Anal. Chem. 1989, 61, 37-40 (13) J. K.: Moffatt. D. J.; Mantsch, H. H.; Cameron, D. G. Appl. . . KauDDlnen. Spe%osc. W81, 35, 271. (14) Marquardt, D. W.; Bennett, R. G.; Burrell. E. J. J . Mol. Spectrosc. 1961, 7, 269. (15) Stelgstra, H.; Jansen, A. P. Anal. Chlm. Acta 1987, 793, 269. (18) Woodruff, H. 8.; Lowery, S. R.; Rkter, G. L.; Isenhour, T. L. Anal. Chem. 1975, 47. 2027.
37
(17) Lowery, S. R.; Isenhow, T. L. J . Chem. Inf. Compuf. Scl. 1975, 75, 212. (18) Dessy, R. E. Anal. Chem. 1984, 56, 1200A. (19) Johnson, D. J.; Compton, D. A. C. Spectroscopy 1988. 3, 47.
RECEIVED for review July 11,1988. Accepted October 3,1988.
Attomole Amino Acid Determination by Capillary Zone Electrophoresis with Thermooptical Absorbance Detection Margaret Yu and Norman J. Dovichi* Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
Attomole quantlties of 4-(dimethylamlno)azobenzene-4’sulfonyl chloride derivatized amino acids are separated by using capillary zone eiectrophoresls In a mixed acetonltrlie/ aqueous buffer system. Detectlon Is performed with an oncolumn thermooptical absorbance detection technique based on a 130mW argon Ion pump laser. Detection llmlts for the concentratlon of analyte Injected onto the column range from 5 X lo4 M for methlonine to 5 X lo-’ M for aspartlc acid. Only 37 a d of methlonine and 450 am01 of aspartlc acid are contained within the subnanollter lnjectlon volume. I t is Interesting to note that these ilmits are a factor of 4 superlor to the best fluorescence detectlon ihnlt reported for chromatographlc separatlon of amlno acids. A subnanollter sample of derivatked human urine was analyzed with thls technique; quantltles of amino aclds contalned within the sample are 3 orders of magnltude greater than the detectlon limit.
The determination of minute quantities of amino acids is an important problem in biological chemistry. Perhaps the most important example comes from the determination of the amino acid sequence of peptide fragments associated with certain rare proteins. This protein sequence information may, in turn, be used in the determination of the DNA sequence and then the location of the gene that encodes the protein. Other examples of amino acid analysis are found in the determination of the total amino acid content of proteins, the detection of amino acids in physiological fluids in the diagnosis of certain metabolic disorders, and the determination of the nutritional value of foods. In general, the amino acids are separated by using a chromatographic or electrophoretic technique and detected spectroscopically. High-sensitivity detection of amino acids usually requires the utilization of a labeling technique. Underivatized amino acids do not absorb visible or near-ultraviolet light. Of course, amino acids may be detected by utilizing refractive index or refractive index gradient detection; however, the detection limit of the refractive index techniques is limited to about M (I). On the other hand, superior detection limits for amino acid analysis are anticipated by formation of either strongly absorbing or fluorescent amino acid derivatives. To date, most of the best amino acid detection limits reported have been those obtained with fluorescence derivatization reagents. Low- to subfemtomole 0003-2700/89/0361-0037$01.50/0
amino acid analyses have been reported using laser-induced fluorescence detection of 9-fluorenylmethylchloroformate (2), 5-dimethylaminonaphthalene-1-sulfonylchloride (DANSYL) (3),and naphthalenedialdehyde (4)derivatives. Two absorbance derivatizing reagents are also used in amino acid analysis: phenyl isothiocyanate (PITC) and 4-(dimethylamino)azobenzene-4’-sulfonyl(DABSYL) chloride (5, 6). PITC is the reagent utilized in the classic Edman degradation scheme for protein sequencing. The derivatized amino acids are detected a t 254 nm. With conventional transmission determination and liquid chromatographic mol of PITC separation, detection limits on the order of amino acid are obtained (5). DABSYL chloride is an interesting reagent for amino acid analysis (6-10). The DABSYL amino acid derivatives have large molar absorptivity in the blue portion of the spectrum, e 2 X lo4 L mol-I cm-I at h 450 nm (9). The spectral properties of the derivatives offer several important advantages for sensitive detection compared with those of PITC. First, the higher molar absorptivity of the DABSYL derivatives produces a proportional improvement in sensitivity and detection limit. Second, the longer wavelength excitation for DABSYL results in a significant reduction in background signal compared with that observed upon excitation at 254 nm for PITC derivatives. Third, the wavelength of maximum absorbance matches well the emission wavelength of both the helium-cadmium and argon ion lasers; high-sensitivity laser-based absorbance techniques may be employed for detection. This research group has been interested in the detection of DABSYL amino acids since 1985 (11-13). The fortuitous overlap of the absorbance spectrum of the derivatives with the 442-nm line of the helium-cadmium laser and the 457.9-nm line of the argon ion laser proved useful in the development of thermooptical detection. The high-sensitivity absorbance detection produced by crossed-beam thermooptical techniques has resulted in very good detection limits. Femtomole amino acid analysis was provided by thermooptical detection and microbore liquid chromatography. Improvements in mass detection followed from use of microbore (0.25-mm-diameter column) liquid chromatography, resulting in amino acid detection limits of 0.75 fmol. Crossed-beam thermooptical detectors provide very small probe volume, typically on the order of a few picoliters. To match better the volume of the detector to that of the separation, it is appropriate to investigate capillary separations.
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Recently, we reported on the combination of capillary zone electrophoresis with crossed-beam thermal lens detection for subfemtomole detection of DABSYL amino acids (13). In that work, several different solvent systems were investigated to optimize the separation of 18 common amino acids. A solvent system of an equal volume mixture of acetonitrile and an aqueous pH 7 phosphate buffer with 5 mM sodium dodecyl sulfate additive produced the separation of all common amino acids except for isoleucine and valine, which coelute, and cysteine and tyrosine, which also coelute. However, those amino acids may be separated in a pH 7 buffer system with neither acetonitrile nor sodium dodecyl sulfate additive. Detection limits for the system were 200 amol of glycine injected onto the column. The sensitivity for thermooptical techniques increases linearly with pump laser power (14,15). The early amino acid analysis technique utilized a low-cost and very reliable 4-mW helium-cadmium laser to provide the pump beam. In the present work, the pump laser is replaced with a 130-mW argon ion laser: the increased sensitivity of the ion laser should produce improved detection limits compared with those of the earlier work. Also, this article presents the result of the application of the capillary zone electrophoresis system for the analysis of human urine for amino acids.
EXPERIMENTAL SECTION The instrument used in the current separation is identical with that employed in earlier work, with one exception: An argon ion laser, operating at 458 nm, is used to provide a 130-mW pump beam (13,16). In brief, the inatrument is constructed from a 1-mW helium-neon probe laser, operating at 632.8 nm, focused with a 16-mm-focal-length microscope objective. The pump beam is chopped mechanically at 47 Hz and focused with a 18-mm-focal-length microscope objective into the capillary. The probe beam intensity is monitored with a small-area silicon photocell. The output of the photocell is conditioned with a current-to-voltage converter (1 Mi2 feedback resistor in parallel with a 47-pF capacitor) and demodulated with a dual-phase lock-in amplifier operating in the amplitude mode with a 1-9 time constant. Alignment of the system differs slightly from that utilized earlier. In this instrument, both the pump and probe laser beams are focused in the capillary tube. The transmitted beam,in both cases, is nearly circular in cross section. The photodiode is located off the beam center at approximately the e-l point in probe beam center intensity. This alignment condition is particularly simple to produce and requires only one critical alignment step: The offset between the pump and probe beams must be varied in small increments to optimize the thermooptical signal. Electrophoresis is driven by a 27000-V power supply and performed in a 115-cm length of 50zm-inner-diameter polyamide-coated fused silica capillary tube. Relatively thin wall tubing is employed with 185-pm outer diameter. The polyamide coating is burned from the detector end with a gentle flame. Thermooptical detection occurs about 10 cm from the ground end of the electrophoresis capillary. The high-voltage, positive electrode is encased in a Plexiglas shield equipped with a safety interlock. At both ends of the capillary, electrical connections to the capillary are made with platinum electrodes immersed in small-volume reservoirs. Injection is performed by using electromigration. At each 5-kV injection applied for 5 s, slightly less than 1 nL of each amino acid is injected, on average (17). The separation buffer is an equal volume mixture of acetonitrile and pH 7, 20 mM phosphate buffer, which contained 5 mM sodium dodecyl sulfate. Eighteen different DABSYL amino acids are prepared by a standard procedure (9). A 1WmL stock solution M DABSYL chloride (Sigma) is prepared in of about 6 X acetone. A M stock solution of each amino acid is prepared in a 0.05 M chloroform saturated aqueous carbonate buffer at pH 8.9. Dilutions and mixtures of the amino acids are prepared in the carbonate buffer. The DABSYL chloride and amino acid solutions are stored at 4 O C . One milliliter of the amino acid solution reacts with 1 mL of DABSYL chloride reagent for 5-15 min at ca. 75 OC in a water bath under gentle stirring. When the reaction is complete, the solution changes color from red to
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Flgure 1. Electropherogram of 18 DABSYL amino acids, 2.5 X lo-' M injected: A, arginine; B, histidine; C, lysine; D, cysteine and tyrosine; E, tryptophan; F, proline; G, phenylalanine; H, leucine; I, methionine; J, isoleucine and valine; K, tyrosine and serine; L, alanine, M, glycine; R, reagent peaks; N, glutamic acid; 0, aspartic acid.
yellow-orange. The solution is evaporated to dryness with a stream of nitrogen gas. The solution is reconstituted with 1mL of distilled water. The derivatized amino acids are stored at -10 "C and thawed and diluted before use.
RESULTS AND DISCUSSION Capillary zone electrophoresis offers advantages for the separation of ions (18,19). The narrow-diameter flow chamber of the capillary facilitates the transfer of heat from the capillary to the environment. Reduction of the temperature rise produced by Joule heating allows use of high applied potential to drive the electrophoresis; since the separation efficiency of the electrophoresis system is proportional to the applied potential, much higher separation efficiency is produced in capillary zone electrophoresis compared with that of conventional electrophoretic systems. Figure 1presents an electrophoretic separation of 18 amino acids. The concentration of each amino acid injected into the capillary is 2.5 X lo4 M. At an injection voltage of 5000 V for 5 s and assuming a linear relationship between applied voltage and flow rate, within the average injection volume of 0.7 nL is 1.7 fmol of each component. Of the 18 aminO acids injected, all are separated except isoleucine and valine, which coelute, and cysteine and tyrosine, which also coelute. However, those amino acids may be separated in a p H 7 buffer system with neither acetonitrile nor sodium dodecyl sulfate additive. A derivative-shaped peak occurs immediately before the arginine peak. This feature is observed consistently in all of the high-sensitivity electropherograms. One possible interpretation of the data is based upon a Schilierin effect ( I ) ; the buffer that contains the sample is slightly different in composition than the electrophoresis buffer due to the presence of traces of carbonate and bicarbonate ions used in the syntheses of the derivatives. These carbonate ions will migrate as a narrow plug through the electrophoretic system. As the ions pass the detector region, the probe laser beam will be deflected slightly on the basis of the concentration gradient of the ions. Although this deflection is not modulated, a significant frequency component will pass through the 3-s time constant of the lock-in amplifier, generating the derivativeshaped peak. Also, a number of spurious peaks are observed in the electropherograms, presumably due to the derivatization of a number of trace impurities in the samples. The capillary zone electrophoresis system produces good separation efficiency. The number of theoretical plates ranges from 140000 for early eluting peaks, such as that for histidine, to 280000 plates for late eluting peaks, such as that for aspartic acid. In fact, the peak width for the amino acids is roughly constant across the electropherogram. For the particular experiment shown in Figure 1, a 3-s time constant was employed on the lock-in amplifier. This time constant, in turn,
ANALYTICAL CHEMISTRY, VOL. 61, NO. 1, JANUARY 1, 1989
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would produce an upper bound of roughly 150000 theoretical plates for a peak with a 20-min retention time. It appears that a shorter lock-in amplifier time constant is required to produce a very large number of theoretical plates in this system. Of course, decreasing the lock-in amplifier time constant will also increase the noise of the measurement. Figure 2 presents the separation of a sample that had been diluted by a factor of 4 compared with that of Figure 1;anal@ concentration injected into the column was 6.25 X lo-' M. Again, a base-line offset is noted slightly before the arginine peak. Although the concentration of this sample is lower than that of Figure 1,greater care was taken in sample preparation; few spurious peaks are noted in the electropherogram. The sloping base line in the electropherogram may be due to a change in temperature within the column. Detection limits for the system are calculated with the method of Knoll (20). Of course, the detection limit may be expressed in several different units. In this article, we are primarily interested in the amount of amino acid required for the analysis; the results refer to the amount of amino acid contained within the injected volume. For example, the concentration detection limit, 3a, for the injected analyte ranges from (5-50) x M of the amino acids. Within the injection volume, only 40 amol of early eluting peaks such as glycine and methionine and 400 amol for late eluting amino acids, such as aspartic acid, are present a t the detection limit. The detection limit for most of the amino acids is less than 100 amol injected onto the column. On the order of 3 fg of glycine in a 4 ppb solution is injected at the detection limit. The signal is linear for at least 3 orders of magnitude over the entire range of concentrations injected for all of the amino acids, from the detection limit to at least lo4 M; correlation coefficients range from 0.9999 for histidine to 0.962 for glutamic acid. Higher concentrations of analyte are expected to give rise to column overload and reduced separation efficiency. These detection limits refer to the amount of analyte injected onto the column. I t is interesting to calculate the absorbance detection limit of the thermooptical system. During the course of separation, of course, the analyte is diluted. For example, the peak concentration of glycine in the detector and at the detection limit is only 3 X 10" M (21). Identifying the capillary diameter with the path length of the absorbance determination and using a value of 2 X lo4L mol-' cm-' for molar absorptivity of the DABSYL derivatives at the
Figure 3. Electropherogram of derivatized human urine: A, arginine: B, histidine; C, lysine: D, cysteine and tyrosine; E, tryptophan: F, proline;
0, phenylalanine and leucine; H, methionine; I, isoleucine and valine: J,
alanine; K, glycine: L, glutamic acid: M, aspartic acid.
