2590
Anal. Chern. 1904, 56, 2598-2600 3
-. "
I _ _
J\Lfi -
01
Ic)
--~--_+-_---_I.
--
+_c_.-L_
0
,
02
03
~-~
04
05
-
-
06
TiME ( s e c )
Figure 3. Temporal resolution of spectral overlaps: (a) raw signal for IO" pg/mL Zn at 213.8 nm showing temporal resolution of W overlap; (b) raw signal for bare wire showing W overlap: (c) wire-corrected signal for pg/mL Zn (a - b). The lifetime of the tungsten wire loop is approximately 100 insertions. After this time precision degrades and the wire becomes brittle. Microscopic examination indicates that surface pitting becomes pronounced after such extended use. Our investigation indicates clearly that the lifetime and performance of the system is significantly degraded if the hot wire is exposed to room atmosphere after being dropped down from the plasma. Clearly this device displays considerable promise in situations which require very low levels of detection or are limited to very small sample volumes. Registry No. W, 1440-33-7; Cu, 1440-50-8; Zn, 7440-66-6.
LITERATURE CITED (1) Salin, E. D.; Horllck, G. Anal. Chem. 1979, 51, 2284. (2) Sommer, D.; Ohis, K. Fresenlus' Z. Anal. Chem. 1980, 97,304.
Horlick, G.; Pettit, W. E.; Todd, B., paper presented at the Annual Conference of The Spectroscopy Society of Canada, St.Jovite, Quebec, Canada, September 26-29, 1982, No. 10. Klrkbright, G. F.; Walton, S.J.; Analyst (London) 1982, 107,276-281. Habib, M. M.; Saiin, E. D. Anal. Chem. 1984, 56, 1186-1188. Nixon, D. E.; Fassel, V. A.; Kniseley, R. N. Anal. Chem. 1974, 46, 210. Human, H. G. C.; Scott, R. H.; Oakes, A. R.; West, C. D. Analyst(London) 1978, 701, 265. Gunn, A. M.; Millard, D. L.; Kirkbrlght, G. F. Analyst (London) 1978, 703,1006. Millard, D. L,; Shan, ti. C.; Kirkbright, G. F. Analyst (London) 1980, 705, 502. Kirkbrlght, G. F.; Snook, R. D. Anal. Chern. 1979, 57, 1938. Mermet, J. M.; Hubert, J. Prog. Anal. At. Spectrosc. 1982, 5 , 1. Crabl, G.; Cavalll, P.; Achilll, M.; Rossl, G.; Omenetto, N. At. Spectrosc. 1982, 3, 81. Axiz, A.; Broekaert, J. A. C.; Lek, F. Spectrochlrn. Acta, Part 8 1982, 378,369. Axlz, A.; Broekaert, J. A. C.; Leis, F. Spectrochlm. Acta, Part 8 1982, 378, 381. Swaldan, H. M.; Christian, G. D. Anal. Chern. 1984, 56, 120. Kltarume, E. Anal. Chem. 4983, 55, 802. Keilsohn, J. P.; Deutsch, R. D.; HleftJe,G. M. Appl. Spactrosc. 1983, 37, 101-105. LI-Xlng, Z.;Kirkbright, G. F.; Cope, M. J.; Watson, J. M. Appl. SpectrOSC. 1983, 37,250-253. Sturgeon, R. W.; Chakrabarti, C. L. Anal. Chem. 1977, 49, 90-97.
Eric D. Salin* R. L. A. Sing Department of Chemistry McGill University 801 Sherbrooke Street, West Montreal, Quebec H3A 2K6, Canada
RECEIVER for review April 30, 1984. Accepted July 9, 1984. This work was made possible by grants from the Natural Sciences and Engineering Research Council of Canada (Grant A1126) and the Government of Quebec (Fonds F.C.A.C. EQ1642). R.L.A.S. acknowledges scholarship support from the Natural Sciences and Engineering Research Council of Canada.
Separation of Amino Acid Enantiomers by High-Performance Liquid Chromatography for Stable Nitrogen and Carbon Isotopic Analyses Sir: Elucidation of the stable isotopic compositions of proteins and peptides is of increasing importance for biochemical and geochemical research. Whereas 613C and 615N values have been reported for protein constituents of modern and fossil samples (1,2),investigations of the nitrogen and carbon stable isotopic compositions of individual amino acids or specific amino acid constituents of peptide and protein hydrolyzates are quite limited (3-5). Living systems consist primarily of L-amino acids. The development of chromatographic procedures for the separation of complex mixtures of amino acids into their respective D and L enantiomers (6, 7) have, however, resulted in confirmation of the presence of free and peptide-bound D amino acids in living systems (8,9).It has also been established that, when an organism dies, its amino acid constituents begin to racemize, eventually approaching equilibrium mixtures of their respective D and L enantiomers (10, 11). This present study reports, for the first time, a procedure for the separation and determination of the stable isotopic compositions of the D and L enantiomers of amino acids. Isotopic integrity (6I3C, 615N) of the individual enantiomers was maintained throughout the separations and analyses. In addition to investigations of kinetic isotope effects, racemization processes and biosynthetic pathways for the formation
of amino acids in living systems, the procedure holds promise for resolving the oftentimes difficult assessment of the extent to which D,L amino acid constituents in a given sample are indigenous or the result of contamination (12).
