Anal. Chem. 1994,66, 3093-3101
Flow Injection Analysis With High-Sensitivity Optical Rotation Detection Charles A. Goss, Douglas C. Wilson, and Wllllam E. Welser' Analyfcal Development Laboratories, Burroughs Wellcome Co., 3030 CornWallis Road, Research Triangle Park, North Carolina 27709
The construction and characterization of a flow injection analysis system with high-sensitivity, laser-based (488 nm), optical rotation detection (FIA-OR) is described. Baseline noise is reduced to 10 pdeg by addition of a pressure pulsation damper between the pump and the autosampler. Sensitivity is increased 100-fold compared to a high-quality conventional polarimeter, and detection of a 43-ng sample of sucrose (25pdeg rotation) is demonstrated. The instrument response is linear over a 500-fold range of concentration to 21 p g of sucrose injected, with the upper limit set by laser b&m distortioncaused by sample refractive index effects. Specific rotation values at 488 nm for 11 organic molecules are measured by FIA-OR with good precision and agree with values obtained by a conventional polarimeter. FIA-OR analysis of enantiomeric punty of (1$2R)-(+)-ephedrie is found to be more discriminating than conventional polarimetry for the analysis of solutions with high enantiomeric excess.
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We report a flow injection' system with high-sensitivity optical rotation detection (FIA-OR) for the determination of optical rotary power and enantiomeric purity of chiral compounds. Our work is motivated by the fact that chiral molecules are currently at the forefront of strategies for the development of safer, more effective drugs2and other chemical agent^.^ For example, there is considerable interest in preparing single enantiomer versions of drugs that are currently marketed as racemates because of the recognition that the opposite enantiomers can have quite different pharmacological effects.2b Optical rotation (OR) measurements are central to the characterization of chiral molecules because this is the only physical property that distinguishes the individual enantiomers. Although polarimetry has traditionally been used to assess the purity of chiral sample^,^ as conventionally performed5 it is a relatively insensitive (- 10-3deg) technique that requires fairly large amounts of sample (typically 1 5 0 mg). In addition, preparing the polarimeter cell for each (1) (a) Ruzicka, J.; Hansen, E. H. Flow Injection Analysis; John Wiley & Sons: New York, 1981. (b) Ruzicka, J.; Hansen, E. H. Flow Injection Analysis, 2nd 4.John ; Wiley & Sons: New York, 1988. (2) (a) Stinson, S.C. Chem. Eng. News 1993, 71(39), 38-65. (b) Stinson, S.C. Chem. Eng. News 1992, 70(39), 46-79. (3) Sheldon, R. A. Chirotechnology: Industrial Sythesis of Optically Actiue Compounds; Marcel Dekker: New York, 1993. (4) (a) USP XXII: The United States Phamacopeia; The United States ~ (b) OffieialMethods Pharmacopeia1Convention: Rockville, MD, 1 9 9 0 ; 1595. of Analysis, 15th ed.; Helrich, K.,Ed.; Association of Official Analytical Chemists: Arlington, VA, 1990. (5) Willard, H. H.; Merritt, L.L., Jr.; Dean,J. A,; Settle, F. A,, Jr. Instrumental Methods of Analysis, 6th ed.; Wadsworth Publishing: Belmont, CA, 1981; pp 412429.
