Anal. Chem. 1994,66, 2630-2635

Chiral Detection in High-Performance Liquid Chromatography by Vibrational Circular Dichroism Chieu D. Tran,' Victor I. Grlshko, and Gullan Huang Department of Chemistry, Marquette Universw, Milwaukee, Wisconsin 53233

A novel chiral detector for high-performance liquid chroma- tography has been developed. This detector is based on the measurement of circular dichroism of chiral effluents in the infrared region, Le., vibrational circular dichroism (VCD). In this instrument, a solid-state spectral tunable (from 2.4 to 3.5 bm) F-center laser was used as the light source. The linearly polarized laser beam was converted into left circularly polarized light (LCPL) and right circularly polarized light (RCPL) at 42 kHz by means of a photoelastic modulator. The intensity of the LCPL and RCPL transmitted through the sample was measured by a liquid nitrogen cooled indium antimonide detector. Double modulation was employed to reduce the noise associated with the laser beam. Specifically, the linearly polarized laser beam, prior to being converted to CPL, was modulated at 85 Hz by a mechanical chopper. Demodulation and amplification were accomplished with the use of two lock- in amplifiers. In its present configuration, the instrument can be used to measure the VCD of 0-H groups. Its sensitivity is so high that it was able, for the first time, to detect chirally (with limits of detection of micrograms) (R)- and (S)-2,2,2- trifluoro-l-(9-anthryI)ethanoland (R)- and (@-benzoin when these compounds were chromatographically separated from the corresponding racemic mixtures by a Chiralcel-OD column. The main advantage of this chiral detector is, however, its universality; Le., it can be used to virtually detect any chiral compounds which has 0-H group (e.g, aliphatic alcohols such as 2-octanol).

Chirooptical analysis has increasingly become an important subject in science as well as in technology. The popularity or rather demand stems from the differences in the physiological and therapeutic effects of enantiomeric forms of many compounds.I4 Very often, only one form of enantiomer is pharmacologically active.14 The other or others can reverse or otherwise limit the effect of the desired enantiomer.I4 For example, methadone has three to five stereoselective receptors which can stimulate differing physiological response^.^ Pro- pranolol, which is one of the most widely used @-blockers, exits in two enantiomeric forms. The enantiomer which is most active in correcting ventricular arrhythmias is much less active as a @-blocker.6 Other examples include the fact that one of the isomers of labetolol is an effective 8-blocker while another is thought to be an a-blocker.6 However, in spite of

(1) Purdie, N. Prog. Anal. Spectrosc. 1987, 10, 345-358. (2) Purdie, N.; Swallows, K. A. AMI. Chem. 1989, 61, 77A-89A. (3) Bobbitt, D. R. In Analytical Applications of Circular Dichroism; Purdie, N.,

Brittain, H. G., Eds.; Elsevier: New York. 1994; Chapter 2, pp 15-52. (4) Chem. Eng. News 1990,68 (19). 38-44; 1992, 70 (28). 46-79. ( 5 ) Armstrong, D. W.; Ward, T. J.; Armstrong, R. D.; Beedey, T. E. Science

( 6 ) Tran, C. D.; Dotlich, J . Chem. Educ., in prcss. 1986, 232, 1132-1 135.

this knowledge, only 61 of the 528 chiral synthetic drugs are marketed as single enantiomers, while the other 467 are sold as racemates4 It is thus hardly surprising that the pharma- ceutical industry needs effective analytical and preparative chiral separation methods.

