=—Guichard recommends saccharification with ten per cent nitric acid (ten cubic centimeters strong acid, ninety cubic centimeters water).[158] This treatment, even after prolonged boiling, gives only a light straw color to the solution which does not interfere with its polarization with a laurent instrument.

In working on cereals four grams of the finely ground material, in which the bran and flour are intimately mixed, are used.

The material is placed in a flask of about 500 cubic centimeters capacity, with 100 cubic centimeters of the dilute acid. The flask is closed with a stopper carrying a reflux condenser. After boiling for an hour the contents of the flask are filtered and examined in the saccharimeter. The dextrose formed is determined by the polarimetric data and the quantity of starch transformed calculated from the dextrose. The following formula is used:

av × 25 × 0.016 A = ------------------. 2 × 52.8

In this formula a = the rotation in angular degrees, v = the volume of the liquid and A = the starch transformed.

In this method no account is taken of the sucrose and other sugars which are present in cereals. In the case of sucrose the left-handed sugar produced by treatment with nitric acid would diminish the rotation to the right and thus introduce an error. On the other hand the dextrose formed from the fiber of the bran would be calculated as starch. If these two errors should be compensating the method might prove practical.

=191. Rapid Estimation Of Starch.=—For the rapid estimation of starch in cereals, cattle foods and brewery refuse, Hibbard recommends a method which is carried out as follows:

The malt extract is prepared by covering ground, dry malt with water containing from fifteen to twenty per cent of alcohol. The object of adding alcohol is to preserve the filtered extract. It exercises a slight retarding effect on the action of the diastase, but prevents the malt extract from fermenting. After standing for a few hours in contact with the malt, the liquid is separated by filtration and is then ready for use. The substance in which the starch is to be determined should be dry enough to be finely pulverized, but previous extraction with ether is omitted. Enough of the material to contain at least half a gram of starch is placed in a flask with fifty cubic centimeters of water and from one to two cubic centimeters of malt extract added. The mixture is at once heated to boiling with frequent shaking to prevent the formation of clots. The addition of the diastase before boiling is to aid in preventing the formation of lumps. After boiling a minute the mixture is cooled to 60° and from two to three cubic centimeters of the malt extract added. It is then slowly heated until it again boils, consuming about fifteen minutes, when, after cooling, it is tested with iodin for starch. If a blue color be produced the operation above described is repeated until it fails to reappear. The mixture is then made up to a standard volume, thrown on a linen filter and an aliquot part of the filtrate, representing from 200 to 300 milligrams of starch, is boiled with five cubic centimeters of hydrochloric acid, of thirty per cent strength, for half an hour. The total volume of the liquid before boiling should be completed to sixty cubic centimeters. By the method above described, it is claimed that the determination of starch in a cereal or similar substance can be completed within two hours. The chief amount of time saved is in the heating with the malt extract, which instead of being continued for two hours, as usually directed, can be accomplished in thirty minutes.[159]

=192. Precipitation of Starch with Barium Hydroxid.=—The tendency of carbohydrate bodies to unite with the earthy bases has been utilized by Asboth as a basis for the quantitive determination of starch.[160]

About three grams of the finely ground sample containing the starch, or one gram of pure starch, are rubbed up in a mortar with water and the detached starch remaining suspended in the wash water is poured off. This operation is repeated until all the starch is removed. In difficult cases hot water may be used. The starch thus separated is heated in a quarter liter flask to the boiling point to reduce it to the condition of paste. When the paste is cold it is treated with fifty cubic centimeters of the barium hydroxid solution, the flask closed and well shaken for two minutes. The volume is then completed to the mark with forty-five per cent alcohol, the flask well shaken and allowed to stand. In a short time the barium-starch compound separates and settles. Fifty cubic centimeters of the clear supernatant liquor are removed with a pipette, or the liquor may be passed through a filter and the quantity mentioned removed for titration of the residual barium hydroxid after the addition of a few drops of phenolphthalein solution.

The quantity of barium hydroxid remaining, deducted from the original quantity, gives the amount which has entered into composition with the starch; the composition of the molecule being BaOC₂₄H₄₀O₂₀, which contains 19.10 per cent of barium oxid and 80.90 per cent of starch.

The set solution of barium hydroxid must be preserved from contact with the carbon dioxid of the air. The burette should be directly attached to the bottle holding the set solution, by any of the usual appliances, and the air entering the bottle must be deprived of carbon dioxid. The water used in the work must be also free of air, and this is secured by boiling immediately before use.

Example.—A sample of flour selected for the analysis weighed 3.212 grams. The starch was separated and reduced to paste in the manner described above. Thirty and four-tenths cubic centimeters of tenth-normal hydrochloric acid were exactly neutralized by ten cubic centimeters of the barium hydroxid solution. After treatment as above described, fifty cubic centimeters of the clear liquor, corresponding to ten cubic centimeters of the added barium hydroxid, required 19.05 cubic centimeters of tenth-normal hydrochloric acid. Then 30.4 - 19.05 = 11.35, and 11.35 x 5 = 56.75, which number corresponds to the total titration of the residual barium hydroxid in terms of tenth-normal hydrochloric acid. This number multiplied by 0.0324, viz., starch corresponding to one equivalent of barium, gave 1.8387 grams of starch or 57.24 per cent of the weight of flour employed.

The barium hydroxid method has been given a thorough trial in this laboratory and the results have been unsatisfactory when applied to cereals. The principle of the process, however, appears to be sound, and with a proper variation of working details, it may become practical.

=193. Disturbing Bodies in Starch Determinations.=—Stone has made a comparison of the standard methods of starch determinations, and the results of his work show that in the case of pure starch all of the standard methods give approximately correct figures. For instance, in the case of a pure potato starch, the following data were obtained:

By inversion with hydrochloric acid, 85.75 per cent; by inversion with oxalic and nitric acids, 85.75 per cent; by solution in salicylic acid, 85.47 per cent; and by precipitation with barium hydroxid, 85.58 per cent.[161]

When these methods are used, however, for the determination of starch in its original state, the widest variations are secured. Stone shows that these variations are due chiefly to the inverting effect of the reagents employed upon the pentosans present. In experiments made with pure xylan obtained from wheat straw, the methods employed gave from 44.73 to 67.16 per cent of material, which would be calculated by the usual methods as starch. Stone also shows that the pentosans are practically unaffected by the action of diastase or malt extract. Pure xylan treated with diastase, under the condition in which starch is converted into maltose and other soluble carbohydrates, fails to give any subsequent reaction whatever with alkaline copper solution. In all cases, therefore, where starch occurs in conjunction with pentose bodies, it is necessary to separate it by diastatic action before applying any of the methods of conversion of the starch into dextrose or its precipitation by barium hydroxid.

=194. Colorimetric Estimation of Starch.=—The production of the intensely blue color which starch gives with iodin has been used not only as the basis of a qualitive method, but also of many attempts at quantitive determination. These attempts have, as a rule, been attended with very unsatisfactory results, due both to the extraordinary delicacy of the reaction and to the fact that starches of different origin do not always give exactly the same intensity of tint when present in the same quantity. At the present it must be admitted that little should be expected of any quantitive colorimetric test.

In case such a test is desired the procedure described by Dennstedt and Voigtländer may be followed.[162] A weighed quantity of the starch-holding material, containing approximately half a gram of starch, is placed in a two liter flask and boiled with a liter of water. After cooling, the volume is completed to two liters and the starch allowed to subside. Five cubic centimeters of the clear supernatant liquor are placed in a graduated cylinder holding 100, and marked in half cubic centimeters. One drop of a solution of iodin in potassium iodid is added and the volume completed to the mark. A half gram of pure starch is treated in the same way and different measured portions of the solution treated as above until the color of the first cylinder is matched. From the quantity of pure starch in the matched cylinder the quantity in the sample is determined. The test should be made in duplicate or triplicate. If a violet color be produced instead of a blue, it may be remedied by treating the sample with alcohol before the starch granules are dissolved.

=195. Fixation of Iodin.=—In addition to forming a distinctive blue color with iodin, starches have the power of fixing considerable quantities of that substance. The starches of the cereals have this power in a higher degree than those derived from potatoes. In presence of a large excess of iodin the starches of rice and wheat have a maximum iodin-fixing power of about nineteen per cent of their weight. When only enough of iodin is employed to enter into combination the percentage absorbed varies from nine to fifteen per cent. The absorption of iodin by starches is a matter of importance from a general chemical standpoint, but as at present determined has but little analytical value. It is evident, however, that this absorption must take place according to definite chemical quantities and the researches of investigators may in the future discover some definite quantitive method of measuring it.[163]

=196. Identification of Starches of Different Origin.=—It is often important, especially in cases of suspected adulteration, to determine the origin of the starch granules. For this purpose the microscope is the sole resort. In many cases it is easy to determine the origin of the starch by the size or the shape and marking of the grains. In mixtures of more than one kind of starch the distinguishing features of the several starches can be clearly made out in most instances. There are, however, many instances where it is impossible to discriminate by reason of the fact that the characteristics of starch granules vary even in the same substance and from year to year with varying conditions of culture.

In many cases the illustrations of the forms and characteristics of starch granules which are found in books are misleading and no reliance can be placed on any illustrations which are not either photographs or drawings made directly from them. In the microscopic study of starches the analyst will be greatly helped by the following descriptions of the characteristic appearance of the granules and the classifications based thereon.[164]

=197. Vogel’s Table of the Different Starches and Arrowroots of Commerce.=—A. Granules simple, bounded by rounded surfaces.

I. Nucleus central, layers concentric. a. Mostly round, or from the side, lens-shaped. 1. Large granules 0.0396-0.0528 mm, rye starch: 2. Large granules 0.0352-0.0396 mm, wheat starch: 3. Large granules 0.0264 mm, barley starch.

b. Egg-shaped, oval, kidney-shaped: Hilum often long and ragged: 1. Large granules 0.032-0.079 mm, leguminous starches.

II. Nucleus eccentric, layers plainly eccentric or meniscus-shaped. a. Granules not at all or only slightly flattened: 1. Nucleus mostly at the smaller end; 0.06-0.10 mm, potato starch: 2. Nucleus mostly at the broader end or towards the middle in simple granules; 0.022-0.060 mm, _maranta starch_.

b. Granules more or less strongly flattened. 1. Many drawn out to a short point at one end. a. At most 0.060 mm long, curcuma starch: b. As much as 0.132 mm long, canna starch: 2. Many lengthened to bean-shaped, disk-shaped, or flattened; nucleus near the broader end; 0.044-0.075 mm, banana starch: 3. Many strongly kidney-shaped; nucleus near the edge; 0.048-0.056 mm, sisyrinchium starch: 4. Egg-shaped; at one end reduced to a wedge, at the other enlarged; nucleus at smaller end; 0.05-0.07 mm, yam starch:

B. Granules simple or compound, single granules or parts of granules, either bounded entirely by plain surfaces, many-angled, or by partly round surfaces.

I. Granules entirely angular. 1. Many with prominent nucleus: At most 0.0066 mm, rice starch: 2. Without a nucleus: The largest 0.0088 mm, millet starch:

II. Among the many-angled also rounded forms. a. No drum-shaped forms present, angular form predominating. 1. Without nucleus or depression very small; 0.0044 mm, oat starch: 2. With nucleus or depression; 0.0132-0.0220 mm. a. Nucleus or its depression considerably rounded; here and there the granules united into differently formed groups; buckwheat starch: b. Nucleus mostly radiate or star-shaped; all the granules free; maize (corn) starch:

b. More or less numerous kettledrum and sugar-loaf like forms. 1. Very numerous eccentric layers; the largest granules 0.022-0.0352 mm, batata (sweet potato) starch: 2. Without layers or rings; 0.008-0.022 mm. a. In the kettledrum-shaped granules the nucleal depression mostly widened on the flattened side; 0.008-0.022 mm, cassava starch: b. Depression wanting or not enlarged. aa. Nucleus small, eccentric; 0.008-0.016 mm, pachyrhizus starch: bb. Nucleus small, central, or wanting. aaa. Many irregular angular forms; 0.008-0.0176 mm, sechium starch: bbb. But few angular forms; some with radiate, nucleal fissure; 0.008-0.0176 mm, chestnut starch.

C. Granules simple and compound; predominant forms, oval, with eccentric nucleus and numerous layers; the compound granule made up of a large granule and one or more relatively small kettledrum-shaped ones; 0.025-0.066 mm, sago starch.

=198. Muter’s Table for the Detection of Starches when Magnified about 230 Diameters.=

[All measurements are given in decimals of an inch.]

Group I: All more or less oval in shape and having both hilum and rings visible.

-------------+----------------+-------------+----------------------- Name. | Shape. | Normal | Remarks. | |measurements.| -------------+----------------+-------------+----------------------- Tous les mois|Oval, with flat | 0.00370 |Hilum annular, near one | ends | to 0.00185 | end and incomplete | | | rings. | | | Potato |Oval | 0.00270 |Hilum annular, rings | | to 0.00148 | incomplete, shape and | | | size very variable. | | | Bermuda |Sack-shaped | 0.00148 |Hilum distinct annular, arrowroot | | to 0.00129 | shape variable, rings | | | faint. | | | St. Vincent |Oval-oblong | 0.00148 |Hilum semi-lunar, rings arrowroot | | to 0.00129 | faint, shape not very | | | variable. | | | Natal |Broadly ovate | 0.00148 |Hilum annular, in arrowroot | | to 0.00129 | center and well marked | | | complete rings. | | | Galangal |Skittle-shaped |About 0.00135|Hilum elongated, very | | | faint incomplete | | | rings. | | | Calumba |Broadly | ” 0.00185|Hilum semi-lunar, faint | pear-shaped | | but complete rings, | | | shape variable. | | | Orris root |Elongated-oblong| ” 0.00092|Hilum faint, shape | | | characteristic. | | | Turmeric |Oval-oblong, | ” 0.00148|Very strongly marked | conical | | incomplete rings. | | | Ginger |Shortly conical,| ” 0.00148|Hilum and rings | with rounded | | scarcely visible, | angles. | | shape variable but | | | characteristic. -------------+----------------+-------------+-------------------------

Group II: With strongly developed hilum more or less stellate.

-------------+--------------+-------------+------------------------- Name. | Shape. | Normal | Remarks. | |measurements.| -------------+--------------+-------------+------------------------- Bean |Oval-oblong |About 0.00135|Fairly uniform. | | | Pea |Like bean | 0.00111 |Very variable in size, | | to 0.00074 | with granules under | | | 0.00111 preponderating. | | | Lentil |Like bean |About 0.00111|Hilum, a long depression | | | seldom radiate. | | | Nutmeg |Rounded | ” 0.00055|The small size and | | | rounded form | | | distinctive. | | | Dari |Elongated | ” 0.00074|Irregular appearance and | hexagon | | great convexity | | | distinctive. | | | Maize |Round and | ” 0.00074|The rounded angles of the | polygonal | | polygonalgranules | | | distinctive. -------------+--------------+-------------+-------------------------

Group III: Hilum and rings practically invisible.

-------------+--------------+-------------+------------------------- Name. | Shape. | Normal | Remarks. | |measurements.| -------------+--------------+-------------+------------------------- Wheat |Circular and | 0.00185 |Very variable in size and | flat | to 0.00009 | very dull polarization | | | in water. | | | Barley |Slightly |About 0.00073|The majority measuring | angular | | about 0.00373 | circles | | distinctive, and a few | | | four times this size. | | | Rye |Like barley | 0.00148 |Small granules, quite | | to 0.00009 | round, and here and | | | there cracked. | | | Jalap |Like wheat | |Polarizes brightly in | | | water. | | | Rhubarb | do. | 0.00055 |Polarizes between jalap | | to 0.00033 | and wheat, and runs | | | smaller and more convex. -------------+--------------+-------------+------------------------- Senega |Like wheat | 0.00148- | | | 0.00009 | Bayberry | do. | 0.00074- | Measurements the | | 0.00011 | only guide. Sumbul | do. | 0.00074- | | | 0.00009 | -------------+--------------+-------------+------------------------- Chestnut |Very variable | 0.00090- |Variable form, and small | | 0.00009 | but regular size, | | | distinctive. | | | Acorn |Round-oval |About 0.00074|Small and uniform size, | | | distinctive. | | | Calabar bean |Oval-oblong | 0.00296 |Large size and shape | | to 0.00180 | characteristic. | | | Licorice |Elongated-oval|About 0.00018|Small size and shape | | | distinctive. | | | Hellebore |Perfectly | 0.00037 |Small, regular size and (green or | rotund | to 0.00009 | rotundity, distinctive. black) | | | | | | Hellebore |Irregular | 0.00055 |Irregular shape and faint (white) | | to 0.00009 | central depression, | | | distinctive. -------------+--------------+-------------+-------------------------

Group IV: More or less truncated at one end.

-------------+--------------+-------------+------------------------ Name. Shape. | Normal | Remarks. | |measurements.| -------------+--------------+-------------+------------------------ Cassia |Round | 0.00111 |Round or muller shaped | | to 0.00018 | granules and faint | | | circular hilum. | | | Cinnamon |Like cassia | 0.00074 |More frequently truncated | | to 0.00009 | than cassia, and | | | smaller. | | | Sago (raw) |Oval-ovate | 0.00260 |Has circular hilum at | | to 0.00111 | convex end and rings | | | faintly visible. | | | Sago | ” | 0.00260 |Has a large oval or (prepared) | | to 0.00111 | circular depression, | | | covering one-third | | | nearly of each granule. | | | Tapioca |Roundish | 0.00074 |A little over fifty per | | to 0.00055 | cent truncated by one | | | facet, and a pearly | | | hilum. | | | Arum |Like tapioca |About 0.00056|Smaller than tapioca and | | | truncated by two facets. | | | Belladonna | do. | |Not distinguishable | | | from tapioca. | | | Colchicum | do. |About 0.00074|Larger than tapioca, | | | and contains many | | | more truncated | | | granules. | | | Scammony | do. | ” 0.00045|Smaller than tapioca, | | | more irregular, and | | | hilum not visible. | | | Cancella |Very variable | 0.00033- |Very variable, form and | | 0.00022 | small size the only | | | points. | | | Podophyllum |Like tapioca |About 0.00040|Like scammony, but has | | | visible hilum in most | | | of the granules. | | | Aconite | do. | ” 0.00037|Like tapioca, but half | | | the size. -------------+--------------+-------------+------------------------

Group V: All granules more or less polygonal.

-------------+--------------+-------------+------------------------- Name. | Shape. | Normal | Remarks. | |measurements.| -------------+--------------+-------------+------------------------- Tacca |Poly- or | 0.00075 |Distinguished from maize | hexagonal | to 0.00037 | by its sharp angles. | | | Oat |Polygonal |About 0.00037|Larger than rice and | | | hilum visible in some | | | granules. | | | Rice | do. | 0.00030- |Measurement using | | 0.00020 | one-eighth or | | | one-twelfth inch power, | | | and then hilum visible. | | | Pepper | do. | 0.00020- | Do. | | 0.00002 | Ipecacuanha | do. |About 0.00018|Some round and truncated | | | granules, adhering in | | | groups of three. -------------+--------------+-------------+-------------------------

=199. Blyth’s Classification.=—Blyth gives the following scheme for the identification of starch granules by their microscopic appearance.[165]

Division I.—Starches showing a play of colors with polarized light and selenite plate:

The hilum and concentric rings are clearly visible, and all the starch granules, oval or ovate. Canna arrowroot, potato, arrowroot, calumba, orris root, ginger, galangal and turmeric belong to this division.

Division II.—Starches showing no iridescence, or scarcely any, when examined by polarized light and selenite:

Class I.—The concentric rings are all but invisible, and the hilum stellate. The bean, pea, maize, lentil, dari and nutmeg starches are in this class.

Class II.—Starches which have both the concentric rings and hilum invisible in the majority of granules: this important class includes wheat, barley, rye, chestnut, acorn, and many starches in medicinal plants.

Class III.—All the granules are truncated at one end. This class includes sago, tapioca and arum, several drugs and cinnamon and cassia.

