1928
Anal. Chem. 1988, 6 1 , 1928-1931
Chemically Bonded Liquid Crystals as Stationary Phases for High-Performance Liquid Chromatography. Effects of Mobile-Phase Composition J o s e p h J. Pesek*
Department of Chemistry, S u n Jose State University, S u n Jose, California 95192 Antoine M. Siouffi
Laboratoire de Chimie Apliquee, Uniuersite d’Aix-Marseille, F13390 Marseille Cedex 13, France
The compound [4-(allyioxy)benzoyi]-4-methoxyphenyi (ABMP), shown to possess liquid crystal properties when bonded to a polyslloxane backbone, was covalently attached to silica and tested chromatographically for similar properties by mobile-phase-composition studies. Methanol, acetonitrile, and tetrahydrofuran are mixed with water in various proportions In order to determine the bonded material’s reversephase behavior. The separation of anthracene and phenanthrene as well as that of carvone and pulegone is used to monitor the mobllephase effects on the stationary phase. I n ail cases a plot of log k’ vs percentage of organic solvent in the mobile phase in not completely linear. At higher percentages of organic solvent the plot is linear without separation of either pair of solutes. At lower percentages of organic solvent the plot is nonlinear and the two pairs of compounds are separated. This observation is attributed to a phase transition in the bonded material. Variable-temperature experiments also support the possibility of a phase transition.
INTRODUCTION The use of liquid crystals as stationary phases in gas chromatography was first established over 25 years ago (1,2). They are used quite extensively for many difficult separations, and a typical review (3) may contain hundreds of references. In contrast, the early studies ( 4 , 5) using liquid crystals as stationary phases for high-performance liquid chromatography were rather incomplete, and no conclusion could be drawn about their potential usefulness. There is, however, one important difference between the use of liquid crystals as stationary phases in gas chromatography and liquid chromatography. In GC the liquid crystal can be coated directly on a solid support or capillary wall. The low volatility of these materials leads to long column lifetimes. In HPLC such a procedure would result in rapid loss of the stationary phase due to solubility in the mobile phase or to removal by shear forces. Therefore, the only practical solution for HPLC is to produce a chemically bonded material that possesses liquid crystal properties. This situation was never properly addressed in the earlier studies, so no conclusion could be made concerning the effectiveness of bonded liquid crystals as stationary phases for HPLC. However, several recent studies (6-8) have shown that certain liquid crystals or structurally similar compounds when bonded to a polysiloxane backbone retain or develop liquid crystalline behavior. Therefore it seems possible that similar compounds bonded to silica may have the same liquid crystalline behavior. An earlier report (9) described the synthetic approach for producing a chemically bonded material that might possess liquid crystal properties. The preliminary tests
of this bonded phase, particularly the thermal and retention index studies, indicated that liquid crystal properties could be present. In this report we further test the bonded material by subjecting it to a varying mobile-phase composition and monitoring the separation of two pairs of solutes. Ordinary reverse-phase behavior should result in a linear relationship between log k‘and the percentage of organic solvent in the mobile phase (10). In contrast, it is known that molecular orientation of crystals is a function of solvent type (11). Therefore, mobile-phase-composition studies might be able to detect phase transitions that occur in the bonded material. These results would provide substantial evidence that a bonded liquid crystal with useful chromatographic properties could be produced. This report is a preliminary study of the solvent effects observed on the ABMP phase which could be used to determine whether further investigations on bonded liquid crystals as stationary phases for HPLC are warranted. Some variable-temperature studies are also included in this report. EXPERIMENTAL SECTION Bonded Liquid Crystal Column. The synthesis of the bonded liquid crystal material has been described previously (9). The bonded phase on Nucleosil300-10was packed into a 150 X 4.6 mm stainless steel column using a Haskel pneumatic amplification pump with methanol as the driving solvent. Materials. Acetonitrile (ACN), tetrahydrofuran (THF), and methanol (MeOH) (Merck) were all chromatographic grade solvents. The water was triply distilled and filtered through a 0.45-pm filter. The solutes used in this study, anthracene, phenanthrene, m o n e , and pulegone (Adrich), were used without further purification. Each compound gave only a single peak in all chromatograms. Apparatus. One liquid chromatography system consisted of a Waters Model 6000A pump, a Rheodyne Model 7125 injector with a 50-pL sample loop, and a Merck variable-wavelength monitor. Variable-temperature experiments were done on a Hewlett-Packard Model 1050 LC system equipped with a Model 1046B fluorescencedetector. Two C-18 columns were employed, both were 150 X 4.6 mm with one containing Lichrosphere Rp-18e (Merck) on 5-pm particles and a second containing the product of Nucleosil 300-10 and octadecyldimethylchlorosilane. Procedures. The solutes anthracene and phenanthrene were monitored at 254 nm while carvone and pulegone were monitored at 230 nm. Fluorescence of anthracene was monitored at 405 nm with an excitation wavelength of 365 nm. The mobile phases were prepared by using standard volumetric glassware and were degassed before use with helium. The void volumes for both the C-18 and liquid crystal columns were determined by nitrate injection. RESULTS AND DISCUSSION The primary pair of test solutes chosen for monitoring retention properties of the bonded liquid crystal was an-
0003-2700/89/0361-1928$01.50/00 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 17, SEPTEMBER 1, 1989
1929
- 2.0
- 1.5
-
-g I
- 1.0 8 0.5
- 0.5 40
I
I
25
30
I
I
I
35
40
45
I
50 . 9
I
55
,
80
,
.
