Determination of Amino Acids and Related Compounds in Honey R. E. LOTHROPAND S. I. GERTLER,Bureau of Chemistry and Soils, Washington, D. C.
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HEX honey is utilized In 1922 R i f f a r t (6) made a Because of interference of reducing sugars for purposes that ret h o r o u g h investigation of the with formation of the characteristic color, the quire heating to relaninhydrin test as a quantitaninhydrin test cannot be used for the detecfion tive m e t h o d f o r determining tively high temperatures, as in or the determination of amino acids and related amino acids and related comthe manufacture of candy or for compounds in honey, A method for determining pounds. He found the method baking, it is found to undergo decomposition a t temperatures to be suitable f o r d e t e c t i n g amino acids and related compounds in the somewhat below that of a mixsmall amounts of amino acids presence of reducing sugars is proposed which ture of dextrose and levulose of colorimetrically, in some cases f is based on precipitation with Neuberg's reagen approximately the same concenin a d i l u t i o n of 1 to 340,000. (mercuric acetate f sodium carbonate). The tration a s h o n e y . In other He also studied the a c t i o n of words, the caramelization temprecipitated mercury amino compounds are the r e a g e n t on a n u m b e r of perature of honey as a rule is substances that might interfere separated by filtration or centrifugation, washed, lower than that of commercial with the reaction. and the free acids recovered by decomposing invert sugar. This low caraAmbler (1) has worked o u t the mercury compounds with hydrogen sulfide. melization temperature of honey a simplified m o d i f i c a t i o n of The ninhydrin test is then applied to the resulting must also be reckoned with when Riffart's m e t h o d and applied solution. h o n e y is h e a t e d to r e t a r d it to the determination of amino granulation or prevent fermentaacids and r e 1a t e d compounds The application of the method to a number of tion. Often such t r e a t m e n t in sugar products. The preshoney samples (after removal of proteins and t e n d s to i m p a i r the' flavor, ence of sucrose does not affect other colloidal material) showed small amounts as well as to produce some disthe r e a c t i o n , and the method of amino acid nitrogen to be present in every case. coloration due to s l i g h t carais very satisfactory and reasonmelization. ably accurate. AtteniDts t o From tests conducted in this laboratory on honeys of apply the ninhydrin test direcily to honey solutions, howdifferent floral sources, considerable differences were iound ever, resulted in the development of a deep brownish red to exist among the various types with respect to their tend- color, and upon dilution no characteristic violet color was ency to caramelize when heated. The low carameliza- obtained. I n a recent article Ambler and Snider (9) showed tion temperature of honey, as pointed out in a previous that the presence of reducing sugars in appreciable amountscommunication (S), is due partly to the presence of certain namely, more than 1 mg. of levulose or 10 mg. of dextrosecolloidal substances. The colloids isolated from honey were altered the typical violet color of the reaction mixture, and found to decompose to an appreciable extent when sub- in cases where larger quantities of these sugars were present jected to temperatures above 50" C. Honey that had been the characteristic color did not appear at all on dilution. Since honey consists largely of dextrose and levulose, treated so as approximately to free it from colloids, however, was still found to be more subject to caramelization on heat- detection of amino acids cannot be carried out by applicaing than commercial invert sugar. Apparently then, other tion of the ninhydrin test directly to the honey itself, and nonsugar substances present in honey besides colloids are some means of separating the amino acids from the sugars responsible for its low caramelization temperature. is necessary. By precipitation of the amino acids from a In a recent paper (5) it was shown that, in addition to honey solution with Neuberg's reagent (mercuric acetate proteins, nitrogenous compounds of amino-acid character sodium carbonate), and subsequent treatment to remove are present in honey. Since reducing sugars apparently the free acids, a good ninhydrin test was obtained, showing interfere seriously with the development of the characteristic that failure to obtain positive tests in the above case was color of the ninhydrin reaction, it was found necessary to due to interference of reducing sugars. separate the amino acids from the reducing sugars of the The procedure for precipitating amino acids with merhoney before application of the test. curic acetate and sodium carbonate according to Neuberg Because the presence of any considerable quantities of and Kerb (4) is carried out as follows: amino acids in honey would tend to promote darkening in To the amino-acid solution made alkaline with sodium carcolor, due to melanoidin formation resulting from the reaction between amino acids and reducing sugars of the bonate is added a 25 per cent solution of mercuric acetate. More carbonate is added as necessary to neutralize the acetic honey, it was thought that differences in behavior of various sodium acid set free, and addition of mercuric acetate is continued until types of honey with respect to caramelization might be due, it causes a permanent yellow coloration of mercuric oxide. The precipitation is completed by addition of 5 t o 8 volumes of partly a t least, to variations in the amino acid content. With this consideration in mind, an attempt has been 98 per cent alcohol. The precipitate is filtered, suspended in water, the mercury removed by treatment with hydrogen made to refine the precipitation method previously reported sulfide. and The amino acids in the resulting solution can then be for the detection of amino acids in honey so that it would detected by use of the ninhydrin test. serve as a quantitative method for the estimation of amino acids and related compounds. Such a method might also Application of the procedure of Neuberg and Kerb to a be useful for the determination of these compounds in other number of honey solutions gave good tests for amino acids saccharin products similar in composition to honey. in each case. Before applying the test, the honey solutions
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ANALYTICAL EDITION
were freed from proteins and other colloidal matter by use of bentonite. Vondrhk (7) has made a study of the precipitation of amino acids from sugarhouse products. He modified the method of Neuberg and Kerb somewhat for his work. He used normal mercuric acetate and sodium carbonate solutions, and showed that by using the correct proportions of each reagent he could practically precipitate quantitatively a known amount of amino acid from a sugar solution. He also found that the relative proportions of the two reagents necessary to produce complete precipitation varied somewhat, depending on the particular amino acid under investigation.