0003-2700/94/0366-3093$04.50/0 0 1994 American Chemical Society
analysis, loading samples, and taking readings involve manual manipulations which consume operator time. FIA-OR is an emerging6v7technology that offers several potential advantages as an alternative to conventional polarimetry. Sensitivitycan beincreased to le'deg by adapting a laser-based micropolarimeter, originally reported by Yeung and for HPLC applications, as the optical rotation detector. Sample requirements are reduced dramatically from several milliliters to 100 ML. Individual analyses will be rapid, on the order of 1-2 min. Finally, sample introduction can be automated, thereby reducing operator involvement and increasing sample throughput. If these potential advantages can be realized in practice, FIA-OR may find important applications in the pharmaceutical and food industries, particularly in quality assurance laboratories where the advantages of automation and rapid analysis time are critical. The use of a laser-based micropolarimeter for FIA-OR was first mentioned by Rice et a1.6 in a discussion of a method to determine specific rotations from peak height data. However, no experimental data were presented because the authors found that Gaussian peak profiles could be more consistently obtained by HPLC-OR. During the course of our work, Liu et al.7 reported using FIA with a dual UV absorbance and OR detectors to determine the amount and enantiomeric purity of a drug in dosage form. Their system employed a commercially available OR detector based on a diode laser operating at 820 nm, which has been discussed in detail previo~sly.~ The present work expands upon previous reports in several important ways. First, we describe in detail the construction, capabilities, and limitations of the most sensitive FIA-OR system yet reported. Fundamental investigations of the instrument performance uncovered a hitherto unknown characteristic of air-based Faraday rotators used for OR detector calibration6,8a-h,10 that could lead to significant
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( 6 ) Rice, P. D.; Shao, Y.Y.;Erskine,
S.R.;Teague,T. G.;Bobbitt, D. R. Talanta 1989, 36, 47 3-478. (7) Liu, G.;Goodall, D. M.;Loran, J. S.Anal. Proc. 1992, 29, 31-33. (8) (a) Yeung, E. S.; Steenhoek, L. E., Woodruff, S.D.; Kuo,J . C . Anal. G e m . 1980,52,1399-1402. (b) Kuo,J. C.; Yeung, E. S . J. Chromatogr. 1981,223, 321-329. (c) Kuo, J. C.; Yeung, E. S.J. Chromatogr. 1982, 229, 293-300. (d) Kuo,J. C.; Yeung, E. S . J. Chromatogr. 1982,253.199-204. (e) Bobbitt, D. R.; Yeung, E. S . AMI. Chem. 1984, 56, 1577-1581. (f) Bobbitt, D. R.; Yeung, E. S.Appl. Spectrosc. 1986,40,407410.(g) Reitsma, B. H.; Yeung, E. S.Anal. Chem. 1987, 59, 1059-1061. (h) Xi, X.; Yeung. E. S . Appl. Spectrosc. 1989, 43, 1337-1341. (9) Lloyd, D. K.;Goodall, D. M.; Scrivener, H. Anal. Chem. 1989, 61, 12381243. (10) (a) Ng, K.; Rice, P. D.; Bobbitt, D. R. Microchem. J. 1991, 44, 25-33. (b) Rice, P. D.; Shao, Y. Y.; Bobbitt, D. R. Talanta 1989,36,985-988. (c) Shao, Y. Y.; Rice, P. D.;Bobbitt, D. R. Anal. Chim. Acta 1989, 221, 239-247.
AnalyticalChemistry, Vol. 66, No. 19, October 1, 1994 3093
OR Detector
A now
I
1-
Acquislt,on
I
Ar+ Laser, 488 nm
Figure 1. Schematic diagram of FIA-OR system. See text for detailed description.
calibration errors if not accounted for. Second, the first specific rotation measurements by FIA-OR are documented and compared with those from conventional polarimetry. The results reveal limitations of the peak height model proposed by Rice et a1.6 when applied to FIA-OR. Finally, equations for enantiomeric purity determinations by FIA-OR are presented along with new results demonstrating that thegreater sensitivity of FIA-OR enables solutions with high (195%) enantiomeric excess to be assayed more accurately than with conventional polarimetry.
EXPERIMENTAL SECTION Reagents. HzO was purified using a Millipore Milli-Q system to a final resistivity of 18.2 MQ. Sucrose (>99%) was from Mallinckrodt. D-Fructose (99%), D-raffinose pentahydrate (98%), (lS,2R)-(+)-ephedrine hydrochloride (99%), (1R,2S)-(-)-ephedrine hydrochloride, (*)-ephedrine hydrochloride (99%), L-(+)-alanine (99%), (S)-(+)-2-aminobutyric acid (98%), (R)-(-)-2-aminobutyric acid (99%), (S)-(+)leucinol (98%), and (R)-(-)-leucinol (98%) were all from Aldrich. L-(+)-lysine hydrochloride (99.9%) was from Fisher. All samples were used as received. General Procedures. All experiments were conducted at the ambient laboratory temperature of 297 2 K. Water was used as the flow solvent in all experiments. Reported uncertainties are one standard deviation. FIA-OR System. The FIA-OR system (Figure 1) consists of a pump, a pressure-pulsation damper, a variable-volume autosampler, and a high-sensitivity optical rotation detector connected in series. Samples were injected by a variablevolume autosampler (Hewlett-Packard Model 1050) into the flowing H20 stream (1 mL/min) provided by a dual-piston pump (Kratos Spectroflow 400) equipped with an internal membrane pulse damper. For most experiments, a reversedphase HPLC column (Vydac RP18 908, Pharmaceutical, 5-pm packing, 250 mm long, 4.6 mm i.d.) was inserted between the pump and autosampler to act as the additional pulse damper shown in Figure 1. The injected sample plug flowed through -200 cm of 0.0254-cm-i.d. tubing to the optical rotation detector. The OR detector is a modified version of a design originally reported by Yeung et alegaThe 2.0-mW, 488-nm output of an argon ion laser (Uniphase Model 2011-1OSL) was directed by mirrors (Newport, Part No. 10D20DM.5)
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Analytical Chemistry, Vol. 66, No. 19, October 1, 1994
and focused by a 350-mm focal length lens (Oriel, Part No. 40400) through a carefully selected Glan-Thompson polarizer (Karl Lambrecht, Part No. MGT25E-8) along the bore (0.5 dm long, 1.5 mm diameter, 88 pL volume) of a custom-built aluminum flow cell centered at the lens focal point. The flow cell windows were selected glass microscope slides (Corning No. 1) attached by silicone adhesive (Dow Corning, general purpose sealant). To facilitate lock-in amplifier detection, the flow cell also functioned as a Faraday modulator. Combining the flow cell and modulator functions improves the modulator characteristics without introducing new scattering centers into the light path. Relative to the original air-based modulator,8a the combined flow cell-modulator produces significantly larger modulation angles with smaller magnet currents, due to the lo3 larger Verdet constant of liquids compared to air. Larger modulation angles have been shown to improve sensitivity,Eaand decreased magnet power lessens noise from ohmic heating.8h A 3-cm-long central section of the cell was wound with -600 turns of 26-gauge magnet wire. The modulator coil was driven by the 8-V, 212Hz ac output of a function generator (Hewlett-Packard 8 1121A), giving peak currents of 190mA and peak magnetic fields of -48 G. This corresponds to peak rotations near 48 mdeg, assuming the Verdet constant at 488 nm (A488) for HzO (3.3 X lo4 deg G-*cm-l).*I The modulated beam then impinged the nulling analyzer crystal (selected to give maximum extinction with the polarizer) mounted in a rotational stage (Aerotech Model ARS 301), producing a sinusoidal light intensity output (424 Hz) that passed through two 1.5-mm-diameter apertures and a 488-nm bandpass filter (Edmund Scientific, Part No. 530907) before being detected by a photomultiplier tube (Oriel Model 77345) biased at 800-900 V by a high-voltage power supply (Fluke Model 415B). A 102-kQ resistor-1-nF capacitor combination converted the photomultiplier output current to voltage, which was subsequently demodulated and amplified by a lock-in amplifier (EG&G Princeton Applied Research Model 5209) with a 1-s time constant. The theory underlying this approach has been discussed elsewhere.8a The essential points are that in the absence of an optically active sample the symmetric modulation gives no net polarization rotation, so the lock-in amplifier output is zero. When an optically active sample traverses the cell, it adds a net rotation a (positive in this example) that increases the intensity of the positive portion of the modulation signal, while decreasing the intensity of the negative half. By comparing the phase and magnitude of the increased light intensity to that of the reference signal, the lock-in amplifier produces a dc output with sign and magnitude proportional to a. An integrated chromatography data system (VG Multichrom) digitized the lock-in amplifier output at 3-4 Hz, stored it on a VAX computer, and provided data processing routines. Conventional Polarimetry. A Rudolph Research Autopol I11 automatic polarimeter (0.001-deg readability) was used with a 1.O dm path length cell. The clean polarimeter cell was filled with pure water, and the blank optical rotation at each of the six available wavelengths (A = 365,405,435,546,589,
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(1 I ) (a) Estimated by interpolation of 492-nm datallb by assuming AkT = l/h2, ignoring the insignificant temperature dependence of AkT. (b) de Mallemann,