85
70
,
75
.
.
BO
85
ORGANIC SOLVENT
Flgure 1. log k’vs percentage of organic solvent in the mobile phase for phenanthrene (X) and antracene (0)on ABMP column. Key: MF, tetrahydrofuran: ACN, acetonitrile; MeOH, methanol.
1.00
1
50
60
Flgure 3. log k’ vs percentage of acetonitrile in the mobile phase on ‘2-18 columns. Merck RP-18e: phenanthrene (X) and anthracene (0) using scale on right side of plot and Cawone (A)and pulegone (.) using scale on left side of plot. C-18 on Nucleosil 300-10: anthracene (0) using scale on left side of plot.
Figure 2 shows a similar plot for the pair of solutes carvone and pulegone in acetonitrile. Like anthracene and phenanthrene, this pair of solutes differs only slightly in structure as shown below:
1
CH3
. 1
0.50
4
80
70
%ACETONITRILE
*::;t, 30
40
carvone
50
% ACETONITRILE
Figure 2. log k’vs percentage of acetonitrile in the mobile phase for cawone (0)and pulegone (X) on ABMP column.
thracene and phenanthrene. This pair can be separated by a number of different stationary phases (12,13) including liquid crystals coated on a capillary column for GC ( 3 ) . Therefore the chromatographic behavior of these solutes may serve as a useful indicator of the presence of liquid crystal properties in the ABMP bonded phase. Figure 1 shows a plot of log k’ for both anthracene and phenanthrene as a function of percentage of organic solvent in the mobile phase using three different solvents. In each case there is a linear portion in which the two solutes are not separated and a nonlinear portion where the k’values of the two compounds begin to diverge. The point of divergence occurs at a higher percentage of organic solvent as the polarity of the solvent increases, i.e. as the solvent strength in the reverse-phase system decreases. For each solvent, the linear portion occurs at relatively high solvent strength and the nonlinear portion at relatively low solvent strength.
CH3
pulegone
The same behavior is observed for this pair of components as for anthracene and phenanthrene, i.e. a linear portion with no separation at high solvent strength and a nonlinear portion with separation at low solvent strength. Figure 3 shows the behavior of both pairs of solutes on a commercial endcapped C-18 phase as well as the retention of anthracene on a (2-18 column made from the identical support used for the liquid crystal phase. In contrast to the behavior of these solutes on the ABMP phase, the ODS phase exhibits linear behavior over the entire mobile-phase-composition range. In addition, both pairs of solutes are separated over the 4040% acetonitrile range on the commercial column. Similar behavior is observed for the C-18 material bonded to Nucleosil300-10, so that the silica support does not appear to have any effect on the retention characteristics of these solutes under these experimental conditions. Therefore, the differences observed between the ABMP and C-18 retention vs mobile-phase composition must be due to the properties of the bonded material and not the silica support. It is evident from an examination of the log k‘variations of either pair of solutes in the organic and aqueous mobile phases studies that there is a fundamental difference between retention on the ABMP phase and that on a conventional reverse-phase material such as C-18. The behavior exhibited by the two pairs of solutes on the C-18 phase (Figure 3) is the predicted response for variations in solvent strength (IO),i.e. log k’varies linearly as a function of percentage of organic solvent in the mobile phase. Therefore the unusual characteristic exhibited by the ABMP phase must be due to some other effect besides or in addition to normal reverse-phase behavior. Silanophobic interactions are probably not responsible for the retention behavior observed since there is no minimum in the plot of log k’ vs percentage of organic solvent in the mobile phase at high solvent strengths (14,15). In addition, all the solutes used in this study as well as N,.h’diethylanaline gave symmetric peaks indicating no substantial
1930
ANALYTICAL CHEMISTRY, VOL. 61, NO. 17, SEPTEMBER 1, 1989
I/’,
,
3 00
,
,
3 10
,
,
.1
110
3 20
~ o ~ / T- ’P1 K
Flgure 4. log fR‘ of anthracene vs l / T on the ABMP phase for acescale on left side of plot) and for acetotonitri1e:water (55/45)(0, nitrikwater (35165) (0, scale on right side of plot) mobile-phase composition.
