Anal. Chem. 2006, 78, 2823-2831
Results with an Apparatus for Pressurized Planar Electrochromatography† Allyson L. Novotny and David Nurok*
Department of Chemistry and Chemical Biology, Indiana UniversitysPurdue University Indianapolis, 402 North Blackford Street, Indianapolis, Indiana 46202 Randall W. Replogle, Gregory L. Hawkins, and Robert E. Santini*
The Amy Facility for Chemical Instrumentation, Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907
Pressurized planar electrochromatography (PPEC) is a fast and efficient planar chromatographic technique. The mobile phase is driven by electroosmotic flow, while the system is pressurized in a manner that allows heat to flow between the sorbent layer and the pressurizing medium. The reproducibility of solute retention was not satisfactory in the initial report describing PPEC. In the current report, this reproducibility is improved by better control of several experimental variables. The pressure at which PPEC is performed is now free of drift, and the temperature at which the layer is preconditioned is maintained to within (1 °C. The best reproducibility of retention is obtained when the plate is soaked in the mobile phase for a defined time before each run. In the original prototype, the temperature of the sorbent layer was not controlled. In the present apparatus, water, at a constant temperature between 3 and 60 °C, is circulated through channels in the two die blocks that pressurize the layer. The highest efficiency is obtained at an intermediate temperature. This behavior is ascribed to high resistance to mass transfer at the lower temperatures and increased diffusion at higher temperatures. Efficiency, as measured by the number of theoretical plates, increases with increasing migration distance. The height equivalent of a theoretical plate diminishes with increasing migration distance, and values as low as 0.0106 mm are obtained under appropriate conditions. This extrapolates to 94 000 plates/m. Manual spotting was used in this report. Evidence is presented that substantially better efficiency would be obtained if the initial spot size were smaller. The efficiency of PPEC in its current form is illustrated by a chromatogram showing the separation of nine solutes in 2 min. PPEC was also performed with TLC plates in a back-to-back configuration, and this doubles the number of samples that can be simultaneously separated.
* Corresponding authors. E-mail: (D.N.)
[email protected]. (R.E.S.)
[email protected]. † This paper is dedicated to the memory of Gregory L. Hawkins who passed away at far too young an age. 10.1021/ac052262v CCC: $33.50 Published on Web 03/22/2006
© 2006 American Chemical Society
Planar electrochromatography (PEC) is a form of planar chromatography in which the mobile phase migrates as a result of electroosmotic flow that is generated by applying an electric field across the sorbent layer of a thin-layer chromatography (TLC) plate.1 While good separations are obtainable in the reversed-phase mode, the technique in its present form suffers from several shortcomings. When there is little Joule heating (e.g., with a low buffer concentration or a low applied voltage), liquid accumulates on the surface of the sorbent layer and this causes streaking of the analytes. When there is too much Joule heating, the separation is terminated due to the layer drying. Good separations are attained when there is a balance between accumulation of liquid on the surface and evaporation from the surface. Even under these conditions, however, the best separations are obtained for relatively short migration distances. The problems associated with either excess heating of the layer or accumulation of liquid on the surface of the layer can be avoided by applying pressure to the surface of the sorbent layer, in a manner that allows heat to flow between the layer and the pressurizing medium. This allows control of the temperature at which PEC is performed. There are at least four different techniques whereby pressure can be applied to the layer. The earliest description of pressurized planar electrochromatography (PPEC) is in a patent2 in which an apparatus is described where force is applied to the layer by a pad containing a pressurized liquid. A sheet of a thermally conducting, electrical insulating material covers the sorbent layer. This allows the temperature of the layer to be controlled by regulating the temperature of the pressurizing liquid. The first literature report of separations performed by PPEC described an apparatus where the layer was held in a vertical position and pressure was applied through a plate driven by an hydraulic ram.3 Three other recent reports have described apparatus for performing PPEC with the plate oriented in the (1) Nurok, D. J. Chromatogr., A 1044, 83-96, 2004. (2) Nurok, D.; Frost, M. C. Arrangement and Method for Performing Chromatography. U.S. Patent 6,303,029 B1, October 2001. (3) Nurok, D.; Koers, J. M.; Novotny, A. L.; Carmichael, M. A.; Kosiba, J. J.; Santini, R. E.; Hawkins, G. L.; Replogle, R. W. Anal. Chem. 2004, 76, 16901695.
