AC Research
Articles Anal. Chem. 1996, 68, 1677-1684
Polarimetry in Capillary Dimensions Darryl J. Bornhop* and Joseph Hankins
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061
Microinterferometeric backscatter is employed to detect optically active molecules in capillary tubes. The capillary polarimetric detector (CPD) is based on the interaction between a polarized laser beam and a capillary tube. The simple optical configuration employs a He-Ne laser, a glass polarizing plate, a fused silica tube, and a CCDbased laser beam analyzer. Side illumination of the capillary produces a 360° fan of scattered light that contains two sets of high-contrast interference fringes. These light and dark spots are viewed on a flat plane in the direct backscatter configuration. It is shown that the modulation depth for the high-frequency component is sensitive to the polarization plane for the illumination source and facilitates the quantitative determination of optical activity for a fluid contained in a capillary. A 90° rotation of exciting plane-polarized light results in a depth of modulation change of ∼80% when unmodified capillaries ranging from 75 to 530 µm i.d. are employed. Signal interrogation in the CPD is based on quantifying the relative intensities (depth of modulation) of adjacent high-frequency interference fringes. Mandelic acid, [r]23 of -153°, is used as the optically active molecule and can be detected at a 2σ detection limit of 1.49 × 10-3 M within a probe volume of about 30 nL. This limit of detection corresponds to 44 pmol or 6.7 ng of mandelic acid. The folded optical arrangement is simple and can be miniaturized with low-cost components. Initial investigations suggest that the CPD can be employed for detection in capillary electrophoresis or for studying the onset and level of solute aggregation. The advantages of miniaturized separation schemes are numerous and include reduced solvent consumption, small sample size requirements, high separation efficiency, increased analysis speed, and reduced analysis cost. Applications for these microseparations include DNA sequencing and the separation of S0003-2700(95)01169-3 CCC: $12.00
© 1995 American Chemical Society
enantiomers.1-3 The benefits of microschemes are clear, but these separation methods are currently limited by the availability of sensitive, small-volume detectors. A particular challenge is the direct detection of chiral species in systems that employ smalldiameter capillaries. The advantages of lasers in chemical analysis are well known and have been demonstrated thoroughly. These advantages include unparalleled sensitivity and selectivity. High spatial coherence, monochromaticity, and high photon flux are three characteristics that make lasers unique sources for chemical analysis. Over the past 5 years, technical advances in lasers have led to reduced cost, enhanced reliability, and the availability of new wavelengths or multiwavelength scanning systems. As a result of technical advances, a number of approaches to highsensitivity microvolume detection have been reported.4-7 Although various detection techniques have been applied to capillary separation schemes, chiral detection has been problematic. Low signal-to-noise ratios, large-volume flow cells, and complex optical arrangements currently limit the use of polarimetry in capillary-based separations. For example, Bobbitt and Yeung constructed a sophisticated device using a modulated argon ion laser to do polarimetry in a 1 µL volume flow cell8 and reported noise levels of 15 × 10-6 °. In a complex optical and electronic configuration, Synovec and Yeung used circular dichroism to measure chiral species,9 and Reitsma and Yeung constructed an optical activity and UV absorbance detector for LC.10 Lloyd et (1) Novotny, M.; Soini, H.; Stefeansson, M. Anal. Chem. 1994, 66, 646A-655A. (2) Bereuter, T. L. LC-GC 1994, 12, 784-766. (3) Monnig, C. A.; Kennedy, R. T. Anal. Chem. 1994, 66, 281R-314R. (4) Dovichi, N. J.; Zarrin, F.; Nolan, T. G.; Bornhop, D. J. Spectrochim. Acta 1988, 43B, 639-649. (5) Hill, H. H., McMinn, D. G., Eds. Detectors for Capillary Chromatography; Chemical Analysis 121; Wiley-Interscience: New York, 1992. (6) Bruno, A. E.; Paulus, A.; Bornhop, D. J. Appl. Spectrosc. 1991, 45, 462467. (7) Yeung, E. S., Ed. Detectors for Liquid Chromatography; Wiley-Interscience: New York, 1986; p 204. (8) Bobbitt, D. R.; Yeung, E. S. Anal. Chem. 1984, 56, 1577-1581. (9) Synovec, R. E.; Yeung, E. S. Anal. Chem. 1985, 57, 2606-2610. (10) Reitsma, B. H.; Yeung, E. S. Anal. Chem. 1987, 59, 1059-1061.
