THE ANALYST. SEPTEMBER, 1904. PROCEEDINGS OF THE SOCIETY OF PUBLIC ANALYSTS. NOTE ON THE ESTIMATION OF SUGARS AND STARCH I N VEGETABLE SUBSTANCES. BY JOHN S. FORD. (Read at the Meeting, June 1, 1904.) THE method described here, whilst more particularly applicable to malted and unmalted grains, applies also more or less directly to other vegetable substances. In the extraction of pre-existent sugars from grains, means must be taken to avoid enzymic action on the sugars themselves, or on the other bodies present. In the case of malts containing invertase and amylase (diastase), amongst other ferments, simple aqueous extraction gives rise, as is well known, to inversion of sucrose, and to production of maltose by the action of amylase on the modified starch. Kjeldahl (Compt. Rend., Carlsberg, 1881) was possibly the first who tried to avoid this, and, amongst other methods he utilized for the inhibition of enzymic action during extraction, was a preliminary treatment with absolute alcohol, the substance being moistened with alcohol, and then heated in a water-bath until dry.This process destroyed the enzymes, and the sugars could then be extracted with water unchanged. O’Sullivan (Jour. Chem. Soc., 1886), in his method of sugar estimation, employed alcohol of different strengths for the extraction of the cereals examined, and by doing so inhibited enzymic change, and also obtained solutions of workable purity. The extraction is, however, tedious, and apparently difficult to bring to corn- ple t ion. Jais (Zed. f u r gesam., Brau, 1893), Jalowetz (Chem.Zeit. Rep. 1-S), Ehrich (Chem. Zeit. Rep., 1894), and Brown and Morris (Jour. Chem. Soc., 1893), recommend destruction of the enzymes by preliminary boiling with alcohol, the first three authors using alcohol of 90 to 94 per cent., and Brown and Morris alcohol of 80 per cent. The writer has employed this method since 1892, and wishes to point out that its utility depends upon the alcoholic concentration of the mixture, and that it is necessary to make allowance for the moisture present in the grain or substance278 THE ANALYST. CuO at 100" C. Grammes per 100 C.C. treated. Boiling with too concentrated alcohol does not destroy the enzymes, and the use of too dilute alcohol is attended with risk of alteration of the starch. The following experiment; carried out with a malt containing 1 per cent.moisture, shows the influence of the alcoholic strength : Ten grammes of the malt were boiled with 100 C.C. of the alcohol for one hour, filtered and washed with alchohol of the same concentration, air-dried, then extracted with 50 C.C. water for ten hours; filtered, and 5 C.C. of the extract added to 50 C.C. soluble starch solution at 40" C . for one hour, then boiled. Five C.C. of each extract were also added to 50 C.C. of the same starch solution at 100" C. The solutions were made up to 100 c.c., and their copper-reducing power determined as usual. CuO a t 40" C. Grammes per 100 C.C. Alcohol. Specilk Gravity, 15*6"C./ i 15'6°C. .SO2 -810 *a23 -833 *848 0.43 0.41 0.39 0.35 0-34 I i 0.75 0.41 0.39 0.35 0.34 Reaction of Aqueous Extract of Residue with Iodine.No coloration. Slight brown. ,, blue. ,, blue. ,, These results indicate that the moat suitable strength of alcohol is about 95 per cent. by volume. In the case of this malt the moisture present may be disregarded, but in natural grain, and other moist vegetable substances containing little sugar, where large quantities of the substance have to be extracted, it is necessary to allow for the water present. For example, 200 grammes of barley, containing 17 per cent. moisture, were boiled with 600 C.C. absolute alcohol for half an hour ; the destruction of the amylase and invertase was complete, and after filtration and washing with alcohol of 90 per cent. or so, the residue was air-dried and extracted with water ; the alcoholic extract, which contained a proportion of the sugars, was evaporated and then added to the aqueous extract, and the sugars estimated as uaual in the combined extract. It may be stated here that the treatment with 95 per cent.alcohol destroys the invertase aa well as the amylase. In the determination of starch by O'Sullivan's method (Jour. Chem. Soc., 1884), this preliminary treatment with alcohol enables the subsequent washing out of the amglans and other bodies to be carried out without loss of starch through diastatic action, a loss inevitable with probably all starches, but more particularly notable in the case of starch from germinated grains. In the case of vegetable substances of an acid nature, the boiling with alcohol even for half an hour gives rise to some inversion of sucrose, if present, and even with malts a slight hydrolysis occurs.If the substance is distinctly acid, a very slight excess of ammonia may be added to the alcoholic mixture before boiling. The actual volume of alcohol taken does not seem to be of much moment, provided there is sufficient to cover the substance and to boil readily; of course, this operation must be carried out with an inverted condense; to keep the concentration constant.THE ANALYST. 279 The destruction of the enzymes is no doubt due to the coagulating effect of the water at the boiling-point of the 95 per cent. alcohol. Boiling with ether 35’ C., petroleum ether 50” C., acetone 56-58” C., benzol 78-80“ C., toluol 106-110” C., as might be excepted, does not destroy the amylase to any great extent. DISCUSSIOX.Dr. SCHIDROWITZ said he must express his astonishment that no mention was made in this paper of the method for preventing diastatic action which had been worked out some years previously by Mr. Ling, and which he (Dr. Schidrowitz) should have thought was well-nigh universally used-namely, to extract with an alkali. This quite effectually prevented any diastatic action, and was far simpler than these complicated boilings with alcohol, etc. Mr. A. R. LING said the alkaline extraction method which he had proposed some years ago for the extraction of the sugars of malt was far simpler than tho treatment with alcohol described by the author, and this process he had developed in a recent paper (Jozun. Inst. Brewing, 1904, x., 238).Moreover, it appeared to him from some experiments made by a German colleague, Herr Mason, with English malts, that even 95 per cent. alcohol might not completely arrest diastatic action ; and Herr Jalowetz, he believed, had proved this. Mr. BEVAN thought that probably there would be some chance of hydrolysis by the action of the boiling alcohol. In trying some years ago to prepare pure crystals of sugar by crystallization from hot alcohol, he had invariably found that some of the sugar was inverted. ADDENDUM. Mr. Ford states that the criticisms of Dr. Schidrowitz and Mr. Ling evidently arise from a misapprehension of the object of this ‘( note,” which is only to point out the necessity of using alcohol of a definite strength when employing this method for the destruction of the enzymes. The alkali method of Mr. Ling is no doubt more suitable for technical work, but for scientific purposes it is open to various objections, and the determination of starch in the extracted residue by O’Sullivan’s method becomes practically impossible. Mr. Bevan’s remarks as to slight hydrolysis of sucrose by the boiling alcohol is, as has been mentioned, a valid objection.