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horizontal plane. Dzido et al.4 applied force using a mechanical screw-driven pressure plate, where the drive screw is tightened to a set torque. They reported good separation quality and good reproducibility of retention. Tate and Dorsey5 used a free weight to pressurize the sorbent layer to ∼1 bar, or, more recently, a commercial high-pressure hydraulic press6 capable of applied pressure of ∼70 bar. This is capable of a pressure range similar to that of the apparatus described in the report below. These authors used the technique to study the equilibration and establishment of electroosmotic flow at 32 positions on the sorbent layer. This was accomplished by fitting a four by eight array of voltage sensing electrodes into the cover plate that contacted the layer. The initial literature report of separations by PPEC3 demonstrated that the method can be ∼24 times faster than conventional TLC and that a two-dimensional array of samples can be simultaneously separated by the technique. An example was given of the separation of nine five-component samples in 1 min on a TLC plate with a pressurized area of 2.5 cm × 10.0 cm. The reproducibility of retention with this apparatus was poor, and a number of possible causes for this were briefly discussed. These included the fact that the temperature of the layer was not controlled, the plates were conditioned in an oven without precise temperature control, and the hydraulic pump used to pressurize the layer exhibited drift over time. These and other issues have been addressed, and improved reproducibility is now obtained with the apparatus and techniques described below. The apparatus used in the above report was constructed on a 1-in.-thick metal base in order to limit mechanical deformation when high thrust was applied. The apparatus weighed more than 50 kg because of this feature. The heavy base plate did not completely prevent elastic deformation under high thrust, and for this reason, side struts were retrofitted to the apparatus.3 The report below discusses a completely redesigned apparatus and the procedures used to obtain improved chromatographic efficiency and enhanced reproducibility of retention. The new apparatus is substantially lighter than the original prototype, and the temperature at which separation is performed can now be regulated. The respective effects of temperature, pressure, and buffer concentration on separation are also discussed. EXPERIMENTAL SECTION Materials Used. The hydraulic pump, gauge, tubing, and ram used for the apparatus were purchased from EnerPac (Milwaukee, WI). Merck LiChrospher RP-18 WF254s plates (Catalog No. 1.05646) were a gift from Merck KgaA (Darmstadt, Germany), and Merck RP-18 F254s plates (Catalog No. 15389-7) were a gift from EMD Chemicals, Inc. (Gibbstown, NJ). In the discussion below, the former are referred to as high-performance plates and the latter are referred to as regular plates. Both types of plates were also purchased from VWR International (Chicago, IL). Acetonitrile, acetic acid, sodium acetate, and methanol were purchased from Fisher Scientific (Pittsburgh, PA). 17R-Acetoxyprogesterone, 2′-acetonaphthone, benzanilide, and o-nitroaniline were a gift from Don Risley of Eli Lilly and Co. A mixture of these (4) Dzido, T. H.; Mroz; Jozwiak, G. W. J. Planar Chromatogr. 2004, 17, 404410. (5) Tate, P. A.; Dorsey, J. G. J. Chromatogr., A 2005, 1079, 317-327. (6) Tate, P. A.; Dorsey, J. G. J. Chromatogr., A 2006, 1103, 150-157.