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al.11 employed a semiconductor diode laser with inherently low flicker noise to simplify the design of a polarimeter while improving sensitivity. They demonstrated that on-line polarimetric analysis can be performed on “dark solutions” in an 8 µL volume flow cell. However, the optical train11 still has two polarizers and requires some form of modulation technique. A sensitive technique based on vibrational circular dichroism producing spectral information has been recently reported.12 Although this technique is powerful, Tran and co-workers employed a somewhat complex optical configuration that is not well suited to miniaturization or flow cell volume reduction. In another effort to improve polarimetric detection capabilities, Mayster and co-workers13 coupled a refractive index detector to a conventional polarimeter for “equalization” and performance enhancement. A standard flow cell with a volume of 40 µL is employed in this “two-for-one” detector configuration. In each report noted above, improvements are made in the performance of chiral detection techniques while analysis volumes are reduced. However, the flow cells employed are far too large for capillary-based separation techniques. While circular dichroism has been performed in picoliter volumes,14 to our knowledge no detection scheme for polarimetry (monitoring nonabsorbing chiral solutes) has been possible “on-column” with capillary-based analysis techniques. The capillary polarimetric detector (CPD) reported here can facilitate high-sensitivity chiral detection directly on an unmodified fused silica capillary. The capillary polarimetric detector is an experimental variation of the microinterferometric backscatter detector (MIBD) recently developed15-17 to measure bulk property changes, such as refractive index and temperature changes, in picoliter volumes. In MIBD, a unique optical property resulting from the interaction of an unfocused laser beam with a small-diameter capillary tube (10500 µm) produces a beam profile that contains a high-contrast interference pattern.16 Measurement of positional shifts in the maxima and minima of the fringes facilitates universal detection in flowing streams at a sensitivity level corresponding to refractive index changes of about 4 parts in 107 within a probed volume of 350 pL (10-12 L). Here, we demonstrate that polarimetry can be performed in nanoliter volumes by using a modified interferometeric backscatter configuration. In this experiment, a linearly polarized light source is used for illumination, and by simply selecting the appropriate orientation of this polarization plane with respect to the central axis of the capillary, optical rotation can be measured. Included in our discussion are details of the construction, operation, and current limitations of the CPD. It is shown that the simple and inexpensive optical train of the CPD allows the direct observation or detection of chiral solutes in unmodified capillary tubes (containing the polymer coating) within a probe volume of 1.2 nL and that further reduction in probe volume is possible. (11) Lloyd, D. K.; Goodall, D. M.; Scrivener, H. Anal. Chem. 1989, 61, 12381243. (12) Tran, C. D.; Grishko, V. I.; Huang, G. Anal. Chem. 1994, 66, 2630-2635. (13) Mayster, F.; Bruno, A. E.; Kuhner, C.; Widmer, H. M. Anal. Chem. 1994, 66, 2882-2887. (14) Nunes, J. A.; Tong, W. G. Anal. Chem. 1993, 65, 2990. (15) Bornhop, D. J. U.S. Patent 5325170, 1994. (16) Bornhop, D. J. Appl. Opt. 1995, 34, 3234-3239. (17) Hankins, J.; Stienmetz, N.; Bornhop, D. J. Unpublished results, 1995.
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Figure 1. Block diagram for the capillary polarimeter. He-Ne laser is linearly polarized, PF is a 10 000:1 glass polarizing filter, ND is a neutral density filter, C is a fused silica capillary, Al block is a mounting block made of aluminum, and CCD is a camera. The output of the CCD is sent to a LBA (laser beam analyzer, not shown), which in turn sends a signal to a personal computer (also not shown).
