Spectrophotometric Determination of Glycine with 2,4,6=Trichloro-s-Triazine Shuichi Suzuki, Yutaka Hachimori, and Uichi Yaoeda Research Laboratory of Resources Utilization, Tokyo Instifute of Technology, Meguro-ku, Tokyo, 152, Japan 2,4,6-TRICHLORO-S-TRIAZlNEis employed as a colorimetric reagent for the spectrophotometric determination of Nbenzoylglycine. N-Substituted derivatives of serine, threonine, tryptophan, and glycine are positive in the color reaction. Serine, threonine, and tryptophan, however, can be easily eliminated by oxidation, without oxidizing glycine. The method can be used for the determination of glycine in the presence of other amino acids. The reaction product is stable in neutral pH, conforms to Beer's law, and has a molar absorptivity of 3.62 X IO4. The method is simple and does not require expensive equipment. N-Mono-substituted derivatives of glycine exhibit color (abbreviated following a reaction with 2,4,6-trichloro-s-triazine to TT) in buffer solution at pH 7.0. Many studies on the spectrophotometric determination of glycine have been reported. In the report of Umberger and Fiorese ( I ) , no data were given for the analysis of an amino acid mixture, and in the method of Jewell et al. (2, 3), the sample containing glycine had to be dried. Other colorimetric methods for the determination of glycine are described in the literature ( 4 ) , but they require a preliminary separation of glycine from other amino acid. The present investigation concerns the determination of glycine and N-mono-substituted derivatives of glycine with TT. The method does not require separation of glycine from other amino acids prior to testing.
EXPERIMENTAL Apparatus. The major apparatus consisted of a Shimazu recording spectrophotometer (Model SV-50) and a Towa Dempa pH meter (Model HM-SA). Reagents. A TT solution was prepared by dissolving 3 grams of TT in 100 ml of dioxane. Benzoylchloride solution was prepared by dissolving 1 gram of benzoylchloride in 25 ml of dioxane. Procedure. STEP I. One milliliter of a sample solution which contained 10-150 pg of glycine, 3 ml of 0.5M bicarbonate-carbonate buffer solution at pH 8.5, and 1 ml of benzoylchloride solution were introduced into a 20-ml test tube. After mixing, this solution was allowed to stand at room temperature for 1 hour. STEP11. One milliliter of this mixture was added to 6 ml of 0.2M phosphate buffer solution at pH 8.0. Three milliliters of TT reagent were then added to the reaction mixture with vigorous stirring until the turbid solution became transparent. Absorbance of the mixture at 382 mp was measured after 10 minutes' standing. RESULTS UV Spectra of the Product. When TT in dioxane was added to the buffer solution of N-benzoylglycine the mixture became turbid, because TT is hardly soluble in water. But (1) C. J. Urnberger and F. F. Fiorese, Clin. Chem., 9, 79 (1963). (2) R. L. Sublett and J. P. Jewell, ANAL.CHEM., 32, 1841 (1960). (3) J. P. Jewell, M. J. Morris, and R. L. Sublett, ibid., 37, 1034 (1965). (4) B. Alexander, G. L. Landrnehr, and A. M. Seligman, J. Biol. Cliem., 160,51 (1945).