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four analytes was prepared in methanol and was used for all but one of the separations in this report. The latter separation includes the following six compounds that were available in our laboratory: 4-cholesten-3-one, hydrocortisone alcohol, benzamide (SigmaAldrich Corp., St. Louis, MO), 4-androsten-17β-ol-3-one acetate (Steraloids, Inc. Wilton, NH), androstenedione (Searle Chemicals Inc., Chicago, IL), and 4-pregen-11R-ol-3,20-dione (Mann Research Laboratories New York, NY). Standard Operating Conditions. In this report, standard operating conditions refer to an applied potential of 6.0 kV, an applied pressure of 63 atm, and a mobile phase consisting of 55% aqueous acetonitrile containing 5 mM acetate buffer at a pH of 4.7. The buffer solution was prepared by mixing equal volumes of 1 M acetic acid and 1 M sodium acetate. The concentration of the solution was then adjusted such that, when mixed (on a volume/volume basis) with acetonitrile and additional water, the mobile phase was at the desired molarity. The water was filtered through a Milli-Q purification system. The reported pH of 4.7 for the mobile phase is a nominal value and refers to the value before the addition of acetonitrile. Preparation of the TLC Plate. The plates were cut into 3.3 cm × 12 cm sections. A 5-mm section of silica was removed from each of the two longer, parallel edges, using an Analtech (Newark, DE) scraper. The two exposed glass surfaces were then coated with a silicone rubber sealant and allowed to cure overnight. Plates were conditioned in a VWR (Cornelius, OR) microprocessor controlled oven model 1330 FM, immediately stored in a desiccator, and used within 24 h. Conditioning was for 20 min at 120 °C for the data used for Figures 6, 8, and 9, as well as for the data in Table 1. These data were collected in the earlier part of the study. All other separations were with high-performance plates conditioned at 160 °C for 20 min. Both the regular and highperformance plates provide a faster separation when conditioned at the higher temperature. Sample spots (10 nL) were applied 4 cm from the bottom of the plate using a 0.5-µL Hamilton (Reno, NV) syringe. This was performed both in a dry and in a prewetted mode. In the dry mode, the sample spot was applied to the plate, and one end of the plate was then dipped into mobile phase to within 1 mm of the sample spot. The plate was then removed, inverted, and the other end dipped into mobile phase to within 1 mm of the spot. The plate was then immediately placed in the Delrin holder, which was quickly assembled and placed in the apparatus. While a small area of the layer is still dry when the plate is first placed in the holder, this area is assumed to be wetted by capillary action by the time PPEC is commenced. In the prewetted mode, the plate was soaked in mobile phase for various lengths of time. The plate was then removed, covered with a glass sheet containing a small hole of ∼2.5-mm diameter, the sorbent beneath the hole blotted with a piece of filter paper, and the sample spot applied to the area beneath the hole. This method resulted in the best reproducibility of retention. Plates were scanned at 254 nm using a model CS 9000U Shimadzu (Kyoto, Japan) Flying-Spot scanner in the fluorescence shadowing mode. This was preceded by a background scan, in a channel close to the path of the analytes.
Table 1. Reproducibility of Migration Distance in PPEC relative standard deviation, % regular platesa
LiChrospher platesb
solute
previous replicatesc
first set replicatesd,e
second set replicatesd,e
third set replicatesd,e
previous replicatesc
first set replicatese,f
17 R-acetoxy progesterone 2′-acetonaphthone benzanalide o-nitroanaline
9.1 7.5 6.1 4.0
4.2 3.0 1.7 1.7
4.1 3.6 3.1 2.8
3.1 2.6 2.0 1.9
3.3 4.6 3.7 2.7
2.7 2.1 1.6 1.6
a In previous report,3 PPEC was for 10 min at 7000 V and 59 atm. The mobile phase was 55% aqueous acetonitrile containing 25 mM acetate buffer at a nominal pH of 4.7. In this report, PPEC was for 10 min at 6000 V and 63 atm. The mobile phase was 55% aqueous acetonitrile containing 5 mM acetate buffer at a nominal pH of 4.7; i.e., standard conditions were used. b In previous report,3 PPEC was for 4 min at 6000 V and 59 atm. The mobile phase was 55% aqueous acetonitrile containing 25 mM acetate buffer at a nominal pH of 4.7. In this report, PPEC was for 6 min at 6000 V and 63 atm. The mobile phase was 55% aqueous acetonitrile containing 5 mM acetate buffer at a nominal pH of 4.7. c Results in previous report3 were for six replicates. The plates were briefly dipped in the mobile phase before PPEC. d Results are for eight replicates. Replicates were performed as a check, whenever any component of the apparatus had been disassembled. e The plates were soaked in the mobile phase for 20 min before PPEC. f Results are for seven replicates.