number of residual silanols. If it is postulated that some type of liquid crystal property (ordering) is possible for the ABMP phase, then an explanation of the solvent strength variations can be formulated. It has been demonstrated that in the presence of a “hostile” (polar) solvent, ordinary brush phases collapse in order to minimize the surface area in contact with the mobile phase (16). In the studies reported here, the mobile phase becomes more “hostile” as the percentage of organic solvent decreases. Figure 1 also demonstrates that this point of collapse is variable, depending on the polarity of the organic modifier. Therefore, the percentage of organic solvent required to reach this critical point is greater for methanol (the most polar of the organic solvents used) than for acetonitrile which is greater than that for T H F (the least polar of the organics used). Under these conditions the collapse could lead to an ordering of the organic moiety on the silica because of the closer proximity of the bonded phase molecules and the partial expulsion of the solvent. This new ordered phase may have a more favorable environment for the solute, which leads to a greater increase in log k’ than predicted from solvent strength considerations alone. The fact that the slope is changing as opposed to attaining a single new value indicates that the process is a gradual change rather than a sudden phase transition. This interpretation is in agreement with the thermal data (9), which shows a phase change over a broad temperature range rather than a sudden sharp transition. Another interesting comparison between ordinary reverse-phase behavior and the retention characteristics of the ABMP phase involves the a values of the solute pairs. As shown in Figure 3, the a value for anthracene and phenanthrene (1.14) and carvone and pulegone (1.36) remains constant on the C-18 phase. For the ABMP phase these values change with solvent composition. With a constant log k’ = 1.5, the a values are 1.15, 1.29, and 1.33 for anthracene and phenanthrene in THF,ACN, and MeOH, respectively. It is interesting to note that a becomes larger as the overall polarity of the mobile phase increases. As discussed previously, the increasing polarity may force the bonded phase into a more ordered environment, which leads to greater efficiency in the separation process. If the a value of carvone and pulegone (1.16) is compared to the value for anthracene and phenanthrene (1.24),then it can be seen that the larger a value occurs for the higher molecular weight pair. This may reflect the overall distance between the bonded moieties, indicating not only that is there is greater degree of interaction for the pair anthracene and phenanthrene (higher percentage of ACN needed to achieve a log k’ = 1) but that the differential in-
teraction between this pair is greater than that for carvone and pulegone. If the spacing between brushes is too large, then the maximum degree of interaction cannot be achieved. The lack of separation for either of these solute pairs a t high percentage of organic solvent is not unusual. Normal phase separations using a variety of stationary phases containing aromatic and substituted aromatic groups result in cy values for anthracene and phenanthrene varying from 1.0 to 1.3 (17). Our own experiments with a theobromine bonded phase resulted in an cy = 1.0 over a solvent composition range of 30-70% ACN in water. Further support for the possibility of a phase transition in the bonded material is shown in Figure 4. At both high and low solvent strength, there is a discontinuity in the plot of log tR’ vs 1/T. The presence of the discontinuity is supported by the fact that the differences between the two straight lines drawn at each solvent strength are much greater than the error ’ Derivative analysis by in the measurement of t ~ (10.7%). comparison of successive experimental points indicates a change in slope so that representation of the data by a single straight line is not possible. The presence of a discontinuity is characteristic of a phase transition and has been observed for this material bonded to polysiloxane (8). The transition temperature observed in these experiments (between 55 and 60 “C) is quite similar to the transition temperature (61 “C) measured on polysiloxane (8). A lowering of the transition temperature might be expected in these studies due to the presence of an impurity (mobile phase). It is interesting to note that the variable-temperature studies reveal a transition occurring in both the linear and nonlinear portions of the solvent strength plot. The difference in slope is probably reflective of the relative amount of ordering that exists in the bonded material. Only additional variable-temperature experiments with more bonded liquid crystal materials utilizing a variety of solutes and mobile phases will elucidate the exact nature of the bonded-phase transitions that are possible on silica. In conclusion, it appears that solvent-induced changes occur in the structure of the ABMP bonded phase that are likely related to their ability to form a liquid crystallike structure. Such mobile-phase effects result in nonlinear plots for log k’ vs solvent strength and could provide a powerful means of controlling retention in complex systems. Current work focuses on determining whether these phenomena are present in other liquid crystal materials bonded to silica as well as formulating other bonded reactions and elucidating surface, temperature, and solvent strength effects.