High-performance liquid chromatography (HPLC) seems to be the instrument of choice for chiral separation because of its efficiency, speed, wide applicability and reproducibility. Various approaches have been made in the last few years to use the HPLC for optical resolution of racemic mixtures.'-' It is now possible to quantitatively perform optical resolution on a number of racemates by LC.7-'1

As the LC separation of enantiomers becomes more prevalent, the demand for detectors that provide information on the chirality of the eluted solutes increases. An ideal detector would be one that produces a complete circular dichroism (CD) spectrum of the eluted solute with about the same sensitivity and speed as a current chromatographic detector.'* These requirements impose severe restrictions on the use of the conventional circular dichroism spectropola- rimeter as a chromatographic detector.12 This is because the CD signals are not only very small but also difficult to measure. Specifically, even at its maximum value, the CD signal is only about 1C2 of an absorbance unit. Furthermore, because it is measured as a small ac signal riding on top of a large dc signal (total absorption), its minimum detectable value is about 10-6 of an absorbance unit.'3 It is thus evidently clear that the sensitivity of the circular dichroism measurements has to be substantially enhanced in order for it to be used as a chiral detector for HPLC. Two different approaches can, in principle, be used, either separately or synergistically, to enhance the sensitivity of the CD measurements. They are based on the use of either novel measurement principles that are entirely different from the conventional transmission measurements or novel instrumentation design that does not rely on conventional optical and electronics components.

The first approach stems from the fact that conventional CD measurements are based on the transmission measure- ments. The sensitivity of these instruments is relatively low because the measured signals are not proportional to the excitation light power. Higher sensitivity can, in principle,

(7) Armstrong, D. W.; Han, S . M. CRC Crit. Rev. Anal. Chem. 1988.19.175-

(8) Hinze, W. L. Sep. Pur$ Methods 1981, I O , 159-237. (9) Armstrong, D. W. Anal. Chem. 1987,59, 84A-91A.

224.

(IO) Armstrong, D. W. Sep. Pur$ Methods 1985, 14, 213-304. ( I 1) H i m , W.L.,Armstrong,D. W.,Eds.OrderedMediainChemicalSeporations;

ACS Symposium Series 342; American Chemical Society: Washington, DC, 1987.

(12) Zukowski, J.; Tang, Y.; Bcrthod, A.; Armstrong, D. W. Anal. Chim. Acra 1992,258, 83-92.

(13) Rau, H. Chem. Rev. 1983,83, 535-547.

2630 Analytical Chemistry, Vol. 66, No. 17, September 1, 1994 0003-2700i94/0366-2630$04.50/0 0 1994 American Chemical Society

be achieved if the CD signals are measured by techniques whose signal intensities are proportional to the light power. In fact, it has been demonstrated recently, that circular dichroism can be sensitively measured by use of either the fluorescence,Iel6 the thermal lens,I7-I9 or the degenerate four- wave mixingZo techniques. The sensitivities of the fluorescence detected circular dichroism ( FDCD),le16 the thermal lens circular dichroism (TL-CD),17-19 and the degenerated four- wave mixing circular dichroism (DGFM-CD)20 were so high that they were able to detect optically active (+)Co(en)3+ at the picogram level.

The second approach is based on the exploitation of the superior properties of lasers; in comparison with conventional light sources, lasers can provide such advantages as higher power, better beam collimation and focusing, and higher degree of spectral and polarization purity. A circular dichroism spectropolarimeter equipped with a laser as light source can, therefore, have better a signal-to-noise ratio and, hence, can be used as a chiral detector for HPLC. In fact, chiral detection of HPLC has been successfully achieved by laser-based circular dichroism spectrop~larimeters.~~-~~

The reported chiral detectors have proven to be effective. Unfortunately, they all suffer limitations; namely, they can only be used for certain types of chiral compounds. Specif- ically, because these detectors are based on the measurement of the CD of the sample in the visible region, they are limited to color chiral samples. Because of the differences in the transparency and anisotropic properties of optical components in the visible and UV region, and also because of the instability of laser in the deep-UV region, it has not been possible to develop a chiral detector for the UV region. Moreover, a UV chiral detector, if it were developed, would not be a universal chiral detector because certain types of chiral compounds, including saturated chiral compounds, do not absorb in the UV region. A detector based on circular dichroism measure- ment in the infrared region can, in principle, be an universal chiral detector because virtually all chiral compounds absorb in this region. Even though the vibrational circular dichroism (VCD) has been theoretically predicted and experimentally verified for some its application to chiral detection has not been realized for a variety of factors, including the