Figure 1. Current apparatus for performing PPEC.
Spot Shape. For all separations discussed in this report, the spot shape was round, or slightly elongated, after PPEC. The only exception was in the reproducibility study (vide infra) where the high-performance plates yielded elongated spots after the sorbent layer was soaked in the mobile phase for 20 min. It was subsequently found that a 10-s soak did not cause spot elongation but that a 1-min soak caused some elongation of the spots after PPEC. Some separations yielded elongated spots even when all precautions were taken. Results from such separations, with the exception of the runs in the reproducibility report noted above, are not included in this report. To maintain good spot shape, it is essential to clean the balland-socket on a regular basis to prevent slight misalignment. It is also necessary to tighten all load-bearing bolts to the same torque level as the apparatus is reassembled after this maintenance procedure. These precautions minimize induced strain that would otherwise produce slightly uneven pressure on the sorbent layer. Apparatus. The apparatus used in this report incorporates several significant improvements to the original prototype. The
apparatus is now more compact and substantially lighter and is shown in Figure 1. In the original design, the pressurizing ram actuated a movable plate attached to a die block, which in turn pressurized the sorbent layer. In an early form of the current apparatus, a preassembled ball-and-socket joint was connected to the die block that applies pressure to the TLC layer. This proved to be unsatisfactory, as irreproducible alignment was observed when the apparatus was subjected to a thrust loading. This misalignment resulted in uneven application of pressure to the plate and resulted in the components of a sample having migration distances that depended on where the sample was spotted with reference to the long edge of the TLC plate. An open ball joint and corresponding socket was substituted for the preassembled ball-and-socket joint, and this is shown in Figure 2. The hydraulic cylinder, piston, connecting rod, and the half of the ball joint attached to the rod is referred to as the ram. The operation of the new ball-and-socket is further discussed in the section on reproducibility. The socket moves along two guide rods and is attached to a die block that pressurizes the layer. The TLC plate assembly is constructed of Delrin and is the same as used in our Analytical Chemistry, Vol. 78, No. 8, April 15, 2006
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Figure 2. Open ball joint and corresponding socket, and die blocks with aluminum nitride sheets. The picture shows the thermocouple on the right and the safety lead and lead to the anode on the upper left.
original report;3 it is pressurized between the movable die block (described above) and a stationary die block. In our original report, a modified pipet, with a slot cut in the bulb, was used as the mobile-phase reservoir. The reservoir is now constructed from Delrin and, during PPEC, is attached to the base of the apparatus in such a position that the end of the TLC plate extends into the reservoir. The reservoir can be removed for cleaning. The anode is a length of platinum wire extending through the wall of the reservoir and oriented along the end of the plate in order to minimize electrical field gradients. An additional modification to the original prototype is that the die blocks that pressurize the layer each have an internal flow path in the shape of an inverted “U” through which water is circulated. Nearly constant temperature is maintained by this means. Unless specifically noted, tap water was used without heating or cooling. The temperature of the water flowing from the tap was constant during a set of experiments, but varied in the range 20-23 °C during the course of this study. Figure 3 shows the shape of the internal channel and is not drawn to scale. The temperature of the stationary die block is monitored by a thermocouple placed 1.4 cm from the face of the die block. It is assumed that the thermocouple readout reflects the temperature of the sorbent layer. Whenever the temperature of a separation is changed, water is circulated for 30 min to allow the instrument to equilibrate before commencing PPEC. Calculations Used. The number of theoretical plates, n, is given by
n ) 5.54(MD/w1/2)2
(1)
where MD is migration distance and w1/2 is the peak width at halfheight. 2826
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Figure 3. Passages for flow of water in die block. Figure is not to scale.