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to give virtually complete precipitation and recovery of the added amino acids. Alcohol is added until a concentration of about 80 per cent is obtained in the solution, from which the acids are precipitated as mercury compounds. Four different amino-acid solutions were prepared of such a concentration that each cubic centimeter contained 0.025 mg. of amino nitrogen. A fifth solution was prepared consisting of equal proportions of the first four amino-acid solutions. Twenty-five cubic centimeters of each solution were added to 25-gram portions of 80 per cent invert sugar solution, and precipitation and subsequent determination of the acids carried out as described in the method finally adopted for use with honey. A blank determination was also made using 25 grams of the invert sugar solution and 25 PRECIPITATION OF AMINOACIDSFROM INVERT CC. of water. Complete recovery of the added amino acids SUGARSOLUTIONS was obtained in every case. With some of the individual Since the composition of invert sugar closely approxi- amino acids the shade of color of the ninhydrin solution mates that of honey, known quantities of amino acids were differed from that of the standard, being slightly bluer with added to pure invert sugar solutions and recovery of the glycine and alanine, and slightly redder with glutamic acid. amino acids attempted by precipitation as mercury com- The color developed by the mixed acids, however, matched pounds. Several different amino acids were used for these the standard almost exactly. It is recognized that certain amino acids such as cystine tests. Apparently, added quantities of amino acids can be completely recovered from invert sugar solutions, pro- do not develop a color with ninhydrin, so that any cystine vided the proper conditions for precipitation are observed. present in a mixture of amino acids would not be estimated Table I shows the effect of varying the amounts of sodium by this method. This is an inherent difficulty of the ninhycarbonate and mercuric acetate on the completeness of drin method of determining amino acids, however, and does precipitation of known quantities of aspartic acid from an not concern the precipitation and separation of amino acids invert sugar solution. .The precipitations were carried out from reducing sugars. I n order to determine the shade and intensity of colors in 20-cc. portions of invert sugar solution containing 1.0 mg. of added amino nitrogen in each. After filtration and developed by various individual aFino acids when treated washing, the mercury precipitates were decomposed with with ninhydrin, solutions containing equivalent concentrahydrogen sulfide to liberate the acids and the ninhydrin test tions of alanine, glycine, valine, leucine, glutamic acid, cystine, phenylalanine, tyrosine, and asparagine were tested was applied to the resulting solutions. by the ninhydrin method, and the colors compared using TABLEI. EFFECTOF VARY IN^ AMOUNTSOF REAGENTE ON aspartic acid standards. The nitrogen in each case, with COMPLETENESS OF PRECIPITATION OF ASPARTIC ACID FROM the exception of cystine, was quantitatively accounted for. INVERT SUGAR SOLUTIONS Cystine gave no color on application of the ninhydrin test. SODIUM AMIXO AMINO A mixture consisting of equivalent proportions of the nine CARBONMERCURIC COLOROF NITROQENNITROQEN ATE" ACETATE" PRECIPITATE ADDED RECOVERED RECOVERY solutions was tested in the same manner. Ninety-two per cc. cc. Mo. Mo. % cent of the amino nitrogen present was accounted for. The 0.625 62.5 White 1.000 2.0 2.0 cystine present in the mixture apparently accounts for this 0.875 87.5 White 1.000 3.0 3.0 Faint pink 1.000 1.000 100.0 4.0 4.0 low recovery of nitrogen. Very slight differences in the 1.000 100.0 Pink 1.000 5.0 5.0 shade of color of the ninhydrin solution compared with the a 1.0 N solution. aspartic acid standards were observed for some of the indiAlthough complete recovery of aspartic acid from invert vidual amino acids. The mixture of nine acids, however, sugar solutions was obtained it was found in case of cer- gave a shade which matched the aspartic acid standards tain of the other amino acids that complete recovery was almost exactly. not possible by this simplified procedure. Neuberg and METHOD Kerb (4) have pointed out that the mercury compounds of some of the amino acids have appreciable solubility in water, I n view of the foregoing results, the following method so that the acids are incompletely precipitated from a water has been used for the determination of amino