Anal. Chem. 1994,66, 2882-2887
Enhanced Polarimetric Detection in HPLC Using a Refractive Index Equalizer Franqols Maystre,? Aifredo E. Bruno,' Christian Kuhner, and H. M. Widmer Analytical Research, Ciba-Geigy Ltd., CH-4002 Basle, Switzerland Pseudorotation effects in polarimetric detectors for HPLC are investigated, and a method to actively reduce these distortions is presented. In this method, the refractive index of the eluents is maintained at a constant value by mixing a variable amount of a second, optically denser phase. To do this, a detector measures the refractiveindex of the eluents and its signal is fed back to control the pumping rate of the second phase. To demonstrate the method, a prototype instrument using a refractiveindex detector with a detection volume of 0.1 pL was constructed. It had a total dead volume of 3 p L and made possible the suppression of refractive index peaks as small as 60 pL. This instrument was used to improve the accuracy of a commercial polarimetric detector in the determination of the enantiomeric excess of (R,S")-l-phenylethanol samples by canceling the pseudorotation. The analysis of chiral compounds has been one of the most important problems in analytical chemistry during the last decade, and a number of powerful techniques have been developed to meet this challenge. In the field of liquid chromatography, new stationary phases with chiral selectivity have been found. Thesechiral stationary phases (CSP) proved to be of practical use in routine analysis. Over the years an increasing number of high-performance liquid chromatography (HPLC) columns packed with these materials have become available. Today, a systematic classification of the CSPs has made the choice of an appropriate chromatographic system easier, so that it can be stated that chiral HPLC allows virtually any mixture of chiral compounds or enantiomers to be separated. In this new field of chiral HPLC, detection can be performed with any type of HPLC detector, with UVvisible detection being most commonly used. In this framework, polarimetric detectors assume a unique place because they cannot only selectively distinguish chiral from achiral substances in achiral HPLC, but also because they produce the opposite response for the chiral forms R and Sin chiral HPLC.' Therefore, the use of polarimetricdetectors yields some fundamental information in the analysis of chiral compounds making them very valuable instruments. A description of the polarimetric instrumentation is beyond the scope of this paper; excellent reviews on this topic can be found el~ewhere.~.~ During the last decade, commercial HPLC polarimeters went through a 10-fold increase in sensitivity to Present address: GMP SA, Postfach, CH-4153, BL1, Switzerland. (1) DiCesare, J. L.; Ettre, L. S. J . Chromarogr. 1982, 251, 1. (2) Lloyd, D.K.; Goodall. D. M. Chirality 1989, 1, 251-264. ( 3 ) Yeung, E. S., Ed. Detectorsfor Liquid Chromafography;Wiley-Interscience: New York, 1986; p 204.
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AnatyticaIChemisiry, Vol. 88, No. 18, September 15, 1994
well below 1 millidegree while the flow cell volumes were reduced by a factor of - 4 to a few tens of microliters. Meanwhile, laser-based laboratory instruments demonstrated the limits to which sensitivity can be pushed, with a few investigations reporting submicrodegree sensitivities in microliter detection cell^.^.^ If the issue of sensitivity can be considered as successfully addressed, the problem of suppressing the spurious signals produced by the rapid changes in the refractive index (RI) of the eluent has received only little attention.6 These pseudorotation7 signals occur as the analyte passes through the detection cell and plague quantitative HPLC measurement using polarimeters. From the point of view of a complete chromatographic system, solutions to this problem can be envisaged at three different levels. The first level concerns the principle of the optical measurement itself. Use of the optical arrangement of a complete polarimeter? which delivers the four Stokes polarization parameters of the medium within the flow cell, would allow true rotation and pseudorotation to be distinguished. However, state-of-the-art optical components do not permit the realization of a complete polarimeter with the sensitivity and the stability required in HPLC. The second level concerns the geometry of the flow cell. It is important to realize here that the polarimetric detection cells which offer the best path length-to-volume ratio are U- or Z-type cells of well-defined cylindrical shape^,^ which leave little room for variation. Experiments with conical cells5 rendered some success, but the literature on this topic is very scarce. Since the disturbing refractive index effects result from a complex dynamic distortion of the optical path, the main difficulty is to devise a cell which will guarantee optimal conditions over all possible flow rates and eluent properties found in HPLC. Finally, the third level is the suppression of the R I variations in the eluents, which solves the problem at its root. The most obvious way to achieve this goal is to select a mobile phase which has the same RI as the samplea7 This can be achieved in some cases and works well when the RI values of the sample and of the indicated mobile phase are close to each other to start with. However, in HPLC, the dominant criterion for selection of the mobile phase is given by separation efficiency. (4) Bobbitt, D.R.; Yeung, E. S. Anal. Chem. 1984, 56, 1577. ( 5 ) Lloyd, D. K.; Goodall, D. M.; Scrivener, H. Anal. Chem. 1989, 61, 1238. (6) MacFarlane, J. D.; Tokieda, T.;Suzuki, H.; Morigushi, S . In Proceeding of the 15fh Inrernarional Symposium on Liquid Chromatography. HPLC '91: Basel, 1993; Poster 227/ 1. (7) DBppen, R.; Voigt, P.; Maystre, F.; Bruno, A. E. Anal. Chim. Acra 1993,282,
47.
