Anal. Chem. IBSO, 62, 2467-2471
2467
Thermal Lens-Circular Dichroism Detector for High-Performance Liquid Chromatography Minren Xu and Chieu D. Tran* Department of Chemistry, Marquette University, Milwaukee, Wisconsin 53233
A novel and ultrasendHve chkal detector for hlshpertonnance Hquid chromatography has been developed. Thb detector is based on the measurement of clrcular dlchrolm of chlral effluents by the thermal lens effect. I n thls Instrument, the chromatographlc effluent was sequentlally exclted by left circularly polarized laser llght (LCPL) and rlght circularly polarked laser llght (RCPL); both of these excltatlon beams were derlved from the same argon Ion laser whose llnearly polarited output was trarwjormed Into ckcularly polarked ight by means of a Pockels cell. The heat generated as a consequence of the sample absorption of the LCPL and RCPL was measured by the probe laser beam colllnearly overlapping wlth the two excltatlon beams. A lockin ampllfler was used to measure the thwmd Iens-ckdar dichroism (TL-CD) dgnal whlch conqmds to the dWference In the thermal lens rlgnals produced by the LCPL and RCPL excltatlon beams. I n addltlon to Its hlgh sensitivity, the advantages of thls TL-CD chiral detector Include Its ablllty to provlde, dlrectly and In real tlme, Information on the chlrallty (Le., circular dlchroism) and optlcal purlty of chlral samples. A detection limn of 7.2 ng was achkved for (-)-trb(ethylenedlamIne)cobalt(II1) ( k ' = 0.45) as well as for the (+)-trls(ethyknedlamlne)cobalt(III)(&' = 1-40) when these two enantiomers wwe chromat~aphkattyseparated from the corrospodng racemlc mixture through the use of bls( p-d-tartrato)dlantknonate( III)Ion pair reversed-phase chromatography. Thb lbnlt of detection was found by uslng a 10-pL flow cell and h a w 5" path length and B m W excltatbn laser beam (A = 514.5 nm) modulated at 2 Hr.
The analysis of chiral drugs has increasingly become an important subject in science as well as in technology. The popularity or rather demand is based on the fact that of 1327 totally synthetic drugs which are currently marketed worldwide, 528 are chiral and capable of existing as two or more optical isomers. Very often, only one form of enantiomers is pharmacologically active. The other or others can reverse or otherwise limit the effect of the desired enantiomer. However, only 61 of the 528 chiral synthetic drugs are marketed as single enantiomers, while the other 467 are sold as racemates. It is thus hardly surprising that the pharmaceutical industry needs effective analytical and preparative separation methods for a variety of enantiomeric compounds (I). Liquid chromatography (LC) seems to be the instrument of choice because of its efficiency, speed, wide applicability, and reproducibility. Various approaches have been made in the last few years to use the LC for optical resolution of racemic mixtures into enantiomers. Perhaps the most notable one is based on the use of cyclodextrin solid stationary phase. In this method, chiral separation is due to the inclusion complex formation between the optically active, doughnutshaped cyclodextrin and the analyte (2-6). In fact, it is now possible to quantitatively perform optical resolution on a number of racemates by LC (2-6). 0003-2700/90/0362-2467$02.50/0
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 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 a conventional circular dichroism spectropolarimeter as a chromatographic detector. This is because the circular dichroism is based on the difference in absorption (AA)of left-handed circularly polarized light (LCPL) and right-handed circularly polarized light (RCPL); i.e., AA = AWL - ARCPL, where A W L and A R C p L are the absorbance of LCPL and RCPL, respectively. These AA values are inherently very small. It has been estimated that the maximum AA is about of an absorbance unit (7-9). Furthermore, because the CD signal is measured as a small ac signal riding on top of a large dc signal (totalabsorption), its minimum detectable value is about lo4 of an absorbance unit (7-9). It is thus particularly important that an ultrasensitive chromatographic chiral detector that is capable of determining circular dichroism of chiral effluents be developed. Taking into account such considerations, we have recently succeeded in developing the first ultrasensitive thermal lens-circular dichroism spectropolarimeter (TL-CD) (10). This instrument is based on the measurement of the difference in the amount of the heat generated (photothermal effect) in an illuminatedchiral sample as a consequence of its absorption of the LCPL and RCPL. Specifically, the chiral sample is sequentially excited by the LCPL and the RCPL, and the corresponding thermal lens signals are monitored by a He-Ne laser overlappingwith the two pump beams inside the sample. The sensitivity of the apparatus is relatively higher than the conventional transmission measurements because in the former the adsorbed energy is measured directly. Furthermore, the use of laser as excitation and monitoring sources enables the CD measurement of samples having very small volumes. In fact, 5 ng of optically active [ C ~ ( e n ) ~ l ~com+I~plex, whose volume