acids and solution. To overcome this they precipitated the acids related compounds in honey: from an alcoholic medium, in which the mercury compounds Twenty-five grams of protein-free honey' are weighed, disare less soluble. As was found later, the addition of alcohol is necessary in order to obtain complete recovery of mix- solved in 25 cc. of water, and the solutjon mixed with 200 cc. of 95 per cent alcohol. The precipitation is carried out by adding tures of amino acids from invert sugar solutions. alternate1 from burets 1-cc. portions of 1.0 N sodium carbonate A number of these difficulties were also encountered in and fresh% prepared 1.0 N mercuric acetate After each addicarrying out the precipitation and subsequent recovery of tion the solution is tested with bromothymol blue paper, and if the amino acids. Filtration of the mercury precipitate is the solution reacts acid an additional I-cc. portion of sodium carbonate is added. The alternate addition of carbonate and slow, and it is difficult to washit free of sugars without use acetate is continued until the precipitate assumes a distinctly of excessive quantities of water. This difficulty was over- yellow or orange color, and shows a tendency to settle. The come by use of centrifugation. The precipitation of mer- final mixture should react slightly alkaline. The precipitate is cury with hydrogen sulfide was also troublesome, colloidal separated by centrifuging the mixture, pouring off the supermercuric sulfide being almost invariably formed. This was 1 The honeys used in these teste had been clarified b y treatment with overcome by addition of a small quantity of hydrochloric bentonite. Testa made on bentonite-treated honeys showed t h a t proteins acid, and precipitating while hot. Centrifugation was also were removed by the treatment. While other means of removing the proteins could be employed, bentonite treatment is recommended because used for removing the precipitated mercuric sulfide. of its simplicity. For treatment with bentonite, a 1 t o 1 honey solution The inclusion of these modifications in the method for is treated with 5 per cent bentonite suspension, 1 volume of bentonite recovering amino acids from invert sugar solutions appears suspension being used for 10 t o 16 volumes of honey solution.
March 15, 1933
INDUSTRIAL AND ENGINEERING CHEMISTRY
natant liquid and washing several times with 20-cc. portions of
80 per cent alcohol, the su ernatant liquid being poured off
after each centrifuging. Ttree such washings are usually sufficient to remove practically all the sugars. The washed precipitate is then suspended in the centrifuge tube in 50 cc. of water, and 5 drops of concentrated hydrochloric acid are added. The tube is immersed in a beaker of boiling water, and hydrogen sulfide passed in for 10 minutes, during which the beaker of water is kept near boiling temperature. The precipitate is centrifuged and the supernatant liquid poured throu h a 7-em. filter paper. The precipitate is stirred with 20 cc. of water, 2 drops of concentrated hydrochloric acid are added, and hydrogen sulfide is passed in for a second 10-minute period as above, in order t o insure complete decomposition of the mercury compounds. The precipitate is centrifuged and the liquid poured through the same filter. The precipitate is stirred up with two 20-cc. portions of water, 1 drop of concentrated hydrochloric acid being added each time to prevent formation of colloidal mercuric sulfide, and centrifuged as before. The filtrate and washings are concentrated in vucuo to about 15 cc. and transferred to a small beaker. Two drops of phenolphthalein are added and dilute sodium hydroxide added drop by drop until the solution is very faint1 alkaline. Very dilute hydrochloric acid is then added until tge solution is just colorless. At this point it should react neutral t o bromothymol blue paper. The solution is made up to 25 cc., and a 2-cc. aliquot representing 2 grams of the original sample is used for determination of amino acids by means of the ninhydrin test. The ninhydrin test is conducted according to the directions of' Ambler ( I ) , using aspartic acid as a standard. The above procedure was used for the determination of amino acids and related compounds in ten samples of honey representing various floral types. Results of these tests are shown in Table I1 The amount of amino nitrogen present varied between 0.0024 and 0.0066 per cent. Buckwheat and tarweed honeys showed relatively high aminoacid nitrogen values, whereas white clover, sage, and orange were among those showing the lowest values. I n a general way a correlation was found to exist between the amount of amino nitrogen as determined above and the tendency of the honey to caramelize on heating, the observations being made on honeys from which colloids had been eliminated. The amounts of amino nitrogen found by the above procedure account for only part of the nitrogen present in honey after proteins and other colloidal materials are removed. Owing to the small amounts present, no attempts were made to isolate individual amino acids from the mix-
