Anal. Chem. 1991, 63,568-574
560
and consequently that this process (which may involve the formation of carbon-carbon double bonds or of cross-linking between the polymer chains) or a similar one might be more common in the case of ion bombardment than in the case of atom bombardment. The exaggerated difference in time dependence behavior for ion beams, as compared to atom beams, observed for PTFE, as compared to PET, certainly suggests that particle-surface electronic interactions are a major contributor to this preferential sputtering of fluorine.
CONCLUSIONS Comparison of spectral time-dependence behavior for P E T and PTFE has provided data concerning the nature of polymer degradation during particle bombardment. In particular, electronic particle-surface interactions have been shown to be a key contributing factor to degradation. In PET, this degradation is by straightforward physical degradation (random segmentation of polymer chains), which is accelerated by the use of charged primary particles. For PTFE, in addition to this process, an additional chemical degradation mechanism operates, which is, to a large extent, the consequence of electronic particle-surface interactions. This involves the preferential sputtering of fluorine and is manifested by the observation of a rise in peaks with a low fluorine to carbon ratio. At doses less than 1013 particles cmV2,this degradation mechanism may be nearly eliminated by the use of primary neutral particles rather than charged particles. Registry No. PTFE, 9002-84-0; PET, 25038-59-9;Ar, 744037-1; polystyrene, 9003-53-6; xenon, 7440-63-3.
LITERATURE CITED (1) Benninghoven, Alfred. I n Ion Formation from Organic SolMs: Benninghoven, Alfred, ed.; Springer-Veriag: Berlin, 1983: pp 64-89. (2) Fenseiau, Catherine. Reference 1, pp 90-100.
(3) Benninghoven, Alfred. J. Vac. Sci. Techno/. 1985. A3, 451-460. (4) Briggs, David. B r . PolymerJ. 1889, 27,3-15. (5) Hearn, Martin J.; Briggs, David. Surf. Interface Anal. 1988, 7 7 , 198-2 13. (6) Briggs, David. Surf. Interface Anal. 1882, 4, 109-115. (7) Kidweil, David, A.; Ross, Mark, M.;Coiton, Richard, J. Int. J . Mass Spectrom. Ion Processes 1987, 78, 315-328. (8) Pachuta, Steven, J.; Cooks, R. Graham. Chem. Rev. 1887, 647-669. (9) Yu, Ming, L. Nucl. Instrum. Methods 1887, 8 7 8 , 542-548. (IO) King, B. V.; Tsong, I . S.T.: Lin, S. H. Int. J . Mass Spectrom. Ion Processes 1987, 78, 341-356. (11) Sigmund, Peter. I n Sputtering by Particle Bombardment I ; Behrisch, Rainer, Ed.; Springer-Verlag: Berlin, 1981; Chapter 2. (12) Murray, P. Terrence; Rabaiais, J. Wayne. J. Am. Chem. Soc. 1981, 703, 1007-1013. (13) Toik, N. H., Traum. M. M., Tully, J. C., Madey, T. E., Eds. Desorption Induced by Electronic Interactions DIET I ; Srpinger-Veriag: Berlin, 1983. (14) Brenig, W., Menzel, D., Eds. Desorption Induced by Electronic Transitions DIET I I ; Springer-Veriag: Berlin, 1985. (15) Avouris, Phaedon; Walkup, Robert E. Annu. Rev. Phys. Chem. 1988, 4 0 , 173-206. (16) Briggs, David; Hearn, Martin, J. Vacuum 1986, 36, 1005-1010. (17) Brown, Alan: van den Berg, Jaap A.: Vickerman, John C. Spectrochim. Acta 1985, 408, 871-877. (18) Eccles, A. John; van den Berg, Jaap A,; Brown, Alan: Vickerman, John C. Appl. Phys. Lett. 1886, 49, 188-190. (19) Hunt, C. P.; Stoddart. C. T. H.: Seah, M. P. Surf. Interface Anal. 1882, 703, 157-160. (20) Wittmaack, K.; Maul, J.; Schultz, F. Int. J . Mass Spectrom. Ion Phys. 1973, 7 7 , 23-35. (21) Wittmaack, K. Vacuum 1982, 32, 65-89. (22) Wittmaack, K. Surf. Interface Anal. 1987, 70,311. (23) Briggs, David; Hearn, Martin J. I n t . J . Mass Spectrum. Ion Processes 1985, 6 7 , 47-56. (24) Leggett, Graham J.; Vickerman, John C.; Briggs, David. Unpublished work (UMIST, 1990). (25) Leggett, Graham J.: Briggs, David: Vickerman. John C. J. Chem. Soc., Faraday Trans. 1980, 8 6 , 1863-1872. (26) Leggett, Graham J. PhD. Thesis, UMIST, 1990.
