Part 30

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

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

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

=156. Precipitation of Furfurol with Pyrogalol.=—Furfurol is thrown out of solution in combination with certain phenol bodies by heating together in an acid solution. Hotter has proposed a method for the determination of furfurol based on the above fact. 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. 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, 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.