378
Anal. Chem. 1990, 62,374-378
(13) Levich, V. G. Physicoch8~iicalHydrodynamics; Prentice-Hall: Englewood Cliffs, NJ, 1962. (14) Szentirmay, M. N.; Martin, C. Anal. Chem. 1984, 56, 1898. Wingard, L. B., Jr. Anal. Chem. 1987, (15) Marrese, C. A,; Miyawaki. 0.; 59, 248. (16) Ewing, A. G.; FeMman, B. J.; Murray, R. W. J. Fhys. Chem. 1985, 89, 1263.. (17) Mauritz, K. A,; Hora, C. J.; Hopfinger, A. J. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 1978, 19, 324.
(18) (19) (20) (21)
Yeager, H. L.; Steck, A. Anal. Chem. 1979, 51, 862. Moore, R. 8.; Martin, C. R. Macromofecules 1988, 21, 1334. Kok, W. Th.; Twos, A. J.; Poppe, H. AM/. Chim. Acta 1989, 228, 31. Laan, E. T. van der Chem. Eng. Sci. 1955, 7 , 187.
for review December
Accepted November
3, 1989.
Thin-Layer Chromatography with Supersonic Jet Fluorometric Detection Totaro Imasaka, Katsunori Tanaka, and Nobuhiko Ishibashi*
Faculty of Engineering, Kyushu University, Hakozaki, Fukuoka 812, Japan
A mixture sample Is developed by a slllca gel on a flexlble thin-layer chromatograph (TLC) sheet. This is mounted on a slMlng sheet roller attached to a supersonic jet nozzle. The flrst dye laser beam (275 nm) Is introduced from a small throughhole to desorb the chemlcal specks on the TLC sheet. The vaporlzed sample is entralned into a carrier gas of argon, and the gas Is then expanded Into a vacuum to form a supersonic let. The sample molecule is detected by fluorescence Induced by the second dye laser beam (366-375 nm). This technlque allows dlrect measurements of the excltatlon spectrum and the chromatogram for the sample developed on the TLC sheet. The amount of sample used for recordlng a spectrum is 4.4 pg. The detection limns are -10 ng In spectrometrlc and chromatographic measurements.
In the environment there are many chemical species with similar structures, whose toxicities drastically change by slight modification of the chemical structure. Therefore, a selective analytical method is essential in their determination. Supersonic jet (SSJ) spectrometry provides a useful means for this purpose, because of sharp line structure in the spectrum (1-3). I t has been used for selective determination of polycyclic aromatic hydrocarbons (PAHs) and biological molecules with similar structures. However, there are so many chemical species in the environment that combination with a separation technique is sometimes necessary prior to the measurement. SSJ spectrometry is a flowing analytical technique, and it is possible to combine it with chromatography. In current works, gas chromatography (GC) is successfully used for this purpose. Methods based on fluorescence (FL) detection (4-8) and multiphoton ionization (MPI) (9-11) have been reported, and picogram quantities of PAHs have already been determined (7). For nonvolatile or thermally labile molecules, liquid chromatography (LC) based on supercritical fluid sample introduction has been used (12). More recently, supercritical fluid chromatography (SFC) is used, instead of LC, for sample separation (13,14). The assignment of the chemical species is performed by measuring a fluorescence spectrum with an optical multichannel analyzer (8) or by measuring a mass spectrum with a time-of-flight mass spectrometer (MS) ( I l ) ,
* Author t o whom correspondence should be addressed.
