Anal. Chem. 1989, 67, 910-912
910
CORRESPOMDENCE Indirect Differential Thermal Lens Detection for the Reversed-Phase Separation of Underivatized Fatty Acids Sir: Recent advances in high-performance liquid chromatography (HPLC) have occurred at a greater pace than the methods for detecting the small amounts of analyte present in such separations. While absorbance detectors are very popular, their application to microbore or capillary column chromatography is difficult at best, due to the necessarily small detection cell volumes required. In addition, several biologically important molecules, such as certain steroids, amino acids, and many fatty acids exhibit limited absorbances in the UV-vis region of the spectrum. Other than derivatization (1-3) to add either a fluorescing or absorbing chromophore to enhance detection, a universal detection scheme such as refractive index (RI) detection must be employed. Even though much has been done to improve the detection limit and reduce the cell volumes to match the available chromatography (4,5),RI detectors are still limited in their use since they preclude the use of gradient elution and are highly temperature and pressure sensitive. Recently, the use of indirect photometric detection for ion chromatography has gained widespread attention (6,7).This same principle was applied by Takeuchi and others (8-10) to the detection of nonelectrolytes for reversed-phase separations. This method provides a universal scheme for detecting analytes at very low concentrations owing to the displacement of a highly absorbing additive (visualization reagent) by the analyte which results in a change in the measured absorbance. While this technique has been shown to be quite useful, it suffers from several drawbacks. First, reasonable detection limits are available only under rigorous experimental conditions (8). In addition, as with RI, solvenbgradient separations are not amenable to this type of system (11). Finally, the signal due to an analyte is highly coupled to its retention time, that is, analytes eluting near the visualization reagent peak yield larger responses than those obtained from eluting at higher or lower retention volumes. In cases where the chromatography has been optimized to produce higher signal for one analyte, the additive peak could easily interfere with other analyte peaks (11). The latter two problems arise due to the affinity of the additive absorbant for reversed-phase columns. In this communication we present a detection method similar to the indirect photometry described above, but with two major distinctions. First, while the additives used in indirect detection have an affinity for the column, we have shown the applicability of a hydrophilic dye as an additive for reversed-phase chromatography. This results not in the displacement of the absorbant from the column but in the replacement of the dye molecules with the analyte in the analyte peak volume, thus reducing the number of dye molecules and therefore the measured absorbance. Second, since dynamic reserve is a key factor in indirect detection (9), a method with an enhanced dynamic reserve should result in a decrease in the detection limit. By use of a differential photothermal lens (TL)spectrometer similar to that described by Harris et al. (12), a large dynamic reserve is obtainable, making UV-vis indirect detection feasible. The direct differential measurement makes this technique amenable to solvent gradient chromatography, while a t the same time 0003-2700/88/0381-0910$01.50/0
stabilizing the system to both pressure fluctuations in the chromatographic system and intensity fluctuations of the pump laser. The probe volumes available for this type of detection scheme are typically less than 50 nL, making it ideally suited for microbore or capillary chromatography.
EXPERIMENTAL SECTION The differential thermal lens spectrometer has been described in detail elsewhere (13). The apparatus was constructed on an 4 X 6 ft X 2.25 in. optical breadboard (Newport Corp., Fountain Valley, CA, Model XS46).The probe optics consisted of a 1-mW helium-neon laser (Uniphase, Sunnyvale, CA, Model 1101) chopped with an in-house-constructed, frequency-stabilized chopper and focused with a 30 cm focal length lens. The sample cells, constructed in-house from 6.2 mm aluminum stock and having a volume of 5 pL, were placed at approximately *3*j2 of the Rayleigh range of the probe beam. The signal was determined by measuring the amount of light passing a 0.02 in. diameter pinhole with a silicon photodiode (Hamamatsu Corp., Middlesex, NJ, Model S1790-04) that was reverse biased at 30 V through a 504 resistor. The output of the photodiode was sent to a lock-in amplifier (Stanford Research Systems, Palo Alto, CA, Model SR510) which was connected to a PC via an IEEE488 interface card (National Instruments, Austin, TX,Model AC-214) for data collection. Light from an argon ion laser &exel, Palo Alto,CA, Model 85.5) operating at 514.5 nm was sent through a ' / JFresnel rhomb (Karl Lambrecht, Chicago, IL, MFR-02-13-580) mounted on a rotational stage (Newport Corp., Model RSA-2) and then through a polarization beam splitter (Karl Lambrecht, MGLA-SW-lo), resulting in two separate pump beams. The rhomb served to rotate the plane of polarization from vertical to approximately 45O with respect to the plane of the table, allowing for adjustment of the intensity of either pump beam. The pump beams, consisting of 35 mW each, were then focused into the individual detection cells with a 25 cm focal length lens. After the initial setup, the system operated for several weeks with only minor adjustment of the pump beam positions. The chromatographic system consisted of a syringe pump (ISCO, Lincoln, NE, Model LC 5000) connected to an injector (Rheodyne, Berkeley, CA, Model 7010) with a 20-pL injection loop and a 25 cm X 4.6 mm i.d. 10-pm ODS column (Alltech Associates, Inc., Deerfield, IL).The two sample cells were connected in seriea by a length of 0.03 in. i.d. tubing having a total volume of 300 wL, about half the analyte peak volume. The eluent, consisting of 95% acetonitrile (Fisher Scientific, Fair Lawn, NJ, HPLC grade) and 5% 0.3 M aqueous solution of phosphoric acid, was degassed under vacuum in an ultrasonic bath. "Pontacyl" Carmine 2B (Du Pont, Wilmington, DE)was added to the eluent to give cm-'. The path length a solution with absorbance of 1.3 X of 0.04 gives an actual background absorbance of 5.2 X lo4. The absorptivity of this additive at 514.5 nm was determined to be 27.0 L g-' cm-l as measured with a conventional spectrophotometer (Carey, Palo Alto, CA, Model 210). Fatty acid standards consisting of decanoic, lawic, and myristic acids (Aldrich Chemical Co., Inc., Milwaukee, WI) were used without further purification. Treatment of the chromatographic data is similar to that described previously by Yeung and co-workers (14,15). Briefly, a running integration was performed on the digitized base-lineadjusted signal from the lock-in amplifier and saved for later analysis. 0 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL.
61, NO. 8, APRIL 15, 1989 911
RESULTS AND DISCUSSION The theory pertaining to indirect absorption detection in ion chromatography has been elegantly presented in Stranahan and Deming (16) and is relatively straightforward in that the observed signal is due to a displacement of an absorbing additive from the column by the anal@. In a similar fashion, the application of this technique to reversed-phase chromatography relies on the same type of column interaction in that the additive has an affinity for the stationary phase. When an analyte is present in the column, the equilibrium between the additive and the stationary phase is perturbed, resulting in either an increase or decrease of the additive in the peak volume as measured by the detector. In contrast, the studies reported here utilize an additive that has no (or very limited) interaction with the stationary phase. Chromatographic studies of the additive, "Pontacyl" Carmine 2B (C2B))verified this to be the case. Under several solvent systems, injections of C2B in varying concentrations showed that in all cases studied, the dye was eluted in the dead volume. By use of such an additive, the signal arises from a dilution or replacement of the visualization reagent by the analyte. In addition, studies of several fatty acids injected with and without the visualization reagent present in the solvent showed no difference in the retention times, ensuring no change in the chromatographic process. It has been stated that indirect absorption detection based upon this type of interaction is not feasible owing to the limited dynamic reserve of absorbance spectrometers (9). Dynamic reserve (DR) is defined as the ratio of the background signal to the minimum measurable change in signal and is indicative of the ability to measure a small change in the presence of a large background signal. This is exactly the situation encountered in indirect detection methods. For example, for a typical absorption measurement the maximum background signal that can be tolerated without increasing the noise values appreciably is about 0.5 absorbance unit (AU). However, a minimum detectable absorbance of approximately 5 x lo-' will yield a DR of loo0 which is not sufficient for most indirect detection applications) particularly with respect to reversed-phase chromatography. We have recently demonstrated an absorbance spectrometer based on the differential thermal lens principle that can tolerate a background absorbance of 0.05 and provide a minimum measurable absorbance of 2 X lo-' under chromatographic conditions (13). These parameters extend the DR of absorbance detection to 2.5 X lo5 while maintaining a detection volume of 32 nL, thus making its application to microcolumn HPLC detection a logical extension. The enhanced DR is due in part to specific design characteristics of the spectrometer and in part to the nature of the photothermal measurement. While the theory and design characteristics have been described in detail elsewhere (13), a general description of the system is provided here to illustrate the distinctive properties of the spectrometer. Since photothermal noise arising from pump-probe interactions in optics such as filters, lenses, or beam splitters is common in single-beam or collinear pump/probe spectrometers, by crossing the pump and probe beams only in the sample, any spurious signals have been eliminated. The oblique crossing angle (OCA) allows for experimental control over the absorbance path length while maintaining a simple and well-behaved detection method. Reduced chromatographic pump and solvent-induced noise is also observed due to the series arrangement of the sample cells (17,18). While this arrangement will yield a derivative-shaped peak, a simple data-processing method described by Synovec et al. (14,15) in which the entire base-line-adjusted chromatogram is integrated will provide a more conventional looking chroma-
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TI& (s;"c, Figure 1. Observed reduction of measured noise due to the dlffsrmtial response: A and C, signal observed when addlng a 9-mW Intensity fluctuation on a 26-mW pump beam over 5 s time intervals in cell one and two, respectively; B, differential response observed when optically pumping the thermal signal in both cells simultaneously under the same conditions listed above.