RECEIVED for review August 27, 1990. Accepted December 7,1990. The financial support of the Science and Engineering Research Council is gratefully acknowledged.
Thermal Gradient Microbore Liquid Chromatography with Dual-Wavelength Absorbance Detection Curtiss N. Renn and Robert E. Synovec* Department of Chemistry, BG-IO, Center for Process Analytical Chemistry, University of Washington, Seattle, Washington 98195
Theoretical relationships are derived relating changes in the refractive index of the mobile phase in liquid chromatography to aperture limited absorbance measurements as applled to a single fiber-optic two-wavelength detector. The detection system was designed for remote sensing in thermal gradient microbore liquid chromatography (TG-pLC). TG-pLC was demonstrated with a reversed-phase separation of an unleaded gasoline sample for a temperature gradient of 25-150 OC over 30 min. The unique two-wavelength difference detection method, along with the single fiber-optic construction, virtually eliminated baseline drift associated with thermal Induced refractlve index ( R I ) aberrations. The detector provides a solution to R I aberrations not only for TEMLC but also for mobile-phase gradient liquid chromatography (MPG-LC) and other flow methods such as flow injection analyds (FIA). The advantages of TG-pLC are presented, including gradient separation capability for MLC, effective control of retention tlme comparable to MPG-LC, and separation efflclency over 72 000 theoretical plates/m using 5-pm packing material. 0003-2700/9 110363-0568$02.50/0
INTRODUCTION From the inception of modern liquid chromatography, UV-vis absorbance detection has played a fundamental role as a diagnostic tool to provide qualitative and quantitative information of complex mixtures ( I ) . The development of new separation techniques, however, has placed additional demands on absorbance detection that have not been previously solved. Mobile-phase gradient liquid chromatography (MPG-LC) ( 2 ) ,supercritical fluid chromatography (SFC) (3), and the more recent technique of thermal gradient LC (TGLC) have placed an additional burden on absorbance detection as a result of large refractive index changes of the mobile phase, interfering with aperture-limited absorbance measurements. To further hamper absorbance detector design, the trend toward microbore LC (pLC) has necessitated small volume flow cells, in the sub to low pL range, in order to preserve the integrity of the chromatographic separation ( 4 ) . Attempts have been made to reduce thc refractive index dependence of absorbance detectors by focusing the light past 0 199 1 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 6, MARCH 15, 1991
the exit of the flow cell, thereby eliminating the optical aperture limitation (5);however, this approach is not readily applicable to long optical path length pLC flow cells, which employ variable or multiwavelength detection. The challenge, therefore, lies in developing detectors compatible with new chromatographic separation techniques. The emphasis of this work is directed toward this challenge, specifically in quantifying and minimizing the effect of RI changes on a new UV-vis absorbance detector as applied to TG-pLC. The motivation for developing TG-pLC is to provide simplified gradient instrumentation which is readily applicable to pLC. TG separations for pLC and capillary LC can be confidently achieved, whereas mobile-phase gradient separations are difficult to perform for pLC and nonexistent for capillary LC (4). From an instrumental point of view, it is much simpler to control the temperature of a mobile phase than to uniformly mix two mobile phases with the precision necessary for optimal separation and detection requirements. Early demonstration of temperature programming with conventional size LC columns (4.6 mm i.d.) was shown by Lawrence and Scott (6) for a normal phase separation with a thermal gradient from 25 to 63 "C. The advent of chemically bonded organic stationary phases allowed Knox and Vasvari (7) as well as Grushka and Kikta (8)to investigate the effect of increasing temperature to reduce analyte retention time, increase peak symmetry, and increase chromatographic efficiency for isothermal separations, indicating the potential benefits for TG-LC. Subsequent work by Majors (9) also indicated slightly elevated temperatures, providing better column efficiency for most chemically bonded organic phases. Kitka et al. (10)developed an inexpensive temperature controller to further promote the simplicity of TG-LC with a demonstration of a thermal gradient separation from 40 to 66 "C. Guiochon et al. investigated the role of temperature in reversed-phase HPLC (RP-HPLC) isothermal separations on both pyrocarbon-containing stationary phases (1I) and chemically bonded RP-HPLC stationary phases (12). More recently, Antia and Horvath (13)presented theoretical relationships for the advantages of high-temperature HPLC (HT-LC) for