On the proteid reaction of Adamkiewicz : with contributions to the chemistry of glyoxylic acid / by F. Gowland Hopkins and Sydney W. Cole.

  • Frederick Hopkins
Date:
[1901]
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Licence: Public Domain Mark

Credit: On the proteid reaction of Adamkiewicz : with contributions to the chemistry of glyoxylic acid / by F. Gowland Hopkins and Sydney W. Cole. Source: Wellcome Collection.

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    3 was heated direct on the water bath till all evolution of CO. had ceased, and then cooled. A trace’of Witte’s peptone was added to the solution, which was free from excess of peroxide, and then strong sulphuric acid. An intense colour reaction was obtained exactly similar to that seen in a normal Adamkiewicz reaction when carried out with acetic acid rich in the chromogenic substance. The solution gave also in the spectroscope an exactly similar absorption band. We found subsequently, however, that glycollic aldehyde, isolated, either in the syrupy or crystalline condition,* and whether in aqueous or acetic acid solution, gave no colour reaction under like conditions, but yielded only a charred product when the sulphuric acid was added. Moreover, acetic acid, however rich in the chromogenic substance, never reduces (after neutralising) alkaline copper solutions. A reduc- tion of ammoniacal silver solutions may be obtained, but never any effect upon Fehling’s solution. : We came to the conclusion, therefore, that the substance sought must be an oxidation product of glycollic aldehyde; and we now found that the latter needs only to be treated by Fenton’s oxidation method carried out at the temperature of the water bath to give a product, which, when free from excess of peroxide, yields in acetic or aqueous solution the proteid reaction abundantly. At this time we made another observation of the greatest assistance to our inquiry, finding that the chromogenic substance is produced in abundance when oxalic acid is reduced in aqueous solution by means of zine and sulphuric acid, or, more conveniently, by sodium amalgam. The ypduction need last for a few minutes only, and a little of the solution, without further treatment, will then be found to give an intense colour with proteid and sulphuric acid, the product showing spectroscopic appearances identical with those of the ordinary Adam- kiewicz reaction. There was now no doubt that a colour reaction, not to be dis- tinguished from that of Adamkiewicz, is yielded by a substance which is at once an oxidation product of glycollic aldehyde and a reduction product of oxalic acid. It was difficult to see how this substance could be other than glycollic acid, glyoxylic acid, or glyoxal. Pure glycollic acid was obtained from Merck. It gave no trace of a colour reaction with proteid solution and sulphuric acid. The product of its oxidation by Fenton’s method reacted, however, in a perfectly typical manner, and Fenton and Jones have found that this product is glyoxylic acid. The latter was therefore prepared from alcohol by the method of Debus. The calcium glyoxylate first obtained was recrystallised thrice. A minute crystal of the salt dissolyed in water, together with a little proteid, gave, upon the addition of strong sulphurie acid, a vivid colour reaction not to be distinguished, spectroscopically or other- wise, from the reaction of Adamkiewicz. Glyoxal, prepared subsequently from the products of the same Debus oxidation, gave no trace of such a reaction.t When glyoxylic acid is added to glacial acetic acid, previously deprived of its chromo- genic power by distillation, further distillation now yields a distillate which reacts typically, and the glyoxylic acid comes-over charac- teristically, like the original chromogenic substance in the earlier fractions. IIL. Glyorylic Acid from Acetic Acid. It now became necessary fo ascertain whether glyoxylic acid is, as a matter of fact, present in such specimens of acetic acid as yield the Adamkiewicz reaction. In seeking for evidence as to this, it was necessary to remember that exceedingly little glyoxylic acid is necessary to the reaction. With an aqueous solution of such strength as will give no more than an opalescence with phenyl hydrazine, the colour reaction with proteid 4s well marked. It was found, however, that oxidation with hydrogen peroxide confers abundant chromogenic power upon acetic acid previously giving no proteid reaction ; and it was our first endeavour to ascertain whether, as a result of this, glyoxylic acid is produced in quantity sufficient for its easier identification. The presence of small quantities of ferrous iron accelerates the oxidation, and is, perhaps, essential to it.t The process occurs most rapidly at boiling temperature, and proceeds most satisfactorily when the acetic acid is repeatedly distilled with the peroxide. The limit of the oxidation is in any case soon reached. Using twenty volumes strength, the peroxide is found to be rapidly destroyed till a volume * Fenton and Jackson, ‘Journ. Chem. Soc.,’ vol. 75, p. 575, 1899. We were indebted to Mr. Hy. Jackson for a supply of the crystalline aldehyde. + Many specimens of commercial glyoxal give the reaction, but only, as we have found, when they contain glyoxylic acid; preparations of glycollic acid may contain traces of the latter, : t We huve found that some specimens of peroxide bring about the oxidation without the addition of iron; others undoubtedly act much less readily, unless a ferrous salt is added. While we have been unable to detect the presence of iron in the former, so small a quantity appears to affect the reaction that it is possible a tiace of the metal present as an impurity may account for the difference. 