low sensitivity of the present IR spectrometer and the even lower sensitivity of the VCD. The first limitation, Le., low sensitivity of the present IR techniques, is mainly due to the low output power of IR sources and the low signal-to-noise ratio of IR detectors. The intensity of the IR light can be substantially enhanced if an IR laser such as a F-center laser is used instead of the conventional Nernst glower. The F-center laser is a solid-state laser which can be tuned spectrally from 2.4 to 3.5 pm. The advantages of the F-center laser, as compared to the Nernst glowers, include higher output intensity (the higher intensity of the laser used in this work was as 45 mW), better beam collimating, and higher degree of polarization. Substantial improvement can be achieved when these advantages are fully exploited: (1) the well- collimated, small-diameter laser beam can be modulated at higher frequency and sensitively detected by a small-diameter IR detector (which has faster response time than conventional large detectors); (2) this laser-based spectrometer is particu- larly suited for samples that have small volume and low concentration (e.g., detector for HPLC); and (3) the well- collimated, highly polarized monochromatic laser beam can be converted completely to circularly polarized light. This enables thelaser-based VCD spectropolarimeter to have higher sensitivity and lower noise as compared to spectropolarimeters that are based on conventional light sources. Additional improvement can also be achieved by use of the low-noise liquid nitrogen cooled indium antimonide IR detector, the low-noise amplifier, and the photoelastic modulator, which has larger acceptance angle and operates at high frequency.

The information presented is indeed provocative and clearly indicates that it is possible to develop a novel chiral detector for HPLC that is truly universal. Such considerations prompted us to initiate this study, which aims to develop a chiral HPLC detector based on the circular dichroism measurements in the IR region. In this paper we will describe the instrumentation development of the first vibrational circular dichroism spectropolarimeter, which is based on use of the F-center laser as the light source, and its application as a chiral detector for HPLC. Preliminary results on the use of this chiral detector on various chiral compounds separated by a Chiralcel-OD chiral column will be reported.

(14) Thomas, M. P.; Patonay, G.; Warner, I. M. Rev. Sci. Instrum. 1986, 57,

(15) Synovcc, R. E.; Yeung, E. S. J . Chromatogr. 1986, 368, 85-93. (16) Christensen, P. L.; Yeung, E. S. Anal. Chem. 1989, 61, 1344-1347. (17) Tran, C. D.; Xu, M. Rev. Sci. Instrum. 1989, 60, 3207-3211. (18) Xu, M.; Tran, C. D. Appl. Spectrosc. 1990, 44, 962-966. (19) Xu, M.; Tran, C. D. Anul. Chem. 1990, 62, 2467-2471. (20) Nunes, J. A.; Tong, W . G. Anal. Chem. 1993.65, 2990-2994. (21) Synovec, R. E.; Yeung, E. S. Anal. Chem. 1985, 57, 2606-2610. (22) Chan, K. C.; Yeung, E. J . Chromargr. 1989,457, 421-426. (23) Rosenzweig, 2.; Yeung, E. S. Appl. Spectrosc. 1993, 47, 2017-2021. (24) Nafie, L. A.; Keiderling, T. A.; Stephens, P. J. J . Am. Chem. SOC. 1976.98,

(25) Diem, M.; Roberts, G. M.; Lee, 0.; Barlow, A. Appl. Spectrosc. 1988, 42, 20-27.

(26) Osborne, G . A,; Cheng, J. C.; Stephens, P. J. Rev. Sci. Instrum. 1973, 44, 10-15.

(27) Freedman, T. B.; Nafie, L. A. In Merhods in Enzymology; Riordan, J. F., Vallcc, B. L., Us.; Academic: New York, 1993; Vol. 226, Chapter 13, pp 470482.

(28) Polavarapu, P. L. In VibrationulSpectra andSrrucrure; Durig, J. R., Sullivan, J. F., Eds., Elsevier: Amsterdam, 1989; Vol. 17B, pp 319-342.

(29) Diem, M. In VibrationalSpectra andStructure; Durig, J. R., Sullivan, J. F., Eds.; Elsevier: Amsterdam, 1990: Vol. 19, pp 1-54.

(30) Nafie, L. A.; Citra, M.; Ragunathan, N.; Yu, G. S.; Che, D. In Analyrical Applications of Circular Dichroism; Purdie, N., Brittain, H. G., Eds.; Elsevier: New York, 1994; Chapter 3, pp 53-89,

1308-1 3 13.

271 5-2723.

EXPER I MENTAL SECTION Figure 1 shows the schematic diagram of the chiral detector

based on vibrational circular dichroism. As illustrated, in this instrument, the IR radiation was provided by an F-center laser (Burleigh Corp., Rochester, NY, Model FCL-10). This infrared solid-state laser, which has output radiation from 2.4 to 3.5 pm, was optically pumped by a krypton laser (Coherent Corp., Palo Alto, CA, Model Innova 70K). The F-center laser was spectrally tuned by use of a computer-controlled stepping motor to rotate the intracavity grating. Because there are of several Brewster angles inside the laser cavity, the output infrared beam is well polarized in the horizontal plane. A lithium niobate polarizer was used, however, to ensure that the beam is completely linearly polarized. The linearly polarized beam was then converted into circularly polarized light and modulated between left circularly polarized light (LCPL) and right circularly polarized light (RCPL) at 42 kHz by means of a photoelastic modulator (PEM, Hinds

Analytical Chemistty, Vol. 66, No. 17, September I , 1994 2691

Kr laser Wavenumber, cm'

I ' J 1.5 3700 3650 3600 3550

J I

t I

Figure 1. Schematic diagram of the vibrational circular dichroism detector for HPLC: PEM, photoelastic modulator; LIA, lock-in amplifier; Preamp, preamplifier.

Instrument, Hillsboro, OR, Model FSA-IR). The chiral light then went through the sample cell, which in this case was a chromatographic microflow cell having a volume of 8 pL and a path length of 8 mm. The light transmitted from the sample was detected by an indium antimonide detector housed in a liquid nitrogen cooled dewar (Graseby Infrared, Orlando, FL, Model IS- 1 .O mounted in a DMSL- 12 dewar). To reduce the noise of the infrared laser beam and to increase the signal- to-noise ratio, a mechanical chopper (Stanford Research Systems Model SR450) was placed between the F-center laser and the polarizer to modulate the IR beam at 85 Hz. Two lock-in amplifiers were used to acquire data in a manner similar to those used by others.2426 Specifically, the output of the In-Sb detector, after being amplified by a low-noise pream- plifier (Princeton Applied Research Model 5 1 13), was connected to a Stanford Research System Model SR 5 10 lock- in amplifier (LIA 1). This lock-in amplifier demodulated and amplified the signal at the frequency of the PEM (i.e., 42 kHz). Its output was then connected to a second lock-in amplifier (LIA 2, Princeton Applied Research Model 5207) whose reference signal was set at 85 Hz (i.e., the frequency of the chopper). The output of this second lock-in amplifier was then connected to a microcomputer through the A/D of the 12-bit interface board (MetraByte DAS-16) to acquire and to record the data.

A Shimadzu isocratic pump (Model LC-600) was used to deliver the eluent. The sample was injected into the system through a Rheodyne Model 7125 sample injection valve equipped with a 20-pL loop. A 4.6 mm X 25 cm stainless steel chiral stationary-phase column (J. T. Baker, Inc., Phillipsburg, NJ, Model Chiralcel-OD) was used for the chiral separations.