isothermal separation of large molecules, where the separation efficiency was predicted to increase similar to SFC due to the reduced viscosity of a high-temperature eluent and increased analyte diffusivity. For example, samples of limited solubility a t room temperatures, i.e., polymers and biopolymers, can be more easily detected at an elevated temperature due to increased solubility. Furthermore, high-speed HPLC for process analysis (14,15) can greatly benefit from reduced eluent viscosity of high-temperature separations in terms of analysis time, separation efficiency, and increased column loading capacity. One of the primary concerns of using temperature to control retention behavior is the thermal stability of the column packing material, thus limiting the practical range of temperatures that ensure a useful lifetime of the column. The solubility of bare silica packing material rises exponentially with temperature (16) and, therefore, is not suitable for separations with polar modifiers. However, bonded organic phases on silica have been shown to be stable over a wide pH range and stable to 160 "C (17). More recently, highly cross-linked polymer supports have been chemically modified to provide RP separation capability (18)for a temperaturestable and pH-stable stationary phase. In addition, carbon packing material investigated by Guiochon et al. (11) also appears very promising for application to TG-LC. Before the benefits of temperature programming can be fully realized, it is first necessary to address detection concerns regarding refractive index (RI) effects in absorbance measurements. Most of the work done in the past (6,10)with
A
569
6
Figure 1. Continuous position sensitive detector (PSD) generatirig photocurrent signals at electrodes A and B where diff = A - B and sum = A 4- B. Dispersed spectra from the grating monochromator illuminates the PSD (shown in bold) with an optical mask (shaded region) applied to select spectral regions A, and A., The wavelength of interest, A,, is selected by adjusting the monochromator, and the optical null is achieved by adjusting the mask to balance the photocurrents at electrodes A and B (23).
TG separations focused on the chromatographic aspects of HPLC, largely ignoring detection concerns. An issue recently addressed by Synovec (19) and McGuffin (20) is the effect of refractive index on aperture-limited absorbance measurements. The effect is commonly manifested as an "injection disturbance" for sharp refractive index profiles (21, 22) or baseline drift for MPG-LC (2). The implication of RI aberrations to absorbance measurements with TG-LC is also manifested in a drifting baseline. Similarly, the common method of pressure programming (3)in SFC also suffers from baseline drift due to density-induced RI aberrations. The temperature range required for effective TG separations demands a robust detection system capable of functioning in a harsh and changing environment, and therefore, a remote fiber optic absorbance detector was designed and investigated for use with TG-pLC separation and detection (19,23). Previous development of the single fiber optic detector used for this work included reduction of the light source contribution to detection noise (19,24,25) and reduction of RI-induced injection disturbances for pLC. In this manuscript, we report the first demonstration of UV-vis absorbance detection for TG-pLC with reduction of baseline drift. In addition, general theoretical relationships are developed relating the RI of an eluent to the relative collected light flux (RCLF) or baseline drift with regard to the fiber optic flow cell developed for this work. The two-wavelength PSD method of absorbance detection corrects for RI aberrations and is a solution not only for TG-pLC but also for MPG-pLC, SFC, and other flow methods such as flow injection analysis (FIA) where the mobile-phase RI changes with time. The remote fiber optic flow cell design allows chromatographic detection directly a t the end of a pLC column to provide sensitive detection of a TG separation of an unleaded gasoline sample (test mixture) via a long optical path length flow cell, 6 mm. The temperature program ranged from 25 to 150 "C, well above the atmospheric boiling point of the mobile phase. Interfacing the optical fibers to a high-pressure flow cell followed by a pressure restrictor allowed high temperature detection. Separation efficiency is evaluated showing substantial improvement over room-temperature isocratic separations.
THEORY The detector developed for this work, shown in Figure 1, was described in previous work (19,23-25) and will be summarized here for subsequent theoretical development. The PSD was designed with two outputs, the sum and the difference (diff), where the signal from each output relative to baseline can be expressed as (23) sum = 2.30310(A1+ A2) (1) diff = 2.30310(X/L)(A, - A2) (2)
570
ANALYTICAL CHEMISTRY, VOL. 63, NO. 6, MARCH 15, 1991 ^_*
ei
A
2 LL
0
I I
K
-90
" ?'