ACKNOWLEDGMENT We acknowledge the assistance of Ms. Sally Swedberg in obtaining the variable-temperature chromatographic measurements. LITERATURE CITED Kelker, H. Z . Anal. Chem. 1983, 798, 254. Kelker, H. 8 e r . Bunsen-GesPhys. Chem. 1983, 8 0 , 698. Wttkiewicz, 2. J . Chromatogr. 1982, 251, 311. Taylor, P. J.; Sherman, P. L. J . L1q. Chromatogr. 1980, 3 , 21. Aratskova, A. A,; Vetrova, 2 . P.; Yoshin, Y. 1. J. Chromatogr. 1988, 365, 27. Jones, 6. A.; Bradshaw, J. S.; Nishioda, M.; Lee, M. L. J . Org . Chem . 1984, 4 9 , 4947. Morkkla, K. E.; Chang, H. C.; Schregenberger. C. M.; Tolbert, 6. J.; Bradshaw, J. S.; Lee, M. L. M C CC. J . High Resolut. Chromatogr. Chromatogr. Common. 1985, 8 , 516. Apfel, M. A.; Finkelman, H.; Janini, G. M.; Laub, R. J.; Luhmann, 8. H.; Price, A.; Roberts, W. L.; Shaw, T. J.; Smith C. A. Anal. Chem. 1985, 5 7 , 651. Pesek, J. J.; Cash, T. Chromatogrephie, in press. Snyder, L. R.; Quarry, M. A; Glajch, J. L. Chromatographia 1987, 2 4 , 33. Ohgawara, M.; Uchida, T. Jpn. J . Appl. Phys. 1981, 2 0 , L237. Jinno, K.; Kawasaki, K. Chromatographia 1983, 77, 445. Jinno, K.; Kawasaki, K. Chromatwaphia 1984, 18, 44. Nahum, A.; Horvath, C. J. Chromatogr. 1981, 203, 53.
Anal. Chem. 1989, 61, 1931-1933 (15) Klaas, B.; Horvath, C.; Melander, W. R.; Nahum. A. J . Chromatogr. 1981, 203, 85. (16) Lochmuller, C. H.; Kersey, M. T. Anal. Chem. 1988, 60, 1910. (17) Bertrand-Motard. Cecile. Ph.D. ~hesis,Unlverslte de Bordeaux, 1988.
RECEIVED for review October 26, 1988. Revised manuscript
1931
received May 8, 1989. Accepted May 20, 1989. J.J.P. was supported by a grant from the French Ministry of Education while at the Universite d’Aix-Marseille. Partial support of this research was also provided by a grant from the National Science Foundation (CHE-8814849).
Laser-Based Indirect Fluorometric Detection and Quantification in Thin-Layer Chromatography Yinfa Ma, Lance B. Koutny, and Edward S. Yeung* Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011
A laser-based Indirect fluorometric detectlon method for thin-layer chromatography Is described. The new technique can be easlty used for quantttatlve measurements because of Its two-dimensional scanning capability. Since It Is based on the Indirect fluorescence mode, unlversai detection Is posslble wIthout derlvatizatlon. Also, it takes only 35 s for acqulrlng a data array of 256 X 64 points with thIs technique to achleve a detection llmtt of 6 pg. Thls Is 100 times lower than when the human eye Is used as a detector for the same samples based on lndlrect fluorometry. The lineartty in indirect fluorometric detectlon Is found to be over 2 orders of magnltude.
