Anal. Chem. 1993, 65, 2990-2994
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Sensitive Circular Dichroism Spectroscopy Based on Nonlinear Degenerate Four-Wave Mixing Jon A. Nunes and William G. Tong’ Department of Chemistry, S u n Diego State University, S u n Diego, California 92182
Degenerate four-wave mixing is demonstrated as an effective and sensitive laser analytical spectroscopic method for circular dichroism measurements. A forward-scattering degenerate fourwave mixing optical setup is used to obtain simple optical alignment, highly efficient wave mixing, and very effective use of low laser power. This nonlinear laser-based circular dichroism method offers many advantages, including easy and efficient optical signal collection, use of very short analyte path lengths (e.g., 0.1 mm), and excellent detection sensitivity that is comparable or better than conventional laser-based or non-laser-based circular dichroism methods. Using an analyte path length of only 0.1 mm, and a probe volume of 98 pL, a circular dichroism mass detection limit of 0.68 pg or 2.8 fmol is reported for (+)Co(en)d+.
INTRODUCTION Circular dichroism (CD) is a popular optical method for determining and analyzing the chirality of molecules. It can be used to measure the differential absorption (At) of rightand left-handed circularly polarized light that chiral molecules exhibit. Of the three most commonly used chiroptical methods, circular dichroism has become the method of choice over polarimetry and optical rotary dispersion (ORD),mainly due to its many advantages for analytical applications.’ Circular dichroism measures both optical rotation and absorbance simultaneously and, hence, is suitable for the analysis of asymmetry of a specific chromophore in an optically active compound. Use of this technique has grown significantly over the past few decades, especially since the introduction of polarization modulation in the optical instrumentation.2 Circular dichroism has many applications in various areas including the study of inorganic and organic molecular structural configuration^,^^^ the study of conformational properties of proteins and other biomole~ules,5~~ and analytical detection of optically active substances in conjunction with liquid and gas chromatographic method~.~-g Circular dichroism spectrometers have long been plagued with detection sensitivity problems because of the inherently
* Author to whom correspondence should be addressed. (1) Purdie, N.; Swallows, K. A. Anal. Chem. 1989, 61,77A-89A. (2) Grosjean, M.; Legrand, M.; Velluz, L. Optical Circular Dichroism; Academic Press Inc.: New York, 1965. (3) Nakanishi, K.; Kuroyanagi, M.; Nambu, H.; Oltz, E. M.; Takeda, R.; Verdine, G. L.; Zask, A. Pure Appl. Chem. 1984, 56, 1031-1048. (4) Sakabe, Y.; Ogura, H. Inorg. Chim. Acta 1991, 189, 225-228. (5) Carrara, E. A.; Gavotti, C.;Catasti, P.; Noma, F.; Berutti Bergotto, L. L.; Nicolini, C. A. Arch. Biochem. Biophys. 1992, 294, 107-114. (6) Vanstokkum, I. H. M.; Spoelder, H. J. W.; Bloemendal, M.; Vangrondelle, R.; Groen, F. C. A. Anal. Biochem. 1990, 291, 11c118. (7) Drake, A. F.; Gould, J. M.; Mason, S. F. J. Chromatogr. 1980,202, 239-245. (8) Westwood, S. A,;Games, D. E.; Sheen, L. J . Chromatogr. 1981,204, 103-107. (9) Premuzic, E. T . ;Gaffney, J. S. J. Chromatogr. 1983,262,321-327.