EXPERIMENTAL SECTION High-Performance Liquid chromatography. An aqueous (pH 5.5), chiral mobile phase consisting of copper acetate (CuAc; 0.004 M) and N,N-di-n-propyl-L-alanine(NNDPA; 0.008 M) was prepared. The synthesis of NNDPA was identical with that previously reported (6). Replicate mixtures consisting of 1.0 mg of D-glutamic acid (D-G~u; NBC, Cleveland, OH) and 1.0 mg of L-glutamic acid (L-G~u; NBC, Cleveland, OH) were then dissolved in 0.2 mL of the c h i d mobile phase. Initial 613C and 615Nvalues for the pure starting materials, Le., D-Glu,L-G~u, NNDPA, and CuAc, are listed in Table I. One-tenth-milliliteraliquots of the D,L-GIusolutions were injected onto a 25.0 cm X 4.6 mm i.d. LC-18 reversed-phase column (Supelco, Inc., Bellefonte, PA). The resin particle size was 5.0 Wm. The column temperature was 0 "C. The D-G~u and L-G~uenantiomers were resolved isocratically with the chiral mobile phase. The buffer flow rate was 0.6 mL/min. The retention times of the amino acid enantiomers were initially established by postcolumn reaction with o-phthaldialdehyde (OPA) and subsequent fluorometricdetection (Figure 1). Next, 0.1-mL aliquots of the D,L-G~u solutions were injected onto the column, and the individual enantiomers were collected directly
0003-2700/84/0356-2598$01.50/0Q 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
Table I. Stable Isotope Values
6l6N?
61sC,*
%o
%o
-6.2 +7.9 +2.3
-25.9 -20.0 -37.4
N.P.‘
-20.3
-6.3 +2.2
-26.1 -33.5
N.P. -6.3
-20.5 -25.7
+2.4
-33.2
Pure Starting Materials L-G~u D-G~u N,N-di-n-propyl-L-alanine (NNDPA) copper acetate (CuAc)
Control Experiments A. solution drying L-G~u + acetic acid eluent NNDPA (0,008 M) + CuAc (0.004 M) B. anion exchange chromatography 1. L-G~u + CuAc fraction 1 (CuAc) fraction 2 (L-G~u) 2. NNDPA + CuAc fraction 1 (NNDPA + CuAc)
Separation of D - G ~ uand L-G~u:Chiral (HPLC) Separation Followed by Anion Exchange Chromatography 1. L-G~u + NNDPA-CuAc fraction 1 (NNDPA CuAc) fraction 2 (L-G~u) 2. D-G~u + NNDPA-CuAc fraction 1 (NNDPA + CuAc) fraction 2 (D-G~u)
+
+2.1 -6.7
-33.2 -26.0
+1.9 +8.3
-32.8 -19.9
“616N reference = air (O%O); 1 standard deviation = f0.2%0. 613C reference = NBS-22 (-29.4%0)(relative to PDB carbonate); 1 standard deviation = &0.1%0, ‘N.P., not present in original sample and not detected in isolated fractions.
mu
zs 8: Zfl 8 K
t
D-Glu L-GIu
2599
of 0.4 M acetic acid. Both fractions of D-G~u and L-G~u that were collected by HPLC were separated from the chiral mobile phase in an identical manner. Complete recovery (essentially 100%) of the individual amino acid enantiomers was confirmed by analyzing aliquots of the eluent fractions by ion-exchange chromatography, using OPA as a postcolumn derivatization reagent (16). The acetic acid fractions containing the isolated D-G~u or L-G~u were evaporated to dryness. The residues were converted to gas and analyzed for their carbon and nitrogen stable isotopic compositions using a Dumas sealed tube combustion in quartz (17, 18). The isotopic compositions are expressed as bNE%o = [Rsample/Rshdard - 11 x io3 where N is the heavier stable isotope of the element E and R is the abundance ratio of the heavy to light isotopes of the element. Values are expressed relative to the appropriate standards (PDB carbonate for 13C and atmospheric nitrogen for I5N) which are assigned a value of 0.0760on their respective scales. Control Experiments. The effects of solution drying and anion exchange chromatography on the stable isotopic composition of glutamic acid and the chiral mobile phase were evaluated. These experiments were conducted to determine whether (1) sample drying released all of the acetate and did not affect the amino acid stable isotopic compositions through decomposition and (2) any individual steps in the analyses resulted in isotopic fractionation of the samples. One-milligram samples of L-G~u were dissolved in 20.0 mL of 0.4 M acetic acid. The solutions were evaporated to dryness, and the residues were analyzed for their stable isotopic compositions. In addition to isotopic analyses of pure NNDPA and CuAc, 30.0-mL aliquots of the aqueous, chiral mobile phase (NNDPA-CuAc) were evaporated t u dryness and analyzed for their stable isotopic compositions. Next, 4.0-mL aqueous solutions consisting of L-G~u and CuAc were placed on the anion exchange column. The fractions containing CuAc and L-G~u were eluted from the column and analyzed for their stable isotopic compositionsas described above. Finally, 4.0-mL aliquots of the chiral mobile phase were placed on the anion exchange column. The chiral mobile phase was eluted from the column and analyzed for its stable isotopic composition as described above.
