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Sub-Microliter Flow-Through Cuvette for Fluorescence Monitoring of High Performance Liquid Chromatographic Effluents L. W. Hershberger, J. B. Callis,” and G. D. Christian Department of Chemistry, University of Washington, Seattle, Washington 98 195
channel (F) where it goes to a waste container. The optical region has an 8-mm diameter quartz window (D) on each of the four sides. Two opposite windows are used for irradiation of the effluent. T h e fluorescent emission is viewed a t 90’ to the excitation radiation through one of the two remaining windows. The quiescent volume between the windows is filled with sheath solvent. Because of laminar flow conditions, the solvent and sample streams do not mix. Instead, the sample is confined to the center of flow, and the result is a “walless” cuvette whose volume may be varied by adjustment of the relative flow rates of sample and sheath fluids. The inlet and outlet probes (L) can be removed for cleaning of the cuvette and adjustment of the gap between the two probes. This gap distance is determined by the thickness of the Teflon ring (H) which provides a seal. T h e nuts ( H ) a t the end of the inlet and outlet probes hold the inlet and outlet tube holders (K) in place with Teflon seals. The cuvette has been constructed to withstand backpressure so as to minimize the effects of bubble formation. Therefore, the windows and seals are able to tolerate a pressure differential. T o accomplish this, each quartz window is held against a Teflon seal by a stainless steel ring fastened to the rest of the detector with four screws.
A new sub-microliter flow-through cuvette suitable for monitoring liquid chromatographic effluents has been developed and tested. The cuvette is based on the sheath flow principle, in which the chromatographic effluent is injected into the center of an ensheathing solvent stream but does not mix with it because laminar flow conditions are maintained. The optical volume of the cuvette is easily varied by adjusting the relative flow rates of sheath and sample. The cuvette has been tested for stability, reproducibility, and dynamic range using porphyrins as test compounds. The detection limit for mesoporphyrin I X dimethyl ester was 8 X lo-‘’ M when the cuvette was operated with an effective detection volume of 53 nL.
Several recent papers observed that the design of present detectors is one of the major limiting factors in HPLC. Knox and Saleem ( I ) suggest that by using smaller diameter particles and shorter columns, the analysis time can be reduced without a loss in resolution. T h e reduction in these parameters is coupled with a reduction in elution volume. Other workers (2, 3 ) have developed methods to reduce liquid chromatography to a miniature scale. Work with microcolumns and flow rates as low as 10 bL/min, gives separation results comparable to larger HPLC columns, but reduces the expense needed for solvents and equipment. With smaller elution volumes, additional constraints on dead volume are placed on detector flow cells. These authors and others (4-6) suggest that injectors, connectors, and tubing can be improved and that the weakest part of liquid chromatographic equipment is detectors. Because of the need for HPLC flow cells with a minimum dead volume, we designed the fluoresence flow cell shown in Figure 1. This flow cell has an optical dead volume variable between 6 and 150 nL, and virtually no “washout” problem. We used laser excitation because of the ease of focusing and the increased power density obtainable. Since our intent was t o demonstrate t h e feasibility of building a sub-microliter cuvette for liquid chromatographic analysis, and to study its stability, reproducibility, linearity, and sensitivity, no attempt was made t o optimize t h e chromatographic conditions.
EXPERIMENTAL Optical. The response of the flow cell to a chromatographic effluent was obtained by the scheme in Figure 3. The optics consisted of a laser to provide the excitation radiation, a photomultiplier tube to measure the fluorescence intensity, and a focusing lens. The laser was a Spectra Physics model 162 argon ion laser operated to provide 8 mW of power at 488 nm. For the stability studies, the laser was a Spectra Physics model 164-05 argon ion laser operated to provide 100 mW at 488 nm. The photomultiplier tube, an EM1 9785A. had S-20 response, and was operated at 1000 V. The laser radiation was focused by means of a 10-cm focal length spherical lens onto the column effluent in the cuvette. The fluorescence emission was collected by a Leitz microscope with a 3.5x, 0.1 n.a. objective and focused onto the face of the PMT. The emission was filtered with a 520-nm cutoff, antifluorescent filter to remove the scattered excitation radiation. A 625-nm cutoff filter provided further rejection of stray radiation and fundamental Raman scattering. Also included in the microscope optics was a mirror to divert the emission to an eye piece for visual monitoring to align the optics and verify proper operation of the flow cell. The PMT current was converted to voltage by a Keithley electrometer and recorded by a Houston Instruments strip chart recorder. The high frequency noise was attenuated by an RC filter with a 1.5-s time constant. Chromatographic. The solvent output from a Micrometrics model 7000 pump was split into two fractions. One fraction went through a metering valve and then to the sheath inlets of the cuvette. The other fraction went through an injection valve, a column, and then to the cuvette. The pump was operated at a pressure of 109 atm and delivered 1.50 mL/min. Adjustment of the valve enabled the diameter of the stream to be varied from 50 to 230 pm, corresponding to effective sample volumes of 6 to 150 nL. For chromatographic analysis, the valve
DETECTOR CONSTRUCTION Our cuvette, diagrammed in Figure 2, is based upon t h e sheath flow principle first introduced by Crossland-Taylor ( 7 ) and later adapted by biophysicists for analysis of mammalian cells (8). T h e column effluent enters the flow cell in the inlet tube (A) which extends 14.5 mm u p into the cylindrical inlet channel (C) and which is centered by a guide near the top. At t h e bottom of the inlet channel, the sheath flow which is usually t h e chromatographic solvent is introduced by two inlets (B). Near the top of the channel the sample leaves the inlet tube (A) and is centered in the sheath flow. The combined flow is aligned in the 2-mm long by 500-bm diameter bore (E) and then jets across a n adjustable, 2-mm to 3-mm gap and finally through a 500-bm exit hole (G) into the exit 0003-2700/79/0351-1444$01 0010
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Solvent Reservoir
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Figure 1. Sub-microliter fluorescence flow-through cuvette for liquid chromatography Figure 3. Schematic diagram of the experimental instrumentation arrangement
Figure 2. Schematic diagram of the sub-microliter fluorescence flow-through cuvette. (A) Effluent entry tube; (B) sheath entry tubes; (C) sheath inlet channel; (D) 8-mm diameter quartz windows: (E) 500-pm diameter inlet alignment bore; (F) exit channel; (G) 500-pm diameter exit alignment bore; (H) Teflon O-ring; (K) inlet or outlet tube holder; (L)inlet or outlet probe; and (M) stainless steel nut
was adjusted to a sample diameter of about 150 pm with a volume of 53 nL. From Poiseuille's law, this implies a sample flow rate of 0.26 mL/min. A backpressure of 14 atm was applied to the flow cell to minimize bubble formation in the optical region. The injection valve was a Micrometrics analytical injection valve with loops of 1, 2, 4, and 8 pL of which the 1-and 2-pL loops were used. The 250 mm X 3.2 mm bore column was packed with 10-pm Spherisorb ODS packing. Reagents. All solvents were used as obtained except for the water which was distilled in glass. The absolute ethanol was U.S.I. reagent grade and the chloroform was Mallinckrodt spectral grade. The water and ethanol were partially degassed before use by stirring under a vacuum for 5 min. The protoporphyrin IX disodium salt was obtained from Calbiochem and the mesoporphyrin IX dimethyl ester (MPDME) was kindly supplied by Martin Gouterman. Enough protoporphyrin was dissolved in ethanol to make a 1.00 X lo4 M stock
Figure 4. Sample flow in the optical region of the flow-through cuvette obtained with a fluorescent microscope. The fluorescent compound is protoporphyrin I X disodium salt
solution. Stock solutions of MPDME were made by dissolving about 2 mg in 100 mL of chloroform. The exact concentration was calculated from the absorption obtained with a Varian Superscan 3 and the molar absorptivity at 399 nm (9). Both stock solutions were stable for several days when stored in the freezer at -6 "C. Dilutions were made with absolute ethanol just before use.
RESULTS AND DISCUSSION Verification of Laminar Flow. Figure 4 is a photograph of the optical region of the cuvette, The fluorescent compound was protoporphyrin dissolved in ethanol and the sheath was ethanol. The sample flow is confined to the center and does not mix appreciably with the sheath solvent. Such behavior is expected since the Reynolds number a t the narrowest orifice (E of Figure 2 ) is calculated to be 42 which is well below the Reynolds number of 2000 needed for onset of turbulent flow.