small magnitude of CD signals. These signals are typically about one-hundredth as large as the analyte’s absorption curve and tend to be immersed in large amounts of background noise. Hence, several groups have worked on improving and advancing the capabilitiesof conventional circular dichroism spectropolarimeters. For example, a rapid-scanning CD spectropolarimeter that provided improved spectral resolution and higher signal-to-noise ratios was demonstrated by utilizing an acoustic optical filter.1° For normal and microbore liquid chromatography, a laser-based CD detector was developed that used high-frequency (500 kHz) polarization modulation to obtain excellent detection limits” for (+)C0(en)3~+.A device for measuring time-resolved CD spectra has been developed that has picosecond resolution.12 Fluorescencedetected CD spectroscopy has been used for the study of the chirality of fluorescent molecules, as a sensitive and selective detector for HPLCl3 and CZE,14 and as a way to measure fluorescence lifetime distribution^.'^ Thermal lens spectroscopy has also been used to increase the sensitivity of CD measurements.16J7 These very significant advances have greatly aided in making CD spectroscopy an important and powerful technique, as many analytical applications demand novel CD detection methods with excellent detection sensitivity.18 Degenerate four-wave mixing (D4WM) is a nonlinear optical technique with many engineering and optics appli~ a t i o n s . ~We ~ -have ~ ~ demonstrated DIWM as a sensitive analytical spectroscopic method with many advantages over conventional techniques, using different atomizers or sample cells including low-pressure hollow-cathode discharge cells,23-% flame at0mizers,~’j-~9 and liquid flows ~ e l l s . ~In ~ *the ~ l gas phase, sub-Doppler spectral resolution allows hyperfine (10)Hatano, M.; Nozawa, T.;Murakami, T.;Yamamoto,T.; Shigehisa, M.; Kimura, S.; Takakuwa, T.; Sakayanagi, N.; Yano, T.; Watanabe, A. Reu. Sci. Instrum. 1981,52, 1311-1316. (11) Synovec, R. E.; Yeung, E. S. Anal. Chem. 1985, 57, 2606-2610. (12) Xiaoliang, X.; Simon, J. D. Reo. Sci. Instrum. 1989, 60, 26142627. (13) Synovec, R. E.; Yeung, E. S. J. Chromatogr. 1986,368, 85-93. (14) Christensen, P.L.; Yeung E. S. Anal. Chem. 1989,61,1344-1347. (15) Wu, K.; McGown, L. B. Appl. Spectrosc. 1991,45, 1-3. (16) Tran, C. D.; Minren, X. Appl. Spectrosc. 1990,44, 962-966. (17) Tran, C. D.; Xu, M. Reu. Sci. Instrum. 1989, 60, 3207-3211. (18) Armstrong, D. W.;Berthod, A.;Tang, Y.;Zukowski, J. Anal. Chim. Acta 1992,258, 83-92. (19) Hellwarth, R. W. J. Opt. SOC.Am. 1977, 67, 1-3. (20) Yariv, A. IEEE J. Quantum Electron. 1978, QE-14, 650-660. (21) Fisher, R. A. Optical Phase Conjugation; Academic Press Inc.: Orlando, FL, 1983. (22) Zel’dovich, B.; Pilipetsky, N . F.; Shkunov, V. V. Principles of Phase Conjugation; Springer-Verlag: New York, 1985. (23) Tong, W. G.; Chen, D. A. Appl. Spectrosc. 1987, 41,586-590. (24) Chen, D. A.; Tong, W. G. J . Anal. Atom. Spectrom. 1988,3,531535. (25) Wu, 2.; Tong, W. G. Spectrochim. Acta B 1992,47B, 3,449-457. (26) Tong, W. G.; Andrews, J. M.; Wu, Z. Anal. Chem. 1987,59,896899. (27) Andrews, J. M.; Tong, W. G. Spectrochim. Acta 1989,44B, 101107. (28) Andrews, J. M.; Weed, K. M.;Tong,W. G. Appl. Spectrosc. 1991, 45, 697-700. (29) Wu, Z.; Tong, W. G. Anal. Chem. 1991, 63, 899-903. (30) Wu, Z.; Tong, W. G. Anal. Chem. 1989,61, 998-1001. (31) Wu, Z.; Tong, W. G. Anal. Chem. 1991, 63, 1943-1947.
