A highly sensitive and selective double-beam laser-based circular dichroism (CD) detector for high-performance liquid chromatography (HPLC) is presented. An all-electronic noise canceller circuit was used to subtract the intensity of the right circularly polarized light from the intensity of the left circularly polarized light. Both of these beams were derived from the same Ar-ion laser operated at 488 nm. The chiral inorganic complexes of tris(ethylenediamine)cobalt(III), Co(en)33+, which are CD active at this wavelength [Δε(488 nm) = 1.9 cm2 mol−1], were chosen as test compounds. The two enantiomers were chromatographically separated from the corresponding racemic mixture through the use of potassium antimonyl(III) tartrate hydrate and ion-pair reversed-phase liquid chromatography. A practical noise-to-signal ratio of 4 × 10−6 for CD measurements was achieved despite the presence of the high background absorption, which is 45 times higher than the CD signal for this particular system. The detector is linear through two orders of magnitude (10−3 M-10−5 M). The deviation between consecutive measurements is less than 5% and the noise level is 1.8 × 10−6 AU. A limit of detection (LOD) of 1.2 × 10−6 M at the flow cell, which corresponds to 25 ng injected material, was achieved. This value is similar to the LOD that was obtained for the same species by using laser-based high-frequency modulation and is only 3.5 times worse than thermal-lens CD detection. Being simpler and less expensive than other laser-based systems, our double-beam HPLC-CD detector provides a three-orders-of-magnitude improvement in sensitivity as compared to that for conventional CD spectrometers.
Zeev Rosenzweig and Edward S. Yeung, "Laser-Based Double-Beam Circular Dichroism Detector for Liquid Chromatography," Appl. Spectrosc. 47, 2017-2021 (1993)