RESULTS AND DISCUSSION The 615N and 6I3C values for the starting materials, control experiments, and chiral separations are listed in Table I. With respect to the control experiments, solution drying and anion exchange chromatographic procedures did not alter the stable isotopic composition of Glu. As expected, the 613C value for the chiral mobile phase (NNDPA-CuAc) was intermediate t o the 613C values for the pure starting materials (NNDPA, CuAc) that were used to synthesize the chiral mobile phase. With respect to procedural blanks, nitrogen that might potentially be derived from column bleed was not detected in fractions containing only CuAc (Table I). Carbon was below detection limits in blank fractions collected from the anion exchange column before and after the elution of Glu. The potential exists, however, for carbon or nitrogen derived from bleed to be below detection limits but to affect a shift in the stable isotopic compositions of smaller samples. Such a bleed might have an isotopic composition that is similar to the reagents used in this study. Maximum contributions from column bleed with stable isotopic compositions similar or identical with the isolated D-G~uand L-G~uenantiomers may be calculated by using the observed slight deviations of the 613C and 616Nvalues of the resolved amino acids from the 613C and 615N values of the starting materials and the following isotopic mixing equation: 6mix = 6A-f S b ( l - f ) , where 6mix is the observed 6I3Cvalue for D-G~uand L-G~uafter separation, 6A is the observed 613C value for the D - G ~ uand L - G ~ upure starting materials, 6B is the hypothetical 613C value for the column bleed, f is the molar fraction of carbon present in the samples, and 1 - f is the molar fraction of carbon present in the bleed. The equation can be similarly solved for nitrogen contribution by substitution of the appropriate 615N and nitrogen molar fraction values. A maximum contribution from
+
2666
Anal. Chem. 1984, 56, 2600-2602
column bleed would be expected if the 613C and 615N values of the bleed were halfway between both the 613C and 615N values of the D-G~uand L-G~uenantiomers, i.e., 613C bleed = -22.95, 615N bleed = +0.85. If a column bleed with such an isotopic composition did exist, it would contribute a maximum of -7 pg of carbon per sample and -2 pg of nitrogen per sample. In view of the wide range of possible isotopic compositions found in natural samples, such an identical match for both isotopes for the bleed is not likely. The maximum effect of the bleed is probably much less. Nevertheless, for extremely small samples with very different stable isotopic compositions, a column bleed such as the one described above could affect the final refinement of 613C and 615N values. For such cases, the results would have to be interpreted with caution. The HPLC procedure described above resulted in the separation of underivatized D - G ~ uand L-G~u(Figure 1) in sufficient quantities (0.5 mg) for stable isotopic analyses. While 0.5 mg might be considered an excessive sample amount for injection onto an analytical column, the difference in retention time for D-G~u and L-G~u(-22 min) facilitated the complete separation and collection of the two components. The 613C and 616Nvalues for the D-G~uand L-G~ucomponents of the mixtures that were (1) separated by HPLC and (2) purified by anion exchange chromatography were, within standard error, identical with the isotopic values of the pure D - G ~ uand L-G~ustarting materials. D,L-Glutamic acid was selected for this initial investigation because of the prevalence of both enantiomers in living systems, e.g., bacterial cell walls, and in fossil materials. The procedure, however, affords a method for obtaining 613C and 615N values for the enantiomers of any of the protein or nonprotein amino acids that can be resolved with HPLC using chiral mobile phases (6, 12). Protein and peptide hydrolyzates contain complex mixtures of L amino acids and, in some instances, substantial quantities of the D enantiomers. Peak coelutions are, however, often a serious problem when attempting to analyze a complex mixture of amino acid enantiomers with HPLC (6). Prior to using the procedures