Part Third

Part Third

PROCESSES FOR DETECTING AND DETERMINING SUGARS AND STARCHES AND OTHER CARBOHYDRATES IN CRUDE OR MANUFACTURED AGRICULTURAL PRODUCTS.

=203. Introduction.=—In the preceding part directions have been given for the estimation of sugars and starches in approximately pure forms. In the present part will be described the most approved methods of separating these bodies and other carbohydrates from crude agricultural products and for their chemical examination. In many respects the processes which in a small way are used for preparing samples for analysis are employed on a large scale for technical and manufacturing purposes. It is evident, however, that the following paragraphs must be confined strictly to the analytical side of the question inasmuch as anything more than mere references to technical processes would lead into wide digressions.

In the case of sugars the analyst is for the most part quite as much in need of reliable methods of extraction and preparation as of processes for analysis. With starches the matter is more simple and the chief methods of separating them for examination were necessarily described in the previous part.

Sugars in fresh plants exist almost entirely in solution. This is true of all the great sources of the sugar of commerce, viz., the palm, the maple, the sugar beet and sugar cane. This statement is also true of fruits and the natural nectar of flowers. By natural or artificial drying the sugar may be reduced to the solid or semisolid state as in the cases of raisins and honey. In certain seeds, deficient in water, sugars may possibly exist in a solid state naturally, as may be the case with sucrose in the peanut and raffinose in cotton seed.

Starches on the other hand when soluble, are probably not true starches, but they partake more or less of a dextrinoid nature. Fine starch particles occur abundantly in the juices of some plants, as for instance sorghum, where they are associated with sugar and can be obtained from the expressed juice by subsidence. But even in such a case it is not certain that the starch enters into the general circulation. It is more likely formed locally by biochemical condensation of its constituents. Starches in a soluble or semisoluble state are transported, as a rule, to the tubers or seeds of plants where they are accumulated in large quantities as a reserve food for future growth. For a study of the plant metabolism whereby starch is produced and for its histological and physiological properties the reader may consult the standard authorities on vegetable physiology.[170]

=204. Sugar in the Sap of Trees.=—Many trees at certain seasons of the year, carry large quantities of sugar in their sap. Among these the maple and sugar palm are preeminent. The sap is secured by cutting a pocket into the side of the tree or by boring into it and allowing the sap to run into an appropriate receptacle through a spile. The content of sugar in the sap of the maple and palm varies greatly. In some cases it falls as low as one and a half and in others rises to as much as six or seven per cent.[171] In most cases the sugar in the maple sap is pure sucrose, but towards the end of the flowing season it may undergo changes of a viscous nature due to fermentation, or inversion, forming traces of invert sugar. In this country the sap of the maple may flow freely on any warm day in winter, but the sugar season proper begins about February 15th in Southern Ohio and Indiana, and about March 25th in Vermont. It lasts from six weeks to two months. The sap flows best during moderately warm, still days, after a light freeze.

In addition to sugar the maple sap contains a trace of albuminoid matters and some malic acid combined with lime. As a rule it can be subjected to polarization without preliminary clarification.

=205. Determination of Sugar in Saps.=—In most cases the sap may be directly polarized in a 200 millimeter tube. Its specific gravity is obtained by a spindle or pyknometer, and the percentage of sugars taken directly from the table on page 73, the degree brix corresponding to the sugar percentage.

On polarizing, the sugar percentage is calculated as follows:

Multiply the specific gravity of the sap by 100 and divide the product by 26.048. Divide the direct reading of the sap on the sugar scale by the quotient obtained above, and the quotient thus obtained will be the correct percentage of sugar in the original solution.

The formula is applicable for those instruments in which 26.048 grams represent the normal quantity of sugar which in 100 cubic centimeters reads 100 divisions on the scale. When other factors are used they should be substituted for 26.048 in the above formula.

The principle of the calculation is based on the weight of the sap which is contained in 100 cubic centimeters, and this is evidently obtained by multiplying 100 by the specific gravity of the sap. Since 26.048 is the normal quantity of sugar in that volume of the solution the quotient of the actual weight divided by that factor shows how many times too great the observed polarization is. The simple division of the polariscope reading by this factor gives the correct reading.

Example: Let the specific gravity of the sap be 1.015 and the observed polarization be 15.0. Then the true percentage of sugar in the sap is found by the equation:

101.5 : 26.048 = 15.0 : x.

Whence x = 3.85 = percentage of sugar in the sap.

The process outlined above is not applicable when a clarifying reagent such as lead subacetate or alumina cream must be used. But even in these cases it will not be found necessary to weigh the sap. A sugar flask graduated at 100 and 110 cubic centimeters is used and filled to the first mark with the sap, the specific gravity of which is known. The clarifying reagent is added, the volume completed to the second mark with water, and the contents of the flask well shaken and thrown on a dry filter. The observation tube, which should be 220 millimeters in length, is then filled with the clear filtrate and the rest of the process is as described above. A 200 millimeter tube may also be used in this case and the observed reading increased by one-tenth.

=206. Estimation of Sugar in the Sap of Sugar Cane and Sorghum.=—In bodies like sugar cane and sorghum the sap containing the sugar will not flow as in the cases of the maple and sugar palm. The simplest way of securing the sap of the bodies named is to subject them to pressure between rolls. A convenient method of obtaining the sap or juice is by passing the cane through a small three-roll mill indicated in the figure. Small mills of this kind have been used in this division for many years and with entire satisfaction. Small canes, such as sorghum, may be milled one at a time, or even two or three when they are very small. In the case of large canes, it is necessary that they be split and only half of them used at once. The mill should not be crowded by the feed in such a way as to endanger it or make it too difficult for the laborer to turn. From fifty to sixty per cent of the weight of a cane in juice may be obtained by passing it through one of these small mills. Experience has shown that there is a little difference between the juice as first expressed and the residual sap remaining in the bagasse, but the juice first expressed may be used for analysis for control purposes as a fair representative of all that the cane contains.

To determine the percentage of juice expressed, the canes may be weighed before passing through the mill and the juice collected. Its weight divided by the weight of the original cane will give the per cent of the juice expressed, calculated on the whole cane. Instead of weighing the juice the bagasse may also be collected and weighed; but on account of the rapidity with which it dries the operation should be accomplished without delay. The expressed juice is clarified with lead subacetate, filtered and polarized in the manner described in former paragraphs. Instead of weighing the juice, its specific gravity may be taken by an accurate spindle and the volume of it, equivalent to a given weight, measured from a sucrose pipette.[172]

A sucrose pipette for cane juice has a graduation on the upper part of the stem which enables the operator to deliver double the normal weight for the polariscope used, after having determined the density of the juice by means of a spindle. A graduation of from 5° to 25° of the brix spindle will be sufficient for all variations in the density of the juice, or one covering a range of from 10° to 20° will suffice for most instances. The greater the density of the juice the less volume of it will be required for the weight mentioned. For general use, the sucrose pipette is graduated on the stem to deliver from forty-eight to 50.5 cubic centimeters, the graduations being in terms of the brix spindle. The graduation of the stem of this instrument is shown in the accompanying figure. In the use of the pipette it is only necessary to fill it to the degree on the stem corresponding to the degree brix found in the preliminary trial.

The quantities of juice corresponding to each degree and fractional degree of the brix spindle are given in the following table; calculated for the normal weight 26.048 grams for the ventzke and for 16.19 grams for the laurent scale. The measured quantities of juice are placed in a 100 cubic centimeter sugar flask, treated with the proper quantity of lead subacetate, the volume completed to the mark, and the juice filtered and polarized in a 200 millimeter tube. The reading of the polariscope is divided by two for the factor 26.048 and by three for the factor 16.19.

TABLE FOR USE OF SUCROSE PIPETTES.

Cubic centimeters Cubic centimeters of juice for 26.048 of juice for 16.19 Degrees factor. Divide Degrees factor. Divide brix. reading by two. brix. reading by three.

5.0 51.1 5.0 47.6 5.4 51.0 5.7 47.5 5.7 50.9 6.3 47.4 6.4 50.8 6.8 47.3 6.9 50.7 7.3 47.2 7.4 50.6 7.8 47.1 7.9 50.5 8.3 47.0 8.4 50.4 8.9 46.9 8.9 50.3 9.5 46.8 9.4 50.2 10.0 46.7 9.9 50.1 10.5 46.6 10.4 50.0 11.0 46.5 10.9 49.9 11.6 46.4 11.4 49.8 12.1 46.3 11.9 49.7 12.7 46.2 12.4 49.6 13.3 46.1 12.9 49.5 13.8 46.0 13.4 49.4 14.3 45.9 13.9 49.3 14.8 45.8 14.4 49.2 15.3 45.7 14.9 49.1 15.9 45.6 15.4 49.0 16.4 45.5 15.9 48.9 17.0 45.4 16.4 48.8 17.5 45.3 16.9 48.7 18.0 45.2 17.4 48.6 18.6 45.1 17.9 48.5 19.1 45.0 18.4 48.4 19.7 44.9 18.9 48.3 20.2 44.8 19.4 48.2 19.9 48.1

In ordering sucrose pipettes the factor for which they are to be graduated should be stated.

It is evident also that with the help of the foregoing table the measurements may be made by means of a burette. For instance, if the degree brix is found to be 19.9, 48.1 cubic centimeters are to be used. This quantity can be easily run from a burette. In order to make the pipette more convenient it has been customary in this laboratory, as practiced by Carr, to attach a glass tube with a stopcock by means of a rubber tube to the upper part of the pipette, whereby the exact level of the juice in the stem of the pipette can be easily set at any required mark.

In the polarization of dilute solutions, such as are found in the saps and juices referred to above, it must not be forgotten that the gyrodynat of the sucrose is increased as the density of the solution is diminished. This change introduces a slight error into the work which is of no consequence from a technical point of view, but becomes a matter which must be considered in exact determinations. To avoid the annoyance of calculating the gyrodynat for every degree of concentration, tables have been constructed by Schmitz and Crampton by means of which the actual percentage of sugar, corresponding to any degree of polarization, is determined by inspection. These tables may be used when extremely accurate work is required.[173]

=207. Measuring Sugar Juices with a Gravimeter.=—A convenient method of weighing sugar juices is the gravimetric process designed by Gird.[174] The apparatus is fully illustrated by Fig. 64. The hydrometer F has a weight of 26.048 grams and its stem is also graduated in degrees brix. The juice is poured into the cylinder A and allowed to stand until air bubbles have escaped. In filling A the finger is held over the orifice G so that the siphon tube B is completely filled, the air escaping at the vent C. After the tube is filled the finger is withdrawn from G and all the liquid which will run out at G allowed to escape. The sugar flask D is now brought under G and the hydrometer F allowed to descend into A. The hydrometer will displace exactly its own weight of liquid. For convenience of reading, the index E may be used which is set five degrees above the surface of the liquid in A. The number of degrees brix read by E is then diminished by five. The hydrometer has been improved since the description given by the addition of a thermometer which, in addition to carrying a graduation in degrees, also shows the correction to be made upon the degree brix for each degree read. It is evident that the hydrometer may be made of any weight, and thus the delivery of any desired amount of juice be secured.

=208. Determination of Reducing Bodies in Cane Juices.=—Sucrose in cane juices is constantly accompanied with reducing sugars, or other bodies which have a similar action on fehling liquor, which interfere to a considerable degree with the practical manufacture of sugar. It is important to determine with a moderate degree of accuracy the quantity of these bodies. These sugars or reducing bodies are of a peculiar nature. The author pointed out many years ago that these reducing bodies were without action on polarized light, and for this reason proposed the name anoptose as one characteristic of their nature.[175] It is also found that these bodies do not yield theoretically the quantity of alcohol which a true sugar of the hexose type would give.[176] It is entirely probable, therefore, that they are quite different in their nature from many of the commonly known sugars. On account of the difficulty of separating these bodies in a pure state their actual copper reducing power is not known. For practical purposes, however, it is assumed to be the same as that of dextrose or invert sugar and the percentage of these bodies present is calculated on that assumption. In the determination of these sugars or reducing bodies, the quantity weighed may be determined by an apparatus entirely similar to the sucrose pipette just described above. The quantity of juice used should be diluted as a rule to such a degree as not to contain more than one per cent of the reducing bodies. For the best work, the juices should be clarified with lead subacetate and the excess of lead removed with sodium carbonate. For technical control work in sugar factories, this process may be omitted as in such cases rapidity of work is a matter of considerable importance and the approximate estimation of the total quantity of reducing bodies is all that is desired.

For volumetric work, the solution of copper and the method of manipulation described in paragraph =117= are most conveniently used.

=209. Preservation of Sugar Juices for Analysis.=—Lead subacetate not only clarifies the juices of canes and thus permits of their more exact analytical examination, but also exercises preservative effects which enable it to be used as a preserving agent and thus greatly diminish the amount of work necessary in the technical control of a sugar factory. Instead, therefore, of the analyst being compelled to make an examination of every sample of the juice, aliquot portions representing the different quantities can be preserved and one analysis made for all. This method has been thoroughly investigated by Edson, who also finds that the errors, which may be introduced by the use of the lead subacetate in the analytical work, may be entirely avoided by using the normal lead acetate.[177]

In the use of the normal lead acetate, much less acetic acid is required in the polariscopic work than when the subacetate is used. The normal lead acetate is not so good a clarifying agent as the subacetate, but its efficiency in this respect is increased by the addition of a little acetic acid. In its use, it is not necessary to remove the lead, even for the determination of the reducing bodies.

For further details in regard to the technical determination of reducing bodies, special works may be consulted.[178]

=210. Direct Determination of Sugars in Canes.=—The methods, which have just been described, of securing the juices of cane by pressure and of determining the sugars therein, do not give the actual percentage of sugar in the cane. An approximate result may be secured by assuming that the cane is composed of ninety parts of juice and ten parts of cellular tissues and other insoluble matters. This assumption is approximately true in most cases, but there are often conditions arising which render the data calculated on the above assumption misleading. In any particular case in order to be certain that the correct percentage of sugar is secured it will be necessary to determine the fiber in the cane. This is an analytical process of considerable labor and especially so on account of the difficulty of securing samples which represent the average composition of the cane. The fibrous structure of the canes, the hardness of their external covering and the toughness of their nodes or joints render the sampling extremely difficult. Moreover, the content of sugar varies in different parts of the cane. The parts nearest the ground are, as a rule, richer than the upper joints and this is especially true of sugar cane. In order, therefore, to get a fair sample, even of a single cane, all parts of it must be considered. Several methods of the direct determination of sugar in canes have been proposed and will be described below.

=211. Methods of Cutting or Shredding the Cane for Analytical Purposes.=—A simple method of cutting canes into small pieces which will permit of an even sampling is very much to be desired. The cutting apparatus shown in Fig. 65 has been long in use in this laboratory. The canes by it are cut into thin slices, but the cutting edge of the knife being perpendicular to the length of the cane renders the use of the instrument somewhat laborious and unsatisfactory. A considerable time is required to cut a single cane and the slices which are formed should be received in a vessel which will protect them as much as possible from evaporation during the process of the work. Instead of the apparatus above a small cane cutting machine arranged with four knives on a revolving disk maybe used. The apparatus is shown in Fig. 66. The cane is fed against the knives through the hole shown in the open front of the apparatus and the knives thus strike the cane obliquely.[179] The knives can be set in the revolving disk at any desired position so as to cut the canes into chips as fine as may be desired. The cossettes furnished by this method may be sampled directly for the extraction of the sugar. In the case of the cossettes from both instruments described above a finer subdivision may be secured by passing them through a sausage cutter.

The best method for shredding canes, however, is to pass them through the apparatus described on page 9. That machine gives an extremely fine, moist mass, which is of uniform nature and capable of being directly sampled.

=212. Methods of Determination.=—Even the finely divided material obtained by the machine just described is not suited to give an instantaneous diffusion for polarization as is done by the finely ground beet pulp to be described further on. For the determination of sugar a proper weight of the cossettes or pulp obtained as described above, taken after thorough mixing, is placed in a flask graduated properly and treated with water.[180]

The flask in which the mixture takes place should be marked to compensate for the volume of the fiber of the cane. When the normal weight of cane is taken for the ventzke scale, viz., 26.048, the flask should be graduated at 102.6 cubic centimeters. If double the normal weight be taken, the flask should be graduated at 205.2 cubic centimeters. This graduation is based on the assumption of the presence of fiber amounting to ten per cent of the weight of the cossettes. The fiber is so nearly the density of the juice obtained as to be regarded as one gram equal to one cubic centimeter. The flask is at first filled almost full of water and then warmed to near the boiling point for an hour with frequent shaking. It is then filled to a little above the mark, the contents well mixed and warmed for ten minutes more with frequent shaking. After cooling, the volume is made up to the mark, well shaken and poured upon a filter. The filtrate is collected in a sugar flask marked at fifty and fifty-five cubic centimeters. When filled to the first mark a proper quantity of lead subacetate is added, the volume completed to the second mark with water, the contents of the flask well shaken, poured upon a filter and the filtrate polarized in the usual way.