RESULTS AND DISCUSSION In addition to the vibrational circular dichroism, the

constructed VCD spectropolarimeter (Figure 1) can also be used to measure the IR absorption of a sample. This can readily be accomplished without any major modification. Specifically, rather than using a combination of chopper and PEM, and two lock-in amplifiers, only the chopper and a single lock-in amplifier (LIA 2) are needed to measure IR absorption of a sample. The sample cell can be either the chromatographic microflow cell, a variable path length CaF2 cell, or a 2 mm path length IR quartz cell. A very small

1 1

(R)-2,2,2.Trifluor01 -(S-anthryl)ethanol

-2 2700 2750 2800 2850

Wavelength, nm

Figure 2. Absorption (A) and vibrational circular dichroism (e) spectra of a 0.10 M solution of 2,2,2-trifluoro-l-(9-anthryl)ethanol In CCI,.

absorption band at 2750.0 nm (not shown) was observed in case of the 2-mm quartz cell. This band is probably due to the residual 0-H groups of the silica. Its presence does not create any difficulty in the present study because it can readily be corrected when the cell is used. Furthermore, as will be described later, the absorption of the cell does not produce any VCD signal. The corrected (for the cell absorption) absorption spectrum of a solution of 0.10 M 2,2,2-trifluoro- l-(g-anthryl)ethanol (TAE) in CCld is shown in Figure 2A. As illustrated, this compound has strong absorption band at 2755.7 nm and (not shown) weak bands at 3227.0 and 3262.5 nm. This spectrum is similar to the reported IR spectrum for 2,2,2-trifluoro- 1 -phenylethan~l,*~ and accordingly, the strong band at 2755.7 nm is attributed to the absorption of the 0-H groups while the weak bands at 3227.0 and 3262.5 nm are due to C-H groups.24

Vibrational circular dichroism spectra of 0.10 M solutions of (R)- and (S)-TAE in carbon tetrachloride are shown in Figure 2B. The (S)-TAE enantiomer exhibits a strong, positive VCD band at about 2755.7 nm. The position of this band corresponds well with the IR absorption band of the 0-H group of the same compound (Figure 2A). It can, therefore, be attributed to the VCD of the 0-H group. Vibrational CD signals of the C-H groups in the 3200-3400-nm region were also observed (not shown). However, the recorded signals are very small. This is probably because these signals are inherently very small and also because the output of the F-center laser is very low in this region (and, therefore, produces more noise). It is pleasing to observe that the (R)- TAE compound has, expectedly, a VCD spectrum that is similar in magnitude but opposite in sign as that of the (S ) - TAE compound.

There was a considerable amount of noise associated with the measured spectra. Possible sources of noise include the fluctuation in the intensity of the F-center laser and the mechanical instability of the motor used to drive the intracavity grating. By comparison of the signal-to-noise ratio (S/N) recorded over a period of time at a single wavelength to the S/N when the motor was scanned over a range of wavelengths,

2632 Analytical Chemistty, Vol. 66, No. 17, September 1, 1994

0 5 10 15 20 I I 0.4

0 2 4 6 8 10

0.3

0.0

-0.3

-0.6

1.2

0.8

0.4

0.0

-0.4

-0.9 ' I 0 5 10 15 20

Time, min Figure 3. Chromatogram showing the separation of (R,+2,2,2-trlfluor+ 149anthryl) ethanol (6.3 X 1 0-2 M) by ChralceCOO column as detected by I R absorption (A) and vibrational circular dichroism (8) at 2755.7 nm. Injection valve was equipped with 20-pL loop; flow rate, 1.5 mL/ min; mobile phase, 49.8:40.0: 10.0:0.2 (v/v/v/v) mixture of hexane/ CHCI~/C~HSCN/N(C~H&.

- 0 . 8 ~ " " " " " 0 2 4 6 8 10

Time, min Figure 4. Chromatogram showing the separation of (R,Spbenroln (6.3 X M) by ChlralceCOD column as detected by IR absorption (A) and vibrational circular dichroism (B) at 2880.0 nm. Injection valve was equipped with 20-pL loop; flow rate, 1 .O mL/mln; mobile phase, 49.8:40.0: 10.00/2 (v/v/v/v) mixture of hexane/CHClS/C2HICN/ N(C2H5)3.

it was found that the mechanical instability of the motor is the major source of noise in this case. The motor used in this work was selected simply becauseof its availability. Replacing it with a high precision motor will undoubtedly reduce a substantial amount of noise. Because the objective of this work is to use the VCD for the chiral detection of HPLC, and the detection is usually performed at one specific wavelength, the noise associated with the motor is not of particular importance.

It was found that the racemic mixture of 2,2,2-trifluoro- l-(g-anthryl) ethanol and of benzoin (BZ) can be separated into the corresponding enantiomers by a cellulose chiral stationary phase (Chiralcel-OD column). The mobile phase used for this chiral separation was a mixture of hexane chloroform propionitrile triethyamine with composition of 49.8: 40.0:10.0:0.2 (v/v/v/v). The flow rate was either 1.5 (for TAE) or 1.0 mL/min (for BZ). Detection, in either the IR absorption (Le., with the chopper and one lock-in) or VCD mode, was accomplished with the developed F-center laser based spectropolarimeter. The wavelength of the F-center laser was set at 2755.7 (for TAE) and 2880.0 nm (for BZ) to detect the absorption and VCD of the 0-H group. The chromatograms obtained for TAE are shown in Figure 3 while those for BZ are shown in Figure 4. For both compounds, the detection by IR absorption agrees well with the VCD detection. By individually injecting pure enantiomers into the column, it was found that, for TEA, the first elution at f R = 6.16 min is the R enantiomer, which has a negative VCD signal at 2755.7 nm. The S enantiomer, eluted at t R = 12.20 min, has, as expected, a positive VCD signal at this wavelength. In the case of benzoin, the order was reversed; namely, the (S)- benzoin which eluted first at t~ = 3.71 min has a positive VCD signal while the R enantiomer at f R = 5.35 min has a negative signal at this wavelength.

The chromatograms of (R,S)-TAE and (R,S)-BZ at different concentrations were also recorded by use of the VCD detector. The results obtained, shown in Figure 5 and 6,

confirms that the VCD signal intensity is indeed proportional to the sample concentration. In fact, the correlation coef- ficients of the calibration curves for all four compounds ( (R)- TAE, (S)-TAE, (R)-BZ, and (S)-BZ) are larger than 0.99. The slopes of the calibration curve for the (R)-TAE and the (S)-BZ are larger than those for the (S)-TAE and (R)-BZ, respectively. This is because calibration curves were con- structed using peak height, and there is considerable broaden- ing in the second peak ((S)-TAE and (R)-BZ) compared to the first peak.

The limit of detection (LOD), defined as twice the peak- to-peak noise of the baseline divided by the slope of the calibration curve, is estimated to be 6.8 X 1 V (for (S)- benzoin) and 8.1 X 10-4 M (for (R)-TAE). Thesecorrespond to the mass detectability of 3.6 and 5.6 pg, respectively. These LOD values are several orders of magnitude higher than those found for other chiral detectors.15J6J9~20~21~23 This is hardly surprising because these techniques, as described previously, are based on the CD measurements in the visible, and visible- CD signals are known to be much higher than those in the IR. The unique feature of this VCD chiral detector is its universal applicability to general types of chiral compounds, in any type of HPLC separation (normal or reversed phase). In its present configuration, this chiral detector can be used to detect any hydroxy chiral compounds, whether of aromatic or aliphatic. In fact, we have successfully used this detector to measure the VCD of such aliphatic alcohols as (R)- and (S)- 2-octanol (it is interesting to note that the VCD signals of (R)- and (S)-2-octanol are about 5 times smaller than those of TAE and BZ. This is probably because the VCD signals, which are due to 0-H group, are larger in TAE and BZ since, in these compounds, the group is held with relatively low conformational mobility). Since aliphatic alcohols such as 2-octanol do not absorb in the visible region, they have not been reported by chiral detection. We are currently inves- tigating an effective chiral separation method so that we can enantiomerically separate and detect these compounds by

AnalyticalChemistty, Vol. 66, No. 17, September 1, 1994 2833

-4.3

\

2 .9 x 0

m 5. Chromatog" of dlfferent concentrations of (R,s).2,2,2-triflw0-149-anthryl) ethanol separated by ChiralceKw) column, detected by the vibrational clrcular dichroism detector at 2755.7 nm.

