70 O’SULLIVAN ON THE PRESENCE OF I X - O I L t h e Presence o j ‘‘ Rafinose ” in B a d e y . By C. O’SULLIVAN, F.R.S. IN the preceding communication, I pointed out that, although the evidence for the presence of certain sugars in barley was moderately conclusive, we conld not be absolutely satisfied until each sugar was isolated and examined by itself. So convinced was I of the truth of this proposition that eight or ten years ago, early in my investigation on the sugars of the cereals, I made an attempt to crystallise them. With that object in view, 2 kilos. of ground barley were extracted with alcohol in the same way as is described for the 200 grams in the last. paper. The solution, freed from alcohol, was evaporated to a syrup, which was dissolved in the least possible quantity of boiling alcohol, sp.gr. 0.83. This solution, on cooling, deposited a syrup which was itgain dissolved in just sufficient alcohol to hold it in solution when cold ; a little ether was then added to produce a slight turbidity, and the whole put aside to crystallise. In a short time, cauliflower-like segregations began to form, and aft’er a few months they ceased to increase. These were collected, washed with strong alcohol, and put aside labelled “ Suci.ose (cane-sugar) from barley,” for no other reason than that they appeared under pretty much the same conditions as those under which Kuhneman (Ber., 8, 202) said he isolated cane-" RAFFINOSE " IN BARLEY. 71 sugar from malt. The quantity I obtained was not large ; it did not amount to more than 1.4 grams, or about 0.07 per cent.of the barley employed. Although t,his was amply sufficient t o determine whether i t was sucrose or not, so certain was I that it was, its crystal- line appearance notwithstanding, that I did not think it necessary to examine it. Working with the barley of the season 1878, I believe, in the same way, I obtained 4.55 grams of the cauliflower-like segregations from 5 kilos. material. This too I put aside, labelled '' Cane-sugar from barley." Recently, while arranging the material f o r the preceding paper, I thought it desirable t o examine more closely the two preparations described above. If they were sucrose, I had no doubt I could, by recrystallisation, obtain recognisable crystals of that substance. Before dissolving them, I examined a little of each preparation under the microscope ; I found the crystals were elongated, flattened prisms terminated by a dome parallel t o the shorter axis, the groups or segregations consisting of the crystals radiating from a centre. Both samples were alike, hence I had undoubtedly t o do with a sub- stance altogether different in crystalline form from sucrose.I pro- ceeded to examine the preparations farther. A determination of the optical activity of the dry matter in the one gave [ a ] j = 125", and in the other [ a ] j = 1 1 4 O . Both preparations were impure, for each contained a considerable quantity of ash, but it was evident that I had the same substance to deal with. I dissolved them together in a little water, filtered from some insoluble matter, and added strong alcohol until a precipitate began t o appear.This solution, on standing, was filled with radiating groups of beautiful silky crystals, which were collected, and washed with alcohol so regulated in strength as to produce no turbidity in the mother-liquid, the sugar being precipitated as a syrup on adding strong alcohol to the concentrated solution. The crystals are well-defined, flat, probably rhombic prisms, FIG. 1. EIb. 2. FIG. 3.72 O'SULLIVAN ON TBE PRESENCE OF terminated by a brachydome. Figs. 1, 2, and 3 give a fair idea of their shape. A second crop of crystals was obtained by concentrating the mother- liquor to a syrup, and again adding a little strong alcohol. On determining the optical activity of these, I found the results did not quite agree; the first crop gave [ a ] j = J 29.8" (c = 4*142), and the second [ a ] j = 132" ( c = 2.392) ; both still contained ash.As, however, the opt>ical activity was so near, and the quantity of sub- stance at my disposal so small, I mixed the two specimens, and sub- mitted the mixture to recrystallisation. A first and second crop of crystals were obtained as in the first instance. I now found that both preparations had practically the same optical activity, viz., for the substance dried in a vacuum over sulphuric acid and then in dry air at 100" until the weight was constant, [a]j = 134-135" (c = 4 to 5); hence, I concluded I had purified the substance. 1st Exp.-O.992 gram of the crystals, first allowed t o remain in dry air until the weight became constant, lost in a vacuum over sulphuric acid 0.142, and then, in dry air at loo", a farther 0.008 gram, making a total loss of 0.150 gram.2nd Exp.-2.061 grams, dried in the same way, lost a total of 0.312 gram. These results iudicate- 1st Exp. 15.12 per cent. water. 2nd Exp. 15.14 ,, 9 , Submitted to combustion in a stream of oxygen, 0.3148 gram dry substance gave- COz = 0.4893 gram, and H,O = 0.1825 ; 0.0020 gram ash was left in the boat; this was chiefly potassium carbonate. The numbers give- Carbon.. . . . . 