where I , is the average photocurrent measured at each collection electrode on the PSD in the absence of an absorbance, A, and A , are absorbances a t wavelengths XI and X2, respectively, and X / L is the distance the beams are separated relative to the total length of the PSD. The sum signal is similar to a single beam measurement providing absorbance information but with no provision for reducing light source fluctuation noise. The diff signal also provides absorbance information at XI and X2 while reducing light source fluctuation noise as previously reported (19,23-25). Both the sum and diff absorbance signals, however, are dependent on the generated photocurrent, I,. The ratio between the diff and sum signal results in low noise absorbance information a t X, and h2, while providing an absolute signal independent of light source intensity ratio = 2.303(X/L)(AI- A,)
(3)
In the work presented in this paper, only the diff signal was used to minimize lamp noise and reduce RI aberrations where the ratio provides no additional benefit in noise or RI aberration reduction. For quantitative analysis, however, the ratio signal should be used for optimal accuracy due to the absolute signal provided, independent of the light source intensity. To predict the effect of RI aberrations due to temperature changes on aperture-limited absorbance measurements, an intrusive sensor model (26) was used which relates transmission of light to the RI of the solvent in the flow cell. The model assumes reflection losses a t the fiber end faces are negligible and light that strikes the wall of the flow cell is scattered and not collected by the exit fiber. These two assumptions were found to be acceptable (19). Figure 2 shows the schematic diagram for the model, where light is transmitted from the entrance fiber through a solvent of refractive index n, and collected by the exit fiber. In the absence of absorbance, a change in refractive index of the solvent changes the divergence cone of both the entrance and exit fibers, thereby changing the light flux received by the exit fiber, resulting in a changing baseline for slow refractive index changes (19,20) or a substantial baseline disturbance for sharp refractive index changes such as injection disturbances (21, 22). For a single wavelength, the relative collected light flux (RCLF) is the amount of light transmitted across the flow cell for a given solvent, relative to a reference solvent, i.e., relative to a stable baseline in a flowing system such as LC or SFC, and is given by (26)
where I , and Irefare the photocurrents generated by the transmitted light for the solvent and reference solvent, respectively, n, and nrefare the refractive index of the solvent and reference solvent, and N.A. is the numerical aperture for the optical fiber at the average wavelength of transmitted light.
150
1'40
130
Flgure 2. Flow cell configuration with respect to the optical fiber and inletloutlet solvent flow geometry. n , = refractive index of the fiber optic core, n, = refractive index of the mobile phase (either n, or nw), 0 = half-angle divergence of the optical fiber in contact with the mobile phase.
RI, n s at 486 nm
Flgure 3. Relative collected light flux for fiber geometry as shown in Figure 2 as a function of the solvent R I , ns,at 486 nm using methanol as the reference solvent, n,,,.
300
400
500
600
WAVELENGTH, nm Flgure 4. Normal dispersion curves for a general solvent at an initial temperature and at an elevated temperature yieMing ns,wtial and nS,-,, respectively. The change in refractive index at Wavelengths A, and A, is given by An A , and An A2, respectively.
Typical N.A. values of 0.10-0.25 allow simplification of eq 4 to a good approximation to yield RCLF = ns2/nref2
(5)
Further simplification of eq 5 can be made by using a linear approximation with adequate accuracy as given by RCLF = 1 2An,/nref (6)
+
where RCLF is proportional to the ratio of the refractive index of the solvent and reference solvent, and An, = n, - nrep The intrusive sensor model is first applied to well-characterized mobile-phase composition changes to test the model with subsequent application to TG conditions. The linear approximation in eq 6 is justified by experimental data as shown in Figure 3 for methanol as the reference solvent. Selection of 486 nm as the monitoring wavelength allowed investigation of the RI dependence of RCLF without interference from trace absorbances present in the pure solvents as previously encountered (19). The measurements were made by using steady-state conditions with the flow cell filled with the respective solvent and monitoring the transmission of light through the fiber optic flow cell with a photodiode. A correlation coefficient of r2 = 0.965 for solvents spanning the entire RI range of common HPLC solvents was calculated for Figure 3, thereby substantiating the linear approximation even though the RCLF is not linear in the initial form, eq 4. To further elucidate the significance of Figure 3 to TG-KLC, a set of normal dispersion curves for an initial and final condition of the mobile phase is shown in Figure 4. For normal dispersion, the RI of the mobile phase increases with decreasing wavelength, where the wavelength dependence is more pronounced at shorter wavelengths. Figure 4 is shown for the general case where no absorbance of the mobile phase occurs in the wavelength regions of interest and the initial and final conditions refer to a temperature change. With an increase in temperature, the density of the mobile phase will
ANALYTICAL CHEMISTRY, VOL. 63, NO. 6, MARCH 15, 1991
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decrease, resulting in a decrease in RI, at the two detection wavelengths. The decrease in RI can then be used in Figure 3 to predict the change in RCLF for the two wavelengths. As predicted from Selmeir's equation, the change in refractive index will be similar for the two wavelengths, and therefore, X1 or X2 can be used as an internal reference to correct for changes in the mobile-phase RI. T o quantitate the sum signal for the dual-wavelength case with respect to RCLF and RI, it is convenient to start with the basic definition of the sum signal (24) sum = I, I, (7)
+
where I , and I 2 denote the photocurrents generated a t wavelengths A, and X2 under one set of mobile-phase conditions. Combining eqs 4 and 7 yields