As discussed by Kaiser,7 the peak width of the initial spot makes a significant contribution to the final spot width and should be considered when calculating the number of “real” theoretical plates. The width at half-height of the initial spot in this report is typically 0.5 mm. As discussed in the relevant section, there is a technique that allows substantially smaller spots to be applied to the layer. If, for instance, the initial spot width were 0.2 mm, then w1/2 would be (with all dimensions in mm)
w1/2 ) 0.2 + (wf - wi)
(2)
where wi is the width at half-height for the initial spot and wf is the corresponding width for the spot after chromatography. (7) Kaiser, R. E. In HPTLC High Performance Thin-Layer Chromatography; Zlatkis, A., Kaiser, R. E., Eds.; J. Chromatogr. Library, Vol. 9; Elsevier Scientific Publishing Co.: Amsterdam, 1977.
The calculation of a “real” value of n in the section on the relationship between efficiency and migration distance was performed using the value of w1/2 given by the above equation. This calculation is the same as used by Kaiser, but with different symbols. The height equivalent of a theoretical plate, HETP, is given by
HETP ) MD/n
(3)
RESULTS AND DISCUSSION Our initial report demonstrated that PPEC can provide both fast and efficient separations, but with inferior reproducibility of retention. The lack of reproducibility was at least partly due to drift in the hydraulic pressure unit, poor temperature control during a separation, inadequate temperature control in the oven during the conditioning of the plates, and poor control of the time for which the plate was soaked to pre-equilibrate the layer. Each of these variables influences the migration distance of an analyte. These issues were addressed by changing the hydraulic actuator used to apply thrust to the apparatus and by acquiring an air circulation oven with specified temperature control within (1 °C for conditioning the plates. As noted above, flow channels were incorporated within pressurizing blocks through which water was circulated to control temperature. The channels are in the shape of an inverted U, and it may be plausibly argued that there would be a temperature differential between the center and the edges of the faces of the blocks. If there is such an effect, it appears to be small. The migration velocity of analytes was independent of whether these were spotted near the long edge, or near the center, of the TLC plate. We attribute this effect to the relatively high thermal conductivity of the aluminum blocks in good thermal contact with the aluminum nitride ceramic, which also exhibits high thermal conductivity. It should, however, be noted that computer simulation using finite element analysis8 predicts that the temperature across the plate would not be entirely uniform. This is a topic for further investigation. Pre-equilibration of the Sorbent Layer. Dzido and coworkers4 have reported that migration distance is substantially increased when the TLC plate is soaked for 24 h before performing a separation by conventional PEC. Tate and Dorsey5,6 have used a different approach and, using an array of electrodes, have studied how the potential along the flow path changes during equilibration of a TLC plate that is initially dry. The reports from both of these groups demonstrate that equilibration of the sorbents used in their experiments is not instantaneous and can be slow. To determine an optimum pre-equilibration time for our system the plates were soaked in the mobile phase for different lengths of time. The fourcomponent analyte mixture was applied to the prewetted layer (see Experimental Section) and then separated by PPEC, under standard conditions, as defined above in the Experimental Section. PPEC was performed for 10 min on the regular plates and for 4.5 min on the high-performance plates. A plot of migration distance versus the pre-equilibration time is shown in Figure 4 for the highperformance plates. A plot of similar shape is obtained for the (8) Personal communication: Krishnan, S. S.; Osman, S.; Burