46 O’SULLIVAN AND TOMPSON : THE ESTIMATION V.--The Estimation of Cane-sugar. By C. ~’SULLIVAS, F.R.S., and FEEDRRTC W. TOYPSON. THE accurate estimation of sucrose in the presence of other carbo- hydrates has always been attended with considerable difficulty ; indeed, in many cases, i t was not found possible until Mjeldahl, in 1881 ublished a paper on the carbohydrates of malt (JfeddeZeZser, 1881, . h i ) . Some time afterwards, in a paper by one of us on the same subject, a method of estimating cane-sugar was given drnost identical with his (Trans.: 1886,49,58). In both cases very accurate results were obtained, and, as the latter paper was published in ignorance of Kjeldahl’s work, it may be regarded as an independent confirmation of it. It is obvious that in solutions such as those obtained from malt, the usual method of estiniating cane-sugar with acid is inapplicable on account of the action of the acid on the othcr carbohydrates.Indeed, this is the case in nearly all solutions obtained from natural sources. This difficulty was overcome by the use of the enzyme, invertase, arid it was eniploycd either in the form of a soluble preparation, or, in the raw state, as brewers’ yeast, together with sufficient thymol to prevent fermentation and growth of yeast. By this nieans the cane-sugar was inverted without any other substance present beiiig altered, and the results thus obtained were perfectly trustworthy. The decrease in optical activity, and the increase i l k cupric reducing power, each formed a measure of the amount of sucrose originally present in the solution.Very concordaiit results were obtained. The estimation of cane-sugar by means of invertase is without doubt a perfectly satisfactory process. The only disadvantage consists in the difficulty of preparing the invertase. Until the recent publica- tion of our paper on invertase (Trans., 1890, 57, 834), this objectioii ’ P‘,,,OF CAKE-SUGAR. 47 vas practically fatal? and it still forms a great drawback to the .universal application of the process, as i t takes at least three weeks t o prepare invertase by our method.* The use of yeast as the hydrolyst has hitherto been held t o require the presence of some such substance as thyniol, to prevent ferments- tion, and this method has not generally been considered to be so i*eliabIe as the former one.It is the puqose of the present paper t o show that the process known commercially as " Tompson's Inversion Process " is perfectly applicable to the estimation of cane-sugar. In this process, ordinary brewers' yeast is employed as the hydrolytic agent without the addition of anything else.' W e shall show that w e have in this rriethod a simple aiid convenient means of estimating cane-sugar independently of other substances in solution, and that she results obtained are accurate and trustworthy. Our procedure is as follows :-The aolntion containiiig the cane- sugar must be approximately neutral (certainly not alkaline), and if the presence of any enzyme is suspected, the temperature should be momentarily raised t o SO" t o destroy it.The optical activity at 15.5" and the cupric reducing power of the solution are determioed,? and then 50 C.C. of it are poured into a beaker and raised to a temperature of 55" in a constant temperature bath. Some ordiuary brewers' yenst, pressed in a towel, is iiow taken. The weight of the pressed yeast should be about l/lOth of the total amount of sugar t o be inverted.: It is thrown into the hot solution, and the whole yent1-y stirred until mixture is complete. The solution is left, for 4 hours in the water-bath: at the end of this time it is cooled t o 1.;*5", a little freshly precipitated aluiniiiium hydrate added to * We take this opportimity of making known the means we have now adopted for keeping a prsparation of invertase as a hydrolytic agent. We have, in oui- fornier paper, given details of tlie preparation of the solution called yeaat liquor.To this we add alcohol uiitil tlie whole conbains about 10-12 per cent. absolute alcohol. Tlic mixtare is a~lowecl to stand it few days, and then tlie cleay solution is filtered off. This solution is kept in a stoppered bottle, and it retains its invert- ing power indefinitely. It may either be nsed in. its undtered state or tho in- veytase may be thrown out of solution by the addition of 3 or 4 times its own bulk of alcohol, and tlie sympy precipitate, which immediately falls out, redissolved in water. We thus get, at a few minutes' notice, a fairly pure, and very strong solution of invertase. -j- It is not necessary t o determine both the optical activity and the cupric re- ducing power, as either factor by itself is sufficient.The o p t i d activity will be found to be the inore convenient, :md, iTe think, the more accurate of the two, unless t h e solution is highly coloured. $ Brewers' yeast varies considerably in inverting power, but we t<hink this ainount is amplc in most cases. The yeast need not necessarily be Presh, as i t does not easily lose its power.48 O'SULLIVAN AND TOXPSON : THE ESTIMATION facilitate filtration (this is not always necessary), and the whole made up to 100 C.C. A portion of this solntion is filtered, and the optical activity observed. The solution is then left in the cold until the next day, when another observation is taken in order to prove that inversion is complete. The cupric reducing power is also esti- mated.The method of calculating the results is the same as when in- vertase is used. F ~ o m the optical activity, the cane-sugar may be calculated as follows :- The following f o r m u l ~ will be found useful. cc = the number of divisions indicated by the polarimeter scale for a' = the same factor in the inverted solution. nz = the number of divisions of tlie polarimeter scale which 200 mm. of a solution containing 1 gram of cane-sugar per 100 C.C. alters on being inverted. I n the case of the Soleil-Ventxke-Scheibler polarimeter which we use, 1 gram of cane-sugar in 100 C.C. indicates + 3.84 div., whereas nfteia inversion it gives - 1.34 dir. Therefore for our experiments rrz = 5.18. P = the weight of cane-sugar present in 100 C.C. of the original solution .From the cupric reducing pozuer we calculate the cane-sugar as 200 mm. of the original solution. f 0110 FVS :- W G = the weight of 100 C.C. of the original solution. (3' = the same factor for the inverted solution. Allowance must, of course, be made here both for dilution and for the 5 per cent. increase of the inverted sugar, but the latter number is so small that it need not be calculated accurately. 20 = the weight of original solution used for the estimation. w' = the same factor for the inverted solution. 7c = the weight of cupric oxide reduced by w. k' - the same factor for z d e p = the weight of cane-sugar present in 100 C.C. of the original It is needless to remark that the values P a n d p should be identical within the limits of error of manipulation.It will be seen from the description we have given that this method of estimating cane-sugar is quick and! simple; it only remains for solut'ion.OF CANE-SUGAR. 49 Results calculated on us to prove its accuracy. I n the following experiments, we ha,ve compared it side by side with estimations made with a preparation of invertase; the accuracy of the latter method is, we believe, un- disputed. Exp. 1.-A blank experiment to test whether the yeast itself imparts any optical activity to the solution. 1 gram pressed yeast was heated in 50 C.C. water to 55" for 4 hours. It was then filtered off by the aid of a little aluminium hydrate. The optical activity of the solution was 0.0 to -0.2 div. in 200 mm. It is obvious that tho optical activity due to the yeast may be neglected.Exp. IT.-Estimation of cane-sugar in a hot water extract of malt. Original solution. Sp. gr., 1079.7. Optical activity after heating t o 80", +67-4 div. in 100 mm. I( of the solution diluted to twice its original bulk, 1.270 grams solution gave 0.1159 gram copper oxide. Inversion was performed in 3 beakers ; each contained 50 C.C. of the solution, and was treated exactly as we have described. Beaker 1.-0*1 gram pressed yeast was employed. Optical activity after 4 hours +63*5 div., next day +63.2 div. in 200 mm. K, 1.320 grams solution gave 0.1405 gram copper oxide. Beaker 2.-A solution of prepared invertase was used. OpticaI activity after 4 hours +63.2 div., next day + 6 3 2 div. in 200 mm. Beaker 3.-2.57'0 grams pure cane-sugar were added, and 0.2 gram yeast was employed.Optical activity after 4 hours +60*4 div., next day +60*0 div. in 200 mm. K, 1.274 grams solution gave 0.2020 gram copper oxide. After making the calculations in the manner we hare described, the results may 'be tabulated as follows :- Grams cane-sugar indicated by the Optical activity. - TABLE I.-l'he Estimation of Cane-sugar i i ~ the Hot-water Extract of Mcclt. 100 C.C. of the solution Amount added t o the solution 7 9 9 9 -I--I- 1'62 1-36 1 -62 2 -62 2 *34 - Yeast none j I n E t e 1 none 2 *530 It will be seen from this table that whilst the results calculated from the decrease of optical activity are perfectly satisfactory, those obtained from the increase in the cupric reducing power are not so VOL. LIX. E50 THE ESTIMATION OF CAKE-SUGAR.Cane-sugar indicated by the good. TEia may be accounted for by the high reducing power of the original solution. Exp. 111.-Some treacle was dissolved i n water with a little loaf sugar ; the cane-sugar was then eskiiiiated as follows :- Original solution. Sp. gr. 1054.2. Optical activity +18.9 div. in 100 mm. Beaker 1.-A solution of prepared invertase was used. Optical :tctivity after 4 hours -6.1 div., next day -6.4 div. in 200 mm. K, 1.309 grams solution gave 0.1800 gvam copper oxide. Beaker 2.-0*4 gram pressed yeast mas employed. Optical activity after 4 hours -6.4 div., next day -6.7 div. in 200 mm. I(, 1.357 grams solution gave 0.186 gram copper oxide. K, 2.312 grams solution gave 0,1124 gram copper oxide. We have tabulated the results.Agent employed. Be*;ker. TABLE II.-TJLe Estirnation of CYame-sugaj* in a Solution of Treacle, showing granzs of Sugar in 100 C.C. of t h Solution. Cane-sugar indicated by the optical activity. I I The agreement between these four results is remarkable. Ezp. 1V.-Some apples were finely divided and boiled in water. The solution was filtered off, arid the cane-sugar estimated. Original solution. Sp. gr. 1.024. Optical activity -11.3 div. in 200 mm. Beaker 2.-A solution of prepared invert'ase was used. Optical activity t,he next day -7.2 div. in 200 mni. Beaker 2.-0.1 gram yeast was used. Optical activity the next day -7.4 div. in 200 mm. TABLE IIJ.-Tlie Estimation of CnrLe-sugnr in, a Solution of Apple- jwice, ::howi,ng grains of Sugay i n 100 C.C. of the Solution. Tnvertsse 0 -60 Sea& 0 -67RICHARDSON: ACTION O F LIGHT ON PURE ETHER, ETC. 51 In this case the solution was too dilute to give perfectly accurate results, and, moreover, it was rather opalescent and difficult to see through. The results of these experiments agree so well together, that no doubt remains as to the accuracy of the process. We may remark that these experiments were not picked out on account of the good results obtained, but were done by theniselves specially t o test the trustworthiness of the process. I n conclusion, we do not hesitate t o say that the method we have described furnishes a simple and accurate means of estimating sucrose in all solutions.