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tures resulting from the mercury precipitation process. Portions of the solution, however, when treated with small amounts of glucose and warmed on the steam bath darkened very quickly and soon assumed a very dark brown color, a t the same time emitting an odor typical of that usually associated with malanoidin formation. A portion of the solution itself and a portion of glucose solution when heated separately in the same manner did not discolor. TABLE11. AMINOACID-NITROGEN CONTENT OF HONEYS OF VARIETYOF FLORAL TYPES PREDOMINANT FLORAL SOURCE OF HONEY
AMINONITROGEN 4," 7"
*
Buckwheat Sumac Cataclaw Orange Tarweed Hawaiian honeydew White sage Alfalfa Sourwood White olover
0.0050 0.0025 0.0022 0.0020 0.0055 0.0045 0.0020 0.0045 0.0025 0.0022
A
AMINONITROGEN (Dry basis) 47"
I"
0.0082 0.0030 0,0027 0.0024 0.0086 0.0058 0.0024 0.0053 0.0029 0.0028
While the results outlined here do not account for nearly all of the nitrogenous constituents of honey that are present as compounds of relatively small molecular weight, they show the existence of compounds of amino-acid character, and the influence they exert on certain properties of honey. A further study is being made of the nitrogenous constituents of honey.
LITERATURE CITED (1) Ambler, Intern. Suoar J.,29, 382, 437, 498 (1927). (2) Ambler and Snider, IND. ENO.CWEM., Anal. Ed., 4, 37 (1932). (3) Lothrop and Paine, IND. ENO.CHEM.,23, 328 (1931). (4) Neuberg and Kerb, Biochem. Z., 40, 498 (1912). ( 5 ) Paine, Gertler, and Lothrop. Unpublished paper presented before the Division of Sugar Chemistry a t the 82nd Meeting of the American Chemical Society, Buffalo, N. Y., dugust 31 to September 4, 1931. (6) Riffart, Biochem. Z., 131, 78 (1922). (7) VondrLk, 2. Zzickerend. Eechoslovak. Rep., 51, 261 (1927). RECEIVEDSeptember 1, 1932. Contribution 123 from the Carbohydrate Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. Presented before the Division of Sugar Chemistry at the 83rd Meeting of the American Chemical Society, New Orleans, La , March 28 to April 1,1032.
Effect of Alkali Treatment on the Yield of Lignin ELWINE. HARRIS,Forest Products Laboratory, Madison, Wis.
S
EVERAL investigators (9,3, 6, 7) have reported that the treatment of wood with dilute alkali removes a portion of the lignin. Their experimental work was done, however, before it was found that rigid control of temperature and time was required for an accurate determination of lignin (4, 6, 8). It was desirable, therefore, to repeat, with proper control of time and temperature conditions, the experiments previously reported in order to determine whether the decrease in lignin yield after alkali treatment was the result of the removal of lignin or of some carbohydrate material readily charred and made insoluble by the action of 72 per cent sulfuric acid. There was also the possibility that a part of the extraneous matter was not removed by the neutral solvents commonly used in the analytical method, that this residue was insoluble in the sulfuric acid and was removed by the alkali treatment, thus lowering the
apparent lignin yield. Cohen and Dadswell (1) have found that certain eucalypts contain large quantities of such extraneous material and that the lignin obtained after extraction with neutral solvents only was contaminated with extractives. In the determination of lignin in these woods they found it necessary to extract with dilute alkali in order to obtain any reasonable figures and they suggest that extraction with alkali precede the lignin determination in all cases. They state that the slight decreases observed by them in lignin yield from hemlock and spruce after preliminary treatment with alkali are also due to extraneous materials and not to the removal of true lignin. When only small differences in lignin yield are caused by alkali extraction it is difficult to decide from the lignin yields alone whether the differences are due t o the actual removal of lignin or to the removal of extractives that would otherwise