Class IV.—In this class all the granules are angular in form and it includes oats, tacca, rice, pepper and ipecacuanha.

=200. Preparation of Starches for Microscopical Examination.=—The approximately pure starches of commerce may be prepared for microscopic examination by rubbing them up with water and mounting some of the suspended particles by one of the methods to be described below.

In grains, seeds and nuts the starch is separated by grinding with water and working through fine linen. The starch which is worked through is allowed to subside, again beaten up with water if necessary and the process continued until the grains are separated sufficiently for microscopic examination. A little potash or soda lye may be used, if necessary, to separate the granules from albuminous and other adhering matter. The analyst should have a collection of samples of all common starches of known origin for purposes of comparison.

The granules are mounted for examination by plain light in a medium of glycerol and camphor water. When polarized light is used the mounting should be in Canada balsam.[166] The reader can find excellent photomicrographs of the more common starches in Griffith’s book.[167]

=201. Appearance in Balsam with Polarized Light.=—Mounted in balsam the starches are scarcely visible under any form of illumination with ordinary light, the index of refraction of the granules and the balsam being so nearly alike. When, however, polarized light is used the effect is a striking one. It is very easy to distinguish all the characteristics, except the rings, the center of the cross being at the nucleus of the granule.

With the selenite plate a play of colors is produced, which is peculiar to some of the starches and forms the basis of Blyth’s classification.

=202. Description Of Typical Starches.=—The more commonly occurring starches are described by Richardson as they appear under the microscope magnified about 350 diameters.[168]

The illustrations, with the exception of the cassava starch, and the maize starch accompanying it were drawn by the late Dr. Geo. Marx from photographs made by Richardson in this laboratory. The two samples excepted were photographed for the author by Dr. G. L. Spencer.

Maranta Starch.—Of the same type as the potato starch are the various arrowroots, the only one of which commonly met with in this country being the Bermuda, the starch of the rhizome of Maranta arundinacea, and the starch of turmeric.

The granules are usually not so varied in size or shape as those of the potato, averaging about 0.07 millimeter in length as may be seen in Fig. 48. They are about the same size as the average of the potato, but are not often found with the same maximum or minimum magnitude, which circumstance, together with the fact that the end at which the nucleus appears is broader in the maranta and more pointed in the potato, enables one to distinguish the two starches without difficulty. With polarized light the results are similar to those seen with potato starch, and this is a ready means of distinguishing the two varieties, by displaying in a striking way the form of the granule and position of the hilum.

Potato Starch.—The starch grains of the potato are very variable in size, being found from 0.05 to 0.10 millimeter in length, and in shape from oval and allied forms to irregular and even round in the smallest. These variations are illustrated in Fig. 49, but the frequency of the smaller granules is not as evident as in some other cases. The layers are visible in some granules with great distinctness and in others hardly at all, being rather more prominent in the starch as obtained from a freshly cut surface. The rings are more distinct, too, near the hilum or nucleus, which in this, as in all tuberous starches, is eccentric, shading off toward the broader or more expanded portion of the granule. The hilum appears as a shadowy depression, and with polarized light its position is well marked by the junction of the arms of the cross. With polarized light and a selenite plate a beautiful play of colors is obtained. The smaller granules, which are nearly round, may readily be confused with other starches, but their presence serves at once to distinguish this from maranta or Bermuda arrowroot starch. Rarely compound granules are found composed of two or three single ones each with its own nucleus.

Ginger Starch.—This starch is of the same class as those from the potato and maranta and several others which are of underground origin. In outline the granules are not oval like those named, but more rectangular, having more obtuse angles in the larger ones and being cylindrical or circular in outline in the smaller, as indicated in Fig. 50. They average nearly the same size as maranta starch, but are much more variable, both in size and form. The rings are scarcely visible even with the most favorable illuminations.

Sago Starch.—This exists in two modifications in the market; as raw and as prepared sago. In the prepared condition it is characterized by a larger circular depression in the center of most of the granules. The rings are not visible. They are mostly circular in form or approaching it, and vary from 0.025 to 0.065 millimeter in diameter, as indicated in Fig. 51.

Pea and Bean Starches.—These starches produce but a slight effect with polarized light. The rings are scarcely visible, and the hilum is stellate or much cracked along a median line, the bean more so than the pea, the latter resembling fresh dough kneaded again into the center as in making rolls, and the former the shape assumed by the same after baking. The grains of both are somewhat variable in size, ranging from 0.025 to 0.10 millimeter in length, as shown in Figs. 52 and 53.

Wheat Starch grains are quite variable in size, varying from 0.05 to 0.010 millimeter in diameter. They belong to the same class as barley and rye, the hilum being invisible and the rings not prominent. The granules are circular disks in form, and there are now and then contorted depressions resembling those in pea starch. They are the least regular of the three starches named and do not polarize actively. The typical forms of these granules are shown in Fig. 54.

Barley Starch is quite similar to that of wheat, but the grains do not vary so much in size, averaging 0.05 millimeter. They have rings which are much more distinct, and very small granules adhering to the largest in bud-like forms, as seen in Fig. 55.