described in this report to isolate the enantiomers of an amino acid for isotopic analyses, it may be necessary t o separate the amino acid in question from a complex mixture that results, for example, during hydrolysis. HPLC procedures have been developed that utilize volatile aqueous buffers to isolate individual, underivatized amino acids from complex mixtures (5, 15). The anion exchange method presented for the separation of D-G~u and L-G~u from the chiral mobile phase (NNDPA) could require modification for the separation of, e.g., neutral amino acid enantiomers from NNDPA. This may be done by (1) raising the initial pH of the anion exchange column or (2) using an alternative cation exchange method (e.g., ref 5) to accomplish the final separation of the amino acid enantiomers from the chiral mobile phase. It is anticipated that the application of these procedures in
conjunction with the procedure described in this report will eventually result in a routine method for the isotopic analysis of the individual enantiomers of any amino acid constituent of a protein or peptide hydrolyzate. ACKNOWLEDGMENT We wish to thank P. E. Hare, T. C. Hoering, and M. L. F. Estep, Geophysical Laboratory, Carnegie Institution of Washington, for providing us with the facilities to conduct this study. Registry No. I5N,14390-96-6;13C,14762-74-4;D-G~u, 6893-26-1; L-G~u, 56-86-0; DL-G~u, 617-65-2. LITERATURE C I T E D DeNiro, M. J.; Epstein, S. Geochim. Cosmochim. Acta 1981, 45, 341-351. Chlsholm, B. S.; Nelson, 0.E.; Schwarcz, H. P. Science 1382, 216, 1131-1 132. Macko, S. A,; Estep, M. L. F.; Engel, M. H.;Hare, P. E. Year BookCarnegie Inst. Washington 1982, 81,417-422. Hare, P. E.; Estep, M. L. F. Year Book-Carnegle Inst. Washington 1983, 82, 410-414. Macko, S. A.; Estep, M. L. F.; Hare, P. E.; Hoering, T. C. Year BookCarnegie Inst. Washington 1983, 82, 404-410. Weinstein, S.; Engel, M. H.; Hare, P. E. Anal. Biochem. 1982, 121, 370-377. Engel, M. H.; Hare, P. E. I n “Chemistry and Blochemlstry of the Amino Aclds”; Barrett, G. C., Ed.; Chapman and Hall: London, 1984; pp 462-479. Corrigan, J. J. Science 1989, 164, 142-149. Williams, D. H. Eighth American Peptide Symposium, Tucson, AZ, 1983, 185 (abstract). Schroeder, R. A.; Bada, J. L. Earth Sci. Rev. 1976, 12, 347-391. Williams, K. M.; Smith, G. G. Origins Life 1978, 8, 91-144. Engel, M. H.; Nagy, B. Nature (London) 1982, 296, 837-840. Abelson, P. H.; Hoering, T. C. R o c . Natl. Acad. Sci. U.S.A. 1961, 47., 623-632 -~ --Gaebler, 0. H.; Vittl, T. G.; Vukmirovich, R . Can. J. Biochem. 1986, 44. 1249-1257. Engel, M. H.; Hare, P. E. Year Book-Carnegie Inst. Washington 1981, 80, 394-397. Hare, P. E.; St. John, P. A.; Engel, M. H. I n “Chemistry and Biochemistry of the Amino Aclds”; Barrett. G. C., Ed., Chaoman and Hall: London, 1984; pp 415-425. Macko, S. A,; Lee, W. Y.;Parker, P. L. J. Exp. Mar. Bioi. Ecoi. 1982, 63, 145-149. Macko, S. A.; Estep, M. L. F.; Hoering, T. C. Year Book-Carnegie Inst. Washington 1982, 8 1 , 413-417.
’
Present address: Memorial University, Department of Earth Sciences, St. John’s, Newfoundland, Canada A I B 3x5.
Michael H. Engel* School of Geology and Geophysics 830 Van Vleet Oval The University of Oklahoma Norman, Oklahoma 73019 Stephen A. Macko’ Geophysical Laboratory Carnegie Institution of Washington 2801 Upton St., N.W. Washington, D.C. 20008 RECEIVED for review January 24,1984. Accepted July 2,1984.
Temperature Programming and Flow Rates in Capillary Gas Chromatography Sir: In a recent article in this journal, Jones et al. (1) discussed the combined effect of temperature program rate and carrier gas flow rate on the efficiency of capillary gas chromatographic separations. They employed a mathematical approach to infer optimum combination of flow rate and program rate values from a series of collected data from
different compound classes at different temperature program and flow rates. There are several aspects of this article that require some comment. In the first instance, the overall impression created by the article is that within each temperature-programmed run the carrier gas flow rate was held constant, due to the repeated
0003-2700/84/0356-2600$01.50/00 1984 American Chemical Society