during the time period when the sample passes through the detector. This approach allows reliable assignment of the chemical species from the well-resolved fluorescence or mass spectrum. Supersonic jet spectrometry combined with chromatography has great selectivity, but it has an inevitable disadvantage described below. In current SSJ spectrometry, a pulsed dye laser is used for excitation or multiphoton ionization of the molecule, because of the wide tuning range and high peak power of the laser. The repetition rate of the pumping laser, e.g. an excimer laser, is practically limited to 10-100 Hz. It is difficult to measure the excitation spectrum in the short time period when the eluents transit a chromatograph detector. Thus only the specified molecule, whose spectral parameters are known prior to the measurement, can be detected with great selectivity, making it difficult to apply chromatography/SSJ spectrometry to unknown samples. In this study we use thin-layer chromatography (TLC) for sample separation in SSJ spectrometry to measure the excitation spectrum and the chromatogram simultaneously. The sample is deposited at the bottom of the chromatograph sheet in a straight line, and it is developed by a solvent. At an appropriate R, value (the ratio of migration distances for the sample to the solvent), the sample is vaporized by the first dye laser beam. The molecules are entrained in a carrier gas, which is expanded into a vacuum to form a supersonic jet. The sample is detected by fluorescence induced by the second dye laser beam. The excitation spectrum is measured by scanning the position of laser desorption (LD). A similar approach using MPI/MS is reported elsewhere (15). However, a portion of the TLC sheet is cut out and attached on the sample holder for laser desorption. Thus the sample location should be known prior to the measurement. The present analytical technique allows repetitive measurements of the excitation spectrum by scanning the position of the TLC sheet, and then it is applicable to unknown samples.
EXPERIMENTAL SECTION Apparatus. A laser ablation/supersonic jet nozzle was originally developed for the studies of metal clusters by Smalley and co-workers (16,17). In the present study the nozzle is modified for the application to the sample developed on the TLC sheet. The nozzle structure is shown in Figure 1, which is essentially a combination of the specially designed component described below and the pulsed nozzle (pulse width, -1 ms) used in our gas-phase experiment (18). A flexible TLC sheet (100mm long, 61 mm wide) is mounted on a sliding sheet roller (diameter, 20
C 1990 American Chemical Society 0003-2700/90/0362-0374$02.50/0
-
Ablation Laser
ANALYTICAL CHEMISTRY, VOL. 62, NO. 4, FEBRUARY 15, 1990 p ~ t o m u ~ i p l iMonochromator er ~
4
Pulse
&
~ ~ x c Integrator ar Recorder 1
Nozzle
Sliding Sh Roller
Vacuum Pump ‘
’
375
275 nm, 1 mJ
,
Pulse Nozzle
iw(
I1
Q
x
7 - ! MTLC Sheet Dye Laser I
Supersonic Jet
,’
Figure 3. Experimental apparatus for TLC/LD/SSJ/FL.
TLC Sheet
Figure 1. Supersonic jet nozzle for thin-layer chromatograph detection. Speed Control Motor
Stepping Motor
/7
In 1’
S,witch
t o Sliding Sheet Rollet
Figure 2. Driving unit for translation and rotation of TLC sheet attached
to supersonic jet nozzle. mm) supported by ball bearings (THK Bearing), which is rotated with gears (Kyoikusha) attached to the ends of two stainless steel rods (diameter, 3 mm). O-rings are fitted to this shaft to contact with the TLC sheet softly. Two ball bearings (THK Bearing) are attached to fit the TLC sheet on the roller, whose holder is mounted on the stage, which is smoothly translated by ball bearings (THK Bearing). A desorption laser beam is introduced from a small through-hole (i.d., 1mm) to vaporize the sample on the TLC sheet. The curvature of the nozzle head surface to contact with the TLC sheet is designed to be 11 mm. The separation between the nozzle head and the TLC sheet is adjusted to 0.2 mm. Figure 2 shows a driving unit for translating and rotating the sliding sheet roller. This is performed by rotating the stainless steel rod by using a stepping motor (Oriental Motor) mounted on the stage, which is translated by rotating a screw with a speed control motor (Oriental Motor). Two switches are positioned at both ends to limit the translational distance. The electroniccircuit for controlling the motors is designed in this laboratory. When the stage arrives at the end switch, the