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be related to its amount. The contribution of the aromatic hydrocarbon to the peak intensity at m / e 253 was neglected. By means of the 50-fold discrimination in the detection of aliphatic hydrocarbons and by means of the characteristic 252 to 253 ratio in peak intensities, a differentiation and quantitative determination of PAH was accomplished in diesel particulates with low amounts of aliphatic hydrocarbons, e.g., after extraction with the most efficient extraction methods. PAHs could not be determined in unextracted diesel particulates, since the amount of aliphatic hydrocarbons exceeded disproportionately the amount of PAHs in this case. Table I1 shows in the fist column the total residue amounts of PAHs and aliphatic hydrocarbons in nanogram/milligram particulates still adsorbed on the particulates after extraction, detected a t the m l e values 252 and 253. The calculated residue amounts of PAHs with the molecular mass 252 are
shown in the second column of Table 11.
LITERATURE CITED (1) Di Lorenzo, A. Chlm. Ind. (Mllan) 1973, 55 (7),573-5713, (2) Di Lorenzo, A. Proc. Int. Clean Air Congr. 4th 1977, 434-437. (3) Majer, J. R.; Reade, M. J. A. Adv. Mass Spectrom. 1970, 5 , 560-562. (4) DI Lorenzo, A. Adv. Mass Spectrom. 1980, 86, 1377-1395. (5) DI Lorenzo, A.; Masi, S.; Guerrinl, R. Rlv. Combust. 1978, 30, 46-51. (6) de Ruiter, E. Erdol Kohle, Erdgas, Pefrochem. 1985, 18, 625-629. (7) Peaden, P. A.: Lee, M. L.: Hirata, Y.: Novotnv. M. Anal. Chem. 1980. 52,2268-2271. (8) Rornanowski, T.; Funcke, W.; Konlg, J.; Balfanz, E. HRC CC, J . Hlgh Resolut. Chromatogr. 1981, 4 , 209-214.
RECEIVED for review July 14,1982. Resubmitted July 21,1983. Accepted July 21,1983. The authors wish to thank the “Fonds zur Forderung der wissenschaftlichen Forschung” for having supported this work (project no. 2388).
Determination of Amino Acids by Liquid Chromatography with Inductively Coupled Plasma Atomic Emission Spectrometric Detection Kazuo Yoshida, Tetsuya Hasegawa, and Hiroki Haraguchi* Department of Chemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
An inductively coupled plasma atomic emission spectrometer (ICP-AES) Interfaced with hlgh-performance liquld chromatography (HPLC) has been applied to the determination of amlno acids. The separation of amino acids was performed by HPLC with a cation exchange column using phosphate buffer. The ICP-AES was used as an element-selectlvedetector for HPLC by observlng C I emlsslon Intensity at 193.09 nm and S I emission Intensity at 180.73 nm. I n the detection of sulfur, a simple argon purge system was used to reduce light absorption by oxygen. The detection limits of 30-50 pg/mL and 1-3 pg/mL as amino acids were obtained by detecting emlssion intensities of carbon and sulfur, respectively.
In recent years, inductively coupled plasma atomic emission spectrometry has been investigated as the detectors for high-performance liquid chromatography (HPLC) (1-11), taking advantage of its versatile capabilities in multielement analysis, low detection limits, wide dynamic ranges, and element specificity (12, 13). The inductively coupled plasma atomic emission spectrometer (ICP-AES) interfaced with HPLC has been, however, applied mainly to analysis of organometallic and metal-coordinated compounds on account of its high sensitivities for metallic elements. From the standpoint of chromatography, it is expected that rapid and selective detection of nonmetallic elements can be made by using an element-specific detector. It has been shown that the ICP-AES is useful as an excitation source for nonmetallic elements as well as metal elements (14). Relatively little attention has been given to the detection of nonmetals, however, because these elements provide their principal resonance lines in the vacuum ultraviolet region. Kirkbright et al. has pioneered the analysis of sulfur, phosphorus, iodine,
arsenic, selenium, and mercury with ICP excited emissions at wavelengths above 170 nm (15,16). Heine et al. extended the spectral region for the measurement of nonmetallic elements with an ICP-AES, which exhibits atomic emission lines in the vacuum ultraviolet region from 120 to 185 nm (17). They described the coupling of the ICP-AES with a vacuum spectrometer and showed the relative intensities and wavelengths of the emission lines observed for oxygen, nitrogen, carbon, sulfur, chlorine, and bromine between 120 and 185 nm. The determination of sulfur with the plasmas has been applied to various sample analyses (18-22) because a low detection limit is obtained for sulfur emission a t 180.73 nm. In this paper an ICP-AES as an element-selective detector for HPLC is described, observing C I emission at 193.09 nm and S I emission a t 180.73 nm. Amino acids are analyzed quantitatively with this HPLC/ICP-AES system. EXPERIMENTAL SECTION Apparatus. A HPLCIICP-AES system for the determination of amino acids is shown in Figure 1. The HPLC system consisted of two solvent delivery pumps (Model LC-3A from Shimadzu Co.), an injection valve (Model 7125 from Rheodyne Co.), and a 4 mm i.d. X 300 mm long stainless steel column packed with a strong cation exchange resin (IEX-210 SC from Toyo Soda Co.). The step gradient elution was performed by changing the mobile solutions from pump A (0.2 M NaH2P04,pH 3.2) to pump B (0.2 M NaH2P04,pH 4.3). The column temperature was maintained at 50 “C with a column oven (Model CTO-2A from Shimadzu Co.). The sample volume injected into the column was 100 rL. The effluent outlet from the column was fed to a cross-flow nebulizer of the ICP spectrometer, which was interfaced with small diameter Teflon tubing (0.5 mm i.d. X 300 mm long). The ICP emission spectrometer consisted of an ICP torch system with a rf generator (Model ICAP-50 from Nippon Jarrell-Ash Co.), a monochromator, a photomultiplier (R 106UH from Hamamatsu TV Co.), a current amplifier (Model 427 from Keithley),and a two-channel recorder. The torch of the ICP spectrometer was modified in the hole diameter and shape of the central tube for the analysis of high
0003-2700/83/0355-2 106$01.50/0 0 I983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983
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Table I. Relative Intensity per Carbon and Sulfur Atom in Various Amino Acids with ICP-AES Detection compound
re1 intens
C 193.09 nm
L-glycine L-alanine L-aspartic acid L-cystine L-glu tamic acid L-met hio nine L-threonine L-valine S 180.73 nm L-cystine L-methionine
Figure 1. Schematic diagram of the HPLC/ICP-AES system: P.A, pump A; P.B, pump 13; MC, mixing coil; I, inlectinn valve; C, column; P, plasma; M, monochromator; PM, photomuitlpller; A, amplifier; R,
recorder. salt content solutions. The monochromator (HR lo00from Jobin Yvon Co.) was of the Czerny Turner type (focal length 1.0 m) with a holographic grating (2400 grooves/mm). The emission signals of carbon and sulfur were recorded on a two-pen strip chart recorder (Model R-10 from Rikadenki Co.). Other experimental conditions were similar to those described in the previous paper (11).
Chemicals. All the chemicals used were of analytical reagent grade. Sodium dihydrogen phosphate and phosphoric acid were purchased from Walko Chemical Co., Tokyo, arid amino acids from Ajinomoto Co., Tokyo. The standard solutiions of carbon and sulfur were prepared by dissolving tris(hydroxymethy1)aminomethane and ammonium sulfate, respectively, in distilled water. All reagents were used without further purification. RESlJLTS AND DISCUSSION Optimization of the HPLC/ICP-AES System. Experimental conditions for the ICP-AES were optimized by aspirating 100 pg/mL standard solutions of carbon as tris(hydroxymethy1)aminometharieand sulfur as ammonium sulfate. The optical observation height above the coil and carrier argon gas flow rate were set so as to obtain the maximum signalto-background ratio. The carbon emission line at 193.09 nm was chosen in the present work because t,he signal-to-background ratio at 193.09 nm was superior to that at 247.86 nm. When detecting carbon with ICP, contamination from carbon dioxide resulted from air leakage and the presence of COZ in sample solutions and the argon gas. In order to reduce the influence of carbon dioxide from air entrained into the plasma, it was preferable to observe the lower plasima position. This is because at the lower observation height, coolant argon gas separates the plasma frorn air. Therefore, the observation height was set at 11 mm above the load coil. Carbon dioxide in the sample solutions was also reduced by purging the solutions with argon gas for over 30 min before analysis. This procedure resulted in the decrease of the background carbon emission to less than 20%. When the standard solution was directly aspirated into the ICP, the detection limit ( S I N = 2) of carbon was 2 pg of C/mL, and the dynamic range was 3.5 orders of magnitude. The detection limit of carbon may be further improved by reducing the blank emission due to carbon dioxide. As for detection of sulfur, since the sensitive resonance lines exist around 180 nm, oxygen in the optical path and in the monochromator-detector housing must be excluded to protect the sulfur emissiori from absorption by molecular oxygen. In the present experiment, the optical path and the monochromator-detector housing were, as previously reported (15), purged with argon gas. Sulfur emission lineis at 180.73,182.04, and 182.63 nm were compared by nebulizing a 100 pg/mL standard solution of sulfur into the plasma. The relative intensities of sulfur lines at 180.73,182.04, and 182.63 nm were in the ratio of 100:82:29. Heine et al. (17)also examined the relative intensities of sulfur lines, and their results were close to the above ratio The most sensitive sulfur line a t 180.73 nm was used for t'he detection of sulfur in the following experiment. The obtrervation height of the plasma was optimal
1.00' 1.01 1.03 0.99 0.97 1.01 0.98 0.97 1.00' 1.01
' Normalized to 1.00. ~
-
~
~~
Table 11. Analytical Figures of Merit in Amino Acid Determination with HPLC/ICP.AES System compound
detection limit, d m L
C 193.09 nm L-alanine 40 L-aspartic acid 40 L-cystine 30 L-glutamic acid 40 L-glycine 50 L-methionine 30 L-threonine 30 L-valine 40
RSD,' %
4.4 3.2 3.5 4.1 3.0 2.9 3.9 3.5
S 180.73 nm
L-cystine L-methionine a
1
3
3.0 2.8
RSD, relative standard deviation.
