Anal. Chem. 1987, 59, 2803-2805
2803
Subfemtomole Detection Limit for Amino Acid Determination with Laser-Induced Crossed-Beam Thermal Lens Detection Thomas G . Nolan Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831
Norman J. Dovichi* Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
Detection llmlts of 0.75 fmol of 18 (dlmethylam1no)azobenzenesulonyl derlvatlzed amlno aclds were achieved by using a 0.25-mm-dlameter reverse-phase chromatography column and a laserinduced crossed-beam thermal lens detector. A pump laser power of 150 mW at 457.9 nm was employed. At the detectlon limn, only 50 analyte molecules are expected within the probe volume at the peak maxlmum.
Recently, a number of separation techniques, including liquid chromatography, have been investigated by using small inner-diameter columns. The capillary separation techniques are interesting for their excellent mass sensitivity, low solvent consumption, and good chromatographic resolution (1-3). Unfortunately, applications of these separation techniques have been limited by detector technology; detectors must provide not only good sensitivity, to quantitate the small amount of material injected onto the column, but also low volume, to eliminate postcolumn band broadening. Lasers are convenient light sources for analysis of small volume samples since laser beams with good spatial coherence may be focused to small spots. For example, laser-induced fluorescence has been used to analyze attomole amounts of material within picoliter probe volumes (4-6). Although the high sensitivity of fluorescence is well recognized, high-sensitivity laser-based absorbance measurements also have been performed. Absorbance measurements are useful because strongly absorbing molecules are much more common than strongly fluorescent molecules. One class of absorbance techniques is based upon the thermal lens (7, 8 ) . In this technique, absorbance of a pump laser beam produces a localized temperature rise, which perturbs the sample's refractive index and defocuses or deflects a second probe beam. Since the temperature rise is proportional to the pump laser power, highly transparent materials may be analyzed with relatively high power lasers. The crossed-beam thermal lens is designed to probe small volume samples (8-15). Here, the pump and probe beams are tightly focused, cross at right angles, and are coplanar. The heated sample acts as a cylindrical lens to defocus the probe beam. The crossed-beam thermal lens offers important advantages compared with other absorbance measurements since the signal is generated only in the intersection volume of the two laser beams, is independent of path length, and is inversely proportional to the pump beam spot size ( 1 1 ) . A tighlty focused pump beam produces both high spatial resolution and high sensitivity; a detection limit of 120 iron 1 , l O phenanthroline molecules has been produced within a 0.2-pL probe volume by using a 100-mW pump laser beam (12). The high spatial resolution and excellent sensitivity of the crossed-beam thermal lens detector are matched to the requirements of capillary chromatography. Subpicomole amounts of five dinitrophenylhydrazones have been separated with a 250-pm diameter chromatographic column and'detected 0003-2700/87/0359-2803$01.50/0
by using a 2-mW pump laser (15). Since the sensitivity of the detector increases linearly with pump laser power, smaller amounts of analyte may be detected with a higher power laser. This paper reports the separation of (dimethy1amino)azobenzenesulfonyl (DABSY)derivatives of amino acids by using a capillary chromatographic column with a 150-mW argon ion laser operating at 457.9 nm. The DABSYL-amino acid derivatives are relatively easy to form, have molar absorptivity of 3 X lo4L M-' cm-' at the pump laser wavelength, and may be separated with conventional liquid chromatography (16). A preliminary study, using the crossed-beam thermal lens detector with a 1-mm-diameter chromatographic column, demonstrated the separation of five amino acids with femtomole detection limits ( 1 7). However, the relatively large volume of peaks eluted from that column dwarf the picoliter probe volume of the detector.
EXPERIMENTAL SECTION The experimental diagram for the crossed-beam thermal lens detector is shown in Figure 1. The pump laser beam is from an argon ion laser operating in the light-regulated mode at 457.9 nm with a power delivered to the sample of about 150 mW. The beam is chopped with a mechanical chopper operating at about 90 Hz. About 5% of the pump beam is split to a reference photodiode, described below. The pump beam in focused with an 18-power microscope objective. The probe beam is provided by a 1-mW, polarized helium-neon laser operating at 632.8 nm. A polarization filter and a 1/4 wave retardation plate are used to minimize retroreflections from reaching the probe laser cavity. The probe beam is focused with a 7-power microscope objective; the probe beam waist is located before the cuvette. The beam propagates through the sample about 25 cm to a mask formed by two razor blades. The mask is aligned parallel with the plane formed by the pump and probe laser beams. The transmitted light is focused onto the signal photodetector with a 50-mm-focal-length lens. Although less sophisticated than the parabolic transmission mask developed by Jansen and Harris (I@, the mask employed in this paper serves also to average spatial noise over its area while acting as a limiting aperture for the thermal lens signal. The sample is contained in a 80-pm square-bore capillary tube connected to the end of the chromatographiccolumn. The probe region is about 1-cm past the end of the column bed. A syringe pump is used to provide flow, Isco Model 393. A 60-nL injection value is connected directly to an 80 cm long, 250-rm-diameterfused-silica column packed with 5-pm-diameter (2-18 stationary phase. A flow rate of about 1-pL/min was employed. The column was prepared by mixing 0.2 g of 5-pm-diameter Spherisorb C18 bonded phase particles with 750 pL of a 1% solution of poly[(oxyethylene)(23)] lauryl ether in acetonitrile. The slurry is packed by increasing the pump pressure from 2000 to 5500 psi over a 5-min period. The columns are left at 5500 psi for 15 min, and then the pressure is allowed to reduce to ambient. Water is run through the column for 2 h at 2500 psi before the column is employed. The signal and reference photodiodes are identical 1-mm2silicon photodiodes conditioned with a current-to-voltage converter constructed from a JFET operational amplifier wired with a 1-MO feedback resister in parallel with a 470-pF capacitor. The crossed-beam thermal lens signal is demodulated with a lock-in 0 1987 American Chemical Society
2804
ANALYTICAL CHEMISTRY, VOL. 59, NO. 23, DECEMBER 1, 1987
:
:
LOCK-IN
Flgure 1. Crossed-beam thermal lens detector for capillary liquid chromatography. M 1 4 4 are front-surface mirrors. PUMP Is an argon ion laser, CHOP is a mechanical chopper, BS is a beam splltter, REF Is a referenced phot&, 18X Is a 18-power microscope objective, He-Ne Is a helium-neon laser, PF is a polarization filter, RP is a '/, wave retardation plate, 7X is a 7 power microscope objective, M Is a mask formed by two razor blades, L is a 50-mm-focal-length lens, and SIG Is the signal photodetector.
amplifier operating in the amplitude mode with a 1.25-9 time constant. The chromatogram is displayed on a strip-chart recorder. A simple gradient elution system was constructed for this separation. Flow of 40160 acetonitrile/pH 5, 0.05 M aqueous acetate buffer was provided by a syringe pump at 1wL/min. A 50-pL gradient injection value was placed in the flow stream between the pump and a 60-nL sample injection valve to produce step increases in mobile-phase strength. The acetonitrile concentration was increased in 5% increments by injecting into the gradient valve the appropriate solvent mixture at 20,36,49,60, 71,82, and 90 min after the sample injection. The final solvent composition, 75/25 acetonitrilelbuffer, was held until the separation was completed.