as small as 8 r L has been detected by using this apparatus (10). In spite of its unique advantages, we have encountered difficulties in our attempts to use the developed apparatus as a chiral detector for high-performance liquid chromatography (HPLC)-optical resolution. The obstacles are not due to the principle of the technique ( 1 0 , I I ) but rather due to the optical confiiation and data acquisition of the developed spectropolarimeter. Specifically,in this apparatus, the LCPL and RCPL excitation beams are derived from the same laser and their amplitude modulation is achieved with the use of a chopper or electronic shutters. These mechanical modulation devices not only introduce the unwanted instability into the system but also restrict its operation to very low modulation frequencies and, hence, necessitate the use of a microcomputer for data acquisition. As a consequence, it is very difficult, if not impossible, to use the apparatus for the real time CD measurement of flowing sample in HPLC. One possibility to overcome these problems is to develop a new 0 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
I
I
He-Ne Laser
1
F
FC
DF
P
t Lock-In
Recor
Flgwe 1. Schematic diagram of the thermal lens-circular dichroism detector for HPLC: P, prism polarizer; PC, Pockels cell; HV, highvoltage power supply; SQ, sfgml generator; L, lens; DF, dichroic mer; FC, microflow cell; F, interference fUtw PH, pkrhde; W , pin photodiode.
optical configuration that facilitates the use of a non-moving-parts electrooptic modulator for modulation and a lock-in amplifier for detection. Such considerations prompted us to initiate the present study, which aims to develop a new thermal lens-circular dichroism spectropolarimeter for the CD detection of HPLC-optical resolution. This spectropolarimeter can be developed by using a novel approach in which a Pockels cell is employed to convert the linearly polarized light into circularly polarized light and also to modulate the chiral light generated. Experimental conditions are performed in such a way that phase-sensitive detection devices such as a lock-in amplifier can be used for data acquisition. The instrumentation and ita application as a CD detector for the HPLC-optical resolution of the racemic mixture of the [Co(en)J3'13- complexes will be reported.
EXPERIMENTAL SECTION Figure 1 shows the schematic diagram of the thermal lenscircular dichroism spectropolarimeter. In this apparatus, a Coherent 70-2 argon ion laser was used as an excitation beam. The laser beam was converted into completely linearly polarized light by a Glan-Thompson ultraviolet prism polarizer (P, Karl Lambrecht Model MUGTSIO). An electrooptic modulator (PC, Conoptics Model 370 transverse-field Pockels cell which includes four crystals of ammonium dihydrogen phosphate (ADP) whose optical axes are set at 45O to the incoming beam propagation direction)was used to transform the linearly polarized laser beam into circularly polarized light. The double refraction (birefringence) was induced into the Pocbls cell through the use of a home-built high-voltage power supply (HV). A signal generator (SG) was used to drive the HV power supply so that the latter can alternatively apply two different voltages, e.g., V1 and Vz,into the cell. These two voltages converted the cell into a quarter-wave plate which alternatively has retardation of +Xi4 and -X/4. As a consequence,the incoming linearly polarized laser bebeam, whose plane polarization was at 4 5 O to the optical axis of the cell, emerged the cell as circularly polarized light whose handedness was altematively modulated (at the m e frequency as the SG) between left and right circularly polarized light (LCPL and RCPL, respectively) (12). The chiral excitation beam was then focused onto the sample cell by means of an achromatic lens with 60 mm focal length. The cell used in this study was a chromatographic microflow cell (FC) having 5-mm path length and 10-pLvolume (ISCO Model 68-0080-011). The probe beam was provided by a Spectra-Physics He-Ne laser (Model 154). The pump and the probe beams were aligned to overlap at the sample cell by a dichroic filter (DF), which reflects the 632.8-nm beam but transmits all other visible wavelengths. The heat generated by the sample absorption of the LCPL and RCPL pump beams changed the intensity of the probe beam. The intensity fluctuation of the probe beam was measured by a PIN photodiode (PD, United Detector Technology PIN 10 DP) placed 2 m from the sample and behind an interference filter (F) and a 2-mm pinhole (PH). A lens with 50-mm focal length was used to focus the probe beam, and its relative distance from the sample was adjusted to
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F-
(dI
5 2 9 m s /=--
Figure 2. Signal generator (a), applied high voltages (b), excitation beam intensity (c), and probe beam intensity (d) as a function of time: (b) applied high voltages ( V , = 56.6 V and V , = 226.9 V) for the 514.5-nm excitation beam; (c) excitation beam (514.5 nm) intensity profile, and (d) probe beam intensity measured on sample of 1.0 X lo4 M (+)-CCo(enh* aqueous solution excited by the excitation beams whose intensity profile is illustrated in part b.