The reducing sugar is determined in an aliquot part of the filtrate by one of the alkaline copper methods.

=213. Determination by Drying and Extraction.=—Instead of the diffusion and polarization method just described, the fine pulp obtained may be dried, the dried residue ground in a drug mill and extracted with aqueous alcohol or with water.

To facilitate the calculation when this method is employed, the water content of a small portion of the well sampled pulp is determined. The rest of the pulp is dried, first for a few hours at a temperature not above 60° or 70°, and then at the temperature of boiling water, either in the open air or a partial vacuum, until all the water is driven off. The dried residue can then be preserved in well stoppered bottles for the determination of sugar at any convenient period. The finely ground dried residue for this purpose is placed in an extraction apparatus and thoroughly exhausted with eighty per cent alcohol. The extract is dried and weighed, giving the total weight of all sugars present. After weighing, the extract is dissolved in water, made up to a definite volume and the reducing sugars determined in an aliquot portion thereof by the usual methods. The weight of reducing sugars found, calculated for the whole extract deducted from the total weight of this extract will give the weight of the sucrose in the sample. From this number the content of sugar in the original cane is determined from the percentage of water found in the original sample.

Example.—In a sample of finely pulped canes the content of water is found to be 76.5 per cent. The thoroughly dried pulp is ground and extracted with aqueous alcohol. Five grams give two and five-tenths grams of the extract. The extract is dissolved in water, made up to a definite volume and the reducing sugars determined in an aliquot part and calculated for the whole, amounting to 150 milligrams. The extract is therefore composed of 2.35 grams of sucrose and 0.15 gram of reducing sugars. The calculation is now made to the original sample which contained 76.5 per cent of water and 23.5 per cent of dry matter, as follows:

5 : x :: 23.5 : 100, whence x = 21.27,

the weight of the original material corresponding to five grams of the dry substance. The original composition of the sample is therefore expressed by the following numbers:

Per cent. Sucrose 11.1 Reducing sugars 0.7 Water 76.5 Fiber (insoluble matter) 11.7

=214. Examination of the Bagasse.=—The method just described for the examination of canes may be also applied to the analysis of bagasses, with the changes made necessary by the increased percentage of fiber therein. On account of the large surface exposed by the bagasse, the sampling, shredding and weighing should be accomplished as speedily as possible to avoid loss of moisture.

The optical examination of bagasses is rendered difficult by reason of the uneven pressure to which the canes are subjected. With fairly good milling in technical work the bagasses will have at least thirty per cent of fiber. The method for the polariscopic examination is therefore based upon that assumption, but the volume of the solution must be changed for varying percentages of fiber in the bagasse. On account of the smaller percentage of sugar, it is convenient to take double or three times the normal weight of the bagasse for examination. Since large sugar flasks are not commonly to be had the diffusion of the bagasse may be conducted in a quarter liter flask. In a quarter liter flask place 52.096 grams of the finely shredded bagasse, very nearly fill the flask with water and extract the sugar as described for canes in the foregoing paragraphs. In the weight of bagasse used there will be, in round numbers, fifteen grams of fiber. When the volume of water is completed to the mark the actual content of liquid in the flask will therefore be only 235 cubic centimeters. Fifty cubic centimeters of the filtrate are placed in a sugar flask marked at fifty and fifty-five cubic centimeters, the proper quantity of lead subacetate solution added, the volume completed to the upper mark, the contents of the flask well shaken, filtered and polarized in a 200 millimeter tube. Let the reading obtained be four degrees and increase this by one-tenth for the increased volume of solution above fifty cubic centimeters. The true reading is therefore four degrees and four-tenths. This reading, however, must be corrected, because the original volume instead of being 200 cubic centimeters, is 235 cubic centimeters. The actual percentage of sugar in the sample examined is obtained by the following proportion:

200 : 235 = 4.4 : x.

The correct reading is therefore 5°.2, the percentage of sugar in the sample examined.

The results obtained by the method just described may vary somewhat from the true percentage by reason of the variation of the content of fiber in the bagasse. It is, however, sufficiently accurate for technical control in sugar factories and on account of its rapidity of execution is to be preferred for this purpose. More accurate results would be obtained by drying the bagasse, and proceeding with the examination in a manner entirely analogous to that described for the extraction of sugar from dried canes by aqueous alcohol. In both instances the reducing sugar is determined in the manner already mentioned.

=215. Determination of Fiber in Cane.=—In estimating the content of sugar in canes by the analysis of the expressed juices, it is important to make frequent determinations of the fiber for the purpose of obtaining correct data for calculation. In periods of excessive drought, or when the canes are quite mature, the relative content of fiber is increased, while, on the other hand, in case of immature canes, or during excessive rainfalls, it is diminished. The chief difficulty in determining the content of fiber in canes is found in securing a representative sample. On account of the hard and fibrous nature of the envelope and of their nodular tissues, canes are reduced to a fine pulp with great difficulty by the apparatus in ordinary use. A fairly homogeneous pulp, however, may be obtained by means of the shredder described on page 9. The canes having been shredded as finely as possible, a weighed quantity is placed in any convenient extraction apparatus and thoroughly exhausted with hot water. The treatment with hot water should be continued until a few drops of the extract evaporated on a watch glass will leave no sensible residue. The residual fiber is dried to constant weight at the temperature of boiling water, cooled in a desiccator and rapidly weighed and the percentage of fiber calculated from the data obtained. On account of the great difficulty of securing a homogeneous pulp, even with the best shredding machines, the determination should be made in duplicate or triplicate and the mean of the results entered as the percentage of fiber. The term fiber as used in this sense, must not be confounded with the same term employed in the analysis of fodders and feeding stuffs. In the latter case the term is applied to the residue left after the successive treatment of the material with boiling, dilute acid and alkali. The analysis of canes for feeding purposes is conducted in the general manner hereinafter described for fodders.

=216. Estimation of Sugar in Sugar Beets.=—The methods employed for the determination of the sugar content of beets are analogous to those used for canes, with such variations in the method of extraction as are made possible and necessary by the difference in the nature of these sacchariferous plants. The sugar beet is more free of fiber and the hard and knotty substances composing the joints of plants are entirely absent from their composition. For this reason they are readily reduced to a fine pulp, from which the sugar is easily extracted. The analytical processes are also greatly simplified by the complete absence of reducing sugars from the juices of healthy beets. The only sugar aside from sucrose which is present in these juices is raffinose, and this is not found in healthy beets, except when they have been injured by frost or long keeping. In practical work, therefore, the determination of sucrose completes the analysis in so far as sugars are concerned. Four methods of procedure will illustrate all the principles of the various processes employed.

=217. Estimation of Sucrose in the Expressed Juice.=—In the first method the beets are reduced by any good shredding machine, to a fine pulp, which is placed in a press and the juice expressed. In this liquor, after clarification with lead subacetate, the sucrose is determined by the polariscope. The methods of measuring, clarifying and polarizing are the same as those described for saccharine juices in paragraphs =83-85=. The mean percentage of juice in the sugar beet is ninety-five. The corrected polariscopic reading obtained multiplied by 0.95 will give the percentage of sugar in the beet.

Example.—The solids in a sample of beet juice, as measured by a brix spindle, are 17.5 per cent. Double the normal weight of the juice is measured from a sucrose pipette, placed in a sugar flask, clarified, the volume completed to 100 cubic centimeters, the contents of the flask well shaken and filtered. The polariscopic reading obtained is 29°.0. Then (29.0 ÷ 2) × 0.95 = 13.8 = percentage of sucrose in the beet.

=218. Instantaneous Diffusion.=—In the second process employed for determining the sugar content of beets, the principle involved depends on the use of a pulp so finely divided as to permit of the almost instant diffusion of the sugar present throughout the added liquid. This diffusion takes place even in the cold and the process thus presents a convenient and rapid method for the accurate determination of the percentage of sugar in beets. The pulping is accomplished by means of the machine described on page 10, or the one shown in Fig. 67. The beet is pressed against the rapidly revolving rasp by means of the grooved movable block and the finely divided pulp is received in the box below. These machines afford a pulp which is impalpable and which readily permits an almost instantaneous diffusion of its sugar content.

=219. Pellet’s Method of Cold Diffusion.=—The impalpable pulp having been obtained, by one of the processes described, the content of sugar therein is determined as follows:[181]

A normal or double normal quantity of the pulp is quickly weighed, to avoid evaporation, in a sugar dish with an appropriate lip, and washed into the flask, which should be graduated, as shown in Fig. 68, to allow for the volume of the fiber or marc of the beet. Since the beet pulp contains, on an average, four per cent of marc, the volume which is occupied thereby is assumed to be a little more than one cubic centimeter. Since it is advisable to have as large a volume of water as convenient, it is the practice of Pellet to wash the pulp into a flask graduated at 201.35 cubic centimeters. If a 200 cubic centimeter flask be used, the weight of the pulp should be 25.87 instead of 26.048 grams. After the pulp is washed into the flask, about six cubic centimeters of lead subacetate of 30° baumé are added, together with a little ether, to remove the foam. The flask is now gently shaken and water added to the mark and the contents thoroughly shaken. If the pulp is practically perfect, the filtration and polarization may follow immediately. The filter into which the contents of the flask are poured should be large enough to hold the whole quantity. It is recommended to add a drop or two of strong acetic acid just before completing the volume of the liquid in the flask to the mark. The polarization should be made in a 400 millimeter tube, which will give directly the percentage of sugar present. It is not necessary to heat the solution in order to insure complete diffusion, but the temperature at which the operation is conducted should be the ordinary one of the laboratory. In case the pulp is not as fine as should be, the flask should be allowed to stand for half an hour after filling, before filtration. An insufficient amount of lead subacetate may permit some rotatory bodies other than sugar to pass into solution, and care should be taken to have always the proper quantity of clarifying material added. The presence of these rotating bodies, mostly of a pectic nature, may be shown by extracting the pulp first with cold water until all the sugar is removed, and afterwards with boiling water. The liquor obtained from the last precipitation will show a decided right-handed rotation, unless first treated with lead subacetate, in which case the polarization will be zero. A very extended experience with the instantaneous cold aqueous diffusion has shown that the results obtained thereby are quite as reliable as those given by hot alcoholic or aqueous digestion.

=220. Flask for Cold Diffusion and Alcohol Digestion.=—For convenience in washing the pulp into the sugar flask, the latter is made with an enlarged mouth as shown in Fig. 68. The dish holding the weighed quantity of pulp is held with the lip in the mouth of the flask, and the pulp washed in by means of a jet of water furnished from a pressure bottle or washing flask. The flask shown is graduated for the normal weight of pulp, viz., 26.048 grams. The marking is on the constricted neck and extends from 100 to 101.3 cubic centimeters. This permits of making the proper allowance for the volume occupied by the marc or fiber, but this is unnecessary for the usual character of control analyses. In the case of healthy, fresh beets, the volume occupied by the marc is nearly one and three-tenths cubic centimeters for the normal polariscopic weight of 26.048 grams of pulp. For the laurent instrument this volume is nearly one cubic centimeter.

=221. Extraction with Alcohol.=—The third method of determining sugar in beets is by alcoholic extraction. The principle of the method is based on the fact that aqueous alcohol of not more than eighty per cent strength will extract all the sugar from the pulp, but will not dissolve the pectic and other rotatory bodies, which, in solution, are capable of disturbing the rotatory power of the sugar present. It is also further to be observed that the rotatory power of pure sucrose, in an aqueous alcoholic solution, is not sensibly different from that which is observed in a purely aqueous liquid. The pulp, which is to be extracted, should be in as fine a state of subdivision as convenient, and the process may be carried on in any of the forms of extraction apparatus already described, or in the apparatus shown in Fig. 69. The extraction tube, of the ordinary forms of apparatus, however, is scarcely large enough to hold the required amount of pulp, and therefore special tubes and forms of apparatus have been devised for this method of procedure. In weighing the pulp for extraction, a quarter, half, or the exact amount required for the polariscope employed, should be used. If the tubes are of sufficient size the full weight may be taken, viz., 26.048 or 16.19 grams for the instruments in ordinary use. Since the pulp contains a large quantity of water, the extraction could be commenced with alcohol of standard strength, viz., about ninety-five per cent. The volume of alcohol employed should be such as will secure a strength of from seventy to eighty per cent when mixed with the water contained in the pulp. The flask receiving the extract should be kept in continuous ebullition and the process may be regarded as complete in about one hour, when the pulp has been properly prepared. The method of extracting beet pulp with alcohol is due to Scheibler, and in its present form the process is conducted according to the methods described by Scheibler, Sickel, and Soxhlet.[182]

If the pulp be obtained by any other means than that of a fine rasp, the extraction of the sugar by the aqueous alcohol takes a long time, and even a second extraction may be necessary. It is convenient to use as a flask for holding the solvent, one already graduated at 100 or 110 cubic centimeters. A flask especially constructed for this purpose, has a constricted neck on which the graduations are made, and a wide mouth serving to attach it to the extracting apparatus, as shown in Fig. 68. When the extract is obtained in this way, it is not necessary to transfer it to a new flask before preparing it for polarization. When the extraction is complete, the source of heat is removed, and when all the alcohol is collected in the flask, the latter is removed from the extraction apparatus, cooled to room temperature, a sufficient quantity of lead subacetate added, the flask well shaken, the volume completed to the mark with water, again well shaken and the contents of the flask thrown upon the filter. It is important to avoid loss of alcohol during filtration. For this purpose it is best to have a folded filter and to cover the funnel immediately after pouring the contents of the flask upon the filter paper, with a second larger funnel. The stem of the funnel carrying the filter paper, should dip well into the flask receiving the filtrate. As in other cases of filtering sugar juices for polarization, the first portions of the filtrate received should be rejected. The percentage of sugar is obtained in the filtrate in the usual way.

Where a weight of pulp equal to the normal factor of the polariscope employed is used, and the extract collected in a 100 cubic centimeter flask, the percentage of sugar is directly obtained by making the reading in a 200 millimeter tube. With other weights of pulp, or other sizes of flask, the length of the observation tube may be changed or the reading obtained corrected by multiplication or division by an appropriate factor. A battery of sickel-soxhlet extractors is shown in Fig. 69.[183]

=222. Scheibler’s Extraction Tube.=—In order to secure a speedy extraction of large quantities of pulp, Scheibler recommends the use of the extraction tube shown in Fig. 70.[184] The apparatus is composed of three concentric glass cylinders. The outer and middle cylinders are sealed together at the top, and the inner one is movable and carries a perforated diaphragm below, for filtering purposes. Near the top it is provided with small circular openings, whereby the alcoholic vapors may gain access to the condenser (not shown). The middle cylinder is provided with two series of apertures, through the higher of which the vapor of alcohol passes to the condenser, while the alcohol which has passed through the pulp and collected between the inner and middle cylinders, flows back through the lower into the flask (not shown) containing the boiling alcohol.

The middle cylinder is provided with a curved bottom to prevent the filtering end of the inner tube from resting too tightly against it.

The tube containing the pulp is thus protected from the direct heat of the alcoholic vapors during the progress of extraction by a thin cushion of liquid alcohol.

=223. Alcoholic Digestion.=—The fourth method of determining sugar in beet pulp, is by means of digestion with hot alcohol. The principle of this method is precisely the same as that which is involved in aqueous diffusion in the cold. The diffusion, however, in the case of the alcohol, is not instantaneous, but is secured by maintaining the mixture of the pulp and alcohol for some time at or near the boiling point. The methods of preparing the pulp, weighing it and introducing it into the digestion flask are precisely those used for aqueous digestion, but in the present case a somewhat coarser pulp may be employed. The method is commonly known as the rapp-degener process.[185]

Any convenient method of heating the alcohol may be used. In this laboratory the flasks are held on a false bottom in a bath composed of two parts of glycerol and one of water. One side of the bath holder is made of glass, as shown in Fig. 71, in order to keep the flasks in view. In order to avoid the loss of alcohol, the digestion flask should be provided with a reflux condenser, or be attached to an ordinary condenser, which will reduce the vapors of alcohol again to a liquid. Unless the weather be very warm, the reflux condenser may consist of a glass tube of rather wide bore and at least one meter in length, as shown in Fig. 71. A slight loss of alcohol during the digestion is of little consequence. A convenient method of procedure is the following.

Double the quantity of the beet pulp required for the ventzke polariscope, viz., 52.096 grams, weighed in a lipped metal dish, is washed, by means of alcohol, into a flask marked at 202.6 cubic centimeters, and the flask filled two-thirds with ninety-five per cent alcohol and well shaken. Afterwards, a proper quantity of lead subacetate is added, and then sufficient alcohol to complete the volume to the mark. The flask is then attached to the condenser, placed in a water-glycerol bath and heated to a temperature of 75° for about forty-five minutes. At the end of this time, the flask is removed from the bath and condenser, cooled quickly with water, alcohol added to the mark and well shaken. The filtration should be accomplished with precautions, to avoid the loss of alcohol mentioned in paragraph =221=. The filtrate is examined in the polariscope in a 200 millimeter tube, and the reading obtained gives directly the percentage of sugar in the sample examined. Half the quantity of pulp mentioned, in a 101.3 cubic centimeter flask, may also be used. A convenient form of arranging a battery of flasks is shown in the accompanying figure.

=224. Determination of Sugar in Mother Beets.=—In selecting mother beets for seed production, it is necessary to secure only those of a high sugar content. This is accomplished by boring a hole about two and a half centimeters in diameter obliquely through the beet by means of the apparatus shown in Fig. 72.

The beet is not injured for seed production by this process, and the pulp obtained is used for the determination of sugar. The juice is expressed by means of the small hand press shown in Fig. 73. Since only a small quantity of juice is obtained, it is advisable to prepare it for polarization in a sugar flask marked at fifty cubic centimeters. The density of the juice, by reason of its small volume, is easiest obtained by the hydrostatic balance, as described in paragraph =53=. In lieu of this, the juice may be quickly weighed in a counterbalanced dish on a balance giving results accurate to within one milligram. The rest of the analytical process is similar to that already described.

=225. Aqueous Diffusion.=—The process of instantaneous aqueous diffusion may also be applied to the examination of mother beets. For this purpose the beets are perforated by a rasp, devised by Keil, shown lying on the floor in Fig. 72, the characteristics of which are shown in Fig. 74. The conical end of the rasp is roughened in such a way as to reduce the beet to an impalpable pulp. This end is fastened by a bayonet fastening to the cylindrical carrier or arm in such a way that, by means of a groove in the conical end of the rasp, the pulp is introduced into the cylinder. The cylinder is provided with a small piston by means of which the pulp can be withdrawn when the cylindrical portion of the rasp is detached from the driving machinery. It is important that the rasp be driven at a high rate of speed, viz., from 1500 to 2000 revolutions a minute. The sample of pulp at this rate of revolution is taken almost instantly, and with skilled manipulators the whole operation of taking a sample, removing the rasp by means of its bayonet fastenings, withdrawing the sample of pulp and replacing the rasp ready for another operation does not consume more than from ten to twenty seconds. From three to four samples may thus be taken in a minute. The samples of pulp as taken are dropped into numbered dishes corresponding to the numbers on the beets. One-quarter of the normal weight for the polariscope is used for the analysis. The pulp is placed in a fifty cubic centimeter flask, water and lead subacetate added, the flask well shaken, filled to the mark with water, again well shaken, the contents thrown on the filter, and the filtrate polarized in a 400 millimeter tube, giving the direct percentage of sugar. For practical purposes the percentage of marc in the beet may be neglected. If the polarization take place in a 200 millimeter tube the number obtained should be multiplied by two for the content of sugar.

In numbering sugar beets which are to be analyzed for seed production, it is found that a small perforated tin tag bearing a number may be safely affixed to the beet by means of a tack. It is not safe to use paper tags as they may become illegible by becoming wet before the sorting of the beets is completed. Where from 1000 to 2000 beets are to be examined in a day, the number of the beets and the dishes corresponding thereto must be carefully controlled to avoid confusion and mistakes.

=226. Determination of Sugars without Weighing.=—An ingenious device for the rapid analysis of mother beets is based upon the use of a machine which cuts from the beet a core of given dimensions and this core is subsequently reduced to a pulp which is treated with cold water and polarized in the manner described above. The cutting knives of the sampler can be adjusted to take a core of any desired size. Since the beets used for analysis have essentially the same specific gravity, the cores thus taken weigh sensibly the same and the whole core is used for the analysis, thus doing away with the necessity of weighing. The core obtained is reduced to a pulp in a small machine so adjusted as to permit the whole of the pulp, when prepared, to be washed directly into the sugar flask. By the use of this machine a very large number of analyses can be made in a single day, and this is highly important in the selection of mother beets, for often 50,000 or 100,000 analyses are to be made in a short time.

=227. Continuous Diffusion Tube.=—To avoid the delay occasioned by filling and emptying observation tubes in polariscopic work, where large numbers of analyses of canes and beets are to be made, Pellet has devised a continuous diffusion tube, by means of which a solution, which has just been observed, is rapidly and completely displaced by a fresh solution. This tube, improved by Spencer, is shown in Fig. 75. The fresh solution is poured in at the funnel, displacing completely the old solution which flows out through the tube at the other end. The observer watches the field vision and is able to tell when the old solution is completely displaced by the clearing of the field, at which time the reading of the new solution can be quickly made. When solutions are all ready for examination an expert observer can easily read, by the aid of this device, from four to five of them in a minute.

=228. Analysis of Sirups and Massecuites.=—The general principles which control the analysis of sirups and massecuites are the same whether these products be derived from canes or beets. In the case of the products of canes, the sirups or massecuites contain chiefly sucrose, invert sugar, and other copper reducing bodies, inorganic matters and water. In the case of products derived from sugar beets the contents are chiefly sucrose, inorganic matters, a trace of invert sugar, raffinose and water. The principles of the determination of these various constituents have already been described.

=229. Specific Gravity.=—The specific gravity of sirups and molasses can be determined by the spindle in the usual way, but in the case of molasses which is quite dense, the spindle method is not reliable. It is better, therefore, both in molasses and massecuites, to determine the density by dilution. For this purpose, as described by Spencer, a definite weight of material, from 200 to 250 grams, is dissolved in water and the volume of the solution completed to half a liter. A portion of the solution is then placed in a cylinder and the quantity of total solids contained therein determined in the usual way by a brix or specific gravity hydrometer. In case 250 grams of the material be used the calculation of the brix degree for the original material is conducted according to the following formula:

G × B × V x = -------------. W

In the above formula x is the required brix degree, V the volume of the solution, B the observed brix degree of the solution, and G the corresponding specific gravity obtained from the table on page 73. When only small quantities of the material are at hand the hydrostatic balance (=53=) should be employed. For this purpose twenty-five grams of the material are dissolved in water and the volume of the solution made up to 100 cubic centimeters. The sinker of the hydrostatic balance is placed in the solution and equilibrium secured by placing the weights upon the arm of the balance in the usual manner. Since the arm of the balance is graduated to give, by direct reading, the specific gravity, the density can be obtained at once.

Example.—Let the position of the weights or riders upon the balance arm be as follows:

(1) at point of suspension of the bob = 1.000 (3) at mark 7 on beam = 0.07 (4) at mark 9 on beam = 0.009 Specific gravity = 1.079

The nearest brix degree corresponding to this specific gravity (=58=) is 19. The total weight of the solution is equal to 100 × 1.079, viz., 107.9 grams. Since the solution contains nineteen per cent of solid matter as determined by the hydrostatic balance, the total weight of solid matter therein is 107.9 × 19 ÷ 100 = 20.5 grams. The total per cent. of solid matter in the original sample is therefore 20.5 ÷ 25 × 100 = 82 and the specific gravity corresponding thereto (page 74) is 1.42934.

The specific gravity of a massecuite may also be determined in pyknometers especially constructed for this purpose.[186]

=230. Determination Of Water.=—The accurate determination of water in sirups and massecuites is a matter of considerable difficulty. The principles of conducting the process (=26=), applicable also to the determination of water in honeys and other viscous liquids, are as follows: In all cases where invert sugar is present the drying should be conducted at a temperature not exceeding 75° or 80°. In dense molasses and massecuites a weighed quantity should be dissolved and made up to a definite volume and an aliquot portion taken for the determination. In order to secure complete desiccation at a low temperature, the drying should be accomplished in partial vacuum (pages 22, 23). The process of desiccation should be conducted in shallow, flat-bottom dishes which may be conveniently and cheaply made of aluminum and the process is hastened by filling the dish previously with thoroughly dried fragments of pumice stone. When the sample does not contain any invert sugar the desiccation can be safely accomplished at the temperature of boiling water. Drying should be continued in all cases until practically constant weight is obtained.

=231. Determination Of Ash.=—Ash is an important constituent of the sirups, molasses, and massecuites from canes and exists in very much larger quantities in the same products from beets. The ash may be determined directly by careful incineration, but it is customary to add a few drops of sulfuric acid, sufficient to combine with all the bases present and be in slight excess. The presence of sulfuric acid is of some advantage in the beginning of the carbonization and renders the process somewhat easier of accomplishment. When sulfuric acid is used, the weight of ash obtained must be diminished by one-tenth to allow for the increased weight obtained by the conversion of the carbonates into sulfates. In general, the principles and methods described on pages 36-40 are to be employed.

=232. Determination of Reducing Sugars in Sirups, Molasses, and Massecuites.=—The quantity of reducing sugars in the products derived from the sugar beet, as a rule, is insignificant. In the products from sugar cane there are large quantities of reducing matters which, in general, are determined by any of the standard methods already given. It has been shown by the author[187] that the juices of healthy sugar canes contain a small quantity of invert sugar, but this statement has been contradicted by Bloufret.[188] It is certain, however, that the reducing bodies derived from the products of manufacture of sugar cane and sorghum deport themselves in a manner somewhat different from pure invert sugar. In the absence of definite information in respect of the constitution of these bodies, the methods applicable to dextrose and invert sugar may be applied.

Since the paragraphs relating to these processes were printed some important improvements in the preparation of the alkaline copper solutions have been made. The copper carbonate solution, as has already been said, is peculiarly suited to the determination of reducing sugars in the presence of sucrose and the modified forms of this solution, and the methods of employing them with invert sugar, dextrose, levulose, and maltose, are described below.

=233. Estimation of Minute Quantities of Invert Sugar in Mixtures.=—The method of Hiller and Meissl, paragraph =142=, may be used for the estimation of small quantities of invert sugar in mixtures. A modified form of Soldaini’s reagent is, however, to be preferred for this purpose. Ost has proposed and tested a copper carbonate solution for the purpose mentioned which gives reliable results.[189] The solution has the following composition:

One liter contains 3.6 grams crystallized copper sulfate. 250.0 ” potassium carbonate. 100.0 ” hydrogen potassium sulfate.

This reagent undergoes no change when kept for a long while, especially in large vessels. Even in smaller vessels it can be kept for a year or more without undergoing any change.

The method of analysis is the same as that described in paragraph =128=, with the exception that the boiling is continued for only five minutes instead of ten, and the quantities of the copper and sugar solutions used are doubled, being 100 and fifty cubic centimeters respectively. In no case must the solution used contain more than thirty-eight milligrams of invert sugar. The quantity of sucrose in the mixture is obtained by polarization (=94=). Ost has also recalculated the reduction values of the common sugars for the strong copper carbonate solution, and the numbers obtained are slightly different from those given on page 142.[190]

For different percentages of invert sugar in mixtures of sucrose, the quantities of invert sugar are calculated from the number of milligrams of copper obtained by the following table:

(A) = Milligrams of copper obtained. (B) = Pure invert sugar. (C) = Invert sugar. (D) = Sucrose.

MILLIGRAMS OF INVERT SUGAR IN MIXTURES OF 5(C) 2(C) 1.5(C) 1.0(C) 0.8 (C) 0.6(C) 0.5(C) (A) (B) 95(D) 98(D) 98.5(D) 99.0(D) 99.2 (D) 99.4(D) 99.5(D) 88 37.9 37.1 36.0 35.4 34.7 34.2 33.9 33.6 85 36.3 35.5 34.5 34.0 33.4 32.9 32.5 32.2 80 33.9 33.0 33.2 31.7 31.2 30.7 30.2 29.9 75 31.6 30.7 30.0 29.5 29.0 28.5 28.1 27.7 70 29.4 28.5 27.8 27.4 26.8 26.4 25.9 25.6 65 27.3 26.3 25.7 25.3 24.7 24.3 23.8 23.5 60 25.2 24.2 23.6 23.2 22.6 22.2 21.8 21.5 55 23.1 22.1 21.6 21.2 20.6 20.2 19.8 19.6 50 21.2 20.1 19.6 19.2 18.6 18.3 17.9 17.7 45 19.3 18.2 17.6 17.2 16.7 16.3 16.0 15.8 40 17.3 16.3 15.7 15.3 14.8 14.5 14.2 14.0 35 15.4 14.5 13.8 13.4 13.0 12.7 12.5 12.3 30 13.5 12.6 12.0 11.6 11.2 11.0 10.8 10.6 25 11.5 10.8 10.3 10.0 9.5 9.3 9.1 9.0 20 9.6 9.1 8.6 8.3 7.9 7.7 7.5 7.3 15 7.7 7.3 6.9 6.7 6.3 6.1 5.8 5.6 10 5.8 5.4 5.1 5.0 4.7 4.5 4.2 3.9

MILLIGRAMS OF INVERT SUGAR IN MIXTURES OF 0.4(C) 0.3(C) 0.2(C) 0.1(C) 0.05(C) 0.02(C) (A) 99.6(D) 99.7(D) 99.8(D) 99.9(D) 99.95(D) 99.98(D) 88 33.3 85 32.0 31.8 80 29.7 29.5 75 27.4 27.2 70 25.3 25.0 65 23.2 22.8 60 21.2 20.8 20.4 55 19.3 18.9 18.5 50 17.4 17.0 16.7 45 15.6 15.3 14.9 40 13.8 13.5 13.2 35 12.1 11.9 11.5 10.3 30 10.4 10.2 9.9 8.8 25 8.8 8.6 8.2 7.3 20 7.1 6.9 6.6 5.8 4.9 15 5.4 5.2 5.0 4.4 3.7 2.0 10 3.8 3.5 3.4 3.0 2.5 1.7

=234. Soldaini’s Method Adapted to Gravimetric Work.=—By reason of their better keeping qualities and because of their less energetic action on non-reducing sugars, copper carbonate solutions are to be preferred to the alkaline copper tartrate solutions for gravimetric determinations of reducing sugars in cane juices and sugar house products, provided the difficulties which attend the manipulation can be removed. Ost has succeeded in securing perfectly satisfactory results with copper carbonate solution by slightly varying the composition thereof and continuing the boiling, for the reduction of the copper, ten minutes.[191] The copper solution is made as follows:

17.5 grams crystallized copper sulfate. 250.0 ” potassium carbonate. 100.0 ” ” bicarbonate.

The above ingredients are dissolved in water and the volume of the solution completed to one liter. The object of the potassium bicarbonate is to secure in the solution an excess of carbon dioxid and thus prevent the deposition of basic copper carbonate on keeping. The manipulation is conducted as follows:

One hundred cubic centimeters of the copper solution are mixed with half that quantity of the sugar solution in a large erlenmeyer, which is placed upon a wire gauze, heated quickly to boiling and kept in ebullition just ten minutes. The sugar solution should contain not less than eighty nor more than 150 milligrams of the reducing sugar, and the quantity of the solution representing this should be diluted to fifty cubic centimeters before mixing with the copper solution. After boiling, the contents of the erlenmeyer are quickly cooled and filtered with suction through an asbestos filter and the whole of the copper suboxid washed into the filter tube. This precipitated suboxid is washed once with a little potassium carbonate solution then with hot water and finally with alcohol, well dried, heated to redness, and the copper oxid obtained reduced to metallic copper in an atmosphere of hydrogen entirely free of arsenic. From the weight of metallic copper obtained the quantity of sugar which has been oxidized is calculated from the tables below.

It is evident that the process given above may be varied so as to conform to the practice observed in this laboratory of cooling the boiling solution sufficiently at once by adding to it an equal volume of recently boiled, cold water, collecting the precipitated copper suboxid in a gooch, and, after washing it, securing solution in nitric acid and the precipitation of the copper by electrolysis.

TABLE SHOWING MILLIGRAMS DEXTROSE, LEVULOSE AND INVERT SUGAR OXIDIZED, CORRESPONDING TO MILLIGRAMS OF COPPER REDUCED.

Copper. Dextrose. Levulose. Invert.