INTRODUCTION Since thin-layer chromatography (TLC) was described by Kirchner and his group ( I ) , many improvements in all aspects of operation have been made, including high quality, high performance, and multimodal TLC plates, accurate and precise spotting techniques, instrumentalized development devices, and sophisticated detectors (2-6). These improvements have transformed TLC into a modern separation technique. Detection and quantification on TLC plates are important considerations. The simplest detection method is based on the human eye aided by a vast array of selective spray reagents, or by the use of plates impregnated with fluorescent indicators to allow components to be detected by absorption a t the excitation or emission wavelength. For quantitative analysis, traditionally the plates were scanned by mechanically driven densitometers (7).This is usually a slow procedure. Recently, various methods have been suggested for improving the detection limit, speed, and scope of application. These include photoacoustic spectrometry @-lo),flame ionization ( I I ) , photothermal deflection (12,13),laser fluorometry (14, 15), mass spectrometry (16-19), and computer-aided videodensitometry (5,20,21).All these detection methods respond to a specific property of the analyte. If the analyte does not possess that specific property, difficulties in detection will arise. There is thus a need for a sensitive universal detection scheme for TLC. Recently, quite a few indirect detection methods for liquid chromatography (LC) have been demonstrated (22-26). The detection mechanisms have been clearly identified (27,28). Briefly, the detector responds to some physical property of the chromatographic eluent. A constant background signal
is then maintained when the analytes are absent. When the analytes elute, displacement of the eluent causes a change in the background signal. So, analytes can be detected indirectly. Following the same principles, the indirect fluorometric detection of anions (29),cations (30),and nonelectrolytes (31) in TLC has also been achieved. However, the results so far have not been optimized. The visual observation of the TLC plates (which are illuminated by a UV lamp) is obviously not sensitive compared to instrumental detection methods. Also, visual observation cannot be reliably used for quantitative measurements. In this paper, we describe a laser-based indirect fluorometric detection scheme for TLC with high sensitivity (picograms) and wide linear dynamic range (more than 2 orders). Reliable scanning is easily performed because the TLC plates and detector are fmed while the excitation laser beam is moved. In this system, a microcomputer controls the X-Y scanning and a t the same time collects the data. This allows a total data acquisition time of 35 s for a data array of 256 X 64. There is also an improvement in the signal-tonoise ratio (S/N) resulting from data averaging.
EXPERIMENTAL SECTION Apparatus. The experimental setup for two-dimensional TLC scanning in laser-based indirect fluorometric detection is shown in Figure 1. A He-Ne laser (Uniphase, Manteca, CA) was used as an excitation light source at 633 nm at a power of 8 mW. In order to maintain a constant laser power, a laser power stabilizer (Cambridge Research and Instrumentation, Cambridge, MA, LS 100) was used. An acoustooptic modulator (Andersen Laboratories, Inc., Bloomfield, CT), which was controlled by a radio frequency (rf) driver, was used to deflect the laser beam. The change in the frequency of rf input to the acoustooptic device causes deflection of the first-order laser beam (32,33). The acoustooptic modulator and a rotating mirror combine to scan the laser beam in the horizontal and vertical directions, respectively. To obtain the optimum spatial resolution, we introduced a cylindrical beam expander with L1and L2 and used long focal length lenses L3 and Lk The focused image onto a microscope eyepiece (12X magnification) was enlarged to an area of 40 X 50 mm (horizontal X vertical) with a laser spot size of about 1.5 mm on the TLC plate. The fluorescence signal was collected by a camera lens (28-105 mm, f2.8-f3.8, Vivitar Corp., Santa Monica, CA), passed through a cutoff filter (to remove scattered 633-nm light), and directed into a R928 photomultiplier tube (PMT) (Hamamatau,Middlesex, NJ) operated at 850 V. The output of the PMT was converted into voltage via a resistor and was fed into a data acquisition system consisting of an analog to digital 1 / 0 interface (Data Translation, Marlborough, MA, DT 2827) and a microcomputer (IBM, Boca Raton, FL, PC/AT). This system was also used to control the rf output and the stepping motor in order to syn-
0003-2700/89/036 1-193 1$01.50/0 0 1989 American Chemical Society