EXPERIMENTAL SECTION The block diagram for the optical configuration is shown in Figure 1. All components are mounted on massive aluminum risers which are bolted to a 4 ft × 4 ft vibrationally dampened optical bread board (Newport Corp., CA). Upon passing the simple conditioning optics (discussed below), a beam from a 4 mW, linearly polarized He-Ne laser (Melles Griot, Irvine, CA) side-illuminates a fused silica capillary tube (PolyMicro Technologies, Phoenix, AZ) that has an outer diameter of 356 µm, an inner diameter of 249 µm, and a polyimide outer coating of ∼19 µm. The relative distances between optical components are as follows: 7.5 cm from the laser’s aperture to a neutral density filter (optical density 0.5-1.5), 2.5 cm to the polarizing plate, and 35 cm from the polarizer to the capillary tube. A commercial camera (Hamamatsu, Japan) based on a CCD detector is mounted on a micrometer-driven translation stage (Newport Corp.) and is positioned just below the illumination beam at a distance of 6.4 cm from the capillary tube. As shown in the side view of Figure 1, by slightly tilting the capillary, the folded optical configuration allows the backscatter radiation emanating from the tube to be directed onto the CCD imager. A laser beam analyzer (LBA 100, Spiricon Inc.) is employed to quantify and display the intensity distribution for a given cross section of the backscatter interference pattern and to quantitatively image the fringes. Functions of the LBA include 3-D intensity mapping, centroid position, and contour profiling. The CCD signals are displayed in real-time on a CRT monitor and stored on a 486 PC (Gateway, Inc., Austin, TX). A glass polarizing plate (Corning Optics, Corning, NY) with an exclusion level of 1:10 000 is used to further purify the polarization of the 1:500 linearly polarized laser beam. The glass polarizer is mounted on a high-precision rotation stage to simplify the selection and orientation of the exciting beam’s polarization plane. A second polarizer, a Glan-Thompson (Melles Griot) is employed to calibrate the rotation stage polarization plate assembly and allows for the accurate selection of the plane of polarization with respect to the central axis of the capillary tube. The rotation stage is configured to read zero when the preferential electric field vector of the laser is vertical or parallel with respect to the capillary central axis. We designate this orientation “parallel” and use “perpendicular” to denote the configuration where the plane of polarization is rotated 90°. A neutral density filter is needed to reduce the intensity of the exciting laser beam so that the fringe pattern impinging on the CCD sensor does not saturate the detector. The total distance from the capillary tube to the laser head is 45 cm. The flow cell consists of a capillary tube mounted
on a massive aluminum block (passive thermal stabilization) painted black, with a small circular hole drilled where the laser beam strikes the tube. The flow cell assembly is mounted on two stacked translation stages for ease in positioning and is tilted at a angle of about 7° relative to normal, as shown in Figure 1. Two frequencies of interference fringes are observed in the CPD. We use static solutions to ascertain experimentally how the position or relative intensity of the high- and low-frequency fringes are dependent on (1) the plane of polarization of the excitation source, (2) the refractive index of the fluid in capillary, or (3) optical activity of the solution in the capillary. First, a solvent is introduced into the capillary. The plane of polarization is fixed, and using the laser beam analyzer, the fringe pattern is sampled. Next, either the polarization plane of the laser or the solution is changed, and the beam profile is sampled. In the laser polarization experiments, the solvent contained in the capillary is H2O throughout. When solution refractive index or optical activity is tested, the laser polarization orientation is fixed. A blank solution of H2O is then injected, the fringe pattern is sampled, a solute containing sample is introduced, the beam profile is sampled again, and the initial blank solution is introduced into the capillary for beam profiling. The fringes are sampled by taking quantitative beam profile pictures or images along the x-axis of the interference pattern with the CCD camera, examining those pictures with the laser beam analyzer, and transferring them to a computer for storage and closer examination. We focus primarily on the central fringe and the three adjacent outlying fringes. For all solution experiments, the laser’s plane of polarization is oriented parallel to the central axis of the capillary tube. As shown below, this orientation produces the maximum depth of modulation for the high-frequency fringes. Sample solutions are introduced to the detection zone with a manual injection valve connected directly to the capillary (Valco, Inc., Houston, TX). Samples are analyzed static after the capillary is filled and the solution has stabilized (>3 s). All chemicals are reagent grade, and solutions are prepared in distilled water that has not been degassed or otherwise modified. Distilled