0003-2700/93/0365-2990$04.00/00 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 21, NOVEMBER 1, lQQ3
structure measurement and stable isotope ratio analysis a t trace concentrations.a For condensed-phase analytes, attomole-level detection sensitivity can be obtained using continuously flowing liquid cells at room temperature.m The D4WM signal is strong enough to yield excellent detection sensitivity even when the excitation wavelength is more than 100 nm away from the absorption wavelength ma~imum.3~ By using a forward-scattering D4WM setup, one can further improve detection limits and simplify optical alignments significantly.32 Attractive characteristics of D4WM as a unique spectroscopic tool include high-resolution sub-Doppler spectral resolution for gas-phase analytes, convenient and efficient optical signal collection, and excellent detection sensitivity. Unique nonlinear dependencies of the observed signal on variousexperimental parameters include its cubic dependence on laser power and its quadratic dependence on absorption coefficient and analyte concentration. Circular dichroism and D4WM are both absorption phenomena, and the special characteristics of D4WM mentioned above can be applied to improve and enhance the conventional CD signal detection. In this paper, we report the first CD measurements by using a nonlinear spectroscopic technique. Enhancements of the CD measurements are obtained by using relatively low laser power levels and by taking advantage of the unique features of the forward-scattering D4WM setup.32 Circular dichroism is defined as the difference in absorbance of left circularly polarized light (LCPL)and right circularly polarized light (RCPL). The absorption-based CD signal can be describedin terms of molar absorptivities for LCPL and RCPL as
Ae = EL - tR
(1)
where e~ and ER are the molar absorptivities for LCPL and RCPL, respectively, and the CD quantity Ae is the differential molar absorptivity. Equation 1can be related to conventional absorbance by substitution into the Beer-Lambert law,
AA = A , - A , = AebC
eLbC - ERbC
(2)
where b is path length (cm), C is concentration (MI, and AL and A Rare the absorbances for LCPL and RCPL, respectively. The D4WM signal intensity can be expressed
(3) where K is a constant, I u ris the total input laser intensity, dnldT is the temperature coefficient of the refractive index, m is the fringe modulation level, e is the absorption coefficient of the nonlinear medium, and k is the thermal conductivity. Equation 3 contains several important characteristics of the D4WM signal as described in detail later.
EXPERIMENTAL SECTION A schematicdiagram of the forward-scatteringD4WM setup for CD measurements is shown in Figure 1. The 488-nm line output of an argon ion laser (Coherent,Model Innova 90-6, Palo Alto, CA) is split by a beam splitter (R/T = 30/70) to form the probe and pump beams. The path length difference between the two beams is kept less than the coherence length of the argon ion laser (5 cm) to maximize D4WM grating sharpness. A twoinpubbeam forward-scattering D4WM optical setups2 is used here in order to simplify the experimentalarrangement. While the three-input-beam backward-scattering D4WM setupm1 allows Sub-Doppler spectral resolution for gas-phase analytes, (32) Wu, 2.; Tong, W. G. A n d . Chem. 1993,65, 112-117. (33) Richard, L.; Maurin, J.; Huignard, J. P. Opt. Commun. 1986,57, 366-370. (34) Simoni, F.; Cipparrone, G.; Duca, D.; Khoo, I. C. Opt. Lett. 1991, 16,360-362.
Epmk
2891
t rmp
v!
E
Argon Ion Laser Polarizer Figure 1. Forward-scattering D4WM circular dichroism experimental setup.