R. In Tables de Consfantes et Donndes Numdriques; IUPAC, Hermann & Co.: Paris, 1951; Vol. 3, p F12.
I Piston Pump without Damper
Piston Pump with Damper
B
~~~4~~~
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50 pdeg
Syringe Pump with Damper
c
t
10 min
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Figure 2. FIA-OR baseline traces recorded over 10 min showing noise decrease by addition of an HPLC column pulse damper: (A) dual-piston pump without column; (B) duakplston pump with column; (C) syringe pump with column. Flow rate, 1 mL/mln.
633 nm) was recorded. The cell was rinsed and filled with the sample solution, and the optical rotations were recorded again.
RESULTS AND DISCUSSION Baseline Noise. Our study began by identifying and controlling sources of instrument noise in three categories: (i) those that affect fundamental instrument sensitivity and those that effect the (ii) short- (Le., several minutes) and (iii) long-term stability of the baseline signal. Fundamental sensitivity in an OR detector of this design is primarily controlled by the extinction characteristics of the optical path, because this corresponds to minimizing the residual rotation, in the same way that stray light limits the sensitivity of optical absorbance detectors. We found that care in selecting, orienting, and mounting the polarizer crystals and cell window components is critical to success in this area, consistent with previous reports.8a It was also important to properly align the flow cell to minimize scatter off the flow cell walls, to avoid strain in the flow cell mounts, and to minimize reflections of the rejected beam from the analyzer. Black marker ink (Sharpie) proved to be a particularly effective antireflection coating. With attention to these details, it was possible to routinely obtain extinction ratios near the 10lOvaluereported by Yeung et a1.8a Short-term stability of the OR detector is critical for optimizing the detectability of the relatively rapid (5-10 s fwhm) FIA signals and is primarily determined by momentary fluctuations in beam power or polarization. Small movement of the flow cell windows caused by pressure pulsations in the liquid flow was identified as the major source of short-term noise. The system is particularly susceptible to this problem because it is necessary to attach the thin windows with relatively flexible adhesive to minimize strain. Window movement can cause either direct depolarization of the beam due toscattering centers in the window or indirect depolarization due to shifting of the beam position on the analyzer. As shown in Figure 2A, the baseline (1 mL/min, 1-s time constant) recorded over 10 min without a pulsation damper shows rapid fluctuations on the order of 50 pdeg. However, a standard CIS HPLC column placed between the pump and the autosampler (see Figure 1)
effectively damped these pulsations, dramatically reducing the baseline noise to 10 pdeg, as illustrated in Figure 2B. The data in Figure 2A,B were obtained using a standard dualpiston HPLC pump, for which some amount of pressure pulsation is naturally expected.12 To test whether further reductions in pulsation noise were possible, we tested the system using a pulse-free syringe pump (1x0 Model 500D). The resulting baseline trace (Figure 2C) shows no significant difference from the piston pump data in Figure 2B, indicating that the column damper effectively eliminates short-term pulsation noise. Additional short-term noise sources include laser flicker, modulator signal fluctuations from function generator instability, vibration-induced optical misalignment, and turbulence in the flow cell. Of these, laser flicker is probably the most important. Laser power stabilizerssh and high-frequency modulation (- 100 kHz)8f have been shown to substantially reduce laser flicker noise, resulting in detectabilities of 2-3 pdeg (1-s time constant), but we have not tested these strategies, in part because the residual inductance in the aluminum flow cell limited the modulation frequency to 51000 Hz. However, the 10-pdeg noise level shown in Figure 2 is comparable to the best reported valuesse for laserbased OR detectors used in HPLC without the use of special laser power stabilizers or high-frequency modulation, thus demonstrating transferability of the OR detector from HPLC to FIA without any loss in performance. Long-term stability (Le., drift) is largely controlled by