speed control motor stops working during 0.6 s, and the stepping motor rotates the shaft five steps, corresponding to 0.4 mm on the TLC sheet. The speed control motor starts moving again backward to change the scanning direction. A typical translational speed is 20 mm/min. This scan controller is installed outside the vacuum chamber, and the shaft is sealed with an O-ring. A block diagram of the experimental apparatus is shown in Figure 3. After an argon gas is injected into the nozzle throat (i.d., 1.5 mm), the first dye laser pumped by the second harmonic of a Nd:YAG laser (Quantel, YG581C-20, TDL50, UVX-2, DCC-3, 20 Hz) is fired to vaporize the sample on the TLC sheet. The laser beam is reflected by a dielectric coated mirror and is exactly focused on the sample surface by a lens (focal length, 30 cm). The
beam position was visually confirmed by looking at it through the dielectric mirror. The laser dye used for desorption was fluorescein 27. The oscillating wavelength was adjusted to 275 nm, which was the shortest wavelength available in the present dye laser system. The pulse energy typically used was -1 mJ. The second dye laser pumped by an excimer laser (Lambda Physik, EMGlOZMSC, FL2002,5 mJ) is synchronously fired at 130 ps after the first dye laser. This is focused by a lens (focal length, 30 cm) 10 mm away from the top of the nozzle throat. The vacuum chamber is evacuated by a pumping system described elsewhere (19). Fluorescence from the sample is measured by a monochromator (Jasco, CT-100,400 groove/“) equipped with a photomultiplier (Hamamatsu, R928). The typical slit width used was 2 mm, corresponding to a spectral resolution of 4.8 nm. The signal is measured by a boxcar integrator (NF Circuit Design Block, BX-530A),the typical gate width being 1ws. The output signal is displayed by a chart recorder (Hitachi, 056). Reagents. The PAHs used in this study, 1-chloroanthracene, 2-chloroanthracene, 9-chloroanthracene, and 9,lO-dichloroanthracene, were purchased from Aldrich. The PAH 9methylanthracene was supplied from Nakarai. The laser dyes BBQ, PBD, and fluorescein 27 were obtained from Exciton. The laser dye 7D4MC was purchased from East” Kodak. The TLC sheet (Merck, Kieselgel60, normal phase) for sample separation consists of a thin silica gel layer (0.2 mm) on a polyester film. Nitrogen dioxide, which was used for an investigation of the cooling effect, was purchased from Takachiho. Procedure. The sample was dissolved in chloroform and was deposited in a straight line at the bottom of the TLC sheet with a glass capillary. The sample was developed 50 mm with a solvent such as n-hexane. This was air-dried and was attached to the sliding sheet roller. After the nozzle was installed in the vacuum chamber, the shaft for translation and rotation of the roller was connected to the motor. The stagnation pressure of the argon gas was adjusted to 200 Torr throughout this experiment.
RESULTS AND DISCUSSION Cooling Effect. The TLC sheet is flexible but cannot be bent sharply into a U-shape. The nozzle throat becomes longer than the bending diameter, as shown in Figure 3. The cooling effect due to jet expansion can be degraded by increasing the length of the nozzle throat; a hydrodynamic flow becomes a molecular flow, which may substantially increase the jet temperature. First, the cooling effect was investigated by changing the length of the nozzle throat, using NOz as a sample; an excitation spectrum was measured by monitoring the v2 band a t around 455 nm. The procedure for the calculation of the rotational temperature from the line profile is described in detail elsewhere (18). The rotational temperature obtained was 3.4-4.1 K for nozzle throat lengths of 0, 1,12, 22, and 39 mm. This fact is consistent with a calculated translational temperature of 3 K obtained by assuming direct jet expansion from the nozzle. Thus,it is tentatively concluded that a nozzle throat shorter than 40 mm induces a minor effect in rotational cooling. In this study the length of the nozzle throat is designed to be 29 mm, promising sharp spectral lines in the SSJ spectrum.
376
ANALYTICAL CHEMISTRY, VOL. 62, NO. 4, FEBRUARY 15, 1990
.-% 4-
(I)
C a, C
4-
e, C e, 0
0
v)
g
3
i i
370
371
372
373
374
375
Wavelength / nm Figure 5. Supersonic jet excitation spectrum for chemical species located at R, = 0.3 in TLC. Fluorescence is monitored at 393.5 nm. A mixture sample of 9-methylanthracene and 9-chloroanthracene is developed by n-hexane.