togram. This method has the added benefit of providing an increase in the measured signal to noise ratio. Since the thermal lens signal is a direct function of solvent characteristics, the series arrangement must be used for solvent gradient chromatography to assure a true differential response. In addition, the differential nature of the thermal lens measurement has been utilized to optically subtract the background signal. This also serves to reduce measured noise due to pump laser intensity fluctuations since any thermal fluctuation due to the pump laser in one cell will be compensated for by an equal but opposite in magnitude fluctuation in the other cell. A dramatic example of this can be seen in Figure 1. Entries A and C result from an experimentally produced fluctuation in the pump laser power to cell one and cell two, respectively. This effect is intended, in an extreme sense, to mirror the intensity fluctuations characteristic of continuous wave (CW) lasers. The response is a result of changing the pump intensity by 25% over a 5-9 time period. However, when both cells are optically pumped simultaneously, the middle signal (B) is observed. It should be noted that total optical noise compensation is not feasible under such extreme conditions due to minor differences in path length, pump/probe crossing angle, or intensity of the pump beams at each cell. However, it is remarkable that the peak-to-peak noise with the 25% added intensity fluctuations as measured by the differential response is only a factor of 3 worse than our best case noise with no added pump laser fluctuations. Thus the assumption that the differentially arranged thermal lens system is capable of minimizing the influence of pump laser noise is well founded. The study and analysis of fatty acids are important areas of chemistry in that they make up a large portion of cellular membranes, they are an important energy store when coupled with acetyl-coA, and they play a key role in the function) storage, and transport of cholesterol. However) the saturated fatty acids do not show appreciable absorption above 210 nm, making their detection difficult. A chromatogram obtained from the separation and indirect T L detection of three biochemically important fatty acids is shown in Figure 2. The top chromatogram is the original signal as measured by the series cell arrangement, while the bottom is the base-lineadjusted integrated result. It is important to note that the ratio of the peak areas for the three different fatty acids correspond exactly, within in-
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912
Anal. Chem. 11989,61. 912-914
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Figure 2. Chromatogram resulting from the injection of 23 pg of decanoic acid (A), 20 pg of lauric acid (e),and 20 pg of myristic acid (C). The top chromatogram is the indirect TL detected signal and the bottom is the result of integrating the base-line-adjustedchromatogram.
tection method will be readily adaptable to microcolumn conditions. Optimization of other experimental variables such as background absorbance or the use of visualization reagents with similar hydrophilic properties but larger molar absorptivities will also result in improvements in the LOD by increasing the DR. This is reasonable because the system is presently operating below the background limitation by 2 orders of magnitude. In conclusion, this communication shows that indirect absorbance detection is feasible for microcolumn chromatography when photothermal methods of detection are applied. This is a result of the large dynamic reserve available in the differential thermal lens spectrometer described as well as the reduced detection volume available with laser probes. While more studies are needed to optimize the experimental parameters both in the chromatography and in the spectrometry, a universal mass LOD of 15 ng of detected and- is a marked improvement for a simple, universal HPLC detection scheme.