equilibration of the flow cell window position with a given flow condition, wandering optical alignment, and temperature fluctuations of the detector components. Once a flow condition has been established, drift from window equilibration simply requires time to dissipate, typically -45-90 min. Noise from building vibrations was significantly reduced by mounting the optical bench on shock-absorbing rubber feet. Thermal gradients within the cell were minimized by operating with low modulator power (- 1 W), to reduce ohmic heating, and relatively low laser power (2 mW). Although increased laser intensity might in principle improve performance by reducing shot noise? in practice we find that higher power (e.g., 10 mW) causes unacceptably large drift we attribute to laser heating induced refractive index gradients within the flow cell. Similarly, it was important to maintain a uniform laboratory temperature, free from drafts, to minimize baseline oscillations (as much as 150 pdeg over 15 min) most likely due to expansion/contraction of the polarizer/analyzer crystals or the cell windows. Minding these procedures, we observe stable baselines with drift on the order of f 3 0 pdeg over 1 h. Calibration. Two methods were used to calibrate the OR detector output. The first employed an air-based Faraday13 cell ( 5 cm long, 0.5 cm id., 1090 turns/cm, No. 30-gauge magnet wire) placed between the polarizer and the flow cell to produce standard optical rotations (awl)that were then converted to an instrument response factor K-1 = awl/HwI, where Hal is the height of the calibration signal. A dc power supply (Tektronix, Model CP5250) drove the calibrator coil to produce 1281.4 G/A, as measured at the solenoid center by a gauss meter (F. W. Bell Model 4048). In theory, using (12) The baseline fluctuations were slightly lower at 0.5 mL/min and larger at mL/min, consistent with the characteristicsof piston pumps. (13) Faraday, M. Philos. Mag. 1846, 28, 294.
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the Verdet constant for air (A:: = 1.44 X lo-' deg G-I cm-I),l4 this corresponds to optical rotations of 9.23 x 104 deg/A. This calibration scheme is similar to systems already described in several publications.6,8J0 However, careful comparison of specific rotations measured using this calibration method with those obtained using a conventional polarimeter revealed an important characteristic of this design, not mentioned previously, that could lead to significant calibration errors if not accounted for. The root of the problem is that air-based calibrator magnets must apply relatively large magnetic fields (typically hundreds of gauss) to produce easily measured rotations (> 100 pdeg), due to the the small Verdet constant of air. Although the magnetic field decays rapidly with distance ( a l / x 3 ) along the axis of the calibrator so1enoid,I5the much larger Verdet constant for liquids relative to air (-2000:l) means that even a small residual magnetic field can act upon the liquid in the flow cell to produce a significant additional optical rotation. For example, a 6 X G field interacting with a 5-cm flow cell filled with water would produce a 10-pdeg rotation, corresponding to 10%error for a nominally 100-pdeg calibrator signal. To understand this effect better, we modeled the system theoretically (see Appendix) and found that the ratio of the total rotation produced by the calibrator magnet acting on both air and the flow cell liquid (atot)to the rotation expected for the calibrator acting on air alone (aair) is given by
Sucrose
t
blank A
A
~
I
1.0 min
Flgure 3. FIA-OR response to nanogram quantities of sucrose demonstrating instrument sensitivity: (A) 10-pL injection of pure H20; (B) 20-, (C) 40-, and (D) 80-pL injections of 2.07 X 10" g/mL sucrose: (E) 10- and (F) 20-pL injections of 2.07 X g/mL sucrose. Flow rate, 1 mL/min.
where A:! and A: are the Verdet constants for air and of the liquid in the flow cell (in deg G-I cm-l), R is the average radius of the calibrator magnet (in cm; 0.9 cm for the one employed here), Leal is the flow cell length (in cm), and x is the center-to-center separation between the calibrator and the flow cell. The most obvious method to reduce a t o t / a a i r is to increase the calibrator-flow cell separation. Assuming water for the liquid, eq 1 predicts that a separation of 45 cm is required to reduce the effect of the interaction to
