SEPARATION AND DETERMINATION OF THE VARIOUS CARBOHYDRATES

OF CEREALS, ETC. STONE'S METHOD.

Stone has thus tabulated the results of a series of analyses of various samples of wheat, flour, corn, and bread, in which he has separated the principal carbohydrates.*

Determination of Cane Sugar.-100 grams of the finely ground material are extracted by boiling under a reflux condenser with 500 cc. of 95% alcohol for three hours, the alcoholic extract is filtered, evaporated nearly to dryness, and then taken up with a small amount of water, to separate the sugar from the oils and waxes dissolved by the alcohol. This aqueous solution is invariably dextro-rotary, and seldom contains any reducing sugar. If the latter is present, it is determined in an aliquot part of the aqueous solution with Fehling's solution, the result being calculated to dextrose. The remainder of the aqueous sugar solution, or the whole of it, if, as is almost always the case, dextrose is absent, is then inverted by heating with hydrochloric acid in the usual manner (page 611) and the sugar is estimated with Fehling's solution, calculating the result to sucrose (page 642).

Determination of Dextrin.-Digest the residue from the above alcoholic extraction from eighteen to twenty-four hours with 500 cc. of cold distilled water, shaking frequently. On filtering, a clear solution is ob

*Jour. Am. Chem. Soc., 19, 1897, p. 183, and U. S. Dept. of Agric., Off. of Exp. Sta., Bul. 34. The percentages of normal starch found by Stone are obviously erroneous, and are for this reason excluded from the table as here given.

tained, which should be tested with iodine for soluble starch. If the latter is not found (which is nearly always the case), the solution is concentrated to a small volume, avoiding a temperature higher than 80° to 90°, and this is boiled under a reflux condenser for two hours with onetenth its volume of hydrochloric acid (specific gravity 1.125). Determine the dextrose by Fehling's solution and calculate to dextrin by the factor 0.9. Or, instead of submitting the concentrated aqueous extract to hydrolysis as above, the dextrin may be roughly determined gravimetrically therein by treating with several volumes of strong alcohol until no further precipitation is produced. The flocculent precipitate thus obtained is collected, dried, and weighed.

Determination of Starch.-Dry in an oven the residue from the preceding treatment and determine its quantitative relation to the original sample; 2 grams are then accurately weighed and subjected to the diastase method of starch determination (page 292).

Determination of Pentosans and Hemicelluloses.-The washed residue, left after filtering off the starch-containing solution from the process of heating with malt extract in the preceding starch determination, is boiled for an hour with 100 cc. of 1% hydrochloric acid, which converts all the pentosans into sugar. Filter, and wash the residue thoroughly, make up the solution to 200 cc., and determine the sugar with Fehling's solution, calculating the results for xylan, assuming that the chief sugar formed is xylose. The reducing power of xylose is assumed to be 4.61 milligrams for each cubic centimeter of Fehling's solution. If the volu metric Fehling method is used, 10 cc. of Fehling's solution are thus equivalent to 0.046 gram xylose. Xylose Xo.88 = xylan.

Crude Fiber (Cellulose, etc.).-The residue from the last dilute acid hydrolysis is boiled with 200 cc. of 1.25% solution of sodium hydroxide for half an hour, filtered, dried, and weighed. It is then ignited, and the weight of the ash deducted from the first weight.

PROTEINS OF CEREALS AND VEGETABLES.

Different cereal and vegetable foods present considerable variations in the character and extent of their protein constituents, and by no means all of the common vegetable foods have been studied in detail.

Osborne, in connection with Voorhees and Chittenden, has made. a careful study of the proteins of many of the cereals, of potatoes, and of peas. A brief outline only will be given in what follows of methods.

for separation of the vegetable proteins. For fuller details the reader is referred to the work of Osborne et al. in the American Chemical Journal, Vols. . 14, and 15, and to the Journal of the American Chemical Society, Vols. 17, 18, 19, and 20.

Proteins Soluble in Water and Dilute Salt Solution.-By the action. of various solvents it is possible to separate the different classes of proteins for examination or analysis. Thus water at first applied extracts certain of the soluble proteins, as does a weak salt solution. Osborne and Voorhees recommend the use of a 10% solution of sodium chloride as the first solvent to apply for separating vegetable proteins, shaking the finely ground material with twice its weight of the salt solution. The salt solution, after filtering, is then subjected to dialysis, the protein matter thus separated out being a globulin, while that not precipitated on dialysis is assumed as the protein matter of the substance soluble in water. Two albumins and a proteose are found in wheat to be thus soluble in water. It the proteins soluble in salt solution are to have their total nitrogen determined, they are completely precipitated from the solution by saturating with zinc or ammonium sulphate.

There are thus two classes of proteins soluble in 10% salt solution: (a) globulins, insoluble in water alone, and (b) albumins and proteoses, which are soluble in water.