water is also used as the blank. Methanol and glycerol are used to qualitatively determine the effect on the high-frequency fringe pattern upon changing the fluid refractive index. Mandelic acid, the R enantiomer at 99.9% purity (Baxter Chemical, St. Louis, MO), has a rotational value, [R]23, of -153° and is used as the chiral probe molecule. Changes in the high-frequency component of the fringe pattern by mandelic acid solutions are used to construct a calibration curve for response to optically active compounds. The percentages of glycerol in distilled water range from 0.01% to 0.07% by increments of 0.02% in volume percent. The concentrations of the solutions of mandelic acid in distilled water are 1.28 × 10-1, 8.52 × 10-2, 4.28 × 10-2, 1.28 × 10-2, 8.45 × 10-3, 4.35 × 10-3, 1.28 × 10-3, and 0.0 M (distilled water). RESULTS AND DISCUSSION The basic optical configuration for CPD is shown in Figure 1. It is similar to that recently employed in the microinterferometeric backscatter configuration used for refractive index and temperature determinations in capillary tubes.16 In each case, the signal is formed as a consequence of interference of radiation (refracted and reflected) produced when a laser beam is directed onto a cylindrical object. Specifically, an unfocused continuous wave laser beam impinges on a fused silica tube of capillary dimensions that contains the fluid or gas to be analyzed. The capillary tube
is tilted slightly to direct the interfering backscattered light rays in a plane above or below the plane of the excitation beam. Interference between the light rays that traverse different optical paths produces a series of light and dark spots (fringes) at the image plane. The fringes formed are found to have high contrast. In our earlier investigations with microinterferometric backscatter detection (MIBD), significant positional shifts were observed when the refractive index of the material in the tube changed; e.g., the position of an intensity maxima moves, sliding along the horizontal either from or toward the center of the pattern. These positional fringe shifts are proportional to the change in refractive index (∆n) and are discussed in detail elsewhere.18 Careful mapping of the MIBD fringes shows that the intensity profile is not entirely smooth. Initially thought to be an anomaly due to tube imperfections, it is now known that these intensity variations are a direct result of a polarization sensitivity. A preliminary model to describe the CPD phenomena which employs five-beam interference is currently under investigation in our laboratory. Here we provide just two results from this theory to give insight into how the CPD works. Figure 2 shows the intensity versus position profiles predicted by this model. In Figure 2A, the electric vector for the polarization component of the beam has been orientated to impinge upon the tube in the parallel configuration. As can be seen in the figure, a set of highfrequency fringes modulates the fringes used to measure bulk properties, such as changes in refractive index (RI) or temperature.18 Upon rotating this polarization component by 90° (the perpendicular configuration), the interference pattern is smoothed, and the high-frequency components are diminished. As described here, a set of “secondary” fringes can be introduced into the beam profile by properly orienting the plane of polarization of the laser relative to the capillary tube central axis. We will denote this set of fringes as the high-frequency fringes and the MIBD fringes as the low-frequency fringes. Figure 3 is the contour plot for a set of high- and low-frequency fringes. This figure clearly shows both modulation components in the beam profile. It should be noted that this report details the results obtained when using 250 µm i.d. capillaries but that similar interference patterns are observed when capillaries with inner diameters as small 50 µm are employed. As shown in Figure 3, the smoothness of the low-frequency interference pattern (i.e., the lack of spikes) is, in general, related to the orientation of the polarization plane of the laser beam. This polarization sensitivity facilitates chiral detection in nanoliter volumes with analytically significant signal-to-noise ratio. The high-frequency component of the fringe pattern is related to the orientation of the laser’s plane of polarization. The contour map of photon intensity impinging on the CCD as a function of position, shown in Figure 3, is taken when the capillary tube is filled with distilled water. Note that, in all color contour plots shown in this paper (Figures 5 and 6), white corresponds to the highest intensity and black the lowest. In order of decreasing magnitude, the intensity values range from white to red through yellow, green, blue, and dark purple to a minimum level represented by black. As we rotate the plane of polarization of the laser from parallel (Figure 3) to perpendicular (relative to the central axis of the capillary), the high-frequency fringes observed in the beam profile decrease significantly and in some cases are (18) Tarigan, H.; Neill, P.; Kenmore, C. K.; Bornhop, D. J. Anal. Chem. 1996, 68, 1762-1770 (in this issue).
