1032
Anal. Chem. W00, 60. 1832-1834
The result predicted or anticipated by eq 8 has been supported by experimental evidence. For example, in SEC analysis of 500 000 g mol-' polystyrene, with both columns, by measuring the detected peak heights for constant injected mass, the detected concentration ratio analogous to eq 8 was found experimentally to be
for three trials for each time regime where 0.3 is the standard deviation calculated by propagation of error of the experiand C ( t R , H S ) proportional to mental data, i.e., with C(~R,SS) detected solute peak height. Although the experimental result, eq 9, is not as optimistic as predicted by eq 6-8, the improvement in detected concentration for superspeed SEC relative to either high- or normal-speed SEC is obvious. This leads to better limits of detection in superspeed SEC, &fold in the example given, albeit with a concomitant loss of chromatographic resolution (1). Yet, this advantage in improved detectability may prove useful in many applications in which resolution is not limiting. Continued work in reducing the limiting band-broadening ratio H l v is required to further this LOD improvement, such as superheated liquid chromatography (SLC) as proposed by Antia and Horvath (7). Potentially, the numerical method of running total integration
may prove useful in optimizing quantitation of chromatographic data (10, 11). We are currently investigating these areas. Registry No. Polystyrene, 9003-53-6.
LITERATURE CITED Rem, C. N.; Synovec, R. E. Anal. Chem. 1988, 6 0 , 200-204. Grushka, E. J. Chromatogr. 1984, 316, 81-93. Erni, F. J. Chromatogr. 1083, 282, 371-383. Callis, J. 8.; Illman, D. L.; Kowalski, B. R. Anal. Chem. 1087, 59, 624A-637A. Harrison, D. J.; Yates, W. R.; Johnson, J. F. J . Appl. Polym. Sci. 1988, 31, 1393-1401. Koenig, J. L. Anal. Chem. 1987, 59, 1141A-1155A. Antia, D. F.; Horvath, C. J. Chromatogr. 1988, 435, 1-15. Karger, B. L. Snyder, L. R.; HONath, C. An Introduction to Separatlon Sclence; Wiley: New York, 1973; pp 135-137. Stewart, J. E. Proc. SPIE-Int. SOC. Opt. Eng. 1084, 529-534. Synovec. R. E.; Yeung, E. S. Anal. Chem. 1988, 58, 2093-2095. Synovec, R. E.; Yeung, E. S. Am/. Chem. 1985, 5 7 , 2182-2167.
Curtiss N. R e m Robert E. Synovec* Department of Chemistry, BG-10 Center for Process Analytical Chemistry University of Washington Seattle, Washington 98195
RECEIVED for review November 17, 1987. Accepted March 30, 1988.
Indirect Fluorescence Detection of Native Amino Acids in Capillary Zone Electrophoresis Sir: Amino acids are but one of several important classes of small chemical compounds in biological chemistry that have an inherent lack of analytically useful physical properties. Amino acids, peptides, fatty acids, sugars, many mono-, di-, and tricarboxylic acids, and phosphorylated intermediates in glycolysis and metabolism show little, if any, UV or visible absorption, fluorescence, or electrochemical activity. As the emphasis of biochemical research shifts to smaller samples where, for example, picomolar quantities of amino acids are analyzed in gas phase protein sequencing (1) or in microliter samples of the extracellular fluid of the mammalian brain (2), the analytical problem becomes even more challenging due to the small volume of sample available for analysis. The determination of the chemical composition of a single cell, which may represent the ultimate analytical challenge in biological chemistry, will only become feasible when appropriate detection systems are developed. Many modern procedures for this kind of analysis are based on chromatographic separations, where detection of the eluted analyte is only accomplished after derivatization of the sample (3). Unfortunately, chemical modifications can be time-consuming and unreliable, result in dilution of the sample, affect the separation process, and are often difficult to implement with very small sample volumes. Additionally, derivatization, by definition, permanently changes the chemical structure of the analyte, which can in turn alter its biological activity. This can be thought of as destructive detection, which may be inappropriate for many biochemical applications, such as the identification of protein or DNA fragments prior to sequencing, or in the preparative isolation and purification of biological materials. Derivatization procedures can be avoided in electrophoresis through the use of indirect detection. Instead of monitoring
the fluorescence of a derivatized analyte molecule directly, a fluoresecent ion is utilized as the main constituent of the electrophoretic buffer. An analyte, if ionic, will interact with the fluorophore and result in either displacement (for ions of like charge) or ion pairing (for oppositely charged ions) with the fluorophore, analogous to ion chromatography ( 4 ) . The signal produced in this way is independent of the spectral properties of the analyte molecule and, therefore, combines the innate sensitivity of the fluorescence technique with a much broader spectrum of analysis. This technique is wellsuited for the analysis of many biochemical compounds, since a great majority of these are charged species. In this work, laser-induced fluorescence spectroscopy is performed on-column to detect the bands separated with capillary zone electrophoresis (CZE). CZE is an instrumental form of zone electrophoresis where chemical species are separated purely on the basis of their electrophoretic mobility, since no supporting gel is utilized. Both anions and cations can be separated in the same run because of the large electroosmotic flow generated in small diameter capillaries (5,6). This technique has already been used successfully in the rapid, efficient separation of dansyl-amino acids (6-10).
