2854 KNECHT AND EIIBBERT THE BE€IAVIOUR OF GLUCOSE AND CCCXCV.-The Behaviour of Glucose and Certain Other Carbohydrates towards Dyestufjs and towards Potassium Ferricyanide in an Alkaline Medium. By EDMUND KNECHT and EVA HIBBERT. ALTHOUGH we are well informed as to what changes take place in the dyestuff when indigo or some other dyestuff is reduced by glucose in presence of alkali no attempts to elucidate the character of the oxidation of the glucose have hitherto been recorded. Many attempts have however been made to ascertain the character of the oxidation products formed by the action of Fehling’s solution and of cupric hydroxide in presence of caustic alkali as well as of other inorganic oxidising agents in an alkaline medium the results of which indicate that the reactions are of a complicated character.The most complete researches on this subject are those of Gaud (Cornpt. rend. 1894 119 604) who employed Fehling’s solution as the oxidising agent and obtained formic oxalic tartronic lactic and glyceric acids along with some catechol and the elaborate work of Nef (AnnaZen 1907 357 214) who using copper sulphate and a slight excess of caustic soda obtained hexonic trihydroxybutyric, glyceric glycollic formic and carbonic acids. By means of red mercuric oxide and barium hydroxide Herzfeld (Annalen 1888, 26 32) obtained gluconic acid as the main product. By using sodium hypoiodite as the oxidising agent Romijn (2. a d . Chm., 1897 36 19) obtained this acid alone and Willstatter and Schudel (Ber. 1918 51 780) showed that the reaction may serve for the estimation of glucose.The authors have shown ( J . SOC. Dyers Col. 1925 41 94) that when glucose is boiled with excess of methylene-blue in presence of caustic soda oxidation takes place very rapidly and to a definite degree (three atoms of oxygen to one mol. of glucose). Further work in this direction has now been carried out. Methylene-blue was not entirely suitable for the purpose on account of its great liability to undergo decomposition by the action of the alkal CERTNN OTHER CARBOHYDRATES TOWARDS DYESTUFFS ETC. 2855 (Bernthsen Annden 1885 230 73) so potassium indigotintetra-sulphonate which reacts in the same way as methylene-blue is much more stable towards alkalis and can be estimated volu-metrically with rapidity and accuracy was used for most of the oxidations given below.The amount of indigotin reduced was invariably equivalent to three atoms of oxygen per mol. of glucose, four atoms of oxygen per mol. of laevulose and as might have been expected 3& atoms of oxygen in the case of invert-sugar. Galactose and glucosamine hydrochloride behaved like glucose each taking up three atoms of oxygen. Maltose was apparently hydrolped, since the molecule (C,2H22011 + H20) reduced an amount of indigo-tin equivalent to six atoms of oxygen. Thioindigodisulphonic acid (Ber. 1907,40,3821) gave with glucose and laevulose results identical with those obtained with potassium indigotintetrasulphonate. Potassium ferricyanide can replace indigotin in the titration of glucose laevulose and glucosamine giving identical results ; the reaction is very rapid being complete in 15 seconds.The extent of the oxidation was in the &st instance determined by titrating the ferrocyanide in the cooled solution midiiied with sulphuric acid with permanganafe to the appearance of a pink colour. The result was corroborated by estimating the excess of ferricyanide by means of standard sodium hydrosulphite with methylene-blue as indicator. Ultimately it waq however preferred to estimate the excw ferricyanide by titration with titanous chloride in presence of a trace of ferric chloride the disappearance of the blue colour indicating the end-point. There was a close agreement of the results in all three cases. In view of the constancy of the results obtained with methylene-blue potassium indigotintetrasulphonate thioindigodisulphonic acid and potassium ferricyanide and the exact stoicheiometric character of the reactions it was surmised that in the case of glucose and galactose the oxidation had resulted in the formation of hexane tetrahydroxy-dicarboxylic acids corresponding to sac-chasic and mucic acids whilst in the case of hvulose the oxidittion would just suffice to produce a mono- and a di-carboxylic acid in equimolecular proportions.In spite of numerous attempts it has, however not been possible to identify any of the oxidation products. The difEculty lies mainly in the removal of the large quantity of colouring matter required to effect the oxidation; for instance, 1 g. of glucose requires more than 16 g.of potassium indigotin-tetrasulphonate which will be present after oxidation is complete, partly as such but mainly in the reduced condition. Also, through the action of the alkali considerable isomerisation o 2856 KNECHT AND EIBBERT THE BEHAVIOUR OF GLUCOSE AND the oxidation products probably takes place and these do not possess the uniformity of the product of oxidation in an acid medium. If glucose which is neutral to phenolphthalein is converted into a dicarboqlic acid (or acids) during the reaction the extent of such conversion should be measurable by titration for the resulting acid would require two equivalents of caustic alkali for its neutralis-ation. On similar grounds the acids resulting from the alkaline oxidation of lamulose should require three equivalents of alkali.It was obviously impossible to use dyestuffs as oxidants to test the validity of this reasoning. Pure potassium ferro- and ferri-cyanides are neutral to phenolphthalein and the latter was employed as the oxidant in the experiments. In carrying out the estimations it had to be taken into consideration that potassium ferricyanide in becoming reduced to ferrocyanide takes up two molecules of potassium hydroxide for each atom of oxygen supplied. The results confirmed exactly the above reasoning. It is known that in the titration of glucose by means of Fehling’s solution the extent of the oxidation as measured by the amount of copper reduced is represented by rather less than 2$ atoms of oxygen per molecule of glucose. On the other hand it has been shown that by employing in place of Fehling’s solution the solution of copper carbonate in potassium carbonate and bicarb-onate advocated by Soldaini (Gazzetta 1876 6 322) the degree of oxidation is almost doubled (see also Ost Ber.1890 23 1035, 3003).* It was therefore considered possible that the use of an alkaline carbonate in the titration of glucose with indigotin might also give rise to a higher degree of oxidation. The results obtained, however were in no way modified by substituting for the potass-ium hydroxide an amount of potassium carbonate and bicarbonate equivalent to that employed in the alkaline copper carbonate solution. In carrying out the titration it is important that the substances be added in the sequence given. If the caustic potash is added to the boiling glucose solution before the indigotin 30 seconds’ boiling is sufficient to lower the result by 50% or more.This seems to be due to the conversion of some of the glucose into lactic acid (Nencki J . p. Chem. 1881 24 498). If the boiling is continued for 2 minutes the amount of alkali neutralised corre-sponds exactly to the formation of two molecules of lactic acid. Laevulose behaves in this respect like glucose. Neither lactic saccharic mucic nor gluconic acid reduced * No satisfactory explanation of this great difference in the behaviour of the two solutions has hitherto been advanced CERTAIN OTHER CARBOBYDRATES TOWARDS DYESTUFFS ETC. 2857 methylene-blue when boiled with this dyestuff in presence of was substituted for methylene-blue or potassium indigotintetra-sulphonate as oxidant both glucose and laevulose took up exactly two atoms of oxygen per molecule.This would correspond to the formation of glycuronic acid from the first and of hydroxygluconic acid from the second of these carbohydrates. Superimposing an inQotin titration on the kitone-blue titration of glucose resulted in a further atom of oxygen being taken up. From its behaviour towards methylene- blue and towards indigotintetradphonic acid, it might have been expected that lamdose after being oxidised with kitone-blue would take up two more atoms of oxygen on superimposing a methylene-blue titration. Only one further atom of oxygen was taken up. b i n d d i n e 2B when employed as the osidising agent supplied only one atomic proportion of oxygen to glucose.By super-imposing a methylene-blue titration no further oxidation took place; and although the oxidation product was not identified, this