the two-input-beam forward-scatteringD4WM setup offers much simpler optical alignment since only two input beams are used, as shown in Figure 1. In order to optimize the D4WM-CD signal, the polarization plane of the argon ion laser beams entering the sample is further purified by using three Glan Thompson prism polarizers. As shown in Figure 1, one polarizer is placed near the laser head, and one polarizer is used in each input beam path near the analyte cell. All polarizers are adjustedto obtain pure linear polarization for the pump beam entering the Pockels cell and for the probe beam entering the sample cell. A Pockels cell (Lasermetrics, Model LMA-4) is placed in the pump beam path behind one of the Glan Thompson polarizers, and it is driven by a home-built variable-frequency high-voltage waveform generator that is referenced to a lock-in amplifier (Princeton Applied Research, Model 5702). The purified plane-polarized pump wave entering the Pockels cell is converted into alternating left and right circularly polarized light at suitable modulation frequency. Careful alignment of the Pockels cell is critical in obtainingthe optimum D4WM-CD signal strength. If the crystal is poorly aligned or the applied voltage is not controlled properly, the polarization will be ellipitical and not purely circular as desired. This will generate an error signal at the reference frequency as described later. The two input laser beams are focused on the analyte flow cell with a 0.1-mm path length (Starna Cells, Inc., Type 48) using a 10-cm focal length lens. The diameter of the focused beam spot is approximately 34 pm, and the input beams intersect in the sample cell with an angle of 1.5. A peristaltic pump (Rainin Instruments, Model Rabbit MiniPuls2) is used to slowly flow (at 0.09 mL/min) the analytes through the cell to ensure that a freeh supply of sample is constantly enriching the probe volume. The signal beam is then directed past a beam trap and through a focusing lens and a 48&nm laser line filter. The photomultiplier tube currentresponse is then sent to a current-to-voltageconverter and then on to the lock-in amplifier. Finally, the D4WM-CD signal is monitored by a strip chart recorder and a personal computer. Optically active (+)- and (-)-tris(ethylenediamine)cobalt(III) complexes, (+)[Co(en)s]I~and (-)[Co(en).& are synthesized and prepared using standard methods.% Racemic mixture solutions of these enantiomers are also prepared. All solutions are prepared using a water-ethanol solvent mixture (1:l by volume) and used immediately after preparation.
RESULTS AND DISCUSSION The nonlinear CD signal is generated via (1)formation of the D4WMgratings and,hence, (2) generation of the scattering signal, (3) proper electrooptical polarization modulation of the input pump beam, and (4) appropriate demodulation of (35) Angelici, R. J. Synthesis and Techniques inlnorganic Chemistry, 2nd ed.; W. B. Saunders Co.: Philadelphia, 1977.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 21, NOVEMBER 1, 1993
ProbeBeam
PumpReam (RCPL Cycle)
-
Strong Signal Ream
Strong Grating
Weak Signal Beam
ProbeBeam
PumpBeam (LCPL Cycle)
I
Weak Grating
Figure 2. Grating and signal response from an optically active analyte to polarization modulation. The grating, and hence the D4WM signal, is stronger for one type of circular light than It is for the other. The difference between these two signals yield the D4WM-CD signal. the output D4WM signal. As shown in Figure 1,a forwardscattering degenerate four-wave mixing (F-D4WM) setup32 uses only two input laser beams traveling in the same direction toward the sample cell. The two input beams are focused and mixed inside the flow cell by the use of a single 10-cm focal length lens, yielding a small laser probe volume of 98 pL. Constructive interference between the input pump and probe beams inside the absorbing nonlinear medium (Le., the analyte) creates a laser-induced thermal grating.32,36The output signal beam is then scattered off of this grating and conveniently detected by a PMT or a photodiode. The conventional CD signal has been generally described as the differential absorbance of LCPL and RCPL by an optically active sample. With the D4WM-CD setup, the CD signal can be defined as the differential D4WM signal generated by differential absorbance of LCPL and RCPL by the analyte. The intensity of a basic D4WM signal beam depends primarly on the sharpness or strength of the D4WM grating from which the signal beam is generated and diffracted. The D4WM grating, in turn, depends on analyte and solvent properties including analyte molar absorptivity and solvent thermal properties. An optically active sample absorbs LCPL and RCPL differently and, hence, generates different levels of grating strengths for LCPL-induced gratings and RCPL-induced gratings. The nonlinear CD signal is then obtained based on the difference in the LCPL-induced D4WM signal and the RCPL-induced D4WM signal. Figure 2 illustrates the relative D4WM signal strengths generated by the LCPL-induced grating and the RCPL-induced grating during one cycle of the Pockels cell modulation frequency. When the analyte is a chiral molecule that absorbs more RCPL than LCPL, upon irradiation with a vertically plane polarized probe beam and a RCPL pump beam, a strong thermal grating is formed in the sample resulting in a strong D4WM signal. Conversely, if the analyte is irradiated with a linearly polarized probe beam and a LCPL pump beam, a weaker D4WM signal is observed due to less efficiently generated gratings. The circular dichroism signal of the analyte depends on the difference between these two signals. A Pockels cell is placed in the path of the vertically plane polarized pump beam to generate and modulate LCPL and RCPL at a desired modulation frequency. By applying an appropriate voltage (i.e., the quarter-wave voltage) to the crystal, the plane-polarized pump beam polarization is (36) Hoffman, H. J. IEEE J. Quantum Electron. 1986, QE-22,552562.