RESULTS AND DISCUSSION The separation of 75 fmol of 18 amino acids is shown in Figure 2. The initial peaks and peaks 3, 4,8, and 18 were
associated with the reagent blank. Peaks 13 and 15 were not identified. The remaining peaks were identified by injecting standards onto the column. All of the amino acids are separated except for histidine and leucine and valine and phenylalanine, which overlap. However, a slightly different gradient allows separation of those amino acids. A nonzero base line, observed in the chromatogram, is associated with highly colored material adsorbed upon the capillary walls. The colored material absorbs the pump laser beam a t the entrance and exit of the detector cuvette, producing localized heating. The heated regions on the cuvette act to focus the probe beam onto the photodetector, generating the background signal. The dips, occurring in the chromatogram before and after each peak, are an artifact of the lock-in amplifier, which, operating in the amplitude mode, presents the absolute value of the thermal lens signal. Elution of colored analyte forms a cylindrical thermal lens, which defocuses the probe beam out of the photodetector, generating a peak. The dips occur as the sum of the thermal focusing signal, produced by the heated windows, and the defocusing signal, produced by the heated sample, goes through zero. It is important to separate the system performance from the chemistry involved in the determination. Invariably, the chromatogram observed for the reagent blank corresponds to the injection of about 10 fmol or 2 X lo-' M of analyte. The blank signal is not surprising since no particular care was taken to minimize contamination of the reagents (19). However, if a blank is injected that consists only of the buffer, detection limits (ZO),30, of 750 am01 of analyte injected onto the column are obtained; only 50 analyte molecules would be present within the 0.2-pL probe volume at the peak maximum. Figwe 3 presents the background chromatogram used in the detection limit calculation. Two spurious peaks are observed in the chromatogram, which appear to be due to sample carry-over from previous injections. Also, a fluctuation in the background signal is observed with an approximately 13-min period. This fluctuation appears to be associated with minute fluctuations in the flow rate of the syringe pump. When operated at this very low flow rate, the position of the synchro-motor employed
am t -
g m Y
W
0
3 N -
k J a
5 - -
a
0 -
0
30
60
90
120
I50
TIME (min.) Figure 2. Chromatogram of 75 fmoi of 18 DABSYL-amino acids. Peak 1 is cysteine, peak 2 is serine, peaks 3,4, 8, and 18 are associated with the reagent blank, peak 5 is aspartic acid, peak 6 is threonine, peak 7 is glutamic acid, peak 9 is glycine, peak 10 is arginine, peak 11 is alanine, peak 12 is proline, peaks 13 and 15 are unidentified, peak 14 Is valine and phenylalanine, peak 16 is methionine, peak 17 is tryptophan, peak 19 is histidine and leucine, peak 20 is isoleucine, peak 21 is tyrosine, and peak 22 is lysine.
ANALYTICAL CHEMISTRY, VOL. 59, NO.
oc
1
I 0
I
30
I 60
I
90
I 120
I 150
I
TIME ( m i d
Figure 3. Chromatogram of the buffer blank. A higher sensitivity for the lock-in amplifier was employed for this chromatogram compared with that in Figure 2.
to drive the piston will be incremented at a very slow rate, which appears to be equal to the fluctuations in the chromatogram. Variations in flow rate can influence the photothermal signal; in fact, the crossed-beam thermal lens signal has been used to map flow rates with detector cuvettes (14, 21).
These amino acid detection limits are among the best reported in the literature. It is interesting that they were obtained with an absorbance detector. Since absorbance is a much more universal property than fluorescence, the high sensitivity of the crossed-beam thermal lens detector should find more universal application than laser-induced fluorescence detectors. Laser-induced fluorescence has been employed for detection of 25 fmol of 9-fluorenylmethyl derivatized amino acids separated by using a 250-pm-diameter column (22). Also, laser-induced fluorescence has been employed for detection of DANSY-amino acids separated by 75-pm capillary electrophoresis at the femtomole level (5). Recently, attomole detection limits were reported for the naphthalenedialdehyde derivatives of amino acids with laser induced fluorescence detection and a 1-mm-i.d. liquid chromatographic column (23). The crossed-beam thermal lens detedor produces detection limits many orders of magnitude superior to those of conventional absorbance techniques for amino acid analysis (23, 24). The small probe volume and excellent sensitivity of the crossed-beam thermal lens yield excellent performance for capillary liquid chromatography detection. This chromatographic system is not optimized. First, the gradient is only an approximation to that employed by Lin (16). An improved gradient will allow faster and better separations. Second, the chromatographic performance is not optimal. In an isocratic mode, this chromatographic system demonstrated a reduced plate height of 4. A crude highpressure pump was employed to prepare the column, which lead to nonuniform packing. Separation of amol quantities of amino acids in tens of minutes will result from both better
23, DECEMBER 1, 1987
2805
column preparation, to produce a reduced peak height of 2, and the use of 3-pm-diameter stationary phase particles, to produce greater resolution. Future work will necessarily consider improved reagent purity to decrease the reagent blank to allow routine analysis near the detection limit. Furthermore, the 250-pm-diameter column is huge compared to the 0.2-pL probe volume of the detedor. The detector volume is best matched to open tubular chromatography with a few micrometer diameter column. Open tubular chromatographic columns provide improved mass detection limits. For example, assuming that the mass detection limit scales as the square of the capillary radius, a 5-pm diameter column should produce detection limits well-below 1 amol. Application of this instrument will be found in the study and sequencing of rare peptides and proteins and the analysis of individual cells and subcellular organelles. Registry No. Cysteine, 52-90-4; serine, 56-45-1; aspartic acid, 56-84-8; threonine, 72-19-5; glutamic acid, 5646-0; glycine, 56-40-6; arginine, 7479-3; alanine, 56-41-7;proline, 147-853;valine, 72-184; phenylalanine, 63-91-2; methionine, 63-68-3; tryptophan, 73-22-3; histidine, 71-00-1;leucine, 61-90-5; isoleucine, 73-32-5; tyrosine, 60-18-4; lysine, 56-87-1.
LITERATURE CITED (1) Novotny, M. Anal. Chem. 1981, 53, 1294A-1308A. (2) Gluckman, J. C.; Hlrose, A.; McGuffln, V. L.; Novotny, M. Chromatograph& 1983. 17, 303-307. (3) Jorgenson, J. W.; Lukas, K. D. Science 1983, 222, 266-272. (4) Dovlchi, N. J.; Martin, J. C.; Jett, J. H.; Keller, R. A. Science 1983, 219, 645-647. (5) Gassmann, E.; Kuo. J. E.; a r e , R. N. Science 1985, 230, 813-815. (6) Hlrschfeld, T. Appl. Opt. 1978, 15, 2965-2966. (7) Long, M. E.; Swoford, R. L.; ,Albrech, A. C. Science 1976, 191. 183-1 85. (8) Dovlchi, N. J. CRC Crlf.Rev. Anal. Chem. 1987, 17, 357-423. (9) Dovlchl, N. J.; Nolan, T. G.; Welmer, W. A. Anal. Chem. 1984, 5 6 , 1700-1704. (10) Nolan, T. G.; Welmer, W. A.; Dovlchi, N. J. Anal. Chem. 1084, 5 6 , 1704- 1707. (11) Nolan. T. G.; Dovichl, N. J. I€€€ Circuits Devices Mag. 1988, 2 . 54-56. (12) Weimer, W. A.; Dovichl, N. J J. Appl. F'bys. 1986, 59, 255-230. (13) Burgi. D. S.; Nolan, T. G.; Rlsfelt. J. A.; Dovichl, N. J. Opt. Eng. 1984, 23,765-787. (14) Weimr, W. A.; Dovichl, N. J. Appl. Opt. 1985, 2 4 , 2981-2986. (15) Nolan, T. G.: Hart, B. K.; Dovichi, N. J. Anal. Chem. 1985, 57, 2703-2706. (16) Lin, J. K. I n CRC Handbook of HfLC for the Separation of Amino Acids, PeptMes, and Proteins; Hancock, W. S., Ed.; CRC Press: Boca Raton, FL, 1984; Vol. 1, pp 359-366. (17) Nolan, T. G.; Bornhop, D. J.; Dovlchi, N. J. JChromafogr. 1987. 384, 189-1 95. (18) Jansen, K. L.; Harris, J. M. Anal. Chem. 1985, 5 7 , 1698-1703. (19) Knecht, R.; Chang. J. Y. Anal. Chem. 1986, 5 8 , 2375-2379. (20) Knoll, J. E. J. chromatogr. Sci. 1985, 2 3 , 422-427. (21) Weimer, W. A.; Dovlchi, N. J. Appl. Specfrosc. 1985, 3 9 , 1009-1013. (22) Einarsson. S.; Folestad, S.; Josefsson, B.; Lagerkvlst, S. Anal. Chem. 1988, 5 8 , 1638-1642. (23) Roach, M. C.; Harmony, M. D. Anal. Chem. 1987, 5 9 , 411-415. (24) Bldllngmeyer, B. A.; Cohen, S.; Tarvln, T. L. J. Chromatogr. 1984, 336, 93-98. (25) Maugh, T. H. Sclence 1984, 225, 42.
RECEIVED for review March 31,1987. Accepted August 7,1987.