Rye Starch is more variable in size, many of the granules not exceeding 0.02 millimeter, while the largest reach 0.06 to 0.07 millimeter. It lacks distinctive characteristics entirely, and is the most simple in form of all the starches. Fig. 56 shows the appearance of the granules under the microscope.

Oat Starch is unique, being composed of large compound masses of polyhedral granules from 0.12 to 0.02 millimeter in length, the single granules averaging 0.02 to 0.015 millimeter. It does not polarize actively, and displays neither rings nor hilum. The illustration, Fig. 57, shows its nature with accuracy.

Indian Corn Starch.—The granules of maize starch are largely of the same size, from 0.02 to 0.03 millimeter in diameter, with now and then a few which are much smaller. They are mostly circular in shape or rather polyhedral, with rounded angles, as shown in Figs. 58 and 61. They form very brilliant objects with polarized light, but with ordinary illumination show but the faintest sign of rings and a well-developed hilum, at times star-shaped, and at others more like a circular depression.

Rice Starch is very similar to that of maize, and is easily confused with it, the grains being about the same size. The grain, however, is distinguished from it by its polygonal form, and its well defined angles, as indicated in Fig. 59. The hilum is more prominent and more often stellate or linear. Several granules are at times united.

Cassava Starch.—This variety of starch is obtained from the root of the sweet cassava, which grows in great profusion in Florida. It is compared with maize starch in Figs. 60 and 61. In the illustration the granules are represented as magnified 150 diameters. The grains of the cassava starch measure about 0.012 millimeter in diameter and resemble very nearly maize starch, except that they have greater evenness of outline.[169]

For further descriptions of starch grains the reader is referred to the work of Griffith, already cited.

These descriptions, it will be seen, do not agree entirely with those of some other authors, but they are based on a somewhat extensive experience.

There are peculiarities of size, shape and appearance of starch granules, which must be allowed for, and the necessity for every investigator to compare a starch which he is desirous of identifying with authentic specimens, must always be recognized.

AUTHORITIES CITED IN PART SECOND.

[23] Vines, Vegetable Physiology.

[24] Berichte der deutschen chemischen Gesellschaft, Band 23, S. 2136; Stone, Agricultural Science Vol. 6, p. 180. Page 59, eighth line from bottom insert “original” before “optical.” Page 60, second line from top, read d instead of l fructose.

[25] Herles, Zeitschrift des Vereins für die Rübenzucker-Industrie, 1890. S. 217.

[26] Tucker; Wiechmann; Sidersky; von Lippman; Tollens and Spencer.

[27] Bulletin No. 28, Department of Agriculture, Division of Chemistry, p. 197.

[28] Physikalisch-Chemische Tabellen, S. 42.

[29] Tucker’s Manual of Sugar Analysis, pp. 100 et seq.

[30] Vid. op. cit. supra, p. 108.

[31] Op. cit. supra, p. 109.

[32] Op. cit. supra, p. 110.

[33] Op. cit. supra, p. 114.

[34] Spencer’s Handbook for Sugar Manufacturers, p. 92.

[35] Landolt’s Handbook of the Polariscope, pp. 95 et seq.

[36] Robb, vid. op. cit. supra, p. 8.

[37] Spencer’s Handbook for Sugar Manufacturers, pp. 22 et seq. Tucker’s Manual of Sugar Analysis, pp. 120 et seq.

[38] Sidersky; Traité d’Analyse des Matières Sucrées, p. 104.

[39] Journal of the American Chemical Society, 1893. Vol. 15, p. 121.

[40] Comptes rendus, 1879. Seance du 20th Octobre 1879; Dingler’s polytechniches Journal, Band 223, S. 608.

[41] Landolt’s Handbook of the Polariscope. p. 120.

[42] Sidersky; Traité d’Analyse des Matières Sucrées, p. 97.

[43] Manual of Sugar Analysis, pp. 143 et seq.

[44] Landolt und Börnstein, Physikalisch-Chemische Tabellen. S. 460.

[45] Bulletin No. 31. Department of Agriculture, Division of Chemistry, p. 232.

[46] Zeitschrift des Vereins für die Rübenzucker-Industrie. 1870, S. 223.

[47] Tucker’s Manual of Sugar Analysis, p. 164.

[48] (bis). Gerlach, Spencer’s Handbook for Sugar Manufacturers, p. 91.

[49] Vid. op. cit. supra, p. 45.

[50] Vid. loc. et op. cit. supra.

[51] Gill; Journal of the Chemical Society, Vol. 24, 1871, p. 91.

[52] Wiley; American Chemical Journal, Vol. 6, p. 289.

[53] Vid. op. cit. supra, p. 301.

[54] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1890. S. 876.

[55] Weber and McPherson; Journal of the American Chemical Society. Vol. 17, p. 320; Bulletin No. 43. Department of Agriculture, Division of Chemistry, p. 126.

[56] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1888, S. 51.

[57] Vid. op. cit. supra, Ss, 699 und 763; 1890. S. 217.

[58] Bulletin de l’Association des Chimistes de Sucrerie et de Distillerie, May, 1890, p. 431.