Figure 3. Straight D4WM signals and D4WM-CD signals for (+)-, (A)-, and (-)C~(en)~~+ solutions(5.0 X M) usinga 250-mW laser intensity and a 1-s lock-in amplifiertime constant. Baseline levels are recorded by blocking one of the input beams,and the signal “peaks”are recorded by sending in both input beams. For straight the D4WM signal, all solutions show equal intensity. For D4WM-CD signal, the (+) and (-) solutions show equal but opposite signals, while the racemic mixture shows no significant signal. Peaks are shown as collected without using any computer noise smoothing schemes. converted to circularly-polarized light and then modulated right and left circular at the frequency of the applied driving waveform voltage. The differential D4WM signal (i.e., the CD signal) is then modulated a t the same Pockels cell frequency and detected conveniently by the lock-in amplifier. Circular dichroism based on D4WM is demonstrated for (+)Co(en)$+, (-)Co(en)33+, and a racemic mixture, (A)Co(en)33+,using the same concentration (5.0 X M) and solvent ratio (1:1 water-ethanol) for all samples. Although pure ethanol is not suitable as a solvent for cobalt complexes, a 1:1water-ethanol solvent mixture offers higher dnldTvalue and, hence, higher thermal grating efficiency and stronger D4WM signal, as demonstrated p r e v i ~ u s l y .As ~ ~expected, we observe stronger D4WM signals of Co(en)s3+in the waterethanol solvent mixture than those in the pure water solvent. Hence, the CD signal based on D4WM could be effectively optimized by using a solvent mixture with the best dnidT value, which, at the same time, does not significantly affect analyte circular dichroism, since in many cases a molecule’s CD is solvent d e ~ e n d e n t . ~ ’ In Figure 3, the D4WM-CD signal and the straight D4WM signal are compared for the three optically active cobalt complexes. Baseline levels are recorded by blocking one of the input beams, and the signal “peaks“ are recorded by sending in both input beams. While the D4WM-CD signal is detected via a polarization-modulated detection scheme using a Pockels cell (at 103-Hz modulation), the straight D4WM signal is collected via an amplitude-modulated detection scheme using a mechanical chopper (35 Hz). For theD4WM-CD signal, a vertically plane polarized probe beam and a RCPLiLCPL modulated pump beam is used. For the straight D4WM signal, both the probe beam and the pump beams are vertically plane polarized, and the pump beam is modulated by a mechanical chopper. The top spectra in Figure 3 are the conventional straight D4WM peaks for the three cobalt complexes. As expected, all three solutions exhibit strong signals that are equal in intensity, since all three samples have identical concentrations and identical (37) Sakaguchi, U.; Nakazawa, H.; Sakai, K.; Yoneda, H. Bull. Chem. SOC.Jpn. 1982,55, 1862-1868.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 21, NOVEMBER 1, 1993 298) absorption coefficients for the linearly polarized light. The bottom spectra in Figure 3 are the D4WM-CD peaks for the three cobalt complexes. As expected, the (+) enantiomer exhibits a positive D4WM-CD peak while the (-1 enantiomer yields the same intensity signal in the opposite direction. The optically inactive racemic mixture solution yields no strong peaks in either direction and maintains a balancedout blank-signal level a t the baseline. Figure 3 demonstrates the selectivity of the D4WM-CD detection scheme for (+) and (-1 enantiomers by yielding CD peaks in the correct directions. Yielding neither a positive peak nor a negative peak for the racemic mixture also indicates that the D4WMCD detection setup has minimum experimental errors that could