Chapter VIII: Preface: To Volume Third (8)

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

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

C₅H₄O₂ + C₆H₆O₃ = C₁₁H₁₀O₅

and

2C₁₁H₁₀O₅ = C₂₂H₁₈O₉ + H₂O.

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

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

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

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

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

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

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

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

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

C₆H₁₂O₆ = 2C₂H₆O + 2CO₂.

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

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

C₁₂H₂₂O₁₁ + H₂O = 2C₆H₁₂O₆

2C₆H₁₂O₆ = 4C₂H₆O + 4CO₂.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The precipitation is accomplished as follows:

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

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

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

_Levulosazone._—This body has the same properties as the dextrose compound.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

DETERMINATION OF STARCH.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

_Example_:—

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

_av_ × 25 × 0.016
_A_ = ------------------.
2 × 52.8

In this formula _a_ = the rotation in angular degrees, _v_ = the volume of the liquid and _A_ = the starch transformed.

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

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

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

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

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

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