LITERATURE CITED Halgunset, J.; Lund, E. W.; Sunde, A. J . Chromafogr. 1982, 237(3),
jection variability, to the ratio of the amount of material injected. In contrast, the signal observed in previously described indirect methods is a function not only of the amount injected but also of the retention time. If the signal arises only from the dilution of the visualization reagent, response of this detector should not rely on any physical or chemical property of the eluent other than injected mass. This universal response would be quite useful for quantitative measurements in that a standard response curve for one analyte could be used for any other analyte detected in a given solvent system. This statement would be valid as long as the analyte and solvent behave as an ideal solution. The response of the detector has been determined to be linear over at least 2 orders of magnitude above the detection limit. The plot of normalized peak height response versus the normalized mass of dodecanoic acid injected had a slope of 0.98, an intercept of 0.06, and a correlation coefficient of 0.990 (5 points). The signals measured in several chromatograms were used to determine an average limit of detection (LOD) of 15 ng detectable in the cell corresponding to 500 ng of injected acid. In terms of the actual probed volume of 32 nL, the amount of decanoic acid detectable in the detection volume a t the detection limit is 90 pg. It should be stressed that the detection volumes demonstrated here are best suited to microcolumn techniques rather than the large diameter columns used in this study. Smaller column sizes can reduce the LOD by a factor of 20 (1 mm i.d. column) due to the reduced peak volumes (9) characteristic of microcolumn chromatography. Preliminary results using 0.5-pL detection cells (1.3 mm path length) have demonstrated that this de-
496-499.
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Watanabe, Y.; Imai. K. Anal. Biochem. 1981, 116(2), 471-472. Hancock, D. 0.; Synovec. R. E. Anal. Chem. 1088, 60, 1915-1920. Bornhop, D. J.; Dovichi, N. J. Anal. Chem. 1986. 58, 504-505. Small, H.; Miller, T. E. Anal. Chem. 1982, 54, 462-469. Downey, B. P.; Jenke, D. R. J . Chromatcgr. Sci. 1987, 25(11), 5 19-524.
Takeuchi, T.; Ishii, D. J . Chromatogr. 1987, 393, 419-425. Takeuchi, T.; Yeung, E. S. J . Chromatogr. 1988, 366, 145-152.
Banerjee, S. Anal. Chem. 1985. 57, 2590-2592. Takeuchi, T.; Ishii, D. J . chrometogr. 1987, 403, 324-330. Dovichi, N. J.; Harris, J. M. Anal. Chem. 1980, 52, 2338-2342. Erskine, S. R.; Bobbitt, D. R. Awl. Spectrosc., in press. Synovec, R. E.; Yeung, E. S. Anal. Chem. 1985, 57, 2162-2167. Synovec, R. E.; Yeung, E. S. Anal. Chem. 1986, 58, 2093-2095. Stranahan, J. J.; Deming, S. N. Anal. Chem. 1982, 54, 1540-1546. Banerjee, S.; Pack, E. J. Anal. Chem. 1982, 54, 324-326. Woodruff, S. D.; Yeung, E. S. J . Chromatcgr. 1983, 260, 363-370.
Steven R. Erskine Donald R. Bobbitt* Department of Chemistry and Biochemistry University of Arkansas Fayetteville, Arkansas 72701 RECEIVED for review October 18, 1988. Accepted January 24, 1989. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of this work. S.R.E. thanks the Analytical division of the American Chemical Society for support through a fellowship sponsored by DOW Chemical Co. D.R.B. acknowledges the support of the Camille and Henry Dreyfus Foundation through a teacher-scholar fellowship. This work was presented in part a t the 27th Annual Eastern Analytical Symposium, Oct 2-7, 1988.
Separation of Two Components of Horse Myoglobin by Isoelectric Focusing Field-Flow Fractionation Sir: In 1984 a theoretical analysis of the focusing field-flow fractionation in channels of various cross-sections was published (I). The focusing principle was originally described by one of the authors (2) in 1982 and approved experimentally in 1986 for a special case of the sedimentation-flotation focusing field-flow fractionation (3). Other methods of focusing field-flow fractionation were suggested but none was realized (4-6). 0003-2700/89/0361-0912$01.50/0
In this communication the first experimental implementation of the isoelectric focusing field-flow fractionation (IEFFFF) is described. In addition to the electric field and pH gradient (3, IEFFFF employs a third active separationaffecting factor, viz., the flow of the liquid through the separation channel with the direction of the flow perpendicular to that of the electric field. The shape of the flow velocity profile formed is influenced by the geometry of the fraction0 1989 American Chemical Society
