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Anal. Chem. 1085, 57,2606-2610
Laser-Based Circular Dichroism Detector for Conventional and Microbore Liquid Chromatography R. E. Synovec and E. S. Yeung* Department of Chemistry and Ames Laboratory-USDOE, Iowa State University, Ames, Iowa 50011
A highly sensttlve and selective laser-based circular dlchroism detector for both conventional and microbore liquid chromatography Is presented. High-frequencypolarkatlon modulation Is employed to reduce the ampiltude noise of the argon Ion laser operated at 488 nm. Presently, the optimum frequency range Is from 500 to 700 kHz. The limit of detection (LOD) for (+)-tris(ethylenedlamlne)cobait( I I I), (+)-Co( en):’, Is 38 ng (k’ = 0.83) In conventional liquid chromatography, while the LOD wlth microbore liquid chromatography Is 5.6 ng (k’ = 1.3) for a 1-s t h e constant when a 500-kHr modulation frequency Is used.
Selective detection and study of optically active chemical species have developed into an important area of analytical research. Circular dichroism (CD) has shown good promise in this area of research (1-4),while nuclear magnetic resonance and optical activity detection have also been very useful (5). Since CD is inherently a “second-order“ absorbance measurement, many investigations have been in the area of CD instrument calibration and performance improvement (1,6, 7). The ability of CD to provide valuable structural information about chemical species in a variety of matrices has driven researchers to overcome instrumental difficulties. Many interesting CD applications have been reported, such as a chiral metal complex inversion study (8),a critical micelle concentration determination (9), solute-induced drug discrimination (IO),nicotine analysis in real samples (11),and opium alkaloid determinations (12),to name a few. These studies all employ CD as a wavelength-scanning spectrometric technique for a sample measured in a “static” mode. This is in contrast to measuring a sample in a ”dynamic” mode, in which the sample concentration changes with time, such as chromatography coupled with CD detection. A gas chromatography-CD system provided detectability down to roughly 10-50 pg (13). High-performance liquid chromatography coupled with circular dichroism detection (HPLC-CD) was demonstrated by interfacing an LC system to a CD spectrometer originally designed for static mode operation (14,15). A 3-pg limit of detection (LOD) for Ltryptophan was reported when HPLC-CD was used (15). Unless stopped-flow techniques are used, single wavelength detection is employed to provide a selective detection system for either a single species or a certain class of CD active compounds. While the original work in HPLC-CD provides ample selectivity (14,15),a future direction in HPLC is toward microbore chromatography (16,17).The inference is that the detection system, be it CD or otherwise, must be compatible with the rest of the microbore system. Thus, it is essential to move away from using a CD spectrometer that has been converted into an LC detector and move toward a detector designed for a smaller volume simultaneous with either maintaining or, preferably, improving analyte detectability (18-20). This direction is most easily taken by designing a
laser-based detector that provides better power throughput, collimation, and focusing superiority, along with spectral and polarization purity, as compared to conventional light sources. These characteristics afforded by a laser-based system are essential in optimizing small volume detection for microbore chromatography (16)using CD detection, which is facilitated by polarization techniques and optics (2). An improvement in mass and differential absorbance detectability must be demonstrated in order to justify the development of a laser-based CD detector. Since the laser is known to operate at relatively high flicker noise levels at lower frequencies (21), the goal of our research was to overcome this difficulty by polarization modulation of the laser light at higher and higher frequencies (ca. 100 kHz to 10 MHz) until an optimum signal-to-noise ratio for the CD detection system was obtained. The results will be reported here and compared to previous work in the area of HPLC-CD analysis. A HPLCCD system would find good utility in the analysis of complex mixtures of either optically active metal complexes (22) or amino acids (23),for example. Because interest in the HPLC analysis of metal complexes has grown recently (24-26),our detection system will be demonstrated with a reversed-phase (RP) ion-pair HPLC separation of metal complexes outlined in one of the earlier papers (26). Both conventional and microbore HPLC-CD will be investigated.
THEORY CD is defined as the difference in absorbance of left circularly polarized light (LCPL) and right circularly polarized light (RCPL). The CD quantity, A€, can be related to the molar absorptivities of LCPL and RCPL as
A€ =
CL
- CR
(1)
Multiplication of both sides of eq 1 by path length, b (cm), and concentration C (M), yields
AA = AebC = CLbC - eRbC = *AL- A R
(2)
where absorbance, Ai, is defined in the conventional way, for the arbitrary subscript i,
Ai = log
(?)
= qbC
(3)
with Io,i the incident beam and Ii the transmitted beam intensities using the base-ten logarithm. Substituting into eq 2 and using eq 3 for i = L for LCPL and i = R for RCPL,
AA = log
[ $1 - [ $1 log
(4)
Since eq 1 dictates that t L > CR for A€ to be positive, this requires I L < I* (for Io,L = Io,R),or AI = IR - I L (5) Since Io,L and Io,R are not exactly the same in practice (due to slight imbalance in the modulation), the difference can be
0003-2700/85/0357-2606$0 1.50/0 0 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
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arbitrarily expressed as
(6)
= Io,L - Io,R
This represents the offset from true null. Rearrangement of eq 4,substitution of eq 5 and 6, and conversion to the natural logarithm give
Taking the exponential of both sides, assuming AA multiplying the right side of eq 7 yield