66.8 367.4 Wavelength / nm Figure 4. Supersonic jet excitation spectrum for 2-chloroanthracene adsorbed on TLC. Fluorescence is monitored at 387.0 nm.
Spectral Features. Supersonic jet excitation spectra were measured for 1-, 2-, and 9-chloroanthracene, 9-methylanthracene, and 9,lO-dichloroanthraceneadsorbed on the TLC sheet. Figure 4 shows the spectrum for 2-chloroanthracene, which is known as a thermally labile molecule to form a polymerized tar at an elevated temperature (12). A rather poor signal to noise ratio (S/N) is due to the low fluorescence quantum yield of 2-chloroanthracene. The line widths of the 0-0 transition peak observed for the above five compounds were 1.9-2.8 cm-'. Amirav et al. calculated the rotational temperature of anthracene to be 15 K from a line width of 3 cm-I (20). Thus it is implied that the molecules are sufficiently cooled by jet expansion, at least to 15 K. Sensitivity. The time period required for the measurement of an excitation spectrum was 15 min. After the sample was laser-desorbed, the TLC sheet was detached from the nozzle. A zigzag trace was found on the TLC sheet, which was 0.1 mm wide and 366 mm long. By extraction of 9,lO-dichloroanthracene adsorbed on the TLC sheet into chloroform, a quantity of sample in a unit area was calculated by measuring the concentration with a conventional fluorescence spectrometer. The result indicates that the density of the sample molecule is 0.12 pg/mm2. During the measurement of an excitation spectrum, 4.4 yg of sample is calculated to be consumed. By a single laser shot 0.24 ng of sample (6 X 10I1 molecules) is desorbed in the present condition, which is slightly less than the values reported elsewhere: 3 X l O I 3 molecules in MPI spectrometry (21) and 2 x 10I2molecules for laser-ablated monomers from a polymer surface (22). In TLC/MPI/MS the detection limits are reported to be -12 ng for indole-3-acetic acid and -3 ng for imipramine, which are obtained by averaging the signals over 10 pulses (1s) (15). These values are comparable to a detection limit of 10 ng for 9,lO-dichloroanthracene obtained in this study, which is estimated from a desorbed amount of sample during the time constant (0.24 ng x 20 Hz x 5 s) and the S / N ratio of the spectrum ( S I N = 7 ) . Background Signal. The sensitivity was determined by several sources of background emission. Strong light scatter originated from particulates ablated from the TLC gel, but could be reduced to negligible levels by using a fluorescence monochromator. Photoemission induced by laser breakdown
-
lasted 20 ys even a t the fluorescence detection port, but it could be also reduced to negligible levels by temporal discrimination. Long-lived photoemission continued more than 130 ys after the desorption laser pulse. The photoemission intensity was not critical to the excitation and fluorescence wavelengths. This was reduced by using a narrow gate width, but it was still difficult to remove completely even at a gate width of 1ps. It is probably due to fluorescence from chemicals adsorbed on the small particulates ablated from the TLC gel. More gentle laser ablation, e.g. by using an infrared COz laser, may reduce formation of particulates, but this approach was not investigated in this study due to unavailability of the equipment. Timing of T w o Laser Pulses. The dependence of the signal intensity on the time period between the firing of the two dye lasers was not investigated quantitatively, due to a poor S / N ratio of the signal. However, the largest signal providing a cooled spectrum was observed only when the timing was exactly adjusted to 130 p s f 2 ns. This fact implies that the sample is localized in the jet pulse and is focused within a short time period. This is consistent with a sample pulse width of -4 ys observed in the previous studies of LD/SSJ and LA/SSJ (22, 23). As pointed out, this is advantageous for improving the sensitivity since the amount of the sample on the TLC sheet is quite limited. In current SSJ spectrometry, it is necessary to use at least several milligrams of sample to optimize experimental conditions. This amount sometimes exceeds a fatal dose for a heavily toxic compound. It is noted that the sample remaining on the TLC is safely