435 152.3 145.9 147.5 430 149.8 143.4 145.3 425 147.3 140.9 143.1 420 144.8 138.4 140.8 415 142.3 135.9 138.5 410 139.8 133.5 136.2 405 137.3 131.1 133.9 400 134.9 128.7 131.6 395 132.5 126.4 129.3 390 130.1 124.1 127.0 385 127.8 121.8 124.8 380 125.5 119.5 122.6 375 123.3 117.2 120.4 370 121.1 115.0 118.2 365 119.0 112.8 116.0 360 116.9 110.6 113.9 355 114.8 108.5 111.8 350 112.8 106.4 109.8 345 110.8 104.3 107.8 340 108.8 102.3 105.8 335 106.8 100.3 103.8 330 104.9 98.4 101.8 325 103.0 96.5 99.9 320 101.1 94.6 98.0 315 99.2 92.8 96.2 310 97.4 91.0 94.4 305 95.6 89.2 92.6 300 93.8 87.5 90.9 295 92.0 85.8 89.2 290 90.2 84.1 87.5 285 88.4 82.4 85.8 280 86.7 80.8 84.1 275 85.0 79.2 82.4 270 83.3 77.6 80.7 265 81.5 76.1 79.1 260 79.8 74.6 77.5 255 78.1 73.1 75.9 250 76.5 71.6 74.3 245 74.9 70.1 72.7 240 73.3 68.6 71.1 235 71.7 67.2 69.5 230 70.1 65.7 68.0 225 68.5 64.3 66.5 220 66.9 62.8 65.0 215 65.3 61.4 63.5 210 63.8 59.9 62.0 205 62.2 58.5 60.5 200 60.7 57.0 59.0 195 59.1 55.6 57.5 190 57.6 54.1 56.0 185 56.0 52.7 54.5 180 54.5 51.2 53.1 175 53.0 49.8 51.6 170 51.5 48.4 50.2 165 50.0 46.9 48.7 160 48.5 45.5 47.3 155 47.0 44.1 45.8 150 45.5 42.7 44.4 145 44.0 41.3 42.9 140 42.5 39.9 41.5 135 41.0 38.5 40.1 130 39.6 37.1 38.6 125 38.1 35.7 37.2 120 36.7 34.3 35.8 115 35.2 32.9 34.3 110 33.7 31.6 32.9 105 32.2 30.3 31.4 100 30.7 29.0 30.0 95 29.2 27.7 28.5 90 27.8 26.4 27.1 85 26.3 25.1 25.6 80 24.8 23.8 24.2 75 23.3 21.5 22.8 70 21.8 20.2 21.4

CORRESPONDING TABLE FOR MALTOSE.

Milligrams Milligrams Milligrams maltose maltose copper anhydrid hydrate obtained. oxidized. oxidized.

435 263.7 277.6 430 259.3 273.0 425 255.0 268.4 420 250.9 264.1 415 247.0 260.0 410 243.2 256.0 405 339.4 252.0 400 235.6 248.0 395 231.9 244.1 390 228.2 240.2 385 224.6 236.4 380 221.1 232.7 375 217.7 229.1 370 214.4 225.6 365 211.1 222.2 360 207.9 218.8 355 204.7 215.4 350 201.5 212.1 345 198.3 208.7 340 195.2 205.4 335 192.0 202.1 330 188.8 198.8 325 185.7 195.4 320 182.5 192.1 315 179.4 188.8 310 176.3 185.6 305 173.3 182.4 300 170.3 179.2 295 167.3 176.1 290 164.4 173.0 285 161.4 169.9 280 158.5 166.8 275 155.5 163.7 270 152.6 160.7 265 149.7 157.6 260 146.8 154.6 255 143.9 151.5 250 141.1 148.5 245 138.2 145.5 240 135.4 142.5 235 132.5 139.5 230 129.7 136.5 225 126.8 133.5 220 124.0 130.6 215 121.2 127.6 210 118.4 124.7 205 115.7 121.8 200 112.9 118.9 195 110.2 116.0 190 107.4 113.1 185 104.7 110.2 180 101.9 107.3 175 99.2 104.4 170 96.4 101.5 165 93.7 98.6 160 90.9 95.7 155 88.2 92.8 150 85.4 89.9 145 82.6 87.0 140 79.9 84.1 135 77.1 81.2 130 74.4 78.3 125 71.6 75.4 120 68.9 72.5 115 66.1 69.6 110 63.4 66.7 105 60.6 63.8 100 57.9 60.9 95 55.1 58.0 90 52.3 55.1 85 49.6 52.2 80 46.8 59.3 75 44.1 56.4 70 41.4 53.5

=235. Weighing the Copper as Oxid.=—In the usual methods of the determination of reducing bodies, the percentage is calculated either volumetrically from the quantity of the sugar solution required to decolorize a given volume of the alkaline copper solution, or the reduced copper suboxid is brought into a metallic state by heating in an atmosphere of hydrogen or by electrolytic deposition. A quicker method of procedure is found in completing the oxidation of the cupric oxid by heating to low redness in a current of air.[192] For this determination the precipitation of the cuprous oxid and its filtration are made in the usual manner. The cuprous oxid is collected in a filtering tube, made by drawing out to proper dimensions a piece of combustion tube, and has a length of about twelve centimeters in all. The unchanged part of the tube is about eight centimeters in length and twelve millimeters in diameter. It is filled by first putting in a plug of glass wool and covering this with an asbestos felt on top of which another plug of glass wool is placed. After the cuprous oxid is collected in the tube it is washed with boiling water, alcohol and ether. The rubber tube connecting it with the suction is of sufficient length to permit the tube being taken in one hand and brought into a horizontal position over a bunsen. The tube is gradually heated, rotating it meanwhile, until any residual moisture, alcohol or ether, is driven off from the filtering material. The layer of glass wool holding the cuprous oxid is gradually brought into the flame and as the oxidation begins the material will be seen to glow. The heating is continued for some time after the glowing has ceased, in all for three or four minutes, the tube and the copper oxid which it contains being brought to a low redness. The current of air passing over the red-hot material in this time oxidizes it completely. The filtering tube, before use, must be ignited and weighed in exactly the same manner as described above. The heat is so applied as not to endanger the rubber tube attached to one end of the filtering tube nor to burn the fingers of the operator as he turns the tube during the heating. After complete oxidation the tube is cooled in a desiccator and weighed, the increase of weight giving the copper oxid. For the atomic weights, 63.3 copper and 15.96 oxygen, one gram of copper oxid is equivalent to 0.79864 gram of copper, and for the weights 63.17 copper and 15.96 oxygen, one gram of copper oxid equals 0.79831 gram of copper. From the amount of metallic copper calculated by one of these factors, the reducing sugar is determined by the tables already given.

=236. Estimation of Dry Substance, Polarization and Apparent Purity for Factory Control.=—For technical purposes the methods of determining the above factors, proposed by Weisberg and applicable to concentrated sirups, massecuites, and molasses, may be used.[193] Five times the half normal quantity of the material, viz., 65.12 grams, are placed in a quarter liter flask, dissolved in water and the flask filled to the mark. In the well shaken mixture, which is allowed to stand long enough to be free of air, the degree brix is estimated by an accurate spindle. For example, in the case of molasses, let the number obtained be 18.8.

Fifty cubic centimeters of the solution are poured into a 100 cubic centimeter flask, the proper quantity of lead subacetate added, the flask filled to the mark with water, its contents filtered, and the filtrate polarized in a 200 millimeter tube. Let the number obtained on polarization be 22°.1. This number may be used in two ways. If it be multiplied by two the polarization of the original sample is obtained; in this case, viz., 44°.2. In the second place, if 44.2 be multiplied by 0.26048 and this product divided by the specific gravity corresponding to 18°.8. viz., 1.078, the quotient 10.68 is secured representing the polarization or per cent of sugar contained in the solution of which the degree brix was 18.8°. From the numbers 18.8 and 10.68 the apparent purity of the solution, 56.8, is calculated, viz., 10.68 × 100 ÷ by 18.8. The original product as calculated above gives a polarization of 44.2 and this number multiplied by 100 and divided by 56.8 gives 77.8, or the apparent percentage of dry matter. The original sample of molasses, therefore, had the following composition:

Degree brix (total solids) 77.8 per cent. Sucrose 44.2 ” Solids, not sucrose 33.6 ” Apparent purity 56.8 ”

It is seen from the above that with a single weighing and a single polarization, and within from ten to fifteen minutes, all needful data in respect of the proper treatment of molasses for the practical control and direction of a factory can be obtained.

In case a laurent polariscope is used, five times the normal weight, viz., eighty-one grams of the raw material are used and the process conducted as above.

SUCROSE, DEXTROSE, INVERT SUGAR, LEVULOSE, MALTOSE, RAFFINOSE, DEXTRIN AND LACTOSE IN MIXTURES.

=237. Occurrence.=—Sucrose and invert sugar are found together in many commercial products, especially in raw sugars and molasses made from sugar cane, and in these products sucrose is usually predominant. They also form the principal saccharine contents of honey, the invert sugar, in this case, being the chief ingredient.

In commercial grape sugar, made from starch, dextrose is the important constituent, while in the hydrolysis of starch by a diastatic ferment, maltose is principally produced. In the manufacture of commercial glucose by the saccharification of starch with sulfuric acid, dextrin, maltose, and dextrose are the dominant products, while in the similar substance midzu ame, maltose and dextrose are chiefly found, and only a small quantity of dextrose.[194] In honeys derived from the exudations of coniferous trees are found also polarizing bodies not enumerated above and presumably of a pentose character.[195] In evaporated milks are usually found large quantities of sucrose in addition to the natural sugar therein contained. These mixtures of carbohydrates often present problems of great difficulty to the analyst, and the following paragraphs will be devoted to an elucidation of the best approved methods of solving them.

OPTICAL METHODS.

=238. Sucrose and Invert Sugar.=—The chemical methods of procedure to be followed in the case of a sample containing both sucrose and invert sugar have been given in sufficient detail in preceding paragraphs (=124, 171=). When, however, it is desirable to study further the composition of the mixture, important changes in the method are rendered imperative. While the estimation of the sucrose and the total invert sugar, or the sum of the dextrose and levulose, is easy of accomplishment the separate determination of the dextrose and levulose is not so readily secured. In the latter case the total quantity of the two sugars may be determined, and after the destruction or removal of one of them the other be estimated in the usual way; or in the mixture the levulose can be determined by the variation in its gyrodynat, caused by changes of temperature.

=239. Optical Neutrality of Invert Sugar.=—The gyrodynat of levulose decreases as the temperature rises (=107=) and at or near a temperature of 87°.2, it becomes equal to that of dextrose, and, therefore, pure invert sugar composed of equal molecules of levulose and dextrose is optically neutral to polarized light at that temperature. On this fact Chandler and Ricketts have based a method of analysis which excludes any interference in polarization due to invert sugar.[196] To secure the polarization at approximately a temperature of 87°, a water-bath is placed between the nicols of an ordinary polariscope in such a way as to hold a tubulated observation tube in the optical axis of the instrument. The ends of the bath, in the prolongation of this axis, are provided with clear glass disks. The space between the cover glasses of the observation tube and the glass disks of the bath is occupied by the water of the bath. When this is kept at a constant temperature it does not interfere with the reading. The observation tube may be of glass, but preferably is constructed of metal plated with platinum on the inside. For the most exact work the length of the observation tube, at 87°, is determined by measurement or calculation. The bath is heated with alcohol lamps or other convenient means. The arrangement of the apparatus is shown in Fig. 75.

In a mixture of sucrose and invert sugar any rotation of the plane of polarized light at 87° is due to the sucrose alone. In a mixture of dextrose and sucrose the polarization is determined, and, after inversion, again determined at 87°. The latter number is due to dextrose alone, and the difference between the two gives the rotation due to sucrose.

=240. Sucrose and Raffinose.=—In raw sugars made from beet molasses considerable quantities of raffinose are found. The method of inversion and polarization in such cases is described in paragraph =100=. In making the inversion by the method proposed by Lindet (=95=), and conducting the polarization on a laurent instrument, a slightly different formula, given below, is used; viz.:

C - 0.4891_A_ S = --------------- 0.810

A - S and R = ----------, 1.54

in which the several letters refer to the same factors as are indicated by them in the formula of Creydt. In the application of the formula just given the normal weight of the mixed raw sugars used is 16.2 grams.[197]

=241. Optical Determination of Levulose.=—The determination of levulose by optical methods alone is made possible by reason of the fact that the gyrodynats of the sugars with which it is associated are not sensibly affected by changes of temperature. The principle of the process, as developed by the author, rests on the ascertainment of the change in the gyrodynat of levulose when its rotation is observed at widely separated temperatures.[198] The observation tube employed for reading at low temperatures is provided with desiccating end tubes, which prevent the deposition of moisture on the cover glasses. The relations of this device to the optical parts of the apparatus are illustrated in Fig. 76.

The protecting tubes are made of hard rubber and the desiccation is secured by surrounding the space between the rubber and the perforated metal axis with fragments of potash or calcium chlorid.

The details of the construction are shown in a horizontal section through the center of the observation tube in Fig. 77. In this figure the observation tube, made of glass or metal, is represented by i, the metal jacket, open at the top in the =V= shape as described, by k. The observation tube is closed by the heavy disk b made of non-polarizing glass. This disk is pressed against the end of the observation tube by the rubber washer a, when the drying system about to be described is screwed on to k. The apparatus for keeping the cover glass dry is contained in the hard rubber tube m and consists of a perforated cylinder of brass e, supported at one end by the perforated disk c and at the outer ends by the arms d. It is closed by a cover glass of non-polarizing glass s and can be screwed on to the system h at n. The space p is filled with coarse fragments of caustic soda, potash, or calcium chlorid by removing the cover glass s. The perforated disk c prevents any of the fragments from entering the axis of observation. When the cover glass s is replaced, it just touches the free end of the perforated metal tube preventing any of the fragments of the drying material from falling into the center at the outer end. When this drying tube is placed in position, the contents of the observation tube i can be kept at the temperature of zero for an indefinite time without the deposition of a particle of moisture either upon the glass b or s.

For determining the rotation at a high temperature the apparatus of Chandler and Ricketts (=238=) may be used or the following device: The polarizing apparatus shown above, Fig. 76, may be used after the =V= shape box is removed from the stand, which is so constructed as to receive a large box covered with asbestos felt an inch thick. The observation tube is held within this box in the same way as in the one just described so that the hot water extends not only the entire length of the tube but also covers the cover glasses. In both cases the cover glasses are made of heavier glass and are much larger in diameter than found in the ordinary tubes for polariscopes. The protecting cylinders of hard rubber are not needed at high temperatures but can be left on without detriment.

The illustration, Fig. 78, shows the arrangement of the apparatus with a silver tube in position, which can be filled and emptied without removing it.

In practice the water is heated with a jet of steam and an even temperature is secured by a mechanical stirrer kept slowly in motion. With such a box it is easy to maintain a temperature for several hours which will not vary more than half a degree. The temperature for reading the hot solutions was fixed at 88°, this being nearly the temperature at which a mixture of equal molecules of levulose and dextrose is optically inactive. In every case the sugar solutions were made up to the standard volume at the temperatures at which they were to be read and thus the variations due to expansion or contraction were avoided. When solutions are read at a high temperature, they must be made with freshly boiled water so as to avoid the evolution of air bubbles which may otherwise obscure the field of vision.

By means of the apparatus described it is easy for the analyst to make a polarimetric reading at any temperature desired. In all cases the observation tube should be left at least a half an hour and sometimes longer in contact with the temperature control media before the reading is made.

The appearance of the field of vision is usually a pretty fair index of the point of time at which a constant temperature is established throughout all parts of the system. Any variation in temperature produces a distortion of the field of vision while a constant fixed temperature will disclose the field of vision in its true shape and distinctness of outline.

Principles of the Calculation.—If 26.048 grams of pure sucrose be dissolved in water and the volume made 100 cubic centimeters, it will produce an angular rotation of 34°.68 when examined in a 200 millimeter tube with polarized sodium monochromatic light. Upon the cane sugar scale of an accurately graduated shadow instrument the reading will be 100 divisions corresponding to 100 per cent of pure sucrose.

In the complete inversion of the cane sugar the reaction which takes place is represented by the following formula:

— + C₁₂H₂₂O₁₁ + H₂0 = C₆H₁₂O₆ + C₆H₁₂O₆.

The minus and plus signs indicate that the resulting invert sugar is a mixture of equal parts of levulose (d fructose) and dextrose (d glucose). We are not concerned here with the fact that a complete inversion of cane sugar is a matter of great difficulty nor with the danger which is always experienced of destroying a part of one of the products of inversion. They are matters which may cause a variation in the analytical data afterward, but do not affect the principles on which the process is based.

In the inversion of 26.048 grams of cane sugar there are therefore produced 13.71 grams of levulose and 13.71 grams of dextrose or, in all, 27.42 grams of the mixed sugars.

The angular rotation which would be produced by 13.71 grams of dextrose in a volume of 100 cubic centimeters and through a column 200 millimeters in length is, with sodium light, 14°.53 equivalent to 41.89 divisions of the cane sugar scale. The specific rotatory power of a dextrose solution of the density given is almost exactly 53, and this number is used in the calculations.

In a mixture of the two sugars under the conditions mentioned and at a temperature of 0° the angular rotation observed is -15°.15 equivalent to 43.37 divisions of the cane sugar scale.

The + rotation due to the dextrose is 14°.53. Therefore the total negative rotation due to levulose at 0° is 15°.15 + 14°.53 = 29°.68. Hence the gyrodynat of levulose at 0° and in the degree of concentration noted is readily calculated from the formula

29.68 × 100 [α]°_{D} = - ------------ = -108.24. 2 × 13.71

Since at 88° (circa) the mixture of levulose and dextrose is neutral to polarized light, it follows that at that temperature the specific rotatory power of levulose is equal to that of dextrose, viz., 53°.

[α]⁸⁸ °_{D} = - 53°.

The total variation in the specific rotatory power of levulose, between zero and 88°, is 55°.24. The variation for each degree of temperature, therefore, of the specific rotatory power of levulose is equal to 55.24 divided by 88, which is equal to 0.628. From these data it is easy to calculate the specific rotatory power of levulose for any given temperature. For instance, let it be required to determine the gyrodynat of levulose at a temperature of 20°. It will be found equal to 108.24 - 0.628 × 20 = 95.68. The required rotatory power is then [a]²⁰ °_{D} = -95°.68.

In these calculations the influence of the presence of hydrochloric acid upon the rotatory power of the levulose is neglected.

Since the variation in angular rotation in the mixture at different temperatures is due almost wholly to the change in this property of the levulose it follows that the variation for each degree of temperature and each per cent of levulose can be calculated. Careful experiments have shown that the variation in the rotatory power of levulose between 0° and 88° is represented by a straight line. For 13.71 grams per 100 cubic centimeters the variation for each degree of temperature is equal to 43.37 ÷ 88 = 0.49 divisions on the cane sugar scale, or 15.15 ÷ 88 = 0°.1722 angular measure. If 13.71 grams of levulose in 100 cubic centimeters produce the deviations mentioned for each degree of temperature, one gram would give the deviation obtained by the following calculations:

For the cane sugar scale 0.49 ÷ 13.71 = 0°.0357 and for angular rotation 0.1722 ÷ 13.71 = 0.01256.

The above data afford a simple formula for calculating the percentage of levulose present from the variation observed in polarizing a solution containing levulose, provided that the quantity of levulose present is approximately fourteen grams per 100 cubic centimeters.

Example.—Suppose in a given case the difference of reading between a solution containing an unknown quantity of levulose at 0° and 88° is equal to thirty divisions of the cane sugar scale. What weight of levulose is present? We have already seen that one gram in 100 cubic centimeters produces a variation of 0.0357 division for 1°. For 88° this would amount to 3.1416 divisions. The total weight of levulose present is therefore 30 ÷ 3.1416 = 9.549 grams. In the case given 26.048 grams of honey were taken for the examination. The percentage of levulose was therefore 9.549 × 100 ÷ 26.048 = 36.66 per cent.

If it be inconvenient to determine the polarimetric observations at temperatures so widely separated as 0° and 88° the interval may be made less. In the above case if the readings had been made at 20° and 70° the total variation would have been only ⁵⁰/⁸⁸ of the one given, viz., 17.05 divisions of the cane sugar scale. The calculation would then have proceeded as follows:

0.0357 × 50 = 1.785.

Then, 17.05 ÷ 1.785 = 9.552 grams of levulose, from which the actual percentage of levulose can be calculated as above.

With honeys the operation is to be conducted as follows:

Since honeys contain approximately twenty per cent of water and in the dry substance have approximately forty-five per cent of levulose, about 38.50 grams of the honey should be taken to get approximately 13.8 grams of levulose.

In the actual determination the calculations may be based on the factors above noted, but without respect to the degree of concentration. If half the quantity of dextrose noted be present its specific rotatory power is only reduced to about 52°.75, and this will make but little difference in the results. In the case of honey 13.024 grams of the sample are conveniently used in the examination, half the normal weight for the ventzke sugar scale. The error, however, due to difference in concentration is quite compensated for by the ease of clarification and manipulation. Alumina cream alone is used in the clarification, thus avoiding the danger of heating the solution to a high temperature in the presence of an excess of lead acetate.

An interesting fact is observed in cooling solutions of honey to 0°. The maximum left hand rotation is not reached as soon as the temperature reaches 0° but only after it has been kept at that temperature for two or three hours. The line representing the change in rotatory power in solutions of honey between 10° and 88° is practically straight but from 10° to 0°, if measured by the readings taken without delay, it is decidedly curved; the reading being less at first than it is afterwards. After three hours the 0° becomes sensibly constant and then the whole line is nearly straight, but still with a slight deficiency in the reading at the 0°. For this reason the computations should be based on readings between 10° and 88° rather than on a number covering the whole range of temperature. Nevertheless, if the solution be kept at 10° for three hours before the final reading is taken, no error of any practical magnitude is introduced.

The calculations given above, for the cane sugar scale, can also be made in an exactly similar manner for angular rotation. The angular variation produced by one gram of levulose for 1° of temperature is 0°.01256. For 88° this would become 1°.10528. Suppose the total observed angular deviation in a given case between 0° and 88° to be 10°.404, then the weight of levulose present is 10.404 ÷ 1.10528 = 9.413 grams.

In the case mentioned 26.048 grams of honey were taken for the examination. The percentage of levulose present, therefore, was 9.413 × 100 ÷ 26.048 = 36.13.

=241. General Formula for the Calculation of Percentage of Levulose.=—Let K = deviation in divisions of the cane sugar scale or in angular rotation produced by one gram of levulose for 1° temperature.

Let T and tʹ = temperatures at which observations are made.

Let R = observed deviation in rotation.

Let W = weight of levulose obtained.

Let L = per cent of levulose required.

R Then L = --------------- ÷ W. K(T - tʹ)

In most genuine honeys the value of R between 0° and 88° is approximately thirty divisions of the cane sugar scale or 10° angular measure for 26.048 grams in 100 cubic centimeters, read in a 200 millimeter tube, or, for 13.024 grams in 100 cubic centimeters read in a 400 millimeter tube.

The method of analysis outlined above has been applied in the examination of a large number of honeys with most satisfactory results. It can also be applied with equal facility to other substances containing levulose.

=242. Sucrose and Dextrose.=—In mixtures these two sugars are easily determined by optical processes, provided no other bodies sensibly affecting the plane of polarized light be present. The total deviation due to both sugars is determined in the usual way. The percentage of sucrose is afterwards found by the inversion method (=92=). The rotation, in the first instance due to the sucrose, is calculated from the amount of this body found by inversion, and the residual rotation is caused by the dextrose. The percentage of dextrose is easily calculated by a simple proportion into which the numbers expressing the gyrodynats of sucrose and dextrose enter. When the readings are made on a ventzke scale the calculations are made as follows:

Weight of sample used 26.048 grams. First polarization 88°.5 Polarization after inversion 10°.5 Temperature 20°.0 Percentage of sucrose 58.4 Rotation due to dextrose 30°.1

Percentage of dextrose:

66.5 : 53 = x : 30.1; whence x = 37.8.

The sample examined therefore contains 58.4 per cent of sucrose and 37.8 per cent of dextrose.

It is evident that the method just described is also applicable when maltose, dextrin, or any other sugar or polarizing body, not sensibly affected by the process of inversion to which the sucrose is subjected, is substituted for dextrose. When, however, more than two optically active bodies are present the purely polariscopic process is not applicable. In such cases the chemical or the combined chemical and optical methods described further on can be employed.

=243. Lactose in Milk.=—By reason of its definite gyrodynat lactose in milk is quickly and accurately determined by optical methods, when proper clarifying reagents are used to free the fluid of fat and nitrogenous substances. Soluble albuminoids have definite levogyratory powers and, if not entirely removed, serve to diminish the rotation due to the lactose.

Milk casein precipitated by magnesium sulfate has the following gyrodynatic numbers assigned to it:[199]

Dissolved in water [a]_{D} = -80° ” ” very dilute solution [a]_{D} = -87°. ” ” dilute sodium hydroxid solution [a]_{D} = -76°. ” ” strong potassium hydroxid solution [a]_{D} = -91°.

The hydrates of albumen have rotation powers which vary from [a]_{D} = -71°.40 to [a]_{D} = -79°-05. From the chaotic state of knowledge concerning the specific rotating power of the various albumens, it is impossible to assign any number which will bear the test of criticism. For the present, however, this number may be fixed at [a]_{D} = -70° for the albumens which remain in solution in the liquids polarized for milk sugar.[200]

Many reagents have been prepared for the removal of the disturbing bodies from milk in order to make its polarization possible. Among the precipitants which have been used in this laboratory may be mentioned:[201]

(1) Saturated solution basic lead acetate, specific gravity 1.97:

(2) Nitric acid solution of mercuric nitrate diluted with an equal volume of water: (=88.=)

(3) Acetic acid, specific gravity 1.040, containing twenty-nine per cent acetic acid:

(4) Nitric acid, specific gravity 1.197, containing thirty per cent nitric acid:

(5) Sulfuric acid, specific gravity 1.255, containing thirty-one per cent sulfuric acid:

(6) Saturated solution of sodium chlorid:

(7) Saturated solution of magnesium sulfate:

(8) Solution of mercuric iodid in acetic acid, formula; potassium iodid, 33.2 grams; mercuric chlorid, 13.5 grams; strong acetic acid, 20.0 cubic centimeters; water 640 cubic centimeters.

Alcohol, ether, and many solutions of mineral salts, hydrochloric and other acids are also used as precipitants for albumen, but none of them presents any advantages.

Experience has shown that the best results in polariscopic work are secured by the use of either the mercuric iodid or the acid mercuric nitrate for clarifying the milk. The latter reagent should be used in quantities of about three cubic centimeters for each 100 of milk. It is evident when it is desired to determine the residual nitrogen in solution, the former reagent must be employed. The quantity of albuminoid matter left in solution after clarification with mercurial salts is so minute as to exert no sensible effect on the rotation of the plane of polarized light produced by the lactose.

For purposes of calculation the gyrodynat of lactose in the ordinary conditions of temperature and concentration may be represented by [a]_{D} = 52°.5 (=107=).

Polarization.—The proper weight of milk is placed in a sugar flask, diluted with water, clarified with the mercuric salt, the volume completed to the mark, and the contents shaken and poured on a filter. The filtrate is polarized in tubes of convenient length. The observed rotation may be expressed either in degrees of angular measurement or of the sugar scale. The weight of milk used may be two or three times that of the normal weight calculated for the instrument employed. Instead of weighing the milk a corresponding volume determined by its specific gravity may be delivered from a burette-pipette (p. 231). For the laurent polariscope three times, and for the half-shadow instruments for lamplight, twice the normal weight of milk should be used. For approximately sixty cubic centimeters of milk the flask should be marked at 105 cubic centimeters in compensation for the volume of precipitated solids or the reading obtained from a 100 cubic centimeter flask, decreased by one-twentieth.

For the laurent instrument the normal weight of lactose is determined by the following proportions:

Gyrodynat of sucrose, 66.5: lactose: 52.5 = x: 16.19.

Whence x = 20.51, that is, the number of grams of pure lactose in 100 cubic centimeters required to read 100 divisions of the sugar scale of the instrument.

For the ventzke scale the normal quantity of lactose required to read 100 divisions is found from the following equation:

66.4: 52.5 = x: 26.048

Whence x = 32.74.

In the one case three times the normal weight of milk is 61.53 and in the other twice the normal weight, 65.48 grams.

=244. Error due to Volume of Precipitate.=—Vieth states that the volume allowed for the precipitated solids in the original process, viz., two and four-tenths cubic centimeters, is not sufficiently large.[202] In such cases it is quite difficult to decide on any arbitrary correction based on the supposed quantities of fat and albuminoids present. A better method than to try to compensate for any arbitrary volume is to remove entirely the disturbing cause or eliminate it by indirect means. To wash the precipitate free of sugar without increasing the bulk of the filtrate unduly would be extremely difficult and tend, moreover, to bring some of the precipitated matters again into solution. It is better, therefore, to eliminate the error by double dilution and polarization (=86=). The principle of this method is based on the fact, that, within limits not sensibly affecting the gyrodynat by reason of different densities, the polarizations of two solutions of the same substance are inversely proportional to their volumes.

For convenience, it is recommended that the volumes of the samples in each instance be 100 and 200 cubic centimeters, respectively, in which case the true reading is obtained by the simple formula given in the latter part of =86=.

In this laboratory the double dilution method of determining the volume of the precipitate is conducted as follows:[203]

In each of two flasks marked at 100 and 200 cubic centimeters, respectively, are placed 65.52 grams of milk, four cubic centimeters of mercuric nitrate added, the volume completed to the mark and the contents of the flask well shaken.

After filtering, the polarization is made in a 400 millimeter tube by means of the triple shadow polariscope described in =75=. From the reading thus obtained the volume of the precipitate and the degree of correction to be applied are calculated as in the subjoined example. The flasks should be filled at near the temperature at which the polarizations are made and the observation room must be kept at practically a constant temperature of 20° to avoid the complications which would be produced by changes in the gyrodynat of lactose and the value of the quartz plates and wedges of the apparatus by marked variations in temperature.

Example.—Weight of milk used in each case 65.52 grams.

Polarimetric reading from the 100 cubic centimeter flask, 20°.84 ” ” ” ” 200 ” ” ” 10°.15 Then 10.15 × 2 = 20.30 20.84 - 20.30 = 0.54 0.54 × 2 = 1.08 20.84 - 1.08 = 19.76 19.76 ÷ 4 = 4.94,

which is the corrected reading showing the percentage of lactose in the sample used.

The volume of the precipitate is calculated as follows:

20.84 ÷ 4 = 5.21, the apparent percentage of lactose present.

Then 5.21: 4.94 = 100: x.

Whence x = 94.82. From this number it is seen that the true volume of the milk solution polarized is 94.82 instead of 100 cubic centimeters, whence the volume occupied by the precipitate is 100 - 94.82 = 5.18 cubic centimeters. So little time is required to conduct the analysis by the double dilution method as to render it preferable in all cases where incontestable data are desired. Where arbitrary corrections are made the volume allowed for the precipitate may vary from two and a half cubic centimeters in milks poor in fat, to six for those with a high cream content.

For milks of average composition sufficient accuracy is secured by making an arbitrary correction of five cubic centimeters for the volume of the precipitate.

SEPARATION OF SUGARS BY CHEMICAL AND CHEMICAL-OPTICAL METHODS.

=245. Conditions of Separation.=—In the foregoing paragraphs the optical methods for determining certain sugars have been described. Many cases arise, however, in which these processes are inapplicable or insufficient. In these instances, the analyst, as a rule, will be able to solve the problem presented by the purely chemical methods which have been previously described, or by a combination of the chemical and optical processes. Not only have the different sugars distinctive relations to polarized light, but also they are oxidized by varying quantities of metallic salts and these differences are sufficiently pronounced to secure in nearly every instance, no matter how complex, data of a high degree of accuracy.

The carbohydrates of chief importance, from an agricultural point of view, are starch and sucrose; while the alternation products of chief importance, derived therefrom by chemical and biological means, are dextrin, maltose, dextrose and invert sugar.

=246. Sucrose, Levulose, and Dextrose.=—The purely chemical methods of separating these three sugars have been investigated by Wiechmann.[204] They are based on the data obtained by determining the percentage of reducing sugars, both before and after the inversion of the sucrose, and before and after the removal of the levulose. For the destruction of the levulose, the method of Sieben is employed, and attention is called to the fact that the complete removal of the levulose by this process is difficult of accomplishment, and is probably attended with alterations of the other sugars present.

=247. Sieben’s Method of Determining Levulose.=—The decomposing action of hot hydrochloric acid on levulose, and its comparative inaction on dextrose are the basis of Sieben’s process.[205] The hydrochloric acid employed should contain about 220 grams of the pure gas per liter, that is, be of twenty-two per cent strength, corresponding to 1.108 specific gravity. If the substance acted on be invert sugar, its solution should be approximately of two and a half per cent strength. To 100 cubic centimeters of such a solution, sixty of the hydrochloric acid are added, and the mixture immersed in boiling water for three hours.

After quickly cooling, the acid is neutralized with sodium hydrate of thirty-six times normal strength. Ten cubic centimeters of the hydrate solution will thus neutralize the sixty of hydrochloric acid which have been used to destroy the levulose. The work of Wiechmann discloses the fact, easily prevised, that the method used for destroying levulose is not always effective and that action of the reagent is not exclusively confined to the levogyrate constituent of the mixture. Nevertheless, data of reasonable accuracy may be secured by this process, which is best carried out as described by Wiechmann. In this connection the possibility of the polymerization of the dextrose molecules, when heated with hydrochloric acid, must not be overlooked.

=248. The Analytical Process.=—The total quantity of invert sugar in a given solution is determined by the methods already given (=136, 141=.)

After this has been accomplished, the levulose is destroyed as described above, and the dextrose determined by any approved method (=136, 140=). In the presence of sucrose, the sum of the reducing sugars is first determined as in =136, 142=. After the inversion of the sucrose, the invert sugar is again determined, and the increased quantity found, calculated to sucrose. The levulose is then destroyed by hydrochloric acid, and the dextrose determined as described above. The quantity of sucrose may also be determined by an optical method (=91, 92, 94=.).

=249. Calculation of Results.=—If we represent by a the weight of metallic copper reduced by the invert sugar present in a solution containing sucrose, and by b that obtained after the inversion of the sucrose, the quantity of copper corresponding to the sucrose is b - a = c. After the destruction of the levulose, the copper reduced by the residual dextrose may be represented by d. The weight of copper equivalent to the levulose is, therefore, b - d = e. From the tables already given, the corresponding quantities of the sugars equivalent to c, d, and e are directly taken. Example:

{ 163.8 milligrams invert sugar. a = 300 milligrams = { 156.5 ” dextrose. { 185.63 ” levulose.

b = 500 ”

d = 275 ” = 142.8 ” dextrose.

c = 200 ” = 106.3 ” invert sugar.

e = 225 ” = 133.89 ” levulose.

The 106.3 milligrams of invert sugar equivalent to c, correspond to 101 milligrams of sucrose. The quantity of dextrose equivalent to 275 milligrams of copper is 142.8. Of this amount 53.15 milligrams are due to the inverted sucrose, leaving 89.65 milligrams arising from the invert sugar and dextrose originally present. This quantity is equivalent to 175 milligrams of copper.

Of the 300 milligrams of copper obtained in the first instance, 125 are due to levulose in the original sample, corresponding to 69.73 milligrams which number, multiplied by two, gives the invert sugar present.

The sample examined, therefore, had the following composition:

Sucrose 101.00 milligrams. Invert sugar 139.46 ” Dextrose 19.92 ” ------ Sum 260.38 ”

On the other hand, if the invert sugar be calculated from the quantity corresponding to the 225 milligrams of copper corresponding to e, the data will be very different from those given above. In this instance of the levulose found corresponding to 225 milligrams of copper, viz., 133.89, 53.15 milligrams are due to the inverted sucrose. Then the quantity due to the invert sugar at first present is 133.89 - 53.15 = 80.74 milligrams. Since half the weight of invert sugar is levulose, the total weight of the invert sugar at first present is 161.48, leaving only 8.91 milligrams due to added dextrose. The difficulties in these calculations doubtless arise from the imperfect destruction of the levulose, and from variations in the reducing action of sugars on copper salts in the presence of such large quantities of sodium chlorid.

=250. Calculation from Data obtained with Copper Carbonate.=—The wide variations observed in different methods of calculations in the preceding paragraph, are due in part to the different degrees of oxidation exerted on alkaline copper tartrate by the dextrose and levulose. Better results are obtained by conducting the analytical work with Ost’s modification of Soldaini’s solution (=128=).

The relative quantities of levulose and dextrose oxidized by this solution are almost identical, and the calculations, therefore, result in nearly the same data, whether made from the numbers obtained with the residual dextrose or from the levulose destroyed. The method of applying this method is illustrated in the following calculation.

Example.—In a mixture of sucrose, invert sugar, and dextrose, the quantities of copper obtained by using the copper carbonate solution were as follows:

Copper obtained before inversion = a = 150 milligrams. ” ” after ” = b = 250 ” ” ” ” destroying lev’e = d = 137.5 ” ” equivalent to inverted sucrose = b - a = c = 100 ” ” ” ” levulose = b - d = e = 112.5 ”

{44.0 milligrams invert sugar. a = 150 milligrams Cu = {45.3 ” dextrose. {42.5 ” levulose. d = 137.5 ” ” = 41.55 ” dextrose. c = 100 ” ” = 29.5 ” invert sugar = 28.025 sucrose. e = 112.5 ” ” = 31.9 ” levulose.

14.75 milligrams of dextrose = 48.5 milligrams Cu. 14.75 ” ” levulose = 51.5 ” ”

137.5 - 48.5 = 89.0 milligrams Cu due to dextrose present before inversion. 89.0 milligrams Cu = 27 milligrams dextrose before inversion. 150.0 - 89.0 ” ” = 61.0 ” Cu due to levulose present before inversion. 61.0 ” ” = 17.8 ” levulose before inversion. 17.8 × 2 = 35.6 milligrams invert sugar present before inversion. 27.0 - 17.8 = 9.2 ” dextrose ” ” ”

Again:

112.5 - 51.5 = 61.0 milligrams Cu due to levulose present before inversion. 61.0 milligrams Cu = 17.8 milligrams levulose. 17.8 ” levulose indicate 35.6 milligrams invert sugar. Dextrose in invert sugar before inversion = 17.8 milligrams. Total dextrose before inversion = 27.0 milligrams. Dextrose above amount required for invert sugar = 27.0 - 17.8 = 9.2 milligrams.

The respective quantities of the three sugars in the solution are, therefore:

Sucrose = 28.025 milligrams. Invert sugar = 35.6 ” Dextrose = 9.2 ”

The calculations made from the later data (=234=) give almost the same results.

=251. Winter’s Process.=—Winter has proposed a method of separating dextrose and levulose in the presence of sucrose based on the selective precipitation produced on treating mixtures of these sugars in solution with ammoniacal lead acetate.[206]

The reagent is prepared immediately before use by adding ammonia to a solution of lead acetate until the opalescence which is at first produced just disappears. The separation is based on the fact that the compound of sucrose with the reagent is easily soluble in water, while the salts formed with levulose and dextrose are insoluble. The separation of the sugars is accomplished as follows:

The ammoniacal lead acetate is added to the solution of the mixed sugars until no further precipitate is produced. The precipitated matters are digested with a large excess of water and finally separated by filtration. The sucrose is found in the filtrate in the form of a soluble lead compound, from which it is liberated by treatment with carbon dioxid. The lead carbonate produced is separated by filtration and the sucrose is estimated in an aliquot part of the filtrate by optical or chemical methods. The precipitate containing the lead compounds of dextrose and levulose is washed free of sucrose, suspended in water and saturated with carbon dioxid. By this treatment the lead compound with dextrose is decomposed and, on filtration, the dextrose will be found in the filtrate, while the lead compound of the levulose is retained upon the filter with the lead carbonate. After well washing the precipitate, it is again suspended in water and saturated with hydrogen sulfid. By this treatment the lead levulosate compound is broken up and the levulose obtained, on subsequent filtration, in the filtrate. The dextrose and levulose, after separation as above described, may be determined in aliquot parts of their respective filtrates by the usual gravimetric methods. Before determining the levulose the solution should be heated until all excess of hydrogen sulfid is expelled.

This method was used especially by Winter in separating the various sugars obtained in the juices of sugar cane. It has not been largely adopted as a laboratory method, and on account of the time and trouble required for its conduct, is not likely to assume any very great practical importance.

=252. Separation of Sugars by Lead Oxid.=—In addition to the combination with the earthy bases, sugar forms well defined compounds with lead oxid. One of these compounds is of such a nature as to have considerable analytical and technical value. Its composition and the method of preparing it have been pointed out by Kassner.[207]

Sucrose, under conditions to be described, forms with the lead oxid a diplumbic saccharate, which separates in spheroidal crystals, and has the composition corresponding to the formula C₁₂H₁₈O₁₁Pb₂ + 5H₂O. The precipitation takes place quantitively and should be conducted as follows:

The substance containing the sucrose, which may be molasses, sirups or concentrated juices, is diluted with enough water to make a sirup which is not too viscous. Lead oxid suspended in water is stirred into the mass in such proportion as to give about two parts of oxid to one of the sugar. The stirring is continued for some time until the oxid is thoroughly distributed throughout the mass and until it becomes thick by the commencement of the formation of the saccharate. As soon as the mass is sufficiently thickened to prevent the remaining lead oxid from settling, the stirring may be discontinued and the mixture is left for twenty-four hours, at the end of which time the sucrose has all crystallized in the form of lead saccharate. The crystals of lead saccharate can be separated by a centrifugal machine or by passing through a filter press, and are thoroughly washed with cold water, in which they are almost insoluble. The washed crystals are beaten up with water into a thick paste and the lead separated as basic carbonate by carbon dioxid. The sucrose is found in solution in the residual liquor and is concentrated and crystallized in the usual way.

Reducing sugars have a stronger affinity for the lead oxid than the sucrose, and this fact is made use of to effect a nearly complete separation when they are mixed together. In order to secure this the lead oxid is added in the first place only in sufficient quantity to combine with the reducing sugars present, the process being essentially that described above. The reducing sugars which are precipitated as lead dextrosates, lead levulosates, etc., are separated in the usual way by a centrifugal or a filter press, and the resulting liquor, which contains still nearly all the sucrose, is subjected to a second precipitation by the addition of lead oxid. The second precipitation obtained is almost pure diplumbic saccharate.

In the precipitation of the sugar which is contained in the beet molasses, where only a trace or very little invert sugar is present, the sucrose is almost quantitively separated, and by the concentration of the residual liquor, potash salts are easily obtained. In this case, after the decomposition of the lead saccharate by carbon dioxid, the residual sugar solution is found entirely free of lead. Where invert sugar is present, however, in any considerable proportions, it is found to exercise a slightly soluble influence on the lead saccharate, and in this case a trace of lead may pass into solution. For technical purposes, this is afterwards separated by hydrosulfuric acid or the introduction of lime sulfid.

Lead oxid is regenerated from the basic lead carbonate obtained by heating in retorts to a little above 260°, and the carbon dioxid evolved can also be used again in the technical process.

=253. Commercial Glucose and Grape Sugar.=—The commercial products obtained by the hydrolysis of starch are known in the trade as glucose or grape sugar. The former term is applied to the thick sirup obtained by concentrating the products of a partial hydrolysis, while the latter is applied to the solid semi-crystalline mass, secured by continuing the hydrolyzing action until the intermediate products are almost completely changed to dextrose. In this country the starch employed is obtained almost exclusively from maize, and the hydrolyzing agent used is sulfuric acid.[208] The products of conversion in glucose are chiefly dextrins and dextrose with some maltose, and in grape sugar almost entirely dextrose. When diastase is substituted for an acid, as the hydrolytic agent, maltose is the chief product, the ferment having no power of producing dextrose. In the glucose of Japan, known as midzu ame dextrin and maltose are the chief constituents.[209]

Commercial glucose is used chiefly by confectioners for manufacturing table sirups and for adulterating honey and molasses.

Commercial grape sugar is chiefly employed by brewers as a substitute for barley and other grains.

In Europe, the starch which is converted into glucose, is derived principally from potatoes. The method employed in conversion, whether an acid or diastatic action, is revealed not only by the nature of the product, but also by the composition of its ash. In the case of diastatic conversion the ash of the sample will contain only a trace of sulfates, no chlorin, and be strongly alkaline, while the product of conversion with sulfuric acid will give an ash rich in sulfates with a little lime and be less strongly alkaline.

The process of manufacture in this country consists in treating the starch, beaten to a cream with water, with sulfuric acid, usually under pressure, until the product shows no blue color with iodin. The excess of acid is removed with marble dust, the sirup separated by filtration, whitened by bleaching with sulfurous acid or by passing it through bone-black and evaporated to the proper consistence in a vacuum. The solid sugar, consisting mostly of dextrose, is made in the same manner, save that the heating with the acid is continued until the dextrin and maltose are changed into maltose. The product is either obtained in its ordinary hydrated form or by a special method of crystallization secured as bright anhydrous crystals. Solutions of dextrose, when first made, show birotation, but attain their normal gyrodynatic state on standing for twenty-four hours in the cold, or immediately on boiling.

=254. Methods Of Separation.=—The accurate determination of the quantities of the several optically active bodies formed in commercial glucose is not possible by any of the methods now known. Approximately accurate data may be secured by a large number of processes, and these are based chiefly on the ascertainment of the rotation and reducing power of the mixed sugars, the subsequent removal of the dextrose and maltose by fermentation or oxidation and the final polarization of the residue. The difficulties which attend these processes are alike in all cases. Fermentation may not entirely remove the reducing sugars or may act slightly on the dextrin. In like manner the oxidation of these sugars by metallic salts may not entirely decompose them, may leave an optically active residue, or may affect the optical activity of the residual dextrin. The quantitive methods of separating these sugars by means of phenylhydrazin, lead salts or earthy bases have not been developed into reliable and applicable laboratory processes. At the present time the analyst must be contented with processes confessedly imperfect, but which, with proper precautions, yield data which are nearly correct. The leading methods depending on fermentation and oxidation combined with polarimetric observations will be described in the subjoined paragraphs.

=255. Fermentation Method.=—This process is based on the assumption that, under certain conditions, dextrose and maltose may be removed from a solution and the dextrin be left unchanged. In practice, approximately accurate results are obtained by this method, although the assumed conditions are not strictly realized. In the prosecution of this method the polarimetric reading of the mixed sugars is made, and the maltose and dextrose removed therefrom by fermentation with compressed yeast. The residual dextrins are determined by the polariscope on the assumption that their average gyrodynat is 193. In the calculation of the quantities of dextrose and maltose their gyrodynats are fixed at 53 and 138 respectively. The total quantity of reducing sugar is determined by the usual processes. The relative reducing powers of dextrose and maltose are represented by 100 and 62 respectively. The calculations are made by the following formulas:[210]

R = reducing sugars as dextrose d = dextrose m = maltose dʹ = dextrin P = total polarization (calculated as apparent gyrodynat) Pʹ = rotation after fermentation (calculated as apparent gyrodynat).

Whence R = d + 0.62_m_ (1) P = 53_d_ + 138_m_ + 163_dʹ_ (2) Pʹ = 193_dʹ_ (3)

From these three equations the values of d, m, and dʹ are readily calculated:

Example: To find d and m:

Subtract (3) from (2) P = 53_d_ + 133_m_ + 193_dʹ_ Pʹ = 193_dʹ_ -------------------------------------- P - Pʹ = 53_d_ + 138_m_ (4)

Multiply (1) by 53 and subtract from (4)

P - Pʹ = 53_d_ + 138_m_ 53_R_ = 53_d_ + 32.86_m_ ------------------------------------------- P - Pʹ - 53_R_ = 105.14_m_ (5)

P - Pʹ - 53_R_ Whence m = ------------------ (6) 105.14

d = R - 0.62_m_ (7)

Pʹ dʹ = ---- (8) 193

Sidersky assigns the values [a]_{D} = 138.3 and [a]_{D} = 194.8 to maltose and dextrin respectively in the above formulas.[211]

Illustration: In the examination of a sample 26.048 grams of midzu ame in 100 cubic centimeters were polarized in a 200 millimeter tube and the following data were obtained:

Polarization of sample in angular degrees 69°.06, which is equal to an apparent gyrodynat of 132.6:

Total reducing sugar as dextrose 33.33 per cent:

Polarization in angular degrees after fermentation 30°.84 = [a]_{D} = 59.2.

Substituting these values in the several equations gives the following numbers:

(1) 0.3333 = d + 0.62_m_ (2) 132.6 = 53_d_ + 138_m_ + 193_dʹ_ (3) 59.2 = 193_dʹ_ (4) 73.4 = 53_d_ + 138_m_ (5) 55.74 = 105.14_m_ (6) m = 0.5301 = 53.01 per cent. (7) d = 3333 - 3286 = 0.0047 = 00.47 per cent. (8) dʹ = 59.2 ÷ 193 = 0.3067 = 30.67 ” ”

Summary: Sample of midzu ame:

Percentage of dextrin 30.67 per cent. ” ” maltose 53.01 ” ” ” ” dextrose 00.47 ” ” ” ” water 14.61 ” ” ” ” ash 00.31 ” ” ----- Sum 99.07 ” ” Undetermined 0.93 ” ”

For polarization the lamplight shadow polariscope employed for sugar may be used, and the degrees of the sugar (ventzke) scale converted into angular degrees by multiplying by 0.3467.

The process of fermentation is conducted as described in the paragraph given further on, relating to the determination of lactose in the presence of sucrose.

=256. The Oxidation Method.=—The removal of the reducing sugars may be accomplished by oxidation instead of fermentation. The process of analysis is in all respects similar to that described in the foregoing paragraph, substituting oxidation for fermentation.[212] For the oxidizing agent mercuric cyanid is preferred, and it is conveniently prepared by dissolving 120 grams of mercuric cyanid and an equal quantity of sodium hydroxid in water mixing the solutions and completing the volume to one liter. If a precipitate be formed in mixing the solutions it should be removed by filtering through asbestos. For the polarization, ten grams of the sugars in 100 cubic centimeters is a convenient quantity. Ten cubic centimeters of this solution are placed in a flask of water marked at fifty cubic centimeters, a sufficient quantity of the mercuric cyanid added to remain in slight excess after the oxidation is finished (from twenty to twenty-five cubic centimeters) and the mixture heated to the boiling point for three minutes. The alkali, after cooling, is neutralized with strong hydrochloric acid and the passing from alkalinity to acidity will be indicated by a discharge of the brown color which is produced by heating with the alkaline mercuric cyanid. The heating with the mercury salt should be conducted in a well ventilated fume chamber.