contribute to a non-CD artifact peak. Artifact non-CD error signals could either add or subtract to the true D4WM-CD peaks, and they could be observed,for example, when the Pockels cell is poorly aligned, or when the Glan Thompson polarizers are not used in the experimental setup. Non-CD artifact peaks could be induced primarily by impure circular polarization (i.e., elliptical light) entering the sample and contributing to the grating formation process. A pure RCPL or LCPL beam contains equal amounts of horizontal and vertical polarization components. When a thermal D4WM grating is generated by constructive interference of two laser beams, one with linear verticalpolarization and one with circularpolarization,the grating is formed mainly by the parallel polarization components of the two beams, and with minimum contribution from the cross-polarized components of the two beams. Hence, the D4WM signal is sensitive to the changes in the polarization state of the input beams. For CD measurements it is necessary to precisely control the purity of the pump beam polarization during the modulation cycle. It is specifically important to ensure that the vertical component of the LCPL has the same intensity as that of the vertical component of the RCPL. Otherwise, a non-D4WM-CD artifact signal could be generated, when a nonoptically active sample, such as racemic (*)Co(en)33+, is used. AB shown in Figure 3, these potential error peaks could be kept to a minimum, and a baseline-level background obtained for a racemic mixture, by purifying the polarization of the linearly polarized input beams, carefully aligning the Pockels cell, and precisely controlling the applied driving waveform voltage for the Pockels cell. Like circular dichroism, D4WM is an absorption-based phenomenon, and hence, analyte molar absorptivity directly contributesin the D4WM signalgeneration process. As shown in eq 3, the straight D4WM signal has a quadratic dependence on absorption coefficient and, hence, on analyte concentration. As shown in Figure 4, the CD signal collected by D4WM is also experimentallyobserved to have a quadratic dependence (slope 2) on the analyte concentration, since the CD signal is simply the difference between the RCPL- and LCPLinduced D4WM signals. Equation 3 also indicates that the straight D4WM signal has a cubic dependence on the total laser input intensity. Figure 6 shows the experimental data verifying a cubic dependence (slope 2.9) of the CD signal collected by D4WM on input intensity. Quadratic dependence on concentration and cubic dependence on laser intensity are the two important properties of the D4WM signal that can be used to verify that the signalis, in fact, the nonlinear D4WM signal. By obtaining proper experimental slope values (i.e., 2 for concentration and 3for laser intensity),one can ensure that there is minimum background noise level present, since the background scattering noise has a linear relationship. One of the reasons the D4WM signal offers excellent detection sensitivity is its nonlinear dependency on laser intensity. For example, a 10fold increase in total laser intensity would result in a 1000-
100.0
L
1 -
-2
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r
1.0
r
(-)Tris (ethylenediamine) cobalt (111)
Argon ion laser 488-nm excitation Slope = 1.96
.-mOD
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I
I
I
I
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I
1
l
l
l
l
concenwauon. 100 0
2e4
z
P (-) Tris (ethylenediamine) cobalt (111) (111) Argon ion laser 488-11117 excitation Slope = 2.9
100
1000
Laser Power (mW)
Flguro 5. Cubic dependence of D4WMCD signal on argon kin laser
intensity for (-)Co(enha+.