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Figure 2. Results for a five-beam interference ray trace of the capillary using two different orientations for the polarization component of the laser beam and a fixed index of refraction for the fluid in the tube. (A) The scattered intensity profile of two and one-half fringes using a parallel configuration. (B) The scattered intensity fringe profile for the perpendicular configuration. The general form and depth of modulation levels compare well with the empirical results.
indiscernible. This change in intensity is directly related to the change in the angle of polarization of the incident beam, allowing polarimetry to be performed within capillary tubes. Even though relatively smooth profiles are possible in the MIBD configuration,16 in none of the experiments reported here are the high-frequency fringes completely eliminated. We believe that inconsistencies in tube diameter, tube coatings, and impurities in the polarization of the source lead to the somewhat nonuniform extinction of the high-frequency fringes. Efforts to produce 100% and 0% modulation are currently underway. Fluid RI changes are often a major source of interference in the measurement of optical activity.7,13,19 In our initial qualitative observations of the CPD, the position and relative spacing of the high-frequency fringes are found to be insensitive to small RI changes. To prove this observation, we mapped the fringes upon injecting solutions of from 0.01% to 0.07% glycerol into the capillary. As expected and predicted theoretically,17,18 a slight shift in the x-position of the low-frequency fringes is seen, yet no motion is detectable for the high-frequency fringes. Rather, it appears that the intensity of the low-frequency fringes moves through the high(19) Llyod, D. K.; Goodall, D. M. Chirality 1989, 1, 251.
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frequency fringes as if the high-frequency fringes are stationary. Neat methanol, which has a significantly different refractive index compared to that of water, causes a large shift in the position of the low-frequency fringes when injected into the tube after distilled water, yet it causes no readily apparent change in the position of the high-frequency fringes used for polarimetric measurements. It is therefore postulated that the high-frequency fringes are not a function of or dependent on the bulk refractive index of the solution in the capillary tube. Independence of the high-frequency component from the low-frequency component should allow two separate determinations (RI and polarimetry) to be made simultaneously and is currently under study. The principle of operation of the CPD is obtained through observation of Figure 4. Displayed in the figure are three sets of interference fringes, each obtained from the same capillary filled with the same aliquot of water. In the first frame, the plane of polarization is selected to be parallel to the central axis of the capillary. In the second frame, the laser and polarizing plate are simultaneously rotated by 45°. Finally, in the last frame, the polarization plane is rotated by an additional 45° to give the 90°, or perpendicular, orientation. This figure shows that, when the plane of polarization of the laser is parallel to the central axis of the capillary tube, the modulation intensity or contrast between the adjacent high-frequency fringes is at a maximum. Upon rotating the principal electric field vector component of the source by 90°, these fringes become blurred, and the contrast is significantly reduced. Therefore, by monitoring the relative intensity changes in the high-frequency fringe pattern, it is possible to monitor the polarization of incident light as it is rotated by 90°. In a conventional polarimeter, the excitation beam is plane polarized, and then it passes a flow cell and an analyzer (polarizer crossed with respect to the initial polarization plane) and is directed onto a detector. When no optical activity is in the cell, there is little or no light collected at the detector. As solute passes through the cell, some light is rotated and impinges on the detector. By analogy, the amount of polarized light reflected at each capillary interface should be dependent on the orientation of incident light. Rotation of this light by a solute contained within the tube should lead to a change in the interference pattern. As shown above, rotating the polarization plane of the excitation beam by 90° leads to the reduction of contrast for the high-frequency interference fringes. This change in intensity is then used as the signal in the CPD and, as shown below, is ultimately related to the amount of optically active solute contained in the capillary tube. Signal response is quantitatively measured by comparing the depth of modulation (DOM) for adjacent high-frequency interference fringes. DOM is defined as the relative intensity of a minimum and maximum for a set of high-frequency interference fringes, as shown in Figure 4. In this figure, H corresponds to the x (vertical)-position of the brightest spot of the fringe set, while L is the x-position for the valley or minimum of the fringe set. The y (horizontal)-position is selected along the short axis of the fringe. This position defines which pixels are used to measure the intensity of the fringe. We define DOM as a percentage value, given by eq 1, where Imax is the maximum intensity for the selected fringe and Imin is the minimum intensity of an adjacent null.
DOM ) ([Imax - Imin]/Imax) × 100%
(1)
Figure 3. Contour plots of the laser beam profile showing the central and two adjacent low-frequency fringes. Contained within the large fringes is the high-frequency (HF) modulation component used to measure polarization. “Parallel” refers to the laser beam plane of polarization oriented parallel to the central axis of the capillary, and “perpendicular” refers to a rotation of this laser polarization plane by 90°. Significantly increased HF modulation is observed in the parallel configuration.