42.66 per cent. Hydrogen.. . 6.47 ,, 6-35 ,, Theory for CgHl6O8. 42-85 per cent. These percentages are sufficient to indicate the empirical formula of the body in the dry state, and, from the amount of water lost by the crystals, we get the formula- or by doubling it to eliminate the $ mol.HzO, CJLO,, 23H20, which probably represents the molecular formula of the substance." RAFFINOSE " IN BARLEY. 73 An aqueous solution containing 1 gram dry substance in 100 C.C. had a sp. gr. 1.003965 ; this shows conclusively that the substance as burned did not contain any water of crystallisation. The dry sugar absorbs water from the atmosphere and becomes a glassy mass. 2.227 grams dry substance in 50 C.C. solution gave aw optical activity = 31.4 divisions of Soleil-Scheibler's instrument ; from this, the specific rotary power for the dry sugar is [ a ] j = + 135.3". It does not reduce copper solution ; 0.1 gram boiled for 25 minutes with 25 C.C. Fehling's solution, properly diluted, yielded only a trace of copper suboxide.Invertase appears t o act upon it, but very slowly : to 25 C.C. of a solution possessing an optical activity = 28.2 divs., 0.015 gram invertase was added, and the mixture digested at 50-52" for three hours ; the optical activity of the cooled solution was = 25.divs. A little more invertase was added, and the digestion continued for another three hours ; the activity had then fallen t o 21 divs. This was a slow process, but does not leave a doubt that invertase has an invertive action. Had sucrose; in the same quantity, been in the solution, it would have been all inverted in the first three hours. A one per cent. solution of sulphuric acid at 100" reduced the optical activity, in one hour, from 28 divs. to 10 divs. In this case, the products of inversion had 5t sp.rt. pr. The amount of material at my disposal did not warrant farther experiments in this direction, so I did not continue then. Treated with 4 parts nitrioacid andtl part water, it yielded a little less than 30 per cent. on dry substance of an insoluble acid, which, from its general behaviour and appearance under the microscope, is muck acid. The filtrate from this acid, neutralised with ammonia and acidified with acetic acid, yielded a precipitate of calcium oxalate on the addition of calcium chloride. From these facts, it is highly probable that galactose is one of the products of inversion, and, from the composition, that two other sugars are produced. These must be sacchsric and oxalic acid yielding sugars. = 43.5", and a K = 80.8. The sugar is fermented by ordinary yeast.I had worked with this sugar some time under the name of " cerenlose," but the recent papers of Scheibler (Rer., 18, 1779) and Tollen (Bey., 18, 2611) leave no doubt on my mind, that it is the " rafinose " of Loiseau (Compt. rend., 82, 1058). Whether the sugar is Berthelot's mellitose as Tollens says raffinose is, I am not prepared to say. Scheibler can say whether my description of the crystals agrees with his observations. My analytical numbers agree well with his, for, although he burned the crystals, and I the dry substance, the VOL. XLIX. G74 ARMSTRONG AND MILLER: THE DECOMPOSITION AND results obtained lead to the same formula. The optical activity observed by me, sp. rt. pr. [ a ] j = 135*3", agrees with the numbers obtained by Scheibler and Tollens, [ a ] j = 114.7" ; they evidently, in this case also, have made the calculation for the crystals, I f o r the dry substance : 114.7" for C18H32016,5H20, is equal to 135.1" for C1,H3,O,,.The sp. gr. of a solution containing 1 gram in 100 C.C. is higher than that given by Tollens ; his figures (Ber., 18, 2616) Ieading to a true sp. gr. 1.003712 for such a solution, my figure is 1.003956. The yield of muck acid is practically the same as observed by Berthelot, Scheibler, and Tollens ; my number is, however, a little higher than that published by Scheibler, 30 per cent. against 26.7, but I cannot say with the quantity I had to work with that my experiment can lay much claim to absolute accuracy. My observations on the products of inversion do not quite agree with those published by Tollens. He says galactose, dextrose, and I~vulose are the pro- ducts. I found, for the products of the action of' sulphuric acid, [aJj = 43*5", and K = 431 ; now, if we suppose that the remaining 15 non-K was unaltered sugar, and the 81 composed of equal parts of galactose, dextrose, and lawnlose, we get a mixture the optical activity of which works out [ a J j = 36*1", a figure sufficiently removed from 43.5" to throw some doubt on the supposition; how- ever, as I have not sufficient substance to settle the point a t present, I must leave it. These facts, then, leave no doubt that I have been dealing with the " raffinose " of Loiseau, which Tollens says is Berthelot's mellitose ; the source whence I isolated ;it seems to me of sufficient interest to record. [&ID = 104" ;