0
3 50
400 Wavelength in mp
450
Figure 1. Absorption spectrum of reaction product of benzoylglycine and TT Concentration of N-benzoylglycine was 2.28 X 10-5M
N-
after stirring for 10 minutes at 25 "C,the solution took on a transparent, yellow color. The absorption spectrum of the reaction product of N-benzoylglycine with TT is shown in Figure 1. The same spectrum was obtained after standing for 3 hours at 25 "C. This product was stable in the solution from pH 3.5 to pH 11.0 at 25 "C. The molar absorptivity of this product was 3.62 X IO4 at 382 mp, the absorption maximum of the spectrum. No absorption bands of the compounds l T and N-benzoylglycine were seen in this region. Calibration Curve. The absorbance of 382 mp of known samples (ranging from 0.5 X 10-4M to 4.0 X 10-4M) of the standard solution of N-benzoylglycine was determined by the procedure of step 11. The calibration curve was a straight-line plot according to Beer's law. Reaction of TT with Various Amino Acids and Their Derivatives. The reactions of glycine and its various derivatives, other than N-benzoylglycine, with TT were carried out by the method described in the experiment. The reaction mixture of N-methyl-, N-acetyl-, N-glycyl-, and N-dimethyl-glycine with TT gave a color similar to that of N-benzoylglycine with TT, although the absorption maxima and absorptivities differed from each other as summarized in Table I. Glycine, glycylalanine, and glycyl residues in proteins gave no colored products as a result of the above reaction. N-Phthalylglycine and glycine ethyl ester also gave no colored products (see Table 11). Other amino acids and their N-benzoylderivatives were investigated as to whether they were positive in color reaction or not. Except for N-benzoyl derivatives of serine, threonine, and tryptophan, none of them produced a colored product with absorbance at from 300 to 450 mp as observed in the reaction of N-benzoylglycine with TT. The absorption
ANALYTICAL CHEMISTRY, VOL. 42, NO. 1, JANUARY 1970
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Table I. Results of Color Test with N-Substituted Derivatives of Amino Acids Compounds which gave positive color reaction, and their Am,, and molar absorptivitiesat absorption maxima Molar absorptivity at absorption Compounds A,, mp maximum, X lo4 N-Benzoylglycine 382 3.62 N-Benzoyl-L-serine 382 3.26 N-Benzoyl-L-threonine 382 1 .09 N-Benzoyl-L-try ptophan 382 3.40 3 54 1.80 N-Acetylglycine N-Acetyl-L-tryptophan 354 1.80 354 2.73 L-Alanylglycine 354 2.10 Glycylglycine N-Methylglycine 405 1.80
Table 11. Results of Color Test with Amino Acids and Their Derivatives Compounds which gave negative color reaction Amino acids Glycine, L-alanine, L-leucine, L-proline, L-serine, L-threonine, L-aspartic acid, L-asparagine, L-glutamic acid, L-glutamine, L-cysteine, L-cystine, L-methionine, L-phenylalanine, L-tyrosine, Ltryptophan, L-lysine, L-histidine, Larginine N-Benzoyl derivatives N-Benzoyl derivatives of amino acids other than those of glycine, L-serine, L-threoof amino acids nine, and L-tryptophan Derivatives of glycine N-Acetylglycine ethyl ester, N-benzoylglycine ethyl ester, N-trimethylglycine, N-phthalylglycine
maxima and absorptivities of colored products were the results of reactions of amino acid derivatives with TT. They are also summarized in Table I. Determinstion of Glycine in the Presence of Serine, Threonine, and Tryptophan. N-Benzoyl derivatives of serine, threonine, and tryptophan also gave similar absorption bands to that of N-benzoylglycine when reacted with TT. If the products serine, threonine, and tryptophan are present in the sample, then it is necessary to eliminate them before attempting to detect glycine. Serine and threonine are known to be decomposed to aldehyde and ammonium when reacted with NaI04in aqueous alkaline solution (5). In order to eliminate serine and threonine from the sample, 1 ml of NaI04 solution (1.04 X lOw3M to 1.04 x 10-2M) was added to a 1-ml mixture which consisted of equimolar serine, threonine, and glycine (1.04 X IO-3M) in aqueous solution and 2 ml of 0.5M bicarbonatecarbonate buffer solution at pH 8.5. The exhaustive elimination of serine and threonine was attained by the addition of a ten-fold excess of N a I 0 4 as compared to the contents of the summation of serine and theonine. The elimination procedure of serine and threonine should be carried out before the benzoylation procedure. In the case of measuring the glycine content in the amino acid mixture containing serine and threonine, it is necessary to use the following procedure (Step 1') in place of Step I in the experiment. (5) B. H. Nicolet and L. A. Slim, J . Bioi. Chem., 139, 687 (1941). 102
Table 111. Results of Determination of Glycine in Amino Acid Mixtures Glycine content Sample O.D. at 382 mp detected (mgiml) 1. Glycine" 0.750 0.156 2. Glycine L-serineb 0.750 0.156 3. Glycine L-threonine 0.755 0.158 4. Glycine L-serine 0.755 0.158 L-threonine 5. Sample IC 0.730 0.152 6. Sample IId 0.750 0.156 Each sample contained 1.04 X 10-3M(0.780 mgjml) of glycine. b The contents of L-serine and L-threonine in samples 2,3, and 4 were 1.04 X lO-aM, respectively. c Sample I was a mixture which contained equimolar amount of glycine, L-alanine, L-serine, L-threonine, L-lysine, L-histidine, L-arginine, L-aspartic acid, L-asparagine, L-glutamic acid, Lglutamine, L-methionine, L-proline, L-cysteine, L-phenylalanine, and L-tyrosine (each one is 2.08 X 10-4M). d Sample I1 was a mixture of glycine (1.04 X 10-3M) and a 100-fold excess of L-alanine (1.04 X 10-'M).
+ +
+
+
5
STEP 1'. One milliliter of NaI04 solution was added to 3 ml of the buffered sample solution ( 2 ml of 0.5M bicarbonate-carbonate buffer p H 8.5, and 1 ml of sample solution which contained 10-150 pg of glycine). The molar ratio of NaI04 and the summation of serine and threonine was 1O:l. The mixture was allowed t o stand for 1 hour at room temperature. Then 1 ml of benzoylchloride solution was added to the mixture, and the new mixture stood a t room temperature for 1 hour. This procedure may replace Step I in the experiment. N-Benzoyltryptophan was oxidized by H20z-dioxane in 0.5M bicarbonate solution according to the method reported previously (6). That is, 1 ml of H202-dioxane(2.5 X 10-ZM to 5.0 X 10-ZM) was added to 2 ml of the mixture of Nbenzoyltryptophan and N-benzoylglycine (2.00 X lO-*M) in 0.5M bicarbonate solution and the mixture was allowed to stand a t room temperature for one hour. After that, the procedure followed Step I1 in the experiment. The appearance of absorbance due to N-benzoyltryptophan was not observed when the sample was treated with more than 2.5 X 10-*M HzOz, but the diminishing of absorbance due to N-benzoylglycine occurred after treatment with more than 10 X 10-*MH202. These results indicated that the interference of tryptophan in the detection of glycine in the sample containing tryptophan was avoided by a treatment of H20z-dioxanebefore the addition of the TT solution. Determination of Glycine in Amino Acid Mixtures. Glycine contents in the sample which are absent from serine, threonine, and tryptophan, can be determined by the procedure described in the experiment without any further pretreatments of the sample. A mixture of glycine (1.04 X 10-3M) and a 100-fold excess alanine (1.04 X l O - ' M ) was chosen as a model sample. The result is shown in Table 111. The glycine content in this mixture was determined with an error of within However, the glycine content of the amino acid mixtures which contained either serine, threonine, or tryptophan or both, could be determined by the method as mentioned above. Samples in Table I11 were prepared as model samples, and
3z.
(6) Y. Hachimori, H. Horinishi, K. Kurihara, and K. Shibata, Biochem. Biophys. Acta, 93,346 (1964).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 1, JANUARY 1970
the quantitative analysis of glycine in these samples was carried out. These results are also shown in Table 111. The glycine contents in these samples were also determined with an error of within 3%. DISCUSSION
Dioxane was used to dissolve TT since T T is stable in dioxane. The increase in the concentration of dioxane in the reaction mixture accelerated the rate of color reaction. But a high content of dioxane in a 0.2M phosphate buffer solution at pH 8.0 resulted in precipitating phosphate and preventing the occurrence of the color reaction. The concentration of dioxane in the reaction mixture was not over 30%. The reaction mixture became turbid when TT in dioxane was added to the buffered solution at pH 8.0 but the mixture became clear as the reaction proceeded. So it was possible to measure the absorbance of the reaction mixture. In addition to N-substituted derivatives of glycine, N-substituted derivatives of serine, threonine, and tryptophan were also positive in color reaction. Only L-type amino acids and their derivatives were used in this paper, but the results obtained from DL-type amino acids and their derivatives were the same as those obtained from the L-type. Among N-substituted derivatives of glycine, N-benzoylglycine was chosen because the molar absorptivity of the color product from N-benzoylglycine and TT was the largest among the N-substituted derivatives of the glycine tested. Also, the benzoylation procedure was relatively simple.
This paper shows that N-benzoyltryptophan, when treated with the HzOz-dioxanemethod, was changed to a substance which was negative in color reaction. T o determine the glycine content in proteins, however, it was necessary to hydrolyze the protein prior to the procedure in this paper. Tryptophans in proteins were decomposed by the acid hydrolysis, so that it was not necessary to use the elimination procedure. Such organic compounds as acetylacetone, diethylmalonate, and citric acid were also positive in this color reaction. These have an active methylene group in the molecule. N-Acetylglycine ethyl ester, however, has also an active methylene group in the molecule, but this was not positive in the color reaction. It could not be concluded that a substance which had active methylene groups in the molecule was always positive in this color reaction. Three milliliters of the colored solution, the absorbance of which was at least 0.1, was required for the measurement of the glycine content by the method described in this paper. The molar absorptivity of the product obtained from Nbenzoylglycine and TT was 3.62 X lo4. Therefore, it may be possible to determine the glycine content in amino acid mixtures quantitatively if there is more than 0.63 pg of glycine in the mixture.
RECEIVED for review June 2, 1969. Accepted September 29, 1969.
Determination of Carbon in Thin Films on Steel Surfaces William R. Lee and Lynn L. Lewis Chemistry Department, Research Laboratories, General Motors Corporation, Warren, Mich. 48090
THEIMPORTANCE of the surface characteristics of materials is widely recognized. In our laboratories, for example, steel surfaces have been studied extensively because of their importance in steel-lubricant and steel-paint systems. Analytical techniques developed for characterizing steel surfaces have been used to provide quantitative data routinely for microgram quantities of sulfur, chlorine, phosphorus, and zinc on ball bearings ( I ) , and additional elements on sheet steel. No technique was available, however, for determining carbon in thin carbonaceous films of varying thickness that originate from oil, grease, etc. The determination of carbon on these surfaces presents a unique analytical challenge. Carbon is also present on and below the steel surface as carbon in solution and as metal carbides, and there is no means of directly measuring the carbon of organic origin. Therefore, the essential feature of an analytical technique is a means for selectively removing the carbon of organic origin from the surface prior to the measurement so that there will be no interference from the carbon in the steel. Previous work on determining the amount of organic residues on metal surfaces has been limited to two studies, both of which are based on heating the sample in oxygen. Combustion conditions were chosen to provide complete oxidation and removal of the organic residue, with the combustion temperature being relatively low to avoid removal of carbon (1) J. L. Johnson, Microchem. J., 8,59-68 (1964).
from the steel. Boggs and Pellissier ( 2 ) heated sheet steel at 500°C in oxygen at low pressure for 10 minutes and measured the carbon dioxide produced using a manometric technique. Solet (3) reported that carbon on the surface of nickel strip could be determined by heating the sample in oxygen at 600 "C and measuring the change of electrical conductivity of a solution whichabsorbed the carbondioxide that was formed. These combustion methods are straightforward and of adequate sensitivity, but they cannot be applied directly to steels covered with films of unknown origin; the proper conditions of time and temperature must be established beforehand for each particular sample. The following equatons illustrate the chemical reactions that are important in the combustion method: C(organie)
+
'12 0 2
= CO
(1)
+ CO M[C] + = MO + CO MO + MC = 2M + CO MC+
=
0 2
MO
(2) (3)
0 2
co +
'12 0 2
=
(4)
coz
(5)
where C(arganio) is a carbonaceous film of organic material, MC is a metal carbide, M[C] is carbon in solution in the metal, (2) W. E. Boggs and G. E. Pellissier, Muter. Res. Std., 1, 627-630 (1961). (3) I. S. Solet, ANAL.CHEM., 38, 504 (1966).
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