EXPERIMENTAL SECTION Reagents. All chemicals were reagent grade unless otherwise noted. Amino acids (native and dansyl derivatives) were obtained from Sigma (St. Louis). Water was deionized (Millipore,Bedford, MA) and all samples were dissolved in the eluent. Apparatus. The experimental apparatus used in this work is illustrated in Figure 1. The anodic, high-voltage end of a high-voltage power supply (50 kV; Spellman Model UHR 50*150) is isolated in a Plexiglas enclosure that is interlocked to provide operator security. Additionally, an electronic timing circuit is used to control the *onntime of sample injection, where sample
0003-2700/86/0360-1832$01.50/0 0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
1833
500 Buffer
3
E
I
3
400
$300 z.
200
Detector 100
0 0
2
6
4
1”
8
10
12
14
(mtnuter)
Flgure 2. Direct fluorescence detection of 200 amol dansyl-amino acids, electropherogram (with digital filtering) of a 1-s (2 nL), 30-kV injection of lo-’ M dansyl-alanine and dansyl-glycine in a 1.1 mM phosphate buffer (pH 7.0). Electrophoresis was performed at 30 kV (1.9 PA). The dansyl fluorescence was collected between 560 and 600 nm using Corning filters 4-65 and 2-69. Flgure 1. Instrumental arrangement for capillary zone electrophoresis with laser fluorescence detection.
is introduced via electromigration of the sample solution onto the capillary (6). An untreated, 100-cm fused-silica capillary (50 pm i.d., SGE) is immersed in buffer solutions at both ends and is connected to the power supply via chrome1 wires. The current passed through the capillary is monitored continuously at the cathodic end. Samples were dissolved in the eluent. Fluorescence Detection. Detection is accomplished “oncolumn” by focusing the 325-nm line of a HeCd laser (8 mW; Liconix Model 4240) with a 4.5-cm focal length quartz lens (Melles Griot; Irvine, CA) directly onto a 50-pm spot in the fused silica capillary (after a small amount of the polyimide coating was burned away to expose the quartz capillary; final detection volume is approximately 100 pL). The capillary was mounted at Brewster’s angle to minimize scattered light and the spot was located 10 cm from the cathodic end of the tube. The resulting fluorescence is collected perpendicular to the plane containing the incident beam and the capillary by imaging the illuminated region onto a photomultiplier tube (Hamamatsu R928) with a 1OX microscope objective. In a darkened room, one can obtain a clear image of the laser beam as it passes through the capillary cell walls and the flowing liquid. For indirect detection, the salicylate fluorescence was isolated with an interference filter at 405 nm and stray light from the cell walls was minimized with spatial filtering. Data Treatment. Data were recorded on a strip chart recorder, or after analog to digital (A/D) conversion (5 Hz; Data Translation Model DT 2827) and digital storage on a personal computer (IJ3M PC-AT). The analyte bands are only a few seconds wide. This presents a unique opportunity to apply digital filtering to the raw data. Specifically, the raw data are Fourier transformed into frequency space. After linear attenuation to zero from 0.024 to 0.48 Hz, the reverse Fourier transform is performed. The signal-to-noise ratio increased by more than a factor of 2 without affecting the resolution of the bands.
RESULTS AND DISCUSSION The sensitivity of this analytical system is best illustrated by the direct fluorescence detection of an injection of 200 amol of dansyl-amino acids in a phosphate buffer (1.1mM, pH 7.0; Figure 2). The high collection efficiency and good stray light rejection of this system result in a detection limit of 10 amol (S/N of 3) of dansyl-alanine and dansyl-glycine. The separation efficiency obtained in this CZE electropherogram is also very high-over 1OOOOO theoretical plates. No change in either the elution time or the separation efficiency was observed when the phosphate buffer was replaced with 1.0 mM salicylate.
Since native amino acids are isoelectric in the pH region between 6 and 7, the pH of the buffer was increased to 9.7 to facilitate separation. At this pH, the carboxyl group is unprotonated and the amino group is near equilibrium, such that most amino acids will have a net negative charge. This is advantageous here because the electroosmotic flow is toward the cathode (opposite in direction to that of the electrophoretic velocity of the amino acids), thereby decreasing analysis time and increasing the separation efficiency (6). Additionally, a small amount of sodium carbonate was added to increase the buffer capacity of the medium and thereby improve the separation. An electropherogram of 200 fmol each of ten amino acids is shown in Figure 3a. Lower detection limits could be obtained by decreasing the concentration of the electrophoretic buffer and the analyte proportionately. This has been demonstrated earlier in liquid chromatography (11, 12). With a salicylate buffer concentration of 200 pM, the corresponding concentration of amino acids could be reduced to 10 pM (20 fmol injected, Figure 3b). It is interesting to note that the S / N ratio is approximately the same for both systems. This implies that the signal a t the lower level is not yet limited by shot noise. Unfortunately, as the buffer concentration was decreased, the conductivity of the solution decreased proportionately, and problems in electrical isolation were encountered at the lowest buffer concentrations used (200 p M 45 kV a t 0.4 pA equal 122.5 GR impedance). These electrical isolation problems must be solved to allow the use of even lower buffer concentrations, which was the limiting factor in the detection limits. The efficiency of separation was only dependent on the ratio of analyte to buffer; essentially the same efficiency was obtained at different absolute buffer concentrations, though the electroosmotic flow rate was observed to increase at lower ionic strengths. Indirect detection has been utilized successfully in many chromatographic systems ranging from ion chromatography (4, 11, 12) to reversed-phase HPLC (13, 14). Indirect fluorescence detection is particularly attractive for “oncolumn” detection in capillary systems, since the sensitivity of the fluorescence measurement is sufficient for use even at very short path lengths while absorption measurement (15) is not. Lasers offer high power at a single wavelength and their coherent, highly collimated output allows the beam to be focused to a very small size (16). Thus, low levels of an ionic, fluorescent species in the eluent can be utilized to visualize even lower levels of charged, nonfluorescing analytes. The
1834
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17,SEPTEMBER 1, 1988
n
-
2
0
I
4
8
6
io
I2
D
5
7
9
I/
Time (minutes)
Figure 3. Indirect fluorescence detection of (a) 200 fmol and (b) 20 fmol underivatized amino acids: (a) Electropherogram (raw data) of a 1-s(2 nL), 45-kV injection of lo4 M amino acids (arginine, proline, ala nine, leucine, phenylalanine, serine, tyrosine, cysteine, glutamic acid, and aspartic acid) in a buffer consisting of 1.0 mM sodium salicylate and 200 pM sodium carbonate, adjusted to pH 9.7 with 0.1 M sodium hydroxide. Electrophoresis was performed at 45 kV (2.3 @A). (b) Electropherogram (with digital filtering) of a 1-s(2 nL), 45-kV M amino acids in a buffer consisting of 200 pM sodium injection of salicylate and 40 pM sodium carbonate, adjusted to pH 9.7 with 0.1 M sodium hydroxide. Electrophoresis was performed at 45 kV (0.4 PA).
combination of these characteristics makes laser-induced fluorescence an ideal choice for indirect detection in CZE. The use of lasers for detection in CZE improved the detection limit by 2 orders of magnitude over conventional light sources, even when used with a double monochromator (7). Part of the improvement in sensitivity results from the greater collection efficiency of the imaging system used in this study. Stray light and fluorescence from the quartz capillary were also readily minimized with spatial filtering of the image produced at the photomultiplier tube. Previous studies utilizing laser illumination and fiber-optic collection of the fluorescence signal (10) reported a detection limit of 2 fmol of dansyl-amino acids injected, approximately 2 orders of magnitude higher than that obtained here. In this work, the detection limit for indirect fluorescence is about 3 orders of magnitude higher than that found for direct detection. This is due to the fact that S/Nin indirect detection is determined by the dynamic reserve of the system (ratio of the background signal to the noise on this signal (4, 11-14)). In the present case, the noise level is limited by laser flicker noise, which only allows a dynamic reserve of approximately 120 (corresponding to a detection limit ( S I N = 3) of about 0.025 the concentration of the fluorophore, or 5 pM).That is, our laser is only stable to one part in 120. The dynamic reserve can be increased dramatically (to approximately 10000) by eliminating flicker noise (which has l/f character) with high-frequency modulation of the incident light in a double beam arrangement (4). So, future work should lead to even better detection limits.
Reduction of noise levels in the laser will not only allow lower detection limits but also improve the separation. As shown previously (6), separation efficiency in CZE is optimal when the ratio of buffer to analyte is in excess of 500:l. The lower separation efficiency found in Figure 3 is probably a result of the distortion of the potential gradient within the sample zone due to overloading (17). As a consequence, not all amino acids could be separated under these conditions. As the ratio of buffer to sample concentration is increased, the potential gradient is restored to that found outside the sample zone, and improvements in the separation efficiency should be possible. In summary, indirect fluorescence detection seems ideally suited for CZE since both techniques work best with charged species. The high separation efficiency of CZE was in desperate need of a sensitive detection scheme that allows detection of a broad range of compounds whose determination would otherwise require involved derivatization procedures. Recent studies have demonstrated that CZE can be used for a broad range of analyses, ranging from monatomic ions to complete viruses and bacteria (18). The use of an indirect detection scheme should serve to increase the utility of CZE to even greater levels. Since the signal is dependent on charge displacement, we can expect similar detection limits for any charged analytes. ACKNOWLEDGMENT The authors thank William Pfeffer for sharing his results on spatial selection of the fluorescence signal. Registry No. L-ARG,74-79-3; L-PRO, 147-85-3;L-ALA,5641-7; L-LEU,61-90-5; L-PHE,63-91-2; L-SER, 56-45-1; L-TYR, 60-18-4;L-CYS,52-90-4; L-GLU,56-86-0; L-ASP,56-84-8; GLY, 56-40-6; L-MET,63-68-3. LITERATURE CITED (1) Murphy, R.; Furness, J. B.; Costa, M. J . Chromatogr. 1987, 408, 388-392. (2) Korf, J.; Venema. K. J . Neurochem. 1983, 40,946-950. (3) Lindroth, P.; Mopper, K. Anal. Chem. 1979, 51, 1687-1674. (4) Mho, S I . ; Yeung, E. S.Anal. Chem. 1985, 57,2253-2256. (5) Hjerten, S.J. Chromatogr. 1985, 347, 191-198. (6) Jorgenson, J. W. and Lukacs. K. D., Science (Washington, D.C.) 1983, 222, 266-272. (7) Green, J. S.;Jorgenson, J. W. J . Chromatogr. 1986, 352, 337-343. (8) Jorgenson, J. W. I n New Directions in Nectrophwetic Methods; Jorgenson, J. W., Phillips, M., Eds.; American Chemical Society: Washington, DC 1987; pp 182-198. (9) Gassmann, E.; Kuo, J. E.; Zare, R N. Science (Washington, DC) 1985, 230, 813-814. (IO) Gozei, P.; Gassmann, E.; Micheisen, H.; Zare, R. N. Anal. Chem. 1987, 59, 44-49. (11) Pfeffer, W. D.;Takeuchi. T.; Yeung, E. S. Chromatographia 1987, 24, 123-1 26. (12) Takeuchi, T.; Yeung. E. S. J . Chromatogr. 1986, 370, 83-92. (13) Bobbitt, D. R.; Yeung, E. S.Anal. Chem. 1984, 56, 1577-1581. (14) Takeuchi, T.; Yeung, E. S. J . Chromatogr. 1986, 366, 145-152. (15) Small, H.; Miller, T. E. Anal. Chem. 1982, 54,462-469. (16) Yeung, E. S. I n Microcolumn Separations; Novotny, M I Ed.; Elsevier: Amsterdam, 1985; pp 135-158. (17) Lukacs, K. D.; Jorgenson, J. W. HRC CC. J . High Res. Chromatogr. Chromatogr. Common. 1985, 8 , 407-411. (18) Hjerten, S.;Elenbring, K.; Kilar, F.; Liao, J.-L.; Chen. A. J. C.; Siebert. C. S.;Zhu. M.-D. J . Chromatogr. 1987, 403,47-61.
Ames Laboratory-USDOE Chemistry Iowa State University Ames, Iowa 50011
Werner G.Kuhr Edward S. Yeung* and Department of
RECEIVED for review February 23, 1988. Accepted April 26, 1988. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract No. W-7405-Eng-82. This work has supported by the Office of Health and Environmental Research.