circumstance strengthens the opinion that the oxidation had resulted in the quantitative formation of gluconic acid. E x P E R I M E N T A L. In principle all the titIratiom are carried out in the same way. A known weight of the carbohydrate is heated with a known volume of a standard solution of the oxidising dyestuff which must be present in considerable excess in a current of nitrogen. When the mixture boils caustic potash is added and the boiling continued for the time specified. The mixture is now a t once acidified and the excess of dyestuff titrated wit'h titanous chloride in a current of carbon dioxide.The quantity of carbohydrate most suitable for a titration is 0-01-0.05 g. and the quantity of caustic potash required is about 0-5 g. or 5 C.C. of a 10% solution. The following special points should be noted in carrying out the titrations. Methylene-blue. A suitable strength of this dyest& (working with a titanous chloride solution of which 1 C.C. = 0.002 g. Fe or thereabouts) is 10 g. of the medicinal product per litre. The time of boiling in presence of alkali should in no case exceed 5 seconds; beyond this limit the methylene-blue loses strength through decomposition and the results become untrustworthy. A suitable strength is 40 g. Potassium ir,digotintetrasuIp~nate 2858 KNECHT AND -BERT rn BEHAVIOUR OF GLUCOSE AND of the crystallised dyestuff per litre.The procedure comkts in adding to an aqueous solution of a definite weight of glucose excess of the standard indigotin solution. The mixture is boiled in a current of nitrogen caustic soda is added and the boiling continued for 8 minute. The mixture is then acidified with dilute sulphuric acid excess of tartrate of soda added and the hot solution titrated with titanous chloride until the blue colour disappears. Ferricyanide. A suitable strength for a standard solution is 50 g. per litre. The quantity of caustic potash added should be more than sufficient to allow for the formation of ferrocyanide and for the neutralisation of the organic acids formed. The time of boiling is 15 seconds. The mixture is rapidly cooled in running water before being acidified with dilute sulphuric acid.The excess ferricyanide is then titrated with standard titanous chloride as described. It is not necessary to employ either nitrogen or carbon dioxide. Boiling for 30 seconds suffices to complete the oxidation after which the solution is acidified with tartaric acid in slight excess before the excess of dyestuff is titrated with titanous chloride. Strength and procedure as for kitone-blue. Rosinduline G also may be used for the purpose. Crystal-scarlet was not found suitable as an alkaline oxidising agent the end-point not being sharp. Like the rosindulines this dyestuff supplies one atom of oxygen to glucose. Titration of Glycuronic Acid.-A solution of pure euxanthic acid (0-201 9.) in sulphuric acid (d 1.735) was diluted boiled and filtered, and the separated euxanthone was washed so that the filtrate contained the whole of the glycuronic acid.The solution thus obtained reduced indigotin equivalent to 26.4 C.C. of Ticla (1 C.C. TiC1 = 0402001 g. Fe) indicating that almost exactly one atom of oxygen had been used. Expressed as percentage of glycuronic acid in euxanthic acid the result gives 45.5% as against 45.9% calculated for Cl,H,,Olo + H,O. Almost the same result was obtained in the osazone process 0.035 g. of the same specimen of euxanthic acid was hydrolysed with sulphuric acid as before and the resulting glycuronic acid converted into the osazone; this required 14.4 C.C. of TiCl (1 C.C. = 0.001924 g. Fe) (Found 4507% calculated on the euxanthic acid employed).Action of Sodium Hydroxide on Glucose and Lceevulose.