fold increase for the D4WM signal. Although this nonlinear dependency codd also yield a higher level of source-lightinduced noise a t the absorption line center as compared to conventional linear laser methods, the increased noise level is more than compensated for by even more enhanced level of signal strength, and the fact that other optical noise sources (e.g., scattering) increase only linearly with laser power while the signal increases nonlinearly. In addition, since the signal collection efficiency is virtually 1007% (unlike fluorescence), the net gain in SIN could be still high compared to many conventional linear laser-based methods. Similarly, the nonlinear dependency of D4WM signals on analyte concentration could be viewed advantageously or disadvantageously since the signal strength changes more rapidly as the concentration increases or decreases. Regardless, D4WM methods yield excellentdetection limits for both fluorescingand nonfluorescingmolecules due to many unique properties of D4WM as described below. In conventional CD spectropolarimetric methods, the detection sensitivity is usually poor, partly because of their reliance on the measurement of a small difference in two large “transmitted” intensities when the absorbance of a RCPL beam (or a LCPL beam) is determined. However, in the nonlinear D4WM-CD method, the absorbance signal for a RCPL beam (or a LCPL beam) is determined by measuring
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 21, NOVEMBER 1. 1993
Table I. Comparison of Laser-Based Methods for CD Measurement of Co(en)ss+Complex cell path concn detectn laser-based CD method length (mm) limit (M) AA detectn limit high-frequency modulated CD (HPLC)
20
high-frequency modulated CD (microbore LC)
10
thermal-lens CD (crossed beams) thermal-lens CD (collinear beams) F-D4WM CD
2 10 0.1
4.3 x 1.6 X 2.7 X 4.7 X 9.4 x 1.1 x 2.9 X
1.6 X 10-5 (injd)osb 6.1 X 10-8“ 5.1 X 10-5(injd)o*b 8.9 X lodo 3.6 X 1 P 1.4 X 10-5 5.5 x 10-70
10-6 106 (injd)o-b 10-6
mass detectn limit (pg) (probe vol)
ref
16“
11
6.5”
11
36” 100 0.68
16,17 17 this work
10-5 (injd)”-b
lodo 10-6 10-7
a Converted from reported data where available (MW 239, Ac = 1.9 L cm-l mol-’, AA = AtbC). “Injected” LODs are listed along with detected LODs for HPLC-interfaced CD experiments.
a “positive” signal intensity of a sharp coherent laser beam, virtually against a dark background. Furthermore, while the conventional CD signal (i.e., molecular ellipticity, [el) has a “linear” dependence on A6 (difference in molar absorptivity for RCPL and LCPL), the coherent D4WM signal beam intensity has a quadratic dependence on molar absorptivity. In addition, since the D4WM-CD has a cubic dependence on excitation laser power, high photon density available from a laser is advantageously used. While many conventional CD optical signal measurements are made in an in-line arrangement, the D4WM-CD signal beam does not propagate in the exact same direction as the pump beams but at a small angle and, hence, allows one to suppress the source-light-induced scattering interference more effectively. One of the most attractive features of a forward-scattering D4WM optical setup is the single-lens-based focusing and wave-mixing arrangement that offers effective use of very short analyte path lengths for absorbance measurements. Using an analyte path length of only 0.1 mm, a probe volume of 98 pL, and a total laser input power of 250 mW, we determine a circular dichroism mass detection limit for (+)C0(en)~3+ to be 0.68 pg or 2.8 fmol (S/N = 2). As shown in Table I, the detection sensitivity levels of the D4WM-CD method, especially the AA LOD and the mass LOD, compare well with conventional CD or other laser-based CD methods. In summary, the use of a nonlinear laser method-based F-D4WM is demonstrated for sensitive circular dichroism measurements in liquid solutions. Detection sensitivity is excellent, showing more than 1 order of magnitude improvement in mass detection limit over previously reported results
for optically active Co(en)+’+. Forward-scattering D4WMCD has many advantages over conventional laser- and nonlaser based CD methods. Unlike backward-scattering D4WM (B-D4WM) setups, a forward-scatteringD4WM (F-D4WM) arrangement uses only two input laser beams, and the optical alignment is much simpler (without major critical optical alignment constraints), the beam focusing and wave-mixing efficiency via a single lens is much higher, and a much smaller laser probe volume and much lower laser power levels can be used. Consequently, the D4WM-CD method offers excellent detectionsensitivity even when very short sample path lengths are used, making it suitable for interfacing with capillary chromatography and capillary electrophoresis systems. In addition, since only a single laser is required in a D4WM-CD setup, the overall system can be made quite compact in this one-color one-laser system. Nonlinear D4WM detection methods offer many potential applications, since the detection sensitivity is excellent for both fluorescing and nonfluorescing analytes, as demonstrated previously.’29-32
ACKNOWLEDGMENT We gratefully acknowledge partial support of this work from the National Institute of General Medical Sciences, National Institutes of Health, under Grant 5-R01-GM41032, and the National Science Foundation under Grant CHE8719843. RECEIVED for review February 8, 1993. Accepted May 26, 1993.