recovered after the measurement. Therefore, even a toxic sample could be measured with negligible levels of contamination in the laboratory. TLC Sample. The sample on the TLC sheet was developed by n-hexne, and the SSJ spectrum was measured at specified R, values. The excitation spectra recorded for the mixture sample of 9-methylanthracene and 9-chloroanthracene are shown in Figures 5 and 6. The wavelength of the fluorescence monochromator was adjusted to 393.5 nm to monitor vibronic bands, at which fluorescence from both the components could be detected within a spectral resolution of 4.8 nm. When the excitation spectrum is recorded a t Rf = 0.3, a single peak appears at 371.16 nm, which corresponds to the 0-0transition for 9-methylanthracene. When the measurement is carried out a t R, = 0.4, a single peak appears at 373.25 nm corresponding to the 0-0 transition for 9-chloroanthracene. No other peaks are observed in this narrow spectral range. As demonstrated, this method allows the measurement of the
ANALYTICAL CHEMISTRY, VOL. 62, NO. 4, FEBRUARY 15, 1990
Rf
Rf
0.2
371
372
373
0.4
0.6
0.8
1
.-h .I-
v)
C
a,
-C
.I-
L
3
377
a,
I I
0
I I
I
I
I
I
C
a, 0
I
374
v)
3 5
g
3
Wavelength / nm Figure 6. Supersonic jet excitation spectrum for chemical species located at R, = 0.4 in TLC. The experimental conditions are identical with those in Figure 5.
excitation spectrum for the chemical species separated by chromatography. This is a distinct advantage of TLC/ LD/SSJ over the other techniques combining SSJ with GC, SFC, or LC. This figure of merit may allow the application of this method to unknown samples. SSJ spectrometry using no chromatograph separation has some difficulties in the assignment of the chemical species in the mixture sample, since there are many possible spectral patterns by a combination of the spectrum. The present TLC technique provides additional selectivity in SSJ spectrometry, and furthermore the assignment of peaks to a specific component might be performed even when the separation resolution is incomplete; if a group of peaks tends to increase or decrease in the same manner when the R, is changed, they might be assigned to the signals originating from the same component. Conventionally, absorption or fluorescence spectrometry has been used in the TLC detector. More recently, a laserbased TLC detector utilizing fluorescence (24,25), thermal lens (26, 27), or mass spectrometry (28) has been reported. However, these methods sometimes suffer from poor spectral selectivity and from lack of information in the assignment. Low-temperature spectrometry has also been used in the TLC detector; fluorescence line narrowing spectrometry (FLNS) is applied to a PAH-nucleoside adduct adsorbed on a TLC plate cooled a t a liquid helium temperature (29). However, the spectral lines are not completely resolved, which may be partly due to a complex structure of the adduct molecule and to the interaction between the sample molecule and the solid substrate. Chromatogram. Figure 7 shows chromatograms measured by adjusting the excitation and fluorescence wavelengths to optimum values for 9-methylanthracene and 9-chloroanthracene, respectively. Only single components are observed in the chromatograms. No other signal peaks are observed at different R, values. It indicates that these compounds are present at R, = 0.3 and 0.4, respectively. In the measurement, 4.9 and 1.7 pg of the above compounds are charged on the TLC sheet, respectively; 9-methylanthracene is slightly overcharged and is appreciably broadened. From the S / N ratio, the detection limit of 9-chloroanthracene is estimated to be 10 ng, which is similar to the case in the measurement of the excitation spectrum. This is comparable to the values for naphthalene derivatives separated by GC using a conventional (noncapillary) separation column combined with a SSJ fluorescence detector (4). The separation resolution is rather poor at present, but it may be improved by using a reversephase sheet, though it is not commercially available yet.
-
i i
Migration Distance / cm Figure 7. Chromatograms for mixture sample of 9-chloroanthracene and 9-methylanthracene. The excitation and fluorescence wavelengths (nm) are (A) 371.16, 390.6 and (B) 373.25, 393.5.