The calculation of the results is conducted by means of the formulas given in the preceding paragraph. In the original paper describing this method, it was stated that its accuracy depended on the complete oxidation of the reducing sugar in a manner leaving no optically active products, and on the inactivity of the reagents used in respect to the dextrin present. These two conditions are not rigidly fulfilled, as is shown by Wilson.[213] According to his data maltose leaves an optically active residue, which gives a somewhat greater right hand rotation than is compensated for by the diminished rotation of the dextrin. Wilson, however, confesses that the dextrin used contained reducing sugars, which would not be the case had it been prepared by the process of treating it with alkaline mercuric cyanid as above indicated. Upon the whole, the oxidation of the reducing sugar by a mercury salt gives results which, while not strictly accurate, are probably as reliable as those afforded by fermentation. The author has attempted to supplant both the oxidation and fermentation methods by removing the reducing sugars with a precipitating reagent, such as phenylhydrazin, but the methods are not sufficiently developed for publication.

=257. Removal of Dextrose by Copper Acetate.=—Maercker first called attention to the fact that Barfoed’s reagent (one part copper acetate in fifteen parts of water, and 200 cubic centimeters of this solution mixed with five cubic centimeters of thirty-eight per cent. acetic acid) reacts readily with dextrose, while it is indifferent to maltose and dextrins. Sieben’s method of removing dextrose is based on this fact.[214] It is found that under certain conditions pure maltose does not reduce either the acidified or neutral solution of copper acetate, while dextrose or a mixture of dextrose and maltose does so readily. It is also shown that the fermentation residue under suitable conditions acts like maltose. Maltose solutions reduce the reagent after boiling four minutes while at 40°-45° they have no effect even after standing four days. The amount of copper deposited by dextrose, under the latter conditions, is found to depend to a certain extent on the amount of free acetic acid present, and as the solutions of copper acetate always contain varying quantities of acetic acid which cannot be removed without decomposition and precipitation of basic salt, the use of an absolutely neutral solution is impracticable. The reagent prepared according to Barfoed’s directions is almost saturated, but a half normal solution is preferable. Sieben proposes two solutions: I, containing 15.86 grams copper and 0.56 gram acetic anhydrid per liter; II, containing 15.86 grams copper and three grams acetic anhydrid per liter. The reduction of the dextrose is secured by placing 100 cubic centimeters of the solution in a bottle, adding the sugar solution, stoppering and keeping in a water-bath at 40°-45° two or three days. An aliquot portion is then drawn off and the residual copper precipitated by boiling with forty-five cubic centimeters of the alkali solution of the fehling reagent and forty cubic centimeters of one per cent dextrose solution, filtered and weighed as usual. The results show that either solution can be used, and that standing for two days at 45° is sufficient. One hundred cubic centimeters of the copper solution are mixed with ten cubic centimeters of the sugar solution containing from two-tenths to five-tenths gram of dextrose, as this dilution gives the best results. No reduction is found to have taken place when solutions containing five-tenths gram maltose or five-tenths gram fermentation residue are used. The data can not be compiled in the form of a table similar to Allihn’s, as it is impossible to obtain a solution of uniform acidity each time, and the solution will have to be standardized by means of a known pure dextrose solution and the result obtained with the unknown sugar solution properly diluted compared with this. This method of Sieben’s has never been practiced to any extent in analytical separations and can not, therefore, be strongly recommended without additional experience.

=258. Removal of Dextrin by Alcohol.=—By reason of its less solubility, dextrin can be removed from a solution containing also dextrose and maltose by precipitation with alcohol. It is impracticable, however, to secure always that degree of alcoholic concentration which will cause the coagulation of all the dextrins without attacking the concomitant reducing sugars. In this laboratory it has been found impossible to prepare a dextrin by alcoholic precipitation, which did not contain bodies capable of oxidizing alkaline copper solutions.

The solution containing the dextrin is brought to a sirupy consistence by evaporation and treated with about ten volumes of ninety per cent alcohol. After thorough mixing, the precipitated dextrin is collected on a filter and well washed with alcohol of the strength noted. It is then dried and weighed. If weaker solutions of dextrin are used, the alcohol must be of correspondingly greater strength. In the filtrate the residual maltose and dextrose may be separated and determined by the chemical and optical methods already described.

CARBOHYDRATES IN MILK.

=259. The Copper Tartrate Method.=—The lactose in milk is readily estimated by the gravimetric copper method described in paragraph =143=. Before the application of the process the casein and fat of the milk should be removed by an appropriate precipitant, and an aliquot part of the filtrate, diluted to contain about one per cent of milk sugar, used for the determination. The clarification is very conveniently secured by copper sulfate or acetic acid, as described in the next paragraph. A proper correction should be made for the volume occupied by the precipitate and, for general purposes, with whole milk of fair quality this volume may be assumed to be five per cent. One hundred grams of milk will give a precipitate occupying approximately five cubic centimeters. In the analytical process, to twenty cubic centimeters of milk, diluted with water to eighty, is added a ten per cent solution of acetic acid, until a clear whey is shown after standing a few minutes, when the volume is completed to 100 cubic centimeters with water, and the whole, after thorough shaking, thrown on a filter. An aliquot part of the filtrate is neutralized with sodium carbonate and used for the lactose determination. This solution contains approximately one per cent of lactose. In a convenient part of it the lactose is determined and the quantity calculated for the whole. This quantity represents the total lactose in the twenty cubic centimeters of milk used. The weight of the milk is found by multiplying twenty by its specific gravity. From this number the percentage of lactose is easily found. In this process the milk is clarified by the removal of its casein and fat. Other albuminoids remain in solution and while these doubtless disturb the subsequent determination of lactose, any attempt at their removal would be equally as disadvantageous. The volume of the precipitate formed by good, whole milk when the process is conducted as above described, is about one cubic centimeter, for which a corresponding correction is readily made.

=260. The Official Method.=—The alkaline copper method of determining lactose, adopted by the Association of Official Agricultural Chemists, is essentially the procedure proposed by Soxhlet.[215]

Dilute twenty-five cubic centimeters of the milk, held in a half liter flask, with 400 cubic centimeters of water and add ten cubic centimeters of a solution of copper sulfate of the strength given for Soxhlet’s modification of Fehling’s solution, page 129; add about seven and a half cubic centimeters of a solution of potassium hydroxid of such strength that one volume of it is just sufficient to completely precipitate the copper as hydroxid from one volume of the solution of copper sulfate. In place of a solution of potassium hydroxid of this strength eight and a half cubic centimeters of a half normal solution of sodium hydroxid may be used. After the addition of the alkali solution the mixture must still have an acid reaction and contain copper in solution. Fill the flask to the mark, shake and filter through a dry filter.

Place fifty cubic centimeters of the mixed copper reagent in a beaker and heat to the boiling point. While boiling briskly, add 100 cubic centimeters of the lactose solution, prepared as directed above, and boil for six minutes. Filter immediately and determine the amount of copper reduced by one of the methods already given, pages 149-155. Obtain the weight of lactose equivalent to the weight of copper found from the table on page 163.

=261. The Copper Cyanid Process.=—It has been found by Blyth that the copper cyanid process of Gerrard gives practically the same results in the determination of sugar in milk as are obtained by optical methods.[216] The milk for this purpose is clarified, by precipitating the casein with acetic acid in the following manner:

Twenty-five cubic centimeters of milk are diluted with an equal volume of distilled water, and strong acetic acid added until the casein begins to separate. The liquid is heated to boiling and, while hot, centrifugated in any convenient machine. The supernatant liquid obtained is separated by filtration and the solid matter thrown upon the filter and well washed with hot water. The filtrate and washings are cooled and completed to a volume of 100 cubic centimeters. This liquid is of about the proper dilution for use with the copper cyanid reagent. The percentage of sugar determined by this reagent agrees well with that obtained by the optical method, when no other sugars than lactose are present. If there be a notable difference in the results of the two methods other sugars must be looked for. The presence of dextrin may be determined by testing a few drops of the clear liquor with iodin, which, in the presence of dextrin, gives a reddish color. Other sugars are determined by obtaining their osazones. For this purpose the filtrate obtained as above should be concentrated until the volume is about thirty cubic centimeters. Any solid matter which separates during the evaporation is removed by filtration. The osazones are precipitated in the manner described in paragraph =147=. On cooling, the almost solid crystalline mass obtained is placed on a filter, washed with a little cold water, the crystals then pressed between blotting paper and dried at a temperature of 100°. The dry osazones obtained are dissolved in boiling absolute alcohol, of which just sufficient is used to obtain complete solution. The alcoholic solution is set aside for twelve hours and the separation of a crystalline product after that time shows that dextrose or invert sugar is present. Milk sugar alone gives no precipitate but only a slight amorphous deposit. The lactosazone is precipitated by adding a little water to the hot alcoholic solution and the crystals thus obtained should be dissolved in boiling absolute alcohol and reprecipitated by the addition of water at least three times in order to secure them pure. The osazones are identified by their melting points, paragraph =172=. The first part of this method does not appear to have any advantage over the optical process by double dilution (p. 278), and requires more time.

=262. Sugars in Evaporated Milks.=—In addition to the lactose normally present in evaporated milks the analyst will, in most cases, find large quantities of sucrose. The latter sugar is added as a preservative and condiment. By reason of the ease with which sucrose is hydrolyzed, evaporated milk containing it may have also some invert sugar among its contents. A method of examination is desirable, therefore, which will secure the determination of lactose, sucrose and invert sugar in mixtures. The probability of the development of galactose and dextrose during the evaporation and conservation of the sample, is not great. The best method of conducting this work is the one developed by Bigelow and McElroy.[217] The principle on which the method is based rests on the fact that in certain conditions, easily supplied, the sucrose and invert sugar present in a sample may be entirely removed by fermentation and the residual lactose secured in an unchanged condition. The lactose is finally estimated by one of the methods already described.

The details of the process follow:

On opening a package of evaporated milk, its entire contents are transferred to a dish and well mixed. Several portions of about twenty-five grams each are placed in flasks marked at 100 cubic centimeters. To each of the flasks enough water is added to bring all the sugars into solution and normal rotation is made certain by boiling. After cooling, the contents of the flasks are clarified by mercuric iodid in acetic acid solution. The clarifying reagent is prepared by dissolving fifty-three grams of potassium iodid, twenty-two grams of mercuric chlorid, and thirty-two cubic centimeters of strongest acetic acid in water, mixing the solutions and completing the volume to one liter. The clarification is aided by the use of alumina cream (=84=). The flask is filled to the mark, and the contents well shaken and poured on a filter. After rejecting the first portion of the filtrate the residue is polarized in the usual manner. Two or more separate portions of the sample are dissolved in water in flasks of the size mentioned, heated to 55°, half a cake of compressed yeast added to each and the temperature kept at 55° for five hours. The residue in each flask is treated as above described, the mercuric solution being added before cooling to prevent the fermentative action of the yeast, and the polarization noted.

By this treatment the sucrose is completely inverted, while the lactose is not affected. The percentage of sucrose is calculated by the formulas given in paragraph =94=, using the factor 142.6. At the temperature noted the yeast exercises no fermentative, but only a diastatic action.

In each case the volume of the precipitated milk solids is determined by the double dilution method, and the proper correction made (p. 278). The lactose remaining is determined by chemical or optical methods, but it is necessary, in all cases where invert sugar is supposed to be present, to determine the total reducing sugars in the original sample as lactose. If the quantity thus determined and the amount of sucrose found as above are sufficient to produce the rotation observed in the first polarization, it is evident that no invert sugar is present. When the polarization observed is less than is equivalent to the quantity of sugar found, invert sugar is present, which tends to diminish the rotation produced by the other sugars. In this case it is necessary to remove both the sucrose and invert sugar by a process of fermentation, which will leave the lactose unchanged.

This is accomplished by conducting the fermentation in the presence of potassium fluorid, which prevents the development of the lactic ferments. For this purpose 350 grams of the evaporated milk are dissolved in water and the solution boiled to secure the normal rotation of the lactose. After cooling to 80°, the casein is thrown down by adding a solution containing about four grams of glacial phosphoric acid and keeping the temperature at 80° for about fifteen minutes. After cooling to room temperature, the volume is completed to one liter with water, well shaken and poured onto a filter. An aliquot part of the filtrate is nearly neutralized with a noted volume of potassium hydroxid. Enough water is added to make up, with the volume of potassium hydroxid used, the total space occupied by the precipitated solid, corresponding to that part of the filtrate, and if necessary, refilter. The volume occupied by the precipitated solids is easily determined by polarization and double dilution. The filtrate, obtained from the process described above, is placed in portions of 100 cubic centimeters each in 200 cubic centimeter flasks with about twenty milligrams of potassium fluorid in solution, and half a cake of compressed yeast. The yeast is broken up and evenly distributed, and the fermentation is allowed to proceed for ten days at a temperature of from 25° to 30°. At the end of this time experience has shown that all of the sucrose and invert sugar has disappeared, but the lactose remains intact. The flasks are filled to the mark with water and the lactose determined by chemical or optical methods. By comparing the data obtained from the estimation of the total reducing sugars before fermentation or inversion and the estimation of the lactose after fermentation, the quantity of invert sugar is easily calculated. The experience of this laboratory shows that invert sugar is rarely present in evaporated milks, which is an indication that the sucrose added thereto does not generally suffer hydrolysis. The mean percentage of added sucrose found in evaporated milks is about forty.

SEPARATION AND DETERMINATION OF STARCH.

=263. Occurrence.=—Many bodies containing starch are presented for the consideration of the agricultural analyst. First in importance are the cereals, closely followed by the starchy root crops. Many spices and other condiments also contain starchy matters. In the sap of some plants, for instance sorghum, at certain seasons, considerable quantities of starch occur. In the analysis of cereals and other feeding stuffs, it has been the usual custom to make no separate determination of starch, but to put together all soluble carbohydrates and estimate their percentage by subtracting from 100 the sum of the percentages of the other constituents of the sample. This aggregated mass has been known as nitrogen-free extract. Recent advances in methods of investigation render it advisable to determine the starch and pentosan carbohydrates separately and to leave among the undetermined bodies the other unclassed substances, chiefly of a carbohydrate nature, soluble in boiling dilute acid and alkali.

=264. Separation of Starch.=—Starch being insoluble in its natural state, it is impossible to separate it from the other insoluble matters of plants by any known process. In bringing it into solution it undergoes certain changes of an unknown nature, but tending to produce a dextrinoid body. Nevertheless, in order to procure the starch in a state of purity suited to analytical processes, it becomes necessary to dissolve the starch from the other insoluble bodies that naturally accompany it. As has been shown in preceding paragraphs, there are only two methods of securing the solution of starch which fully meet the conditions of accurate analysis. These are the methods depending on the use of diastatic ferments and on the employment of heat and pressure in the presence of water. These two processes have been described in considerable detail in paragraphs =179-181=. It is important, in starch determinations, to remove from the sample the sugar and other substances soluble in water and also the oils, when present in large quantities, before subjecting it to the processes for rendering the starch soluble.

=265. Desiccation of Amyliferous Bodies.=—The removal of sugars and oils is best secured in amyliferous substances after they are deprived of their moisture. As has already been suggested, the desiccation should be commenced at a low temperature, not above 60°, and continued at that point until the chief part of the water has escaped. The operation may be conducted in one of the ways already described (pp. 12-27). There is great difference of opinion among analysts in respect of the degree of temperature to which the sample should be finally subjected, but for the purposes here in view, it will not be found necessary to go above 105°. Before beginning the operation the sample should be as finely divided as possible, and at its end the dried residue should be ground and passed through a sieve of half a millimeter mesh.

=266. Indirect Method of Determining: Water in Starch.=—It is claimed by Block[218] that it is necessary to dry starch at 160° in order to get complete dehydration. Wet starch as deposited with its maximum content of water has nine molecules thereof, viz., C₆H₁₀O₅ + 9H₂O. Ordinary commercial starch has about eighteen per cent of water with a formula of C₆H₁₀O₅ + 2H₂O.

The percentage of water may be determined by Block’s feculometer or Block’s dose-fécule. The first apparatus determines the percentage of anhydrous starch by volume, and the second by weight.

Block’s assumption that starch can absorb only fifty per cent of its weight of water is the basis of the determination.

A noted weight of starch is rubbed up with water until saturated, the water poured off, the starch weighed, dried on blotting paper until it gives off no more moisture and again weighed. Half of the lost weight is water, from which the original per cent of water can be calculated. This at best seems to be a rough approximation and not suited to rigorous scientific determination.

=267. Removal of Oil and Sugar.=—The dried, finely powdered sample, obtained as described above, is placed in any convenient extractor (=33-43=) and the oil or fat it contains removed by the usual solvents. For ordinary purposes, even with cereals, this preliminary extraction of the oil is not necessary, but it becomes so with oily seeds containing starch. The sugar is subsequently removed by extraction with eighty per cent alcohol and the residue is then ready for the extraction of the starch. In most cases the extraction with alcohol will be found sufficient. In some bodies, for instance the sweet potato (batata), the quantity of sugar present is quite large, and generally some of it is found. If not present in appreciable amount, the alcohol extraction may also be omitted. The sample having been prepared as indicated, the starch may be brought into solution by one of the methods described in paragraphs =179-181=, preference being given to the aqueous digestion in an autoclave. The dissolved starch is washed out of the insoluble residue and determined by optical or chemical methods =186-194=.