Figure 4. Quantitative illustration of how modulation depth changes as the plane of polarization of exciting light is rotated from 0° to 90°. At parallel polarization, the depth of modulation is 71.4%, and for perpendicular polarization, DOM is 6.37%.
DOM values have been determined for the fringes produced by parallel and perpendicular orientation. In fact, the DOM values obtained by 90° rotation would inevitably define the limit of detection (LOD). Note from Figure 4 that the DOM for the parallel configuration is 71.4%, and that for the perpendicular configuration the DOM value has dropped to 6.37%. Our investigations with tubes of different diameters, different wall thicknesses, and varying coating thicknesses indicate that the absolute depth of modulation is affected by inconsistencies in the optical properties of the capillaries. Practical use of the optical train is primarily dependent on the ability to reproducibly obtain a large change in the modulation depth for a polarization change. This
prerequisite is easily accomplished using high-quality fused silica capillaries like those manufactured for CE. A simple calculation can be done to estimate the LOD in terms of absolute rotation. The laser beam analyzer was used to obtain absolute values for the intensities of adjacent fringes. These peak values are compared to the minima that define the peaks for calculation of the DOM, as described above. These are plotted as a function of the change in rotation (∆degrees) to yield a slope. Next, an estimation of noise was obtained by evaluating the intensity change of several minima at integration times from 10 ms to 1 s. Using the value at 500 ms, the value for σ, or the noise in the determination, is found. Assuming the LOD is 2σ/slope, Analytical Chemistry, Vol. 68, No. 10, May 15, 1996
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Figure 5. Map for backscatter interference pattern for (A) water, (B) 1.28 × 10-1 M mandelic acid, and (C) water. The color contours are representations of measured photon intensity. In order of increasing intensity, these colors are black, purple, blue, green, yellow, orange, red, pink. and white.
CPD can be used to measure absolute rotation changes on the order of 1.3 x 10-4 °. Related investigations17,18 lead us to believe that this value can be improved by a factor of 21. To test the concept that an optically active solute can rotate the plane of polarization of light as it traverses the small optical path of the capillary, we first fix the polarization plane of the source at parallel, producing a set of high-contrast, high-frequency fringes (HFs), and record the interference pattern (Figure 5A). Next, the beam profile is interrogated (Figure 5B) after water is replaced with a 1.28 × 10-1 M aqueous solution of (R)-mandelic acid, an optically active carboxylic acid. Third, we reintroduce H2O into the capillary and sample the beam profile. As shown in Figure 4, when starting with the polarization parallel to produce the maximum DOM, the level of high-frequency modulation for any pair of adjacent HFs in the beam profile is significantly changed when introducing the optically active solute. Although some of the fringes show more sensitivity to optical rotatory power than others (each set of HFs is not perfectly well behaved), we can choose several sets that provide significant sensitivity. In this regard, we can focus on the low-frequency fringe set, second from the central fringe. This fringe set is expanded and presented in Figure 6. In the first frame of Figure 6, water is contained in the capillary, and a high value (about 80%) is obtained for the DOM, as expected for a parallel configuration. When the optically active solute (mandelic acid) is introduced into the capillary, the DOM for any set of adjacent HFs is reduced significantly to a value of about 12%. This DOM reduction can be used to detect optical activity in capillary volumes and suggests a phenomenon where the impinging polarized light has been rotated by the solute, causing a change in the interference with the rays not in contact with the fluid (front surface reflections). Finally (bottom of Figure 6), upon reintroducing water into the capillary, the interference pattern is nearly identical to the pattern initially seen for H2O. Thus, the presence of an optically active solute in the capillary causes a relative change in the DOM of a set of HFs and can serve as a measure of the change in optical rotatory power of that solute. 1682 Analytical Chemistry, Vol. 68, No. 10, May 15, 1996