Separation of Albumins, Proteoses, and Globulins.-Starting with the aqueous solution containing the albumins and proteoses, if present, the former are best separated according to Osborne and Vorhees by fractional coagulation, effected by heating at different temperatures, those that precipitate out at a temperature under 65° being first filtered out, and the filtrate submitted to a higher temperature not exceeding 85°. The two portions thus separated may be collected in filters, and their nitrogen separately determined.

The proteose may be precipitated from the filtrate by saturating with ground salt, or by adding, first salt to the extent of 20%, and finally acetic acid.

The globulins, precipitated in the original 10% salt solution by the process of dialysis as described, may themselves be separated by employing salt solution of varying strength as solvents.*

Proteins Soluble in Dilute Alcohol, but Insoluble in Water. The residue from the treatment with 10% sodium chloride is digested with 75% alcohol at about 46° C. for some time and filtered. The residue is further

* Am. Chem. Jour., 13, p. 464.

digested at about 60° with 75% alcohol three separate times. The evaporated filtrates contain the alcohol-soluble proteins. In this class are the hordein of barley, the gliadin of wheat and rye, and the zein of corn.

Proteins Insoluble in Water, Salt Solution, and Dilute Alcohol. It is customary to determine the nitrogen in the final residue without further attempt to separate the remaining protein matter. It is, however, possible to further extract with alkaline and acid solvents, if desired, which process, however, changes the nature of the proteins from that in which they originally exist in the substance.

Character and Amount of Proteins in Wheat.* The proteins of wheat, according to Osborne, are five in number, as follows:

The term gluten is applied to the protein content of wheat flour insoluble in water, the value of flour for baking bread depending on the amount present. Gluten contains the two definite proteins, gliadin and glutenin. Crude gluten, as obtained by washing the dough in the analytical process (page 331), is a complex mixture of many bodies, containing, besides the two proteins above named, small quantities of cellulose, mineral matter, lecithin, and starch.

Separation and Determination of Wheat Proteins.-Teller's Method.†Non-gluten Nitrogen.-Two grams of the finely divided sample are mixed with about 15 cc. of 1% salt solution in a 250-cc. flask. The flask is shaken at intervals of ten minutes during one hour, after which it is filled to the mark with the salt solution and allowed to stand two hours. The supernatant liquid is then filtered through a dry filter into a dry flask. leaving most of the solid material in the flask, passing the first part through twice, if necessary, for a clear filtrate. With a pipette, exactly 50 cc. of clear filtrate are run into a 500-cc. Kjeldahl digestion-flask, 20 cc. of the usual reagent sulphuric acid for the Gunning process (p. 58) are added, and the contents of the flask brought to a gentle boil. After the water has

* Am. Chem. Jour. XV, 392-471; XVI, 524.

† Ark. Exp. Sta. Bul. 42, p. 96.

been driven off and the acid has stopped foaming, the potassium sulphate is added and the digestion completed. From the per cent of nitrogen thus obtained 0.27% is deducted, this figure corresponding to the amount of gliadin soluble in 1% salt solution under the above conditions. The remainder is the percentage of non-gluten nitrogen.

Gluten Nitrogen.-This is obtained by difference between the total nitrogen and the non-gluten nitrogen as above obtained, or by deducting the combined nitrogen of the edestin, leucosin, and the amido-nitrogen from the total nitrogen.

Edestin and Leucosin.-Edestin is a globulin belonging to the vegetable vitellins, and is precipitated from salt solutions by dilution, or by saturation with magnesium or ammonium sulphate, but not by saturating with sodium chloride. It is not coagulated below 100° C., but is partly precipitated by boiling. Leucosin is an albumin, coagulating at 52°, but precipitates from salt solution by saturating with sodium chloride or magnesium sulphate.

To 50 cc. of the clear salt extract, obtained as described under nongluten nitrogen, 250 cc. of pure 94% alcohol are added in a Kjeldahl 500-cc. digestion-flask, the contents thoroughly mixed, and allowed to stand over night. The precipitate is collected in a 10-cm. filter, which is returned to the flask and the nitrogen determined. This represents the nitrogen of the combined edestin and leucosin. These proteins may, however, be separated by coagulating the leucosin at 60°, and precipitating the edestin by adding alcohol to 50 cc. of the clear filtrate, determining the nitrogen separately in each precipitate.

Amido-nitrogen.—Allantoin, asparagin, cholin, and betaine are nitrog enous bases present in wheat.

Ten cc. of a 10% solution of pure phosphotungstic acid are added to 100 cc. of the clear salt extract as above obtained, thus precipitating all the proteins, which are allowed to settle preferably over night. Filter, and determine the nitrogen in the clear filtrate. The filtrate should be tested with a little of the phosphotungstic acid reagent to make sure that all the proteins have been separated. In some cases, as in bran for instance, more than 10 cc. of the reagent are necessary.

Gliadin is dissolved most readily from flour by hot dilute alcohol, but is entirely insoluble in absolute alcohol. One gram of the material is extracted with 100 cc. of hot 75% alcohol, by shaking the mixture thoroughly in a flask, and heating for an hour at a temperature just below