+
sum = Iref(RCLF, RCLF,)
(8)
where Zref is the photocurrent using the reference solvent, and RCLF, and RCLF2 are the relative collected light flux a t wavelengths X1 and X2, respectively, in the context of Figure 3. Changing the solvent refractive index by changing the temperature, mobile-phase composition, or pressure results in new relative collected light flux conditions, RCLFBand RCLF4, for wavelengths X1 and X2, respectively, to yield
sum,,,, = Iref(RCLFI- RCLF,
+ RCLF, - RCLF4)
(9)
where sumcurris the signal measured relative to the baseline condition before and after the change in solvent conditions, i.e., baseline-corrected sum signal (23). Substitution of eq 6 for each RCLF with subsequent simplification yields sumcorr
=
2zref(AnA,
(10)
+ AnAp)/nref
where AnA, and AnApare the change in refractive index from an initial to final condition at X1 and h2 as depicted in Figure 4. To cast eq 10 into absorbance data, it is necessary to recall that sum,,, is the change in baseline mobile-phase conditions and, therefore,
(11) sum,,,, = AI, + AIz where AI, = I I - Z3 and AI2 = I2 - 14.I3 and I4 are the photocurrents at the final mobile-phase conditions for A, and Xz, respectively. By use of an approximation for small absorbances, Mi = 2.303IiAi
(12)
where Ai is an apparent absorbance due to a refractive index change at wavelength Xi. Combining eqs 10-12 yields sum,,,, = 2.303[I,A, IzAz] = 2zref(An~, A?ZA,)/n,,f
+
+
(13) It is important to recognize that Iref/nrefis a constant for a given flow cell geometry, i.e., path length, fiber N.A., and optical fiber diameter. A convenient method of calibration for Iref/nref is to use baseline mobile-phase conditions a t one wavelength where Iref= I , and nRf= n,. Also, employing the optical null condition for highest noise reduction in the diff and ratio signals yields I, = Zz, where the apparent absorbance is given by Abs,,, = A1 + A2 = 2 ( A n ~+ , An~,)/2.303n, (14) A similar derivation, not shown for brevity, for the apparent absorbance in the difference signal yields Absdjff = A1 - A2 = 2 ( L / x ) ( A n ~-, AnAZ)/2.3O3n1 (15) The magnitude of the refractive index signal registered by the diff signal will be greatly reduced compared to the sum signal. The explanation can be seen in Figure 4 where AnA, and AnAz are the change in refractive index from the initial to the final
Figure 5. Experimental schematic diagram for the single fiber optic 2 1 PSD absorbance detector with TG-pLC. HgL = mercury-xenon lamp, OF = optical fiber (shown as bold line), FC = flow cell (510 pm
i.d. X 6 mm), GM = grating monochromator, PSD = position sensitive detector (two outputs, A + B = sum, and A - B = diff), P = syringe pump, I V = pLC injection valve, pLC = pLC column, R = pressure restrictor, H = heater, TC = temperature controller, PC = personal computer. temperature. Since Anh,and Anh2are similar in magnitude, the difference between Anx,and Anhzwill be small, resulting in a small baseline drift for the diff signal. In practice, X, and X2 are chosen so that only A, is absorbed and X2 provides the in situ reference to reduce light source intensity noise as previously reported (23)and, for the objective of this paper, to reduce baseline drift.
EXPERIMENTAL SECTION The experimental schematic diagram of the dual-beam PSDbased absorbance detector as employed for pLC is shown in Figure 5 and as described in previous work (19,23)with the following experimental modifications made for TG-pLC. An important consideration for TG separations is the configuration of the chromatographic apparatus and the choice of separation technique. Microbore HPLC is ideal for TG separations due to low volumetric flow rates, 25-100 wL/min, which promote rapid heating and equilibration of the mobile phase and injected sample, allowing the pump and injector to be maintained at room temperature, greatly simplifying instrumentation requirements. The small mass and small external diameter of the microbore column also promotes uniform heating of the column, minimizing radial temperature gradients when a temperature program is used. With the choice of pLC for TG separations, careful attention must be made to minimize extra column band broadening due to injection, flow cell, and connecting tubing volume. A critical design factor was the placement of the flow cell in the heating unit, thereby reducing connecting tubing volume and eliminating the need to adjust the mobile phase to equilibrium temperature conditions of the flow cell. Other experimental modifications include two fused silica lenses (Newport, 25 mm diameter, 50 mm focal length, Fountain Valley, CA) used to collect and focus the spectrum of the Hg-Xe lamp into a UV-vis transmissive optical fiber (Fiberguide Industries, Superguide-G UV-vis Fiber, Sterling,NJ) to obtain increased light intensity at the two detection wavelengths, 230 and 295 nm. The optical fiber as specified by the manufacturer consisted of a 400/480-pm fused-silica core/clad, 505-pm polyimide jacket, with a numerical aperture (N.A.) of 0.22. The monochromator (American Holographics, Chemspec 100M, Littleton, MA) used for this work was supplied with a reciprocal linear disperson of 6.1 nm/mm, yielding a 75-nm range across the photoactive surface of the 1 X 12 mm PSD. The protective glass window on the PSD was carefully removed to allow use in the UV spectral range. A L / X ratio of 1.14 was experimentally determined and used in eqs 2, 3, and 15. A temperature-programmed reversed-phase separation of unleaded gasoline was included to demonstrate the utility of thermal gradient separations by using the dual-wavelength PSD absorbance detector. A 1-mm X 250-mm, 5-pm, alumina-based C18 packed column (ES Industries, Gamma Bond y18, Marlton, NJ)
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 6, MARCH 15, 1991 -003.0
a
I
0 0
5
15
10
TIME
20
. 0
hinl
5
0 10
15
TIME
0
20
25
0 30
35
(min)
Temperature-programmed separation with detection via the sum signal for the Same unleaded gasoline sample and mobile-phase composition as used for Figure 6b. The temperature program is shown as a dashed line, ranging from 25 to 150 O C . Flow rate = 25 pL/min and mobile-phase composition = 40 % /60 % methanol/water by volume with the total separation time = 33 min. Figure 7.