give maximum thermal lens signals. The output of the photodiode was amplified and demodulated by a lock-in amplifier (Stanford Research Systems Model SR-510)whose output was connected to a recorder. A Shimadzu isocraticpump (Model LC-600) was used to deliver the eluent. The sample was injected into the system through a Rheodyne's Model 7125 sample injection valve equipped with a 20-pL loop. A 4.6 mm X 25 cm stainless steel column packed with Cls, 5-pm spherical material (Custom LC, Inc., Houston, TX) was used. Chromatograms obtained with the developed thermal lens-circular dichroism detector were compared with those measured by using a conventionalabsorption detector (Shimadzu Model SPD-6AV UV-visible variable wavelength detector). The racemic mixture and optically active forms of the [Co(en),j3+13-complexes were synthesized and resolved as described previously (9). Bis(p-d-tartrato)diantimonate(III),Kz[Sbz(dtart),], was obtained from Aldrich and used as received. Methanol, propanol, and acetonitrile were purchased from Burdick and Jackson (Muskegon,MI) and used as such. Deionized water was distilled from an all-glass distillation apparatus. Mobile phase was degassed and fiitered through a Spartan nylon filter with 0.45 pm pore size.
RESULTS AND DISCUSSION The profiles of the signal generator and the actual high voltages which were applied onto the Pockels cell as a function of time are shown in parts a and b of Figure 2. In this case 514.5 nm was the wavelength for the excitation wavelength. As shown in the figure, the signal generator drove the highvoltage power supply in such a way as to enable the latter to alternatively supply two different voltages, i.e., VI = 56.6 V and V , = 226.9 V, onto the Pockels cell at a frequency of 1.89 Hz.As a result of the electrooptical effect, which was induced by these two voltages, the ADP crystals in the Pockels cell became birefringent and acted as a quarterwave plate. The magnitude of the retardation induced by the 56.6-V applied
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
voltage is the same as that caused by the 226.9-V applied voltage (Le., quarter-wave retardation) except that the former voltage provided the +A14 retardation whereas the latter supplied the -AI4 retardation. As a consequence, the Pockels cell was able to alternatively transform (at frequency of 1.89 Hz) the incoming linearly polarized laser beam into left circularly polarized light (LCPL) and right circularly polarized light (RCPL). The corresponding intensity of the light emerging from the Pockels cell is shown in Figure 2b. As depicted, intensity of the RCPL is the same as that of the LCPL. This is as expected because except for the polarization, the effect of the Pockels cell at the two applied voltages on the incoming beam is the same. The intensity of the probe beam, obtained with a sample M (+)-C~(en),~+ being excited by the aforeof 1.0 X mentioned RCPL and LCPL, is shown in Figure 2d. As illustrated, intensity of the probe beam was substantially lower when the sample was excited by the LCPL (226.9 V applied voltage) than when it was excited by the RCPL (56.6 V applied voltage).' Because thermal lens signal measures the heat generated as a consequence of the sample absorption of the LCPL and RCPL, the results obtained seem to indicate that the sample has higher absorptivity for the LCPL than for the RCPL. This is as expected because the sample in this case was (+)-C~(en),~+, which has a positive CD signal at 514.5 nm 6.e. ALCPL> ARCPL). It is noteworthy to add that in the present apparatus the sample was continuously excited by either the LCPL or the RCPL. There was no dark period where it is not excited by any beam. As a consequence, the sample was unable to return to the original thermal state but was forced to remain in the steady state where the rate of heat generated by absorption equals the rate of the heat conducting out to the bulk solution. This configuration was used because of the following advantages: (1)higher signal intensity as compared to the configuration where there is a dark period between two consecutive excitation periods to enable the sample to return to its original thermal state; (2) there are only two signals detected by the photodiode, namely the steady-state signals correspond to the LCPL and RCPL excitation. Therefore, a lock-in amplifier can be used for the direct measurement of the difference between these two signals, i.e., direct measurement of the thermal lens-circular dichroism. Thermal lens-circular dichroism (TL-CD) signals of 1.0 X M aqueous solutions of (+)-Co(en),,+, (-)-C~(en),~+ and ( * ) - C ~ ( e n ) ~were ~ + measured a t four available excitation wavelengths, i.e., 457.9, 476.5, 488.0, and 514.5 nm. The dissymmetry factors, g m D (10,13,14), which were calculated by using the signals obtained from this apparatus, were compared with the gCD values calculated from the CD signals of the same samples measured on a conventional CD spectropolarimeter (JASCO). Good agreement between the gTL