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
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Figure 6. Fluorescence response to an injection of 1 pL of a 4.16 X lo-' M solution (133 pg) of mesoporphyrin I X dimethyl ester. The eluting solvent was 90% absolute ethanol and 10% water
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Figure 5. Linearity of fluorescence response for the injection of 1 or 2 p L of different concentration solutions of mesoporphyrin I X dimethyl ester. The eluting solvent was 90% absolute ethanol and 10% water
Stability. T o check the steady-state response of the cuvette, the sheath and sample flows were each delivered separately from nitrogen pressurized containers. The flow rates of sheath and sample were set by adjusting the nitrogen pressure applied to each container. Since no backpressure was applied to the flow cell and a column was not used, the pressures needed were much lower than described earlier. For a 1.0 X M protoporphyrin solution run for 3 min at 0.133 mL/min, the response was found to vary by less than 2 % . Reproducibility. The cuvette was studied for reproducibility by injecting 4.97 ng of MPDME onto the column and then monitoring the effluent. The MPDME was eluted with a mixture of 90% absolute ethanol and 10% water. For a series of ten injections over a 3-h period, the eluted peak area was found to have a percentage range of 13.6% and a coefficient of variation of 4.25%. Linearity. T h e response of the detector to different concentrations was checked by the same experimental set-up. Thirteen different amounts of MPDME ranging from 53 pg to 53 ng were injected. Each injection was repeated at least three times and the results are shown in Figure 5 . The error bars for each point represent one standard deviation from the mean, and if no error bars are shown, the standard deviation is less than the size of the data point. A linear least squares fit to the data gives a slope of 1.093 f 0.013 where the 0.013 is the standard deviation of the slope. The correlation coefficient is 0.999. Detection Limit. Figure 6 shows a representative elution peak near the detection limit for MPDME which has a molar absorptivity of 7.9 X lo3L/mol-cm at 488 nm and a quantum efficiency of 10% (10). Using the method described by Diebold and Zare (11),the maximum concentration of a 53-pg injection was calculated to be 1.2 x M and the signal to noise ratio (S/N) was 3. The minimum detectable level with a S/N of 2 was 8 X lo-'' M. A t this concentration there were 2.5 X lo7 molecules in the optical region of the cuvette and 5 X lo5 molecules in the laser beam. Discussion. We believe the flow cell offers several advantages for use in HPLC detectors. The main advantage is its very small optical dead volume which is variable between 150 and 6 nL. The flow cell therefore shows potential use in
detectors designed for situations where low dead volume is important. We believe that in situations where a low flow rate is needed that the flow cell would maintain laminar flow conditions. One of us (J.B.C.) has worked with similar laminar flow cells using flow rates as low as 10 pL/min. A second advantage is the placement of the cuvette's optical windows away from the sample flow. The largest amount of scattered light occurs a t the window interfaces because of the large difference in refractive index between the quartz and air or solvent. Since the window is placed 5 mm from the sample flow, the scattered excitation radiation focused by the microscope optics is greatly reduced. A t the sample stream, the difference in refractive index between the sample and sheath solvent is much lower; therefore, the scattering is much less. A third advantage arises because the sample does not come in contact with the windows of the flow cell. This is particularly useful in analyses where the injected sample is dirty and eluted components tend to collect on and contaminate the windows. Laminar flow cells would also be useful in several other unique applications. As has been shown by Crossland-Taylor ( 7 ) and others ( B ) , laminar flow is uniquely applicable to particle analysis. Air particulates could be lined up and individually passed by a laser beam for monitoring the emission of each particle. A critical requirement for laminar flow cells is the necessity for stable flows of both sheath and effluent since the sample volume is dependent on the relative flow rates of the two. We were able to greatly reduce this problem by obtaining the sheath and effluent flows from the same pump.
ACKNOWLEDGMENT The authors express their gratitude to the chemistry department machine shop, and especially to Ed McArthur for construction of the flow cell and to Ken Barber for cutting the quartz windows. The authors also express gratitude to David Johnson and Chu-Ngi Ho for their helpful suggestions. LITERATURE CITED (1) Knox, J. H.; Saleem, M. J . Chromatogr. Sci. 1969, 7, 614-22. (2) Ishii, D.; Asai, K.; Hibi, K.; Jonokuchi, T.; Nagaya, M. J . Chromatogr. 1977 74., 157-68 -. (3) Tsuda, T.; Novotny, M. Anal. Chem. 1978, 5 0 , 271-75. (4) Kirkland, J. J.: Yau, W. W.; Stoklosa, H. J.; Dilks, C. H., Jr. J. Chromatogr. Sci. 1977. 15. 303-16. (5) Martin, M.; Eon, C.; Guiochon, G. J . Chromatogr. 1975, 108, 229-41. (6) Knox, J. H. J . Chromatogr. Sci. 1977, 15, 352-64. (7) Crossland-Taylor, P. J. Nature (London). 1953, 171, 37-38. (8) Wolbarsht. M. L.. Ed. "Laser Applications in Medicine and Biology", VoI. 2; Plenum: New York, 1974: Chapter 5. Eisevier Publiina ComDanv: (9) Falk. J. E. "PorDhYrins and MetalloDorDhyrins": . . . . . New York, 1964: p 233. (IO) Goutermn, M.: Khalil, GamaKddin, J. M i . Spectrosc. 1974, 53, 88-100. (11) Diebold, G. J.; Zare, R. N. Science 1977, 19, 1439-41.
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RECEIVED for review December 11, 1978. Accepted April 20, 1979. This research was supported in part by NIH Grant zGM22311.