-An aqueous solution of 0.5045 g. of glucose was boiled with 25 C.C. of N-sodium hydroxide for 2 minutes and the excess of alkali then titrated with N-sulphuric acid and phenolphthalein. The amount of caustic soda neutralised was 5.6 c.c. which is equal Kitone-blue. A suitable strength is 10 g. per litre. RosinduZine 223 CERTAIN OTHER CARBOHYDBATES TOWLELDS DYESTIEFIB ETC. 2859 to 44.4% on the weight of the glucose (calc. for N-NaOH, 0.5115 Gram of h d o s e similarly treated required 5.7 C.C. of Y-sodium hydroxide which represents 4404% on the weight of the laevulose. In another experiment the boiling with caustic soda waa continued for 5 minutes; this did not affect the result.Acidi'metric Titration of the Oxidation Products of Glucose and Lmlose.4lucose (0-5 g.) was oxidised with 7-5 g. of potassium ferricyanide and 50 C.C. of N-potassium hydroxide. Titration with N-sulphuric acid and phenolphthalein then showed that 1.24 g. of potassium hydroxide had been neutralised in the reaction. The amount required according to the equation is 1-245 g. On similar treatment of 0.5 g. of lmulose 1.45 g. of potassium hydroxide were neutralised (calc. 1.40 g.). Indig&i?a Titration supem'mped on Kitone-blue Titration.-0-02084 Gram of glucose required kitone-blue equivalent to 13.6 C.C. of T i 3 (1 C.C. = 0.001916 g. Fe) which (for 2 atoms of oxygen supplied) corresponds to 0.02094 g. of glucose or 100.4%. In the superimposed indigotin titration an amount of indigotin equivalent to 6.9 C.C.of TiC1 was required corresponding (for one additional atom of oxygen) to 0.02123 g. of glucose or 101.3%. 4445%). Titre of TiCl ( 1 C.C. 3 =- x10-r TiCI, Sugar. Oxidant. (g.). g . Fe). (c.c.). Glucose ......... ......... ......... ......... ......... ......... Laehose ...... 9 , 9 9 9 9 9 , ...... ...... ...... ...... Sucrose (inv.). .. Galactose ...... Arabinose ...... Glucosamine hydrochloride Glucoesmine hydrochloride Glycuronic acid in euxanthic acid ............ 9 , 9 9 ... ... 9 ...... M I Th F R K-b M I Th F K-b M I F M I I I F I 0.03956 0.0400 0.03733 0.02029 0.0400 0~0200 0.04006 0.02023 04400 0.0390 0-0200 0.0291 0.0227 0.0227 0.0388 0-03304 0*0400 0.05232 0.02505 0-2010 27.42 18.38 19-23 19-23 19.98 19-98 20.81 19-15 19.23 19-23 19-98 15-72 20-01 20.01 17-24 18.45 20-66 19.23 17-49 20.01 26.7 40.5 36-2 19.7 12-4 12.5 48- 1 26-2 51-5 50.5 26.2 42.5 25.9 26.0 42-0 33-4 42.4 42.3 22.2 26.4 sugar %of found sugar (g.).taken. 0.03922 99.15 0.03988 99.7 0.037286 99.8 0.02032 99.8 0.03984 99.6 0.020076 100.4 0.04023 100.4 0.02016 99-7 0-03976 99.4 0.03898 99.9 0-03033 100.3 0-02914 100-1 0,02261 99-6 0.02269 99.9 0-03879 99.8 0.03300 99.8 0-03911 97.8 0.05219 99.7 0.02493 99.5 049150 45.5 M methylene-blue ; I potassium indigotintetrasulphonate ; Th thio-indigodisulphonic acid ; F potassium ferricyanide ; R rosinduline ; K-b kitone-blue 2860 KNECHT AND HIBBERT THE BEHAVIOUR OF GLUCOSE ETC.Various amounts of glucose with potassium indigotintetrasulph-onate as oxidising agent. Glucose taken C.C. of TiCl, k-). d. 0.052675 50.1 0.04214 40.2 0.02 107 20.0 0.010535 10.1 04052675 5.0 1 C.C. TiCl = Glucose found yo of glucose k.1- taken. 0.052505 99.7 0.042 1 29 99.9 0.02096 99.5 0.0 10585 100.4 0.00534 99.6 0.001956 g . Fe. Various amounts of glucose with potassium ferricyanide as oxidising agent. 0*0900 65.3 0-0798 99.8 0.0500 41.0 0.0500 100-05 0*0400 32.6 0.0399 99.7 0.0198 16.2 0.0198 100.0 0.0099 5.1 0.0099 100.0 1 C.C. TiCl = 0.002272 g.Fe. Sir mniaq. 1. It is shown that in an alkaline medium glucose lmdose, and certain other carbohydrates are oxidised to a definite degree by methylene-blue and by potassium indigotintetrasulphonate. Glucose glucosamine and galactose take up under the conditions, exactly three and lzvulose four atomic proportions of oxygen. Potassium ferricyanide can replace the dyestuffs in these estim-ations. 3. When boiled for two minutes with excess of caustic soda in an atmosphere of nitrogen both glucose and laevulose neutralise an amount of alkali corresponding exactly to the formation of two molecules of lactic acid. 3. When glucose is boiled with excess of potassium ferricyanide and caustic potash the amount of alkali neutralised (over and above that required to form potassium ferrocyanide) represents exactly three equivalents of caustic potash. In the case of Itemlose, the amount of caustic potash neutralised is approximately four equivalents. 4. Glycuronic acid can be estimated quantitatively by titration with potassium indigotintetrasulphonate. The volumetric osazone titration method may also be employed for the purpose. 5. In presence of caustic alkali glucose is also oxidised quanti-tatively by kitone-blue and by rosinduline. In the former case, two and in the latter one atomic proportion of oxygen is supplied by these dyestuffs. MUNIC~AL COLLEGE OF TECHNOLOGY, UNIVERSITY OF M~NCHESTER. [Received J d y 23rd 1925.