So, by simply side-illuminating a capillary with an unfocused, linearly polarized He-Ne laser beam, we can, for the first time, measure the optical activity of a fluid within a tube of capillary dimensions. For the CPD to be a useful analytical tool, such as a detector for capillary electrophoresis, the relationship between signal and solute concentration must be established. A response or calibration curve for (R)-mandelic acid is presented in Figure 7, where the DOM is plotted as a function of solute concentration. With the polarization of the laser orientation set at parallel to maximize the contrast (max DOM) in the high-frequency fringes, the change in DOM is found to decrease as the optical rotatory power or concentration of mandelic acid increases. This change with varying solute concentration is incremental and appears, upon first examination, to be logarithmic. Although more detailed investigations are needed to confirm this hypothesis, it is believed that the nonlinear response observed at relatively high concentrations of analyte is attributed to aggregation of the carboxylic acid molecules. Such aggregations often produce deviations in spectral or optical properties in solution20-22 and are commonly seen in highly hydrogen-bonding systems, particularly in enatiomeric mixtures of carboxylic acids.20 If we assume that the optical rotatory power is directly proportional to the concentration of an unbound solute, then at the inflection point in the curve, a significant change in the number density of unbound species is realized. It is known that, at some level of aggregation, the optical properties of a solute will change. If this perturbation in optical activity happens at the onset of 1:1 association, we can estimate from our plot that this ratio of molecules will occur at concentrations above about 4.28 × 10-2 M. In any event, molecular association will change the optical rotation capabilities of the system and can serve as a measure of (20) Eliel, E. L.; Wilen, S. H.; Mander, L. N. Stereochemistry of Organic Compounds, 2nd ed.; Wiley-Interscience: New York, 1994; pp 1071-1079. (21) Van Holde, K. E. Physical Biochemistry; Prentice-Hall: Englewood Cliffs, NJ, 1971; pp 213-218. (22) Marshall, A. G. Biophyscial Chemistry; John Wiley: New York, 1978; pp 7085.
Figure 7. Calibration curve for mandelic acid. As described in the text, DOM is a measure of the relative intensity change for adjacent maxima and minima for a high-frequency fringe. Two linear regions are observed, and the nonlinearity at high solute concentration is believed to be due to solute aggregation.
Figure 6. Expanded plot for the second low-frequency fringe shown in Figure 4. Note how the high-frequency modulation is changed upon introducing an optically active molecule. The top plot is for water contained in the tube, the middle plot is for a 1.28 × 10-1 M mandelic acid solution, and the bottom plot is for water again. Note the change in depth of modulation and the reproducibility of the fringe upon introducing the original fluid. For the meaning of the colors, see Figure 5.
these molecular changes. Further work is now underway to establish equilibrium constants for such solute systems and to apply this technique to the investigation of binding of small molecules or ions to macromolecules.
Closer observation of the curve might suggest two linear regions, one for the five least concentrated solutions and one for the three most concentrated solutions. Here we choose to employ the steep-sloping low-concentration region as a linear curve for the calculations of LOD and dynamic operation range. From this region of the calibration plot and an estimate of the noise determined over a 3 min period, an approximate detection limit can be calculated. At the 2σ level, we estimate the concentration LOD for mandelic acid to be 1.49 × 10-3 M. The probe volume for a 0.6 mm diameter beam and a 0.250 mm i.d. capillary is about 29.5 nL. Within the probe volume, there is about 44 pmol or ∼6.7 ng of solute present. Although the concentration detection limits reported here are somewhat higher than those previously reported,8,11,13 the mass detection limits are superior to any reported for polarimetric detection by nearly 3 decades,8,10-13 yet they fall short of those obtained for absorbing species when using CD schemes.9,14 Further, the system described here is simple and robust and can probe capillaries directly without modification (i.e., removal of the polyimide protective coating). It is noteworthy and quite encouraging that (1) the highest slope (DOM vs concentration) is obtained in the most desired region, at low solute concentrations, and (2) the absolute depth of modulation decreases as the concentration of solute increases, suggesting that, when properly calibrated, the CPD can be used to measure the actual angle of rotation of unknown chiral species. As a point of reference, a commercial polarimeter normally has a volume of 1.5 µL, and the best attempts to reduce these volumes to the nanoliter regime have been largely unsuccessful, with volumes ranging from 1 to 8 µL.8-13 Even though the 250 µm tube used in this investigation is too large for CE, capillaries as small as 75 µm have been used to produce this interferometric backscatter phenomenon15-17 with minimal reduction in signal amplitude. Therefore, the CPD should be readily applicable to high-sensitivity polarimetry in capillaries with inner diameters as small as 50 µm. Analytical Chemistry, Vol. 68, No. 10, May 15, 1996