[59] Neue Zeitschrift für Rübenzucker-Industrie, Band 19, S. 71.

[60] Journal of the Chemical Society, Transactions, Vol. 57, pp. 834, et seq.

[61] Op. cit. supra, p. 866.

[62] Op. cit. supra, 1891, p. 46.

[63] Neue Zeitschrift für Rübenzucker-Industrie, Band 19, S. 71.

[64] From γῦρος and δῦνᾶτός (δύνᾶμις).

[65] Landolt’s Handbook of the Polariscope, p. 125.

[66] Vid. op. cit. supra, pp. 48 et seq.

[67] Berichte der deutschen chemischen Gesellschaft, 1877, S. 1403.

[68] Die landwirtschaftlichen Versuchs-Stationen, Band 40, S. 307.

[69] Spencer’s Handbook for Sugar Manufacturers, p. 80; Landolt’s Handbook of the Polariscope, p. 216; Tollens’ Handbuch der Kohlenhydrate.

[70] Annalen der Chemie and Pharmacie, May, 1870.

[71] Tucker’s Manual of Sugar Analysis, p. 208.

[72] Rapport fait a la Société d’Encouragement d’Agriculture; Journal de Pharmacie et de Chimie, 1844. 3d serie, Tome 6, p. 301.

[73] Annalen der Chemie und Pharmacie, Band 39, S. 361.

[74] Jahrbücher für praktische Heilkunde, 1845, S. 509.

[75] Archives für Physiologische Heilkunde, 1848, Band 7, S 64.

[76] Rodewald and Tollens; Berichte der deutschen chemischen Gesellschaft, Band 11, S. 2076.

[77] Chemical News, Vol. 39, p. 77.

[78] The Analyst, Vol. 19, p. 181.

[79] Gaud; Bulletin de l’Association des Chimistes de Sucrerie et de Distillerie, Apr. 1895, p. 629; Comptes rendus, 1894, Tome 119, p. 604.

[80] Annalen der Chemie und Pharmacie, B. 72, S. 106.

[81] Journal of Analytical and Applied Chemistry, Vol. 4, p. 370.

[82] Wiley; Bulletin de l’Association des Chimistes de Sucrerie et de Distillerie, April, 1884.

[83] Vid. op. cit. supra, 1895, p. 642; Comptes rendus, Tome 119, 1894, p. 650.

[84] Annual Report, United States Department of Agriculture, 1879, p. 65; Zeitschrift für Analytische Chemie, Band 12, S. 296; Mohr Titrirmethode, sechste auflage, S. 508.

[85] Comptes rendus, 1894, Tome 119, p. 478.

[86] Gazetta Chimica Italiana, Tome 6, p. 322.

[87] Sidersky; Traité d’Analyse des Matières Sucrées, p. 148.

[88] Vid. op. cit. supra, p. 149.

[89] Neue Zeitschrift für die Rübenzucker-Industrie, Band 22, S. 220.

[90] Zeitschrift des Vereins für Rübenzucker-Industrie, 1889, S. 933.

[91] Vid. op. cit. supra, 1887, S. 147.

[92] Berichte der deutschen chemischen Gesellschaft, Band 23, No. 14, S. 3003; Zeitschrift des Vereins für die Rübenzucker-Industrie, 1891, S. 97.

[93] Ost; vid. op. et loc. cit. supra.

[94] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1890, S. 187.

[95] Chemical News, Vol. 39, p. 77.

[96] The Analyst, 1894, p. 181.

[97] Chemical News, Vol. 71, p. 235.

[98] Journal de Pharmacie et de Chimie, 1894, Tome 30, p. 305.

[99] Pharmaceutical Journal, (3), 23, p. 208.

[100] Vid. op. cit. supra, (3), 25, p. 913.

[101] Sidersky; Bulletin de l’Association des Chimistes, Juillet, 1886 et Sept. 1888.

[102] Bodenbender and Scheller; Zeitschrift des Vereins für die Rübenzucker-Industrie, 1887, S. 138.

[103] Vid. op. cit. supra, 1889, S. 935.

[104] Ewell; Manuscript communication to author.

[105] Journal für praktische Chemie, 1880, Band 22, 46; Handbuch der Spiritusfabrication, 1890, S. 79; Zeitschrift des Vereins für die Rübenzucker-Industrie, 1879, S. 1050; Ibid, 1883, S. 769; Ibid, 1889, S. 734.

[106] Handbuch der Spiritusfabrication, 1890, 79.

[107] Wein; Tabellen zur quantitativen Bestimmung der Zuckerarten, S. 13. (The caption for the table on page 159 should read as on page 160.)

[108] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1889, S. 735.

[109] Bulletin No. 43, Department of Agriculture, Division of Chemistry, p. 209.

[110] Chemiker-Zeitung, 1893, S. 548.

[111] Wein; Tabellen zur quantitativen Bestimmung der Zuckerarten, S. 35.

[112] Berichte der deutschen chemischen Gesellschaft, Band 16, S. 661.

[113] Vid. op. cit. supra, Band 22, S. 87.

[114] Chemisches Centralblatt, 1895, Band 2, S. 66.