4 has been added about equal to that of the acetic acid taken ; after this the reaction becomes very slow. We proceeded as follows :—A litre of glacial acetic acid was mixed with an equal bulk of twenty-volume peroxide and some ammonio ferrous sulphate added (half a gramme per litre, or less). The mixture was slowly distilled nearly to dryness, and the distillate returned and again distilled. The second or third distillate usually showed freedom from peroxide when tested with chromic acid; if not, distillation was repeated. One-tenth of the final distillate was set aside, and the remainder neutralised with potash. The still acid portion being then mixed with the rest, the whole was distilled as low as possible, avoiding, however, any separation of potassium acetate in the retort. The distillate always gave an abundant proteid reaction, and if any trace of free peroxide had been left at the previous stage, it always disappeared during the distillation of the partially-neutralised mixture as just described. A small trace of free peroxide will interfere with the proteid reaction. On adding phenyl hydrazine hydrochloride (without acetate) to the distillate thus obtained, a light yellow precipitate begins to separate almost at once, and after standing it becomes con- siderable in amount, and is crystalline. But although, as we were able to show, the hydrazone of glyoxylic acid is present in this precipitate, it is mixed with a considerable proportion of a compound much less soluble in acetic ether and in hot water. If the original precipitate be recrystallised from a minimal quantity of acetic ether, the substance which separates first consists of perfectly colourless glistening plates, which after recrystallising from acetic ether may assume the form of rosetted prismatic needles. These melt sharply at 184°. The nature of this substance became clear after the publication of certain recent observations. Gerhard Ollendorff has shown that formic aldehyde is formed when glycollic acid is oxidised with per- oxide of hydrogen, and Fenton* calls attention to the fact that gly- oxylie acid must in this case be the intermediate product. The product we obtained from acetic acid was undoubtedly the compound of formaldehyde described by Wellington and Tollens.+ A portion repeatedly recrystallised from acetic ether and showing a constant melting point (184°) was analysed. 0-147 gramme gave 27°4 c.c. moist N, at 12°,and 758mm. N = 22-07 per cent. Another preparation, recrystallised from a mixture of alcohol and toluol, melted at 182—183°; of this 0-211 gramme gave 39°3 ¢.c. moist N, at 14°, and 758 mm. N = 21°87 per cent. Calculated for ie IL. C,sHisN,. 22°07 21-87 22°22 This hydrazone can be obtained pure in the above manner with great ease if not more than 4 to 5 grammes of phenylhydrazine hydro- chloride have been added to the final distillate obtained after oxidising, as above, 1 litre of acetic acid, nearly neutralising the mixture and distilling. We prepared the compound from formaldehyde, and found it to agree with our product in every particular. Formaldehyde certainly does not yield the proteid reaction, and its formation when acetic acid is treated as described seems to be in itself evidence for the formation of glyoxylie acid during the process, as it is difficult to see how it could arise during the oxidation of acetic acid if not from a preliminary formation of glyoxylic acid with subsequent loss of carbon dioxide. But its formation adds greatly to the difficulty of obtaining pure the hydrazone of glyoxylic acid itself, especially as the precipitate produced by phenylhydrazine undoubtedly contains, in addition to the compound of Wellington and Tollens, smaller amounts of the deriva- tives described by J. W. Walker. After the nature of this bye-product was recognised we modified our procedure by neglecting the earlier portions of the final distillate which contains, of course, the greater part of the formaldehyde. Phenylhydrazine hydrochloride added to the latter half, or two-thirds, of such a distillate yields a precipitate which forms more slowly than that obtained when the whole is dealt with. After twenty-four hours it is usually crystalline and of a yellow colour, growing darker with further standing. We found it easier to obtain a product with a constant melting point by recrystallising from hot water rather than from an organic solvent, prolonged heating with the water being at any stage avoided. This treatment involves considerable loss, however, and we obtained only about 4 decigrammes of the hydrazone after oxidising 3 litres of acetic acid. This, however, had all the characters of glyoxylic hydrazone, and melted sharply at 137°. 0-204 gramme gave 30-4 ¢.c. moist N at 16° and 750mm. N = 17:14 per cent. Calculated for CsHs0.N. = 17:07. * Fenton, ‘Journ. Chem. Soc.,’ 1900, vol. 77, p. 1296. + ‘Deutsch. Chem. Ges. Bericlite,’ 1835, vol. 18, p. 3330
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