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Using our preliminary experimental data, we can estimate the sensitivity of the device for chiral detection in capillary tubes. If a 10 V signal is associated with the maximum intensity for a fringe, we can calculate that a change corresponding to 9 × 10-6 ° rotation will be detectable in a 150 pL volume. The current detection method, based on using a laser beam analyzer to compare a set of adjacent fringes, is limited to analyzing static samples and would not be applicable to flowing streams. Two approaches are currently under investigation. A simple and inexpensive approach employs a spatial mask and a Si photodetector, positioned so that an intensity measurement can be made for one set of high-frequency fringes. The second technique is more complicated and is based on a recent report on smart sensing using an integrated circuit and a charge-coupled device.23 These are just two of the techniques that show promise for monitoring the changes in beam profile related to optical rotatory power of solutes as they traverse the detection zone. CONCLUSIONS It is shown that it is feasible to detect optical activity by monitoring the relative change of intensity (depth of modulation) of a pair of adjacent fringes that are produced when a planepolarized beam of radiation is used to side-illuminate a fused silica capillary tube. Using the fringe analysis methods currently under investigation, the capillary polarimetry detector can potentially be used as a detector in capillary electrophoresis and capillary HPLC. In the CPD experiment, laser beam focusing is not required, alignment of the capillary is particularly simple, and the protective polymer coating is left on the capillary, maintaining structural integrity. Nanogram sensitivity makes the device competitive with current conventional polarimetric techniques, yet this sensitivity is achieved within nanoliter probe volumes. Investigations currently underway include the further development of a descriptive model based on five-beam interference, the application to separation techniques, the study of signal generation, the quantitation of absolute optical rotation, and the distinction of R versus L racemers. Further, we will be using the CPD for noninvasive optical studies in hydrogen bonding and solute aggregation. (23) Seitz, P.; Spirig, T.; Vietze, O.; Engelhardt, K. Opt. Eng. 1995, 34, 22992308.
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As a note, it is believed that the instrument can also measure absolute rotation values in small volumes in addition to relative polarization changes as demonstrated above. Although the absolute rotation sensitivity is currently modest (on the order of 10-4 °), we have recently been able to improve the contrast ratio for adjacent fringes and, with an improved detection scheme, can predict significant improvements in detection limits. A simple estimation and calculation suggests that optical rotation can be measured at the level of 9 × 10-6 °. A comment regarding probe volume is also in order. Current chiral detectors based on polarimetry, not on circular dichroism (commercial microscale systems), have volumes in the 10 µL range (10-6 L). Past attempts toward miniaturization of polarimeters have produced some success,7-13 yet these systems still have volumes in the 1-8 µL regime. With the CPD, the probe volume is about 30 nL (10-9 L) for a 250 µm i.d. capillary, nearly 3 decades in volume reduction. Further reduction in probe volume to 110 pL (10-12 L) is predicted since we have observed the microinterferometric backscatter phenomena in capillaries as small as 75 µm i.d (17). Capillary polarimetric detection appears to be universally applicable to all optically active molecules. Finally, using recently established methods,24,25 miniaturization to the size of a single chip appears possible for the CPD. ACKNOWLEDGMENT This research was funded in part through grants provided by Texas Tech University, College of Arts and Sciences Research Enhancement Fund, and by The Dow Chemical Co. Neven Steinmetz, Abdolreza Darigan, and C. Kyle Kenmore are acknowledged for their assistance in preparing the figures for this manuscript. Special thanks are extended to Spiricon Inc. of Logan UT for the donation of the LBA-100. Received for review December 4, 1995. February 19, 1996.X
Accepted
AC951169B (24) Lee, S. S.; Lin, L. Y.; Pister, K. S. J.; Wu, M. C.; Lee, H. C. Grodzinski, IEEE Photon. Technol. Lett. 1994, 6, 1031-1033. (25) Lin, L. Y.; Lee, S. S.; Pister, K. S. J.; Wu, M. C. Appl. Phys. Lett. 1995, 66, 2946-2948. X Abstract published in Advance ACS Abstracts, April 1, 1996.