.-c v)
C
e,
.I-
C
-e, C 0
a,
oRB O
Further improvement may be obtained by using high-performance thin-layer chromatography (HPTLC). Resolution on Excitation Spectrum. Since the real sample contains many chemical species, it is sometimes difficult to resolve all the components by chromatography. Even in such cases, it is possible to resolve the components on the excitation spectrum in TLC/LD/SSJ spectrometry. Figure 8 shows the excitation spectrum measured for the mixture sample developed by the mixed solvent of n-hexane and chloroform (2:l). In this condition, 9-chloroanthracene and 9-methylanthracene are not resolved on the TLC sheet. In room-temperature spectrometry they provide almost identical excitation and fluorescence spectra, and thus it is difficult to determine these components selectively by TLC equipped with a conventional fluorometric detector. As shown in Figure 8, these components are clearly resolved on the SSJ excitation spectrum, since they provide sharp 0-0 transition peaks at different wavelengths. This figure of merit may be ascribed to good spectral selectivity in TLC/LD/SSJ spectrometry.
378
Anal. Chem. 1990, 62,378-386
LITERATURE CITED Hayes, J. M.; Small, G. J. Anal. Chem. 1983, 55, 565A. Johnston, M. V. TrAC, Trends Anal. Chem. 1984, 3 , 58. Lubman. D. M. Anal. Chem. 1987, 59, 31A. Hayes, J. M.; Small, G. J. Anal. Chem. 1982, 54, 1202. Pepich, 8. V.; Callis. J. B.; Danielson, J. D. S.; Gouterman, M. Rev. Scl. Instrum. 1986, 57, 878. (6) Pepich, B. V.; Callis, J. B.; Burnes, D. H.; Gouterman, M.; Kalman, D. A. Anal. Chem. 1988. 58, 2825. (7) Stiller, S. W.; Johnston, M. V. Anal. Chem. 1987. 5 9 , 567. (8) Imasaka, T.; Tanaka, K.; Ishibashi, N. Anal. Scl. 1988, 4 , 31. (9) Imasaka, T.; Shigezumi, T.; Ishibashi, N. Analyst 1984, 109, 277. (IO) Imasaka, T.; Okamura, T.; Ishibashi, N. Anal. Chem. 1986. 5 8 , 2152. (11) Imasaka, T.; Tashiro, K.; Ishibashi, N. Anal. Chem. 1988, 58, 3242. (12) Imasaka, T.; Yamaga, N.; Ishibashi, N. Anal. Chem. 1987, 59, 419. (13) Simons, J. K.; Sin, C. H.; Zabriskie, N. A,; Lee, M. L.; Goates, S. R. J. Microcol. Sep. 1989, 1 , 200. (14) Goates, S.R.; Sin, C. H.; Simons, J. K.; Markides, K. E.; Lee, M. L. J. Microcol. S e p . 1989. I , 207. (15) Li, L.; Lubman, D. M. Anal. Chem. 1989, 67, 1911. (16) Powers, D. E.; Hansen, S. G.; Geusic, M. E.; Puiu, A. C.; Hopkins, J. B.; Dietz, T. G.; Duncan, M. A,; Langridge-Smith, P. R. R.; Smalley, R. E. J . Chem. Phys. 1982, 8 6 , 2556.
(1) (2) (3) (4) (5)
(17) Hopkins, J. B.; Langridge-Smith, P. R. R.; Morse, M. D.; Smailey, R. E. J. Chem. Phvs. 1983, 78. 1627. (18) Hayashi. T.; fmasaka, T.; Ishibashi, N. Chem. P h p . 1988, 709, 145. (19) Imasaka, T.; Fukuoka, H.; Hayashl. T.; Ishibashi, N. Anal. Chlm. Acta 1984, 756, 111. (20) Amirav, A,; Horwitz, C.; Jortner, J. J . Chem. Phys. 1988, 88, 3092. (21) Li, L.; Lubman, D. M. Rev. Sci. Instrum. 1988, 59, 557. (22) Imasaka, T.; Tashiro, K.; Ishibashi, N. Anal. Chem. 1989, 6 1 , 1530. (23) Arrowsmith. P.; de Vries, M. S.;Hunziker, H. E.; Wendt, H. R. Appl. Phys. B 1988, 46, 165. (24) Berman, M. R.; Zare, R. N. Anal. Chem. 1975, 47, 1200. (25) Bicking, M. K. L.; Kniseley, R . N.; Svec, H. J. Anal. Chem. 1983, 55, 200. (26) Chen. T. I.; Morris, M. D. Anal. Chem. 1984, 56, 19. (27) Fotiou, F. K.; Morris, M. D. Anal. Chem. 1987, 59, 185. (28) Finney, R. W.; Read, H. J. Chromatogr. 1989, 471, 389. (29) Cooper, R. S.;Jankowiak. R.; Hayes, J. M.; Pei-qi, L.; Small, G. J. Anal. Chem. 1968, 60, 2692.
RECEIVED for review August 14, 1989. Accepted November 22, 1989. This research is supported by Grants-in-Aid for Scientific Research from the Ministry of Education of Japan and by Kurata Foundation.
Optimization of Separations in Supercritical Fluid Chromatography Using a Modified Simplex Algorithm and Short Capillary Columns Jeffrey A. Crow and Joe P. Foley*
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803
A modlfied elmplex algorithm has been used to optimize supercritical fMd chromatography (SFC) separations of samples contahhg nonhomdogous, nondigomerk components. Short capUlary cdunns are employed In the initial separations, with a transfer to a longer column for greater effklency as needed. Two chromatographic response functions, based on peakvalley ratio or threshold resolution criteria, were found to be suitable for SFC. Two- and three-variable simplexes utilizing (I) density gradient rate and temperature, (li) slmultaneous pressure and temperature gradient rates, or (ill) Initial density, de&y gradlent rate, and temperature provided good results ?or the samples In thls study. Convergence to the global optimum was shown for case I by restarting the simplex In another part of the parameter space. A synthetic mixture of three difficult-to-separate sesquiterpene lactones was separated by optimization on a short column using the three-parameter simplex and then transferrlng the method to a longer column.
INTRODUCTION More often than not, the initial separation of a given sample is unsatisfactory, usually because the desired resolution between all the peaks of interest is insufficient. To improve the separation in an efficient manner, an optimization procedure with well-defined goals is strongly recommended ( I ) . The goals set may vary depending on how many peaks are of interest, the resolution required, the importance of analysis time, and other considerations. The point a t which an optimization procedure is terminated depends on the quality of the separation desired; there is a distinct difference between an acceptable and an optimum separation. The decision upon which optimization is usually based takes into account a 0003-2700/90/0362-0376$02.50/0
minimum resolution in some maximum time frame (2). Many chromatographic response functions (CRF’s) have been developed and used based on this idea (3, 4 ) . Each of the three terms in the fundamental resolution equation ( 5 ) can be optimized to improve the separation. Retention (k’j and the selectivity ( a )should first be optimized via changes in the density of the mobile phase, the temperature, and the gradient rates. Optimization of these two parameters is clearly the first step, since it will indicate if the current mobile phase/stationary phase combination is adequate for the separation being considered. The efficiency of a column, N , is determined by its length and the nature of the stationary phase, including column diameter or particle size. Given the square root dependency of resolution on N , large changes in parameters controlling N (e.g., column length or linear velocity) will result in only moderate changes in resolution and thus should only be considered if changes in the parameters controlling selectivity and retention in SFC (composition, density, temperature (or their respective gradients, if employed)) do not suffice. Supercritical fluid chromatography (SFC) displays GCand/or LC-like behavior, depending on both the solutes and the experimental conditions. Some components may partition by their vapor pressures while others partition by solventlike properties of the mobile phase (6). As the experimental conditions are changed, the behavior of some or all of these components may be reversed. Elution order may also depend on such properties as basicity and steric hindrance (7). Finally, many of the parameters that control retention and selectivity are moderately to highly synergistic (8). For these reasons, univariate optimization strategies (sequential optimization of one parameter at a time) or intuitive approaches are often ineffective in locating a true optimum (3),hence the need for a simultaneous multivariate approach in order to obtain the best possible separation. 1990 American Chemical Society