=268. Preparation of Diastase for Starch Solution.=—The methods of preparing malt extract for use in starch analysis have been described in paragraph =179=. If a purer form of diastase is desired it may be prepared by following the directions given by Long and Baker.[219] Digest 200 grams of ground malt for twenty-four hours with three parts of twenty per cent alcohol. Separate the extract by filtration and to the filtrate add about one and a half liters of ninety-three per cent alcohol and stir vigorously. After the precipitate has subsided the supernatant alcohol is removed by a syphon, the precipitate is brought onto a filter and washed with alcohol of a strength gradually increasing to anhydrous, and finally with anhydrous ether. The diastase is dried in a vacuum over sulfuric acid and finally reduced to a fine powder before using. Thus prepared, it varies in appearance from a white to a slightly brownish powder. Made at different times and from separate portions of malt, it may show great differences in hydrolytic power.

=269. Estimation of Starch in Potatoes by Specific Gravity.=—A roughly approximate determination of the quantity of starch in potatoes can be made by determining their specific gravity. Since the specific gravity of pure starch is 1.65, it follows that the richer a potato is in starch the higher will be its specific gravity. The specific weight of substances like potatoes is conveniently determined by suspending them in water by a fine thread attached to the upper hook of a balance pan. There may be a variation of the percentage of other constituents in potatoes as well as of starch, and therefore the data obtained from the following table can only be correct on the assumption that the starch is the only variable. In practice, errors amounting to as much as two per cent may be easily made, and therefore the method is useful only for agronomic and commercial and not for scientific purposes. The method is especially useful in the selection of potatoes of high starch content for planting. The table is constructed on the weight in grams in pure water of 10000 grams of potatoes and the corresponding per cents of dry matter and starch are given. It is not always convenient to use exactly 10000 grams of potatoes for the determination, but the calculation for any given weight is easy.[220]

Example.—Let the weight of a potato in air be 159 grams, and its weight in water 14.8 grams.

Then the weight of 10000 grams of potatoes of like nature in water would be found from the equation 159: 10000 = 14.8: x.

Whence x = 931 nearly.

In the table the nearest figure to 931 is 930, corresponding to 24.6 per cent of dry matter and 18.8 per cent of starch. When the number found is half way between the numbers given in the table the mean of the data above and below can be taken. In other positions a proper interpolation can be made if desired but for practical purposes the data corresponding to the nearest number can be used.

TABLE FOR CALCULATING STARCH IN POTATOES FROM SPECIFIC GRAVITY.

10000 grams of potatoes weigh in Per cent Per cent water. dry matter. starch. Grams.

750 19.9 14.1 760 20.1 14.3 770 20.3 14.5 780 20.7 14.9 790 20.9 15.1 800 21.2 15.4 810 21.4 15.6 820 21.6 15.8 830 22.0 16.2 840 22.2 16.4 850 22.4 16.6 860 22.7 16.9 870 22.9 17.1 880 23.1 17.3 890 23.5 17.7 900 23.7 17.9 910 24.0 18.2 920 24.2 18.4 930 24.6 18.8 940 24.8 19.0 950 25.0 19.2 960 25.2 19.4 970 25.5 19.7 980 25.9 20.1 990 26.1 20.3 1000 26.3 20.5 1010 26.5 20.7 1020 26.9 21.1 1030 27.2 21.4 1040 27.4 21.6 1050 27.6 21.8 1060 28.0 22.2 1070 28.3 22.5 1080 28.5 22.7 1090 28.7 22.9 1100 29.1 23.3 1110 29.3 23.5 1120 29.5 23.7 1130 29.8 24.0 1140 30.2 24.4 1150 30.4 24.6 1160 30.6 24.8 1170 31.0 25.0 1180 31.3 25.5 1190 31.5 25.7 1200 31.7 25.9 1210 32.1 26.3 1220 32.3 26.5 1230 32.5 26.7 1240 33.0 27.2 1250 33.2 27.4 1260 33.4 27.6 1270 33.6 27.8 1280 34.1 28.3 1290 34.3 28.5 1300 34.5 28.7 1310 34.9 29.1 1320 35.1 29.3 1330 35.4 29.6 1340 35.8 30.0 1350 36.0 30.2 1360 36.2 30.4 1370 36.6 30.8

=270. Constitution of Cellulose.=—The group of bodies known as cellulose comprises many members of essentially the same chemical constitution but of varying properties. The centesimal composition of pure cellulose is shown by the following numbers:

Carbon, 44.2 per cent Hydrogen, 6.3 ” ” Oxygen, 49.5 ” ”

corresponding to the formula C₆H₁₀H₅.

According to the view of Cross and Bevan, cellulose conforms in respect of its ultimate constitutional groups to the general features of the simple carbohydrates, but differs from them by reason of a special molecular configuration resulting in a suppression of the activity of constituent groups in certain respects, and an increase in activity of others.[221]

=271. Fiber and Cellulose.=—The carbohydrates of a plant insoluble in water are not composed exclusively of starch. There are, in addition to starch, pentosan fibers yielding pentose sugars on hydrolysis and furfuraldehyd on distillation with a strong acid. The quantitive methods for estimating the pentosan bodies are given in paragraphs =150-157=. The method to be preferred is that of Krug (=155=).

In the estimation of cattlefoods and of plant substances in general the residue insoluble in dilute boiling acid and alkali is called crude or indigestible fiber.

The principle on which the determination depends rests on the assumption that all the protein, starch and other digestible carbohydrates will be removed from the sample by successive digestion at a boiling temperature with acid and alkali solutions of a given strength. It is evident that the complex body obtained by the treatment outlined above is not in any sense a definite chemical compound, but it may be considered as being composed partly of cellulose.

=272. Official Method of Determining Crude Fiber.=—The method of estimating crude fiber, adopted by the Association of Official Agricultural Chemists, is as follows:[222]

Extract two grams of the substance with ordinary ether, at least almost completely, or use the residue from the determination of the ether extract. To this residue, in a half liter flask, add 200 cubic centimeters of boiling 1.25 per cent sulfuric acid; connect the flask with an inverted condenser, the tube of which passes only a short distance beyond the rubber stopper into the flask. Boil at once, and continue the boiling for thirty minutes. A blast of air conducted into the flask may serve to reduce the frothing of the liquid. Filter, wash thoroughly with boiling water until the washings are no longer acid, rinse the substance back into the same flask with 200 cubic centimeters of a boiling 1.25 per cent solution of sodium hydroxid, free or nearly free of sodium carbonate, boil at once and continue the boiling for thirty minutes in the same manner as directed above for the treatment with acid. Filter into a gooch, and wash with boiling water until the washings are neutral, dry at 110°, weigh and incinerate completely. The loss of weight is crude fiber.

The filter used for the first filtration may be linen, one of the forms of glass wool or asbestos filters, or any other form that secures clear and reasonably rapid filtration. The solutions of sulfuric acid and sodium hydroxid are to be made up of the specified strength, determined accurately by titration and not merely from specific gravity.

The experience of this laboratory has shown that results practically identical with those got as above, are obtained by conducting the digestions in hard glass beakers covered with watch glasses. The ease of manipulation in the modification of the process just mentioned is a sufficient justification for its use.

=273. Separation of Cellulose.=—Hoppe-Seyler observed that cellulose, when melted with the alkalies at a temperature as high as 200°, was not sensibly attacked.[223]

Lange has based a process for determining cellulose on this observation.[224]

The process, as improved by him, is carried out as follows:

From five to ten grams of the substance are moistened with water and placed in a porcelain dish with about three times their weight of caustic alkali free of nitrates and about twenty cubic centimeters of water. The porcelain dish should be deep and crucible shaped and should be placed in an oil-bath, the temperature of which is easily controlled. The contents of the dish are stirred with the thermometer bulb until all foaming ceases and the temperature of the mixture is then kept at from 175° to 180° for an hour. After the melt has cooled to 80° about seventy-five cubic centimeters of hot water are added to bring it into solution and it is then allowed to cool. The solution is acidified with sulfuric and placed in large centrifugal tubes. After being made slightly alkaline with soda lye, the tubes are subjected to continued energetic centrifugal action until the cellulose is separated. The supernatant liquid can be nearly all poured off and the separated cellulose is broken up, treated with hot water and again separated by centrifugal action. The cellulose is finally collected upon the asbestos felt, washed with hot water, alcohol and ether, dried and weighed. With a little practice it is possible to complete the separation of cellulose in two and one-half hours.

=274. Solubility of Cellulose.=—Cellulose resembles starch in its general insolubility, but, unlike starch, it may be dissolved in some reagents and afterwards precipitated practically unchanged or in a state of hydration. One of the simplest solvents of cellulose is zinc chlorid in concentrated aqueous solution.

The solution is accomplished with the aid of heat, adding one part by weight of cotton to six parts of zinc chlorid dissolved in ten parts of water.

A homogeneous sirup is obtained by this process, which is used in the arts for making the carbon filaments of incandescent electric lamps.

In preparing the thread of cellulose, the solution, obtained as described above, is allowed to flow, in a fine stream, into alcohol, whereby a cellulose hydrate is precipitated, which is freed from zinc hydroxid by digesting in hydrochloric acid.

Hydrochloric acid may be substituted for water in preparing the reagent above noted, whereby a solvent is secured which acts upon cellulose readily in the cold.

A solution of ammoniacal cupric oxid is one of the best solvents for cellulose. The solution should contain from ten to fifteen per cent of ammonia and from two to two and a half of cupric oxid.

In the preparation of this reagent, ammonium chlorid is added to a solution of cupric salt and then sodium hydroxid in just sufficient quantity to precipitate all of the copper as hydroxid. The precipitate is well washed on a linen filter, squeezed as dry as possible and dissolved in ammonia of 0.92 specific gravity. The cellulose is readily precipitated from the solution in cuprammonium by the addition of alcohol, sodium chlorid, sugar, or other dehydrating agents. Solutions of cellulose are used in the arts for many purposes.[225]

=275. Qualitive Reactions for Detecting Cellulose.=—Cellulose may be identified by its resistance to the action of oxidizing agents, to the halogens and to alkaline solutions. It is further recognized by the sirupy or gelatinous solutions it forms with the solvents mentioned above. The cellulose hydrates precipitated from solutions have in some instances the property of forming a blue color with iodin.

A characteristic reaction of cellulose is secured as follows: To a saturated solution of zinc hydrochlorate, of 2.00 specific gravity, are added six parts by weight of potassium iodid dissolved in ten parts of water and this solution is saturated with iodin. Cellulose treated with this reagent is at once stained a deep blue violet color.[226] For the characteristics of cellulose occurring in wood the researches of Lindsey may be consulted.[227]

=276. More Rarely Occurring Carbohydrates.=—It is not possible here to give more space to the rarer forms of carbohydrates, to which the attention of the agricultural analyst may be called. Nearly a hundred kinds of sugars alone have been detected in the plant world. For descriptions of the properties of these bodies and the methods of their detection and determination, the standard works on carbohydrates may be consulted.[228]

AUTHORITIES CITED IN PART THIRD.

[170] Vines; Physiology of Plants. Nägeli; Beiträge zur näheren Kenntniss der Stärkegruppe.

[171] Bulletin 5, Department of Agriculture, Division of Chemistry, pp. 191 et seq.: Bulletin 25, New Hampshire Experiment Station.

[172] Spencer; Handbook for Sugar Manufacturers, p. 31.

[173] Vid. op. cit. supra, pp. 102, 108.

[174] Journal of the American Chemical Society, Vol. 16, p. 677.

[175] Botanical Gazette, Vol. 12, No. 3.

[176] Bulletin de l’Association des Chimistes de Sucrerie et de Distillerie, Tome 13, p. 133.

[177] Journal of Analytical and Applied Chemistry, Vol. 4, p. 381.

[178] Spencer’s Handbook for Sugar Manufacturers, pp. 30 et seq.

[179] Bulletin de l’Association des Chimistes de Sucrerie et de Distillerie, Tome 13, p. 292.

[180] Vid. op. cit. supra, Tome 2, p. 369.

[181] Dosage du Sucre Cristallisable dans la Betterave, pp. 117 et seq.: Journal of the American Chemical Society, Vol. 16, p. 266.

[182] Neue Zeitschrift für Rübenzucker-Industrie. Band 3, S. 342; Band 14, S. 286: Zeitschrift des Vereins für die Rübenzucker-Industrie, 1876, S. 692: Dingler’s Polytechnisches Journal, Band 232, S. 461.

[183] Sidersky: Traité d’ Analyse des Matières Sucrées, p. 304.

[184] Neue Zeitschrift für Rübenzucker-Industrie, Band 14, S. 286.

[185] Zeitschrift des Vereins für die Rübenzucker-Industrie, Band 32, S. 861.

[186] Spencer’s Handbook for Sugar Manufacturers, p. 42.

[187] Bulletin No. 4 of the Chemical Society of Washington, pp. 22, et seq.

[188] Vid. op. et loc. cit. 7.

[189] Chemiker-Zeitung, Band 19, S. 1830.

[190] Vid. op cit. supra, S. 1784.

[191] Vid. op. cit. supra, S. 1829.

[192] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1895, S. 844.

[193] Journal des Fabricants de Sucre, 1895, No. 33.

[194] Journal of the American Chemical Society, Vol. 2, p. 387: Agricultural Science, Feb. 1892.

[195] American Chemical Journal, Vol. 13, p. 24.

[196] Tucker; Manual of Sugar Analysis, p. 287: Wiechmann; Sugar Analysis, p. 51.

[197] Sidersky; vid. op. cit., 14, p. 197.

[198] Journal of the American Chemical Society, Vol. 18, p. 81: Allen; Commercial Organic Analysis, Vol. 1, p. 291.

[199] Handbuch der Physiologisch- und Pathologisch-Chemischen Analyse, S. 286.

[200] Kühne und Chittenden; American Chemical Journal, Vol. 6, p. 45.

[201] Vid. op. cit. supra, p. 289.

[202] Analyst, Vol. 13, p. 64.

[203] Journal American Chemical Society, Vol. 18, p. 438.

[204] School of Mines Quarterly, Vols. 11 and 12.

In a later method (School of Mines Quarterly, Vol. 13, No. 3) Wiechman describes the separation of the sugars by one polariscopic and two gravimetric determinations, one before and one after inversion. The polariscopic examination is made in a ten per cent solution at a temperature of 20°. The gyrodynats of sucrose, dextrose and levulose at the temperature mentioned are fixed at 66.5, 53.5 and -81.9 respectively. The gravimetric determinations are conducted according to the methods already described. In the formulas for calculating the results a represents sucrose, b reducing sugars, x the dextrose, y the levulose, and d the observed polarization expressed in degrees angular measure. The gyrodynats of sucrose, dextrose and levulose divided by 100 are represented by s, d and l. The calculations are made from the following formulas:

(as + xd) - yl = p. (as + xd) = p + yl. xd = p + yl - as.

p + yl - as x = -----------------. d

In this calculation the gyrodynat of levulose is about ten degrees lower than that of most authorities.

[205] Vid. op. cit., 23, Band 24, S. 869.

[206] Vid. op. cit. supra, 1888, S. 782.

[207] Neue Zeitschrift für Rübenzucker-Industrie, Band 35, S. 166.

[208] Journal of the American Chemical Society, Vol. 2, p. 399: Science, Oct. 1, 1881: Proceedings American Association for the Advancement of Science, 1881, p. 61: Sugar Cane, Vol. 13, p. 533, pp. 61-66.

[209] Wiley and McElroy; Agricultural Science, Vol. 6, p. 57.

[210] Chemical News, Vol. 46, p. 175.

[211] Vid. op. cit, 14, p. 352.

[212] Vid. op. et loc. cit., 41.

[213] Vid. op. cit., 41, Vol. 65, p. 169.

[214] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1884, S. 854.

[215] Bulletin 46, Division of Chemistry, U. S. Department of Agriculture, p. 60.

[216] The Analyst, Vol. 20, p. 121.

[217] Journal American Chemical Society, Vol. 15, p. 668.

[218] Comptes rendus, Tome 118, p. 147.

[219] Journal of the Chemical Society, Transactions, 1895, p. 735.

[220] Die agrikultur-chemische Versuchsstation, Halle, a/S., S. 114.

[221] Cellulose, p. 77.

[222] Vid. op. cit., 46, p. 63.

[223] Zeitschrift für physiologische Chemie, Band 13, S. 84.

[224] Zeitschrift für angewandte Chemie, 1895, S. 561.

[225] Vid. op. cit., 52, pp. 8 et seq.

[226] Vid. op. cit. supra, p. 15.

[227] Composition of Wood, Agricultural Science, Vol. 7, pp. 49, 97 and 161.

[228] Tollens; Handbuch der Kohlenhydrate: von Lippmann; Chemie der Zuckerarten.

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