[115] Comptes rendus; Tome 112, No. 15, p. 799.

[116] Vid. op. cit. supra, Tome 94, p. 1517.

[117] Journal of the Chemical Society, June, 1888, p. 610. (In the formulas for lactose and arabinose read H₂₂ and H₁₀ respectively.)

[118] American Chemical Journal, Vol. 11, No. 7, p. 469.

[119] Chemisches Centralblatt, 1889, No. 7.

[120] American Chemical Journal, Vol. 17, No. 7, pp. 507, 517.

[121] Comptes rendus, Tome 118, p. 426.

[122] Justus Liebig’s Annalen der Chemie, 1890. Band 257, S. 160.

[123] Journal of Analytical and Applied Chemistry, Vol. 7, pp. 68 et seq.

[124] Flint and Tollens; Berichte der deutschen chemischen Gesellschaft, Band 25, S. 2912.

[125] Vid. op. cit. supra, Band 23, S. 1751. (Read Günther.)

[126] Journal of Analytical and Applied Chemistry, Vol. 5, p. 421.

[127] Vid. op. cit. supra, p. 426.

[128] Berichte der deutschen chemischen Gesellschaft, Band 24, S. 3575.

[129] Journal of Analytical and Applied Chemistry, Vol. 7, p. 74.

[130] Chemiker-Zeitung, Band 17, 1743.

[131] Vid. op. cit. supra, Band 18, N. 51, S. 966.

[132] Monatshefte für Chemie, Band 16, S. 283; Berichte der deutschen chemischen Gesellschaft, Referate Band 28, S. 629.

[133] Papasogli; Bulletin de l’Association des Chimistes de Sucrerie et de Distillerie, Juillet 1895, p. 68.

[134] Gans und Tollens; Zeitschrift des Vereins für die Rübenzucker-Industrie, Band 38, S. 1126.

[135] Berichte der deutschen chemischen Gesellschaft, 20, S. 181; Zeitschrift des Vereins für die Rübenzucker-Industrie, 1891, S. 895.

[136] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1891, S. 891.

[137] Chemiker-Zeitung, 1888, No. 2; Zeitschrift des Vereins für die Rübenzucker-Industrie, 1888, S. 347.

[138] Fischer; Berichte der deutschen chemischen Gesellschaft, Band 20, S. 821; Band 21, Ss. 988; 2631.

[139] Zeitschrift für physiologische Chemie, Band 11, S. 492.

[140] Vid. op. cit. supra, Band 12, No. 4, Ss. 355 et seq; No. 5, Ss. 377 et seq.

[141] Berichte der deutschen chemischen Gesellschaft, Band 20, S. 540.

[142] Sitzungsberichte der Mathematisch-Naturwissenschaften in Wien, Band 93, Heft 2, S. 912.

[143] Tollens; Handbuch der Kohlenhydrate; von Lippmann, Chemie der Zuckerarten.

[144] Wilder Quarter-Century Book, 1893; Abdruck aus dem Centralblatt für Bakteriologie und Parasitenkunde, Band 18, 1895, No. 1; American Journal of Medical Sciences, Sept., 1895.

[145] Griffiths, Principal Starches used as Food; Nägeli’s Beiträge zur näheren Kenntniss der Stärkegruppe.

[146] Zeitschrift für Physiologische Chemie, Band 12, Ss. 75-78.

[147] Maercker; Handbuch der Spiritusfabrikation, 1890, S. 90.

[148] Chemiker-Zeitung, Band 19, S. 1501.

[149] Paragraphs =28-32=, this volume.

[150] Vol. 2, p. 204.

[151] Chemisches Centralblatt, 1877, Band 8, S. 732.

[152] Chemiker-Zeitung, Band 19, S. 1501.

[153] Vid. op. cit. supra, S. 1502; Moniteur Scientifique, 1887, p. 538.

[154] Chemiker-Zeitung, Band 19, S. 1502.

[155] Vid. op. cit. supra, 1895, S. 1727.

[156] Chemiker-Zeitung, Band 19, S. 1502.

[157] Jahresberichte der Agrikulturchemie, 1892, S. 664.

[158] Journal de Pharmacie et de Chimie, 5ᵉ, Série, Tome 25, p. 394.

[159] Journal of the American Chemical Society, Vol. 17, p. 64.

[160] Repertorium der Analytischen Chemie, 1887, S. 299.

[161] Journal of the American Chemical Society, Vol. 16, p. 726.

[162] Förschungs Berichte über Lebensmittel, Hamburg; Abs., The Analyst, Vol. 20, p. 210.

[163] Rouvier; Comptes rendus, Tome 107, pp. 272, 278; Tome 111, pp. 64, 186; Tome 120, p. 1179.

[164] Bulletin 13, Department of Agriculture, Division of Chemistry, pp. 154 et. seq.

[165] Foods, Their Composition and Analysis, p. 139.

[166] Richardson, Vid. op. cit. 142, p. 158.

[167] Principal Starches used as Food, Cirencester, Baily & Son, Market Place.

[168] Vid. op. cit. 142, pp. 158 et seq.

[169] Bulletin 44, Department of Agriculture, Division of Chemistry, p. 14.

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