Interference of Reducing Sugars in Ninhydrin Reaction for Amino Acids and Related Compounds as Applied to Carbohydrates J. A. AMBLERAND J. B. SNIDER,Bureau of Chemistry and Soils, Washington, D. C.
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N ATTEMPTING to apply the ninhydrin reaction for amino acids (1) to some samples of honey it was noted
completed as usual with 2 cc. of the phosphate buffer solution (pH 6.9) and 1 cc. of ninhydrin solution. For comparithat,although one of the honeys showed a deep blue color son, 1 gram of the sucrose was similarly dissolved in aspartic soon after the reaction mixture was placed in the boiling- acid solution and made up with the buffer and ninhydrin water bath, a t the end of the half-hour reaction period the solutions. All the tubes were heated for one-half hour, mixture was deep brownish red, and on diluting it for the and the colors produced were noted every 5 minutes. measurement, no trace of the characteristic violet color was From these experiments it is evident that reducing sugars to be found. The transitory blue color was not observed are objectionable in the ninhydrin test when more than 10 in the other samples of honey being tested, and in no case mg. of glucose or 1 mg. of fructose are present. The greater was a positive test for amino acids obtained. Because of activity of fructose was further shown by the fact that all this it seemed advisable to study the effect of d-glucose and the tubes containing this sugar, with the exception of the of d-fructose on the reaction. one containing 1 mg., developed a red color during the first I n the previous study of the application of this reaction 5 minutes of heating, whereas the tubes containing glucose, to sugars and sugarhouse products ( I ) , it was found that 4 after being heated for 10 minutes, showed merely a gradamg. of &glucose per cc., or a total of 8 mg. in the reaction tion of purple corresponding to the quantity of glucose present, mixture has no effect on the reaction. The effect of increas- and the one with 10 mg. was still purple when removed from ing proportions of both d-glucose and d-fructose has now been the bath a t the end of half an hour. . studied, and the results obtained are given in Table I. The experiment with 400 mg. of fructose and 0.20 mg. of nitrogen was watched more carefully than the others. TABLE I. EFFECTOF INCREASING PROPORTIONS OF REDUCINQ I n less than 2 minutes after it was placed in the bath it had SUGAR IN NINHYDRIN REACTION developed a deep blue color. I n about 15 minutes slight R ~ D U C I N G WT. NITROGEN SUQAR TAKENTaken Found REMARKS effervescence was noted, and the blue color gradually disMg. Me. Mg. appeared until in 25 minutes the color was a deep red. When d-Gluoose 10 0.02 0.02 No interferenoe 20 o.oz+ Slightly redder and darker than diluted to 100 cc., the color was a very dark brown with a standard 60 Redder and darker still slight purplish nuance, and the solution contained flocculated Redder and darker still 80 material. This experiment clearly indicated that fructose Redder and darker still 100 Lighter than preceding and muddy 150 destroys the blue ninhydrin-amino acid compound when the 200 Lighter than preoeding and muddy Muoh lighter and muddy 400 latter has been formed, in addition to possible destruction Pale muddy violet 500 of ninhydrin itself. This experiment also explains the for&Fructose 1 0.01 O.Ol+ Slightly redder than standard 2 O.Ol+ Slightly redder than standard mation and disappearance of the blue color in the test on 3 Redder and darker than preceding the honey mentioned in the first paragraph. Redder than preoeding 4 Redder than preoeding 5 I n testing white sugars which have become “creepy,” it Redder than preoeding $ Redder than preoeding often happens that a light brown color is obtained instead Redder than preceding i of the violet color of the ninhydrin-amino acid compound. Red-violet 9 10 0.02 Redder and darker than standard One such sugar was found to contain 4.2 per cent invert Redder and darker still 20 sugar. I n the test, since 1 gram of sugar was used, 42 mg. Similar to 600 mg. of glucose 60 Muddy yellow 80 of invert sugar were present. With low concentrations of Paler muddy yellow 100 Still paler muddy yellow 150 amino acids such as are normally encountered in white 200 Light orange sugars, this quantity of invert sugar would be sufficient to Darker orange 400 Deeper orange 500 destroy the violet color and produce a brown one. Very dark brown (see text) 400 0.20 Methods of detecting, and of possibly determining, this Gluoose + fruoSame colors as with fruotose 2 to t o s e ( l t o 1 ) 4 t o 2 0 0.01 10 mg. type of nitrogen compound in honeys are under investigation. The indicated amounts of the two sugars were weighed LITERATURE CITED into clean test tubes with sufficient amino acid-free granulated sugar to give a total weight of 1 gram of total sugars. The (1) Ambler, Intern. Sugar J.,29, 382, 437, 498 (1927). sugars were then dissolved in 2 cc. of a solution of aspartic RECEIVEDJuly 6 , 1931. Contribution 113, Carbohydrate Divisian, Bureau acid of known concentration, and the reaction mixture was of Chemistry and Soils.
RESTORATION OF BRONZE AEQIS. The practice among early Egyptians of using various materials in effective combination is well illustrated by the inlaid bronze aegis recently acquired and restored by the Boston Museum of Fine Arts. While restoration of the object was under way, analyses of the metals were made by the Chemistry Department of the Massachusetts Institute of Technology. The main body of the object consists of a copper alloy, composed chiefly of copper and lead, while the inlays are of electrum, gilded silver, and true bronze. Tho piece dates from approximately the Twenty-second Dynasty,
or about 945 B. C., and some speculation has arisen as to why a copper and lead alloy was used instead of true bronze. Although Egypt had an abundance of copper from the earliest times, tin was not known in appreciable amounts until late and was then impmted. The copper-lead compound may, therefore, hale been used for reasons of economy. On the other hand, DOWSDunham, assistant curator in charge of Egyptian Art at the Boston Museum, suggests that this pnrticular alloy may have been employed to enhance the contrast between the body aiid the d a y s , which were all originally of a golden color.