Table I. Detection Limits for Several Amino Acids Given as a Comparison of Capillary Zone Electrophoresis and Thermooptical Detection with State-of-the-Art Liquid Chromatography and Fluorescence Detection.
amino acid methionine glycine alanine tryptophan glutamic acid aspartic acid
detection limit, amo14 thermooptical fluorescence detectionb detectionC 37 42 58 71 81 421
420 240 300 540 240 240
Detection limits correspond to the amount of reagent injected onto the column that produces a signal that is 3 times larger than the standard deviation of the background signal. bPresent work, detection limit calculated by using the method of Knoll (18). Reference 4. Note that the detection limits reported in ref 4 are based upon a S I N ratio of 2: those results have been multiplied by 1.5 for comparison with the current results. laser wavelength, the absorbance detection limit of the system This absorbance detection limit is roughly 1000 is 3 X times superior to that for conventional transmission measurements and reflects the high sensitivity produced by thermooptical absorbance techniques for small-volume analysis. As an example of the analysis of a physiological sample, Figure 3 presents the electropherogram produced when dilute human urine is labeled with DABSYL and injected onto the column. The peaks are labeled in accordance with the observed retention time from the amino acid standards. Several peaks are observed in the electropherogram with retention times that do not correspond to those of the amino acids. Most likely, these additional peaks are produced by peptides and proteins present in the sample. Although the sample was diluted 1:4 with buffer, the analyte peaks are roughly lo4 timeshigher than the detection limit. Only 200 pL of urine was injected onto the column; very small natural samples may be analyzed. These detection limits are among the best for amino acid determination. Table I lists the detection limit for the system along with the best published fluorescence detection limit for chromatographic separation of several representative amino acids ( 4 ) . The fluorescence detector system used both a high-power laser to excited fluorescence (900 mW at 457 nm)
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and a cooled photomultiplier tube with photon counting for detection. Similar detection limits have been produced for amino acid detection using capillary zone electrophoresis and laser-induced fluorescence detection (3). The present system was a thermooptical absorbance detector with a 130-mW pump beam. It may be surprising to find that an absorbance detection technique gives rise to detection limits that are superior to those produced by state-of-the-art fluorescence measurements in liquid chromatography (3, 4). To achieve good mass detection limits, it is useful to utilize a separation technique within narrow-bore columns. However, fluorescence measurements within narrow-diameter capillary tubes are a challenging task. Reflection and refraction at the curved capillary surface lead to the formation of a fan of scattered laser light in the plane perpendicular to the tube (22,23). This scattered laser light acts as a major source of background signal in fluorescence measurements. On the other hand, the scattered laser light is easily eliminated from the thermooptical measurement and produces an insignificant contribution to the background signal. The high sensitivity of crossed-beam thermooptical absorbance detectors results in excellent detection capabilities when very small volume samples must be analyzed. It should be noted that recent work in this laboratory has demonstrated subattomole amino acid analysis using laser-induced fluorescence detection in the sheath flow cuvette and capillary zone electrophoresis separation (24);to obtain superior detection limits in fluorescence, it appears necessary to utilize a flow cell with excellent optical characteristics. Future work will evaluate the performance of capillary zone electrophoresis and thermooptical detection for detection of the 4(dimethylamino)azobemene-4’-isothiocyanate(DABITC) amino acid derivatives (25, 26). The isothiocyanate group allows use of DABITC in an Edman degradation scheme of proteins. However, to apply the DABITC reagent with capillary zone electrophoresis for protein sequence determination, it will be necessary to match the sample volume produced in the degradation step with the volume required for electrophoresis. One approach is to concentrate the sample produced in the degradation step to match the requirements of the electrophoresis. Unfortunately, concentration of the sample will lead to a concomitant concentration of impurities. It may prove necessary to redesign the degradation system to match the nanoliter sample volume required by the electrophoresis. If the method is successful, attomole quantities of proteins may be sequenced, a 3 orders of magnitude improvement over current capabilities in amino acid analysis.
Since submission of this paper, several reports have appeared that describe high-sensitivity fluorescence detection of amino acids in capillary zone electrophoresis. Jorgenson reported 3-am01 detection limits for labeled amino acids (27). Kuhr and Yeung report detection limits of 10 amol for the DANSYL derivative of alanine and glycine (28). Last, this research group has obtained detection limits of 0.009 amol for the fluorescein isothiocyanate derivative of amino acids (24). At least for the last two cases, the fluorescence detector was designed to minimize the effects of scattered light at the capillary walls.
ACKNOWLEDGMENT We thank Edward Yeung for supplying information about recent work in fluorescence detection of amino acids.
LITERATURE CITED (1) Pawliszyn, J. J. L i q . Chromatcgr. 1987, 70, 3377-3392. (2) Einarsson, S.; Folestad, S.;Josefsson. B.; Lagerkvist, S.Anal. Chem. 1988, 58, 1638-1643. (3) Gozel, P.; Gassmann, E.; Michelsen, H.; Zare, R. N. Anal. Chem. 1987, 59,44-49. (4) Roach, M. C.; Harmony, M. D. Anal. Chem. 1987, 59, 411-425. (5) Bidllngmeyer, B. A.; Cohen, S.A.; Tarvin, T. L. J. Chromatogr. 1984, 336, 93-104. (6) Chang, J-Y.; Knecht, R.; Braun, D. G. Biochem. J . 1981, 547-555. (7) Knecht, R.; Chang, J-Y. Anal. Chem. 1988, 58,2375-2379. (8) Lin, J-K.; Wang, C-H. Clin. Chem. 1980, 579-583. (9) Lin, J-K.; Chang, J-Y. Anal. Chern. 1975, 4 7 , 1634-1638. (10) Lin, J-K.; Lai, C-C. Anal. Chem. 1880, 5 2 , 630-635. (11) Nolan, T. G.; Hart, B. K.; Dovlchi, N. J. Anal. Chem. 1985, 57, 2703-2705. (12) Nolan, T. G.; Dovichi, N. J. Anal. Chem. 1987, 59, 2803-2805. (13) Yu, M.; Dovichl, N. J. Mikrochim. Acta, in press. (14) Nolan, T. G.;Weimer, W. A.; Dovichi, N. J. Anal. Chem. 1984, 56, 1704-1707. (15) Dovichi, N. J. Prcg. Anal. Spectrosc. 1988, 7 1 , 179-207. (16) Bornhop, D. J.; Dovichi, N. J. Anal. Chem. 1987, 5 9 , 1632-1634. (17) Huang, X.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1988, 60, 377-380. (18) Mlkkers, F. E. P.; Everaert. F. M.; Verbeggen, Th. P. E. M. J. Chromatogr. 1979, 7669, 11-15. (19) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 5 3 , 1298-1302. (20) Knoll, J. E. J. Chromatogr. Scl. 1986, 23, 422-425. (21) Meyer, V. R. J. Chromatogr. 1985, 344, 197-209. (22) Folstad, S.;Johnston, L.; Josefsson, B.; Gab, B. Anal. Chem. 1982, 54, 925-929. (23) Lyons, J. W.; Faulkner, L. R. Anal. Chem. 1982, 54, 1960-1964. (24) Cheng, Y. F.; Dovichi, N. J. Sclence 1988, 242, 582-564. (25) Chang, J. Y.; Creaser, E. H.; Bentley, K. W. Biochem. J. 1978, 753, 607-61 1. (26) Chang, J-Y. Biochem. J. 1981, 199, 557-564. (27) Jorgenson, J. Paper presented at the I X International Symposium on Capillary Chromatography, Monterey, CA, May 16-19, 1988. (28) Kuhr, W. G.; Yeung, E. S.Anal. Chem. 1988, 6 0 , 1832-1834.
RECEIVED for review June 2,1988. Accepted October 3,1988. This work was funded by the Natural Sciences and Engineering Research Council of Canada.