a t 14 mm above the load coil in the case of sulfur. When the standard solutions were nebulized directly into the ICP-AES, the detection limit ( S I N = 2) of sulfur was 0.02 pg of S/mL, and dynamic range was 4 orders of magnitude. Citrate buffers are generally used as the mobile phase for the separation of amino acids with HPLC (23-25). Citrate solution, however, is not suitable as the buffer when an ICP-AES is used as the detector of carbon. In this study, 0.2 M sodium dihydrogen phosphate solutions were chosen as the mobile phase for the separation of amino acids. Quantitative Analysis. The relative responses per carbon and sulfur atoms for various amino acids obtained with ai HPLC/ICP-AES system are listed in Table I. These relative responses were calculated by taking the ratio of emission peak. area to number of objective atoms in each amino acid. As can be seen from Table I, the relative responses of these amino acids showed good agreement within the standard deviation These results suggest that it is possible to determine all of' these amino acids by using one calibration curve obtained with the carbon or sulfur standard solutions, when peak areas by carbon or sulfur emission intensities are measured for the determination of amino acids. The detection limits and relative standard deviations for each amino acid are summarized in Table 11. These detection limits were obtained as the concentration of each amino acid which provided a peak height corresponding to twice the base line noise level. The relative standard deviations were measured a t concentrations of 500 pg/mL of each amino acid. The calibration curves of carbon obtained by peak heights showed linear relationships in the concentration range of' 50-500 pg of C/mL for all amino acids examined. As for sulfur, the calibration curves obtained with peak heights
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983
I
C 193.09 n m
S 180.73 n m
I
f
to identify objective compounds, when peak overlappings occur. Such an example has been practically demonstrated in the case of chromatographic separation of L-valine and L-cystine. General applications of the HPLC/ICP-AES system to other compounds containing carbon or sulfur, which cannot be directly detected with a colorimetric or fluorometric method, possibly may be made. ACKNOWLEDGMENT The authors thank Keiichiro Fuwa a t the University of Tokyo for his valuable discussion and kind encouragement throughout the present experiment. Thanks are also due to Masatoshi Minami and Satoshi Ozawa for their helpful discussion in the determination of sulfur. Registry No. L-Alanine, 56-41-7; L-aspartic acid, 56-84-8; L-cystine, 56-89-3; L-glutamic acid, 56-86-0; L-glycine, 56-40-6; L-methionine, 63-68-3; L-threonine, 72-19-5; L-valine, 72-18-4. LITERATURE C I T E D
0
10
20
I
T i m e (min)
Flgure 2. Element selective chromatograms for amino acids with the HPLC/ICP-AES system obtained with carbon and sulfur detectlons: mobile phase (step gradient method), 0.2 M NaH,PO,, pH 3.2 (0-10 0.2 M NaH,PO,, pH 4.3 (10-20 min); flow rate of mobile phase, min) 1.4 mL/min; sample volume, 100 pL; sample, 50 p g of each amino acid; (a) L-aspartic acid, (b) L-threonine, (c) L-glutamic acid, (d) L-glycine, (e) L-alanine, (f) L-cystine, (9) L-valine, (h) L-methionine.
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showed linear relationships in the range of 5-500 pg of S/mL for L-cystine and L-methionine. Chromatographic Separation. Typical chromatograms obtained with a HPLC/ICP-AES system are illustrated in Figure 2, in which the separation of amino acids was performed by using a step gradient elution. These chromatograms demonstrate that amino acids are easily determined by detecting carbon or sulfur with the ICP emission spectrometric method. As can be seen in Figure 2, even when the peak of L-cystine is overlapped with the peak of L-valine in the chromatogram by carbon emission measurement, L-cystine can be determined by observing sulfur emission. This is one of advantages that resulted from the chromatograms with element-selective detection. CONCLUSIONS Generally, a colorimetric method using ninhydrin reagent and a fluorescence methods using o-phthalaldehyde (OPA) (26) or (dimethy1amino)naphthalene-1-sulfonyl chloride (Dns-C1) (27) reagent have been investigated for the determination of amino acids. The colorimetric or fluorescence methods, however, require the postcolumn reactions. In analyses with a HPLC/ICP-AES system, such reactions are not necessary, since the objective elements are directly detected by ICP-AES detection with high sensitivity. Moreover, as the ICP-AES has a capability of multielement analysis with low detection limits, multielement detection may be helpful
(1) Fraley, D. M.; Yates, D.; Manahan, S. E. Anal. Chem. 1979, 57, 2225-2229. (2) Gast, C. H.; Kraak, J. C.; Poppe, H.; Maessen, F. J. M. J . Chromatogr. 1978, 785,549-561. (3) Morlta, M.; Uehiro, T.; Fuwa, K. Anal. Chem. 1980, 52, 349-351. (4) Hausler, D. W.; Taylor, L. T. Anal. Chem. 1980, 53, 1223-1227. (5) Morlta, M.; Uehiro, T.; Fuwa, K. Anal. Chem. 1981, 53, 1806-1808. (6) Morita, M.; Uehiro, T. Anal. Chem. 1981, 53, 1997-2000. (7) Fraley, D. M.; Yates, D. A,; Manahan, S. E.; Stalling, D.;Petty, J. Appl. Spectrosc. 1981, 35, 525-531. (8) Whaley, B. S.; Snable, K. R.; Brower, R. F. Anal. Chem. 1982, 5 4 , 162-1 65. (9) Heine, D. R.; Denton, M. B.; Schlabach, T. D. Anal. Chem. 1982, 5 4 , 81-84. (10) Gardner, W. S.; Landrum, P. F.; Yates, D. A. Anal. Chem. 1982, 5 4 , 1196-1198. (11) Yoshida. K.: Haraauchi. H.: Fuwa. K. Anal. Chem. 1983. 55. 1009-1012. (12) Boumans, P. W. J. M.; de Boer, F. J. Spectrochim. Acta, Part6 1972, 278. 391-414. (13) Fassel, V. A,; Kniseley, R. N. Anal. Chem. 1974, 4 6 , 1110A-1120A. (14) Windsor, D. L.; Denton, M. B, Appl. Spectrosc. 1978, 32,336-371. (15) Kirkbright, G. F.; Ward, A. F.; West, T. S . Anal. Chim. Acta 1972, 62, 241-251. (16) Klrkbright, G. F.; Ward, A. F.; West, T. S. Anal. Chim. Acta 1973, 6 4 , 353-362. (17) Helne, D. R.; BaMs, J. S.; Denton, M. B. Appl. Spectrosc. 1980, 34, 595-598. (18) Ellebracht, S. R.; Fairless, C. M.; Manahan, S. E. Anal. Chem. 1978, 50, 1649-1651. (19) Swain, P. D.;Ellebracht, S. R. Anal. Chem. 1979, 51, 1605-1609. (20) Treybig, D. S.;Ellebracht, S.R. Anal. Chem. 1980, 52, 1633-1636. (21) . . Lee. J.: Pritchard. M. W. Soectrochim. Acta. Part 6 1981, 368, 591-594. (22) Miles, D.L.; Cook, J. M. Anal. Chim. Acta 1982, 141, 207-212. (23) Moore, S.;Spackman. D. H.; Stein, W. H. Anal. Chem. 1958, 30, 1186-1190 . .- - . .- -. (24) Spackman, D. H.; Stein, W. H.; Moore, S. Anal. Chem. 1958, 30, 1190-1 206. (25) Berridge, B. J.; Chao, W. R., Jr.; Petters, J. H. Anal. Biochem. 1971, 41, 256-264. (26) Vmagat, H.; Kucera, P. J . Chromatogr. 1982, 239, 463-474. (27) Koroleva, E. M.; Maltsev, V. G.; Belenkl, B. G. J. Chromatogr. 1982, 242, 145-152.
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RECEIVED for review March 29, 1983. Accepted July 1, 1983. This research has been supported by the Grant-in-Aid for Environmental Science (No. 57030018) from the Ministry of Education, Science and Culture, Japan (1982).