Fburo 6. Chromatmm of dlfferent concentrations of (R,S)-benzoln separated by ChlralceLOD column, detected by the vibrational clrcular dichroism detector at 2880.0 nm.

HPLC. It is important to add that this detector is not limited only to the detection of chiral alcohols. With the use of a better grating, which can provide higher laser output in the long-wavelength region (up to 3500.0 nm), the detector can be used for the detection of any compounds which have C-H group. Because all organic compounds contain at least one (or more) 0-H and/or C-H group, the detector is, in principle, universal; Le., it is applicable to the chiral detection of virtually all chiral organic compounds.

As described earlier, the major limitation of the VCD chiral detector reported here is its low sensitivity. However, its sensitivity can, in principle, be improved by decreasing the

noise and/or increasing the signal intensity. Major sources of noise in this case are the fluctuations in the laser intensity and in the chromatographic pump. Several methods are currently available to stabilize the intensity of lasers. However, as we have demonstrated recently, the most effective methods to stabilize the amplitude of a single wavelength or a multiwavelength laser beam are those based on an electrooptic modulator (Le., Pockels cell)31 or an acoustooptic tunable filter (AOTF).32-34 These methods have shown to provide up to 11 1 times reduction in the noise of a Ti-sapphire laser. (31) Tran, C. D.; Furlan, R. J. Appf. Spectrosc. 1993,17,235-238. (32) Tran, C. D. Anal. Clrem. 1992,64971A-981A.

2634 Ana~IChetnistry, Vo/. 66, No. 17, September 1, 1994

Therefore, they should substantially reduce the fluctuation in the intensity of an F-center laser which, similar to the Ti- sapphire laser, is a solid-state tunable laser. It is important to point out that the stabilization by either a Pockels cell or an AOTF effectively reduce the fluctuation in the intensity of the laser, regardless of the origin of the n ~ i s e . ~ l - ~ ~ That is, the noise can be due to the instability of the laser and/or the aforementioned fluctuation generated by the stepping motor. Therefore, vibrational spectra of chiral samples can also be sensitively measured when the laser is stabilized. The use of low-noise, high dynamic reserve digital lock-in amplifiers may also reduce the noise in the chiral detector. Substantial noise reduction can also be achieved when a stable, pulse-free chromatographic pump such as a syringe pump is used. This is because it was found that turning on the chromatographic pump led to a twofold increase in the noise of the baseline. There is also a possibility that the noise is due to the thermal fluctuation produced by the mobile-phase absorption of the infrared laser radiation. However, this is not the case because the baseline obtained with the sample is similar to that without the sample. (33) Tran, C. D.; Furlan, R. J. Appl. Spectrosc. 1992, 46, 1092-1095. (34) Tran, C. D.; Furlan, R. J. Rev. Sci. Instrum. 1994,65, 309-314.

Alternatively, the sensitivity of this chiral detector can also be improved by using a different method to measure the VCD signals. Specifically, rather than using the conventional transmission method to measure the VCD, as in the present work, the thermal lens effect can be used to measure the VCD signal and toimproveits sensitivity. Thesensitivityis enhanced because, as we have demonstrated recently for the visible regionJ7-19 the sensitivity of the TL-CD is proportional to the excitation laser power. It is, therefore, higher than the transmission CD methods. Experiments are now in progress to explore these possibilities.

ACKNOWLEDGMENT The authors are grateful to the National Institutes of

Health, National Center for Research Resources, Biomedical Research Technology Program for financial support of this work. Acknowledgment is also made to the NIH BRSG (Grant RR07196) and Marquette University, Committee on Research, for partial support of this work.

Recelved for review M a y 16, 1994. Accepted June 18, 1994.0

Abstract published in Aduance ACS Abstracts, July 15, 1994.

Analytical Chemistry, Vol. 66, No. 17, September 1, 1994 2681