0.100
b A E
TIME (minl Sum signal for a reversed-phase separation of unleaded gasoline with detection at A, = 230 nm and A, = 295 nm. A 1-mm X 250-mm C18 column was used at 25 pL/min at room temperature with the injection disturbance, I , occurring at 5 min and defines the dead time or the void volume of the column. The same peak (analyte A) is marked in all chromatograms to provide a convenient reference to compare retention time and peak width for different solvent conditions. No analytes were detected after 40 min due to the weak mobile phase. Mobile-phase composition for 6a = 70%/30% methanol/water by volume. Mobile-phase composition for 6b = 40% /60% methanoVwater by volume. Figure 6.
was used for the separation with the column effluent passing to the flow cell, all maintained in thermal contact with a 1-cm X 5-cm X 30-cm aluminum block bonded to a 500-W heating strip (Wellman, SS2181, Shelbyville, IN) and controlled by a temperature controller (FIAtron, TC-55, Oconomowoc, WI). The calculated volume of the flow cell was 1.2 pL, which was adequate for microbore chromatography. The temperature of the column was monitored independently of the temperature controller by using a thermocouple and recorded on a strip chart recorder (Fisher, Record all Series 5000, Pittsburgh, PA) set at 10 mV full scale sensitivity. Fiberglass insulation was wrapped around the column, flow cell, and heating strip to promote uniform heating. A 250 psig pressure restrictor (Upchurch, U-462, Oak Harbor, WA) was connected to the exit of the flow cell via 1 m of stainless steel HPLC tubing to allow the mobile phase to cool to room temperature before entering the pressure restrictor. The pressure restrictor was necessary for chromatographic separations at temperatures above the atmospheric boiling point of the eluent. The solvents used for Figure 3 and the HPLC separations of the test mixture were HPLC-grade (EM Science, Omnisolv, Cherry Hill, NJ) with compositions as specified in the figure captions.
RESULTS AND DISCUSSION After the experimental design and construction was completed, the detection system was evaluated by chromatographic separations of an unleaded gasoline sample using isothermal and TG-pLC conditions with detection via the sum signal. Subsequent optimization of detection was achieved
by employing the diff signal. Liquid chromatography with UV detection at 230 nm was chosen to provide selectivity for aromatic and unsaturated components in the unleaded gasoline sample. A separation of the unleaded gasoline sample is shown in Figure 6a to demonstrate proper functioning of the dual-wavelength detector under isocratic conditions. Separation conditions are not optimal as evident by the poor resolution obtained in Figure 6a. A typical solution to the lack of resolution is to decrease the strength of the mobile phase as shown in Figure 6b. The same peak, labeled analyte A, was arbitrarily chosen in both the 70% /30% and 40%/ 60% methanol/water separations to provide a convenient reference point to compare analysis time and peak width. Unfortunately, decreasing the strength of the mobile phase greatly increased the retention time and caused excessive peak broadening due to unfavorable kinetic and thermodynamic partitioning factors (9). A more suitable solution to optimize both the resolution and analysis time is to dynamically change the retention behavior of the analytes by changing the mobile-phase strength during the separation. Unfortunately, gradient mobile-phase programming for microbore separations is difficult to perform ( 4 ) due to the small elution volumes required for microbore separations, on the order of 1OC-4000 pL. Another shortcoming of MPG-pLC is the complexity and expense of the instrumentation generally requiring the use of two syringe pumps, a pLC mobile-phase mixer, and a gradient controller. Our solution is to change the retention behavior via temperature programming, simplifying the instrumentation, requiring only one pump, heating strip, and temperature controller. The concept of thermal gradient separations is essentially the same as the mobile-phase gradient strategy, to start with a weak eluent to achieve the desired resolution for early eluting analytes and then dynamically change the retention behavior by increasing the temperature to remove strongly retained analytes in a timely manner as shown in Figure 7, for the sum signal. The temperature profile is shown as a dashed line, following the same trend as the baseline drift. As shown in Figure 7 for a change in temperature from 25 to 150 "C, the drift in the sum signal indicates the magnitude of the problem associated with the use of aperture-limited absorbance measurements. The results for the sum signal, similar to a conventional single-beam absorbance measurement, is a substantial baseline drift or an apparent absorbance of 0.097 AU monitoring wavelengths 230 and 295 nm. The apparent absorbance was calculated via A = -log (Z/Z,J, where Zo and Z equal sum/2 at the beginning and end of the chromatogram, respectively. Z and Zo were
~
~
ANALYTICAL CHEMISTRY, VOL. 63, NO. 6, MARCH 15, 1991
-a
573
Table 1. Ratio of the Capacity Factors at Two Different Temperatures, with the Temperature Labeled as the Subscript in “C
0.100
3
m,“ kcal/mol
kdk50
k2dkI5
k25/k100
k2dk150
W
0.5 1.0 2.5 5.0 7.5 10.0
1.07 1.14 1.39 1.92 2.67 3.70
1.13 1.27 1.83 3.36 6.17 11.3
1.19 1.40 2.34 5.46 12.77 29.84
1.28 1.65 3.48 12.13 42.22 147.03
U
z a
0,050 0 ffl
m a a
0.000
TIME
(minl
Figure 8. Baseline drift reduced via the diff signal for the temperature-programmed separation of the unleaded gasoline sample, collected simultaneously with the sum signal shown in Figure 7. Separation conditions are given in Figure 7 caption. defined as sum/2 to be consistent with conventional dualbeam absorbance measurements where only the sample beam is absorbed by an analyte and therefore only the sample beam intensity is used to calculate the absorbance value. Even though there is baseline drift in the sum signal, this is a substantial improvement over what could be achieved with an external flow cell design, without sacrificing separation efficiency. The sum signal, however, does contain valuable RI information that can be used to determine the mobilephase composition, based upon baseline “drift” throughout an entire mobile phase or thermal gradient separation. By use of eq 14,the refractive index of the mobile phase changed from 1.3425 at 25 “C to 1.1925 at 150 O C , indeed a substantial change in refractive index due to a change in temperature. T o cast the data from Figure 7 in an alternative form, the refractive index change can be used in the Lorentz-Lorenz (1) relation to estimate the change in density from 0.9347 to 0.5459 g/mL. Similar problems of baseline drift are seen for SFC by using gradient density programming with UV absorbance detection ( 3 ) . Detection is obviously not optimized with the sum signal as the drifting baseline provides difficulties for quantitation and, therefore, the diff signal was collected simultaneously with Figure 7 as shown in Figure 8 to provide a chromatogram nearly independent of baseline drift. As predicted by eq 15 in the context of Figure 4, the diff signal in Figure 8 provides a solution to the baseline drift problem exhibited by traditional single-wavelength absorbance measurements, such as the sum signal. The baseline drifted by only 0.0078 AU, a factor of 12 better than the sum signal. The key to the success of the dual-beam system is that both the sample and reference beams are directed through the same fiber and flow cell, and therefore, the reference beam experiences the same environmental changes as the sample beam. Clearly, the baseline is more stable, allowing confident identification and quantitation of analyte peaks and yields a larger dynamic range of detection. A continuous PSD was chosen for this work due to the excellent signal-to-noise characteristics and simplicity of operation. In principle, one is not limited to the PSD and can expand the variety of detectors to include digital devices such as the photo diode array (PDA) or charge coupled detectors (CCD) to provide complete spectral information with excellent sensitivity and resiliency to baseline drift or RI disturbances as provided by the in situ absorbance difference measurement. In practice, “extra” pixels where no absorbance is expected to occur could be used as the reference wavelength(s1 to provide the optical null condition necessary for effective signal stabilization.
AH and AS are assumed to be invariant with temperature.