( 8 ) Azzam, R. M. A.; Bashara, N. M.Ellipsomerry and Polarized Lighr; NorthHolland: Amsterdam, 1988; p 153.
0003-2700/94/0366-2882$04.50/0
0 1994 American Chemical Society
coefficients of refraction9 7,
=
2 sin 8’ cos 8 sin(8 + 8’) cos (8 - 8’)
(3)
2 sin 8’ cos 8
Flgure 1. Model of a U-shaped flow cell used in most polarimetric detectors. An eluting peak entering the cell creates two zones of refractive indices nl and nB separated by an interface oriented at an angle 8. The light propagates along the z axis of the cylinder, whereas its polarization is described in the x-y plane.
(4)
where 8 is the angle of incidence with the interface plane and 8‘ is the refraction angle given by Snell’s law, Le., n2 sin 8’ = nl sin 8
(5)
If the second zone (2) within the cell produces a total optical rotation a,the overall transmission matrix T of the cell becomes In this paper, we present a novel instrument dubbed the “refractiveindex equalizer”,which suppressesthe RI variations An in the eluentsjust before the polarimetric detector. In this instrument, the eluents coming from the separation column pass through a mixer before reaching a RI detector. The signal from this detector feeds a servocontroller unit which drives the rate of a dispenser also connected to the mixer, such as to correct for the RI changes which cause pseudorotation. The dispenser contains a nonperturbing low- or high-RI liquid which is mixed with the eluents in order to correct for the RI changes that cause pseudorotation. Using this technique, the accuracy of a commercial polarimetric detector used for the determination of the enantiomeric excess of (R,S)-1 -phenylethanol samples was considerably enhanced. THEORY Pseudorotation. The changes in the polarization properties of a light beam passing through a U-shaped flow cell filled with an inhomogenous medium is described using a simple model. Figure 1 defines the geometrical parameters of importance in a cylindrical flow cell. The light propagates along the z axis, which coincides with the axis of the flow cell. The state of polarization of the light is given by the trajectory of the electrical field vector in the x-y plane, normal to the propagation axis. On the entrance window, the polarization is assumed to be linear with the azimuth $, so that the incoming electrical field vector can be described by use of the Jones formalism by (see p 13 in ref 8)
E, = (‘Os sin d
)
Let the appearance of a peak within the flow cell be described by the creation of two discrete homogenous zones 1 and 2 with respective RIs of nl and n2, separated by a planar interface. If the interface is not perpendicular to the direction of propagation, the transmission coefficients for the x and y components of the field vector (Le., the TE and TM waves) are not equal. The medium is thus uniaxially linearly dichroic and its transmission matrix D reads (see p 75 in ref 8)
In this matrix,
T~
and
T,
are the amplitude transmission
7, cos a - T,, sin a = (T,, sin a 7,, cos a
T=
)
where R(a) is the rotation matrix. T permits derivation of the expression of the output polarization vector E,,,, which reads 7,
E,, = TE, =
T~
cos a cos 4 - T,, sin a sin 4 sin a cos 6 + T,, cos a sin
)
(7)
Using eq 7, it can be shown that Eoutcorresponds to a linear state of polarization, and that for T.-T,, = AT