Having successfully addressed detection concerns with the ability to reduce RI aberrations in absorbance measurements, it was interesting to investigate the effect of temperature on the unleaded gasoline separations and to provide a framework for potential benefits of TG reversed-phase separations. Peak A in Figure 8 is eluted much earlier with the temperature program, k = 1.2, than with the same mobile phase using a 25 “C isothermal separation, k = 2.2. Furthermore, strongly retained components not eluted from the isothermal separation, Figure 6b, are easily eluted with the temperature program. The thermal gradient separation is complete in 40 min, while the room-temperature separation with the same mobile-phase composition is only 30% complete. The temperature dependence of the capacity factor can be expressed as (12)
Ink = AH/RT- AS/R +In f
(16)
where AH and AS are the enthalpy and entropy, respectively, of an analyte partitioning from the stationary phase to the mobile phase, R is the universal gas constant, T i s the temperature in kelvin, and f is the phase volume ratio of the stationary to mobile phase. The capacity factor, k , is calculated from k = (tR - t o ) / t owhere , tR is the elution time of an analyte and tois the dead time or elution time of an unretained analyte. An experimental method of determining AH is to perform the separation at different temperatures and construct a van’t Hoff plot, In k versus 1/T, where the slope of the line is AH/R and the y intercept is In V, - AS/R. Since the change in capacity factor versus temperature is the parameter of interest for this work, eq 16 can be put in an alternative form to yield the capacity factor ratio, k l / k 2
where k l and k2 are capacity factors for an analyte a t temperatures TI and T2(K), respectively. For a given AH, it is then possible to predict the ratio of the capacity factors for an analyte at different separation temperatures. As predicted by eq 17, the capacity factor ratio for analytes with large AH values should be more strongly affected by temperature changes, whereas the capacity factor ratio for analytes with small AH values should be relatively insensitive to temperature changes. Also, large AH values for reversed-phase separations correlate with large capacity factors and thus long retention times, indicating temperature as an effective control of retention equilibria. Predicted capacity factor ratios for reversed-phase separation enthalpies are given in Table I, with a typical range of 0.5-10 kcal/mol for isocratic RP-HPLC separations. An isothermal separation at 150 OC with 40%/60% methanol/water was also obtained in order to explore the range of temperature-programmed separations. The chromatogram, not shown for brevity, is qualitatively similar to the 70%/30% methanol/water separation, Figure 6a, exhibiting short analysis time and good efficiency but poor resolution. Analyte
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 6, MARCH 15, 1991
Table 11. Isothermal versus Temperature-Programmed Separation of the Unleaded Gasoline Test Mixture separation, "C
tR, min
W,, min
N, plates/m
25 25-150
22.7 21.8
2.7 0.65
5 700 72 000
separation can be attributed to the initial concentration of the analytes onto the column due to the weak mobile phase, particularly for strongly retained components. As the temperature is raised, the analytes desorb from the stationary phase and migrate down the column with more favorable kinetics due to the temperature increase and viscosity decrease (9).
A had a capacity factor of 0.41 at 150 "C for the 40%/60% methanol/water separation, yielding a capacity factor ratio of 5.4 for kZ5/klm,a large change in retention volume. By use of eq 17, AH = 3.3 kcal/mol, and for more strongly retained analytes, larger AH values, a much greater capacity factor ratio will result as indicated by Table I. Thus, TG-pLC is an effective method of gradient LC separation, precisely in the chromatographic region where the ability to perform gradient separations is necessary, at long retention times. To compare TG-LC with MPG-LC, a brief summary of temperature change and mobile-phase composition change is presented for the unleaded gasoline sample. A change in methanol concentration from 70% to 40% resulted in a 7-fold change in capacity factor as shown in Figure 6 for analyte A. This is approximately a 2-fold change in k for every 10% change in methanol (27). Alternatively, the separations at 25 and 150 "C with the same mobile phase resulted in a capacity ratio of 5.4, clearly showing the ability of TG-pLC to perform comparably with MPG-LC for reversed-phase separations. Another benefit of the TG-pLC which warrants investigation is the apparent efficiency that is obtained for the temperature-programmed separation compared to the isothermal separation as shown in Table 11. Clearly, no efficiency has been sacrificed by the temperature program. The efficiency was calculated by N = ( t , / t J Z ,where N is the number of theoretical plates, t , is the retention time, and t, is the standard deviation of the peak. Even by mobile-phase gradient separation standards, 72 000 theoretical plates/m is an impressive achievement for 5-pm packing. Since chromatographic efficiency is dependent on the retention time, the same time period was chosen for both chromatograms to provide a reasonable comparison of measured chromatographic efficiency. With the TG-pLC separation, the peak at 21.5 min is as narrow as the earliest eluting peaks in the chromatogram. In part, the excellent efficiency of the temperature-programmed
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RECEIVED for review September 4,1990. Accepted December 20, 1990. We thank the NSF Center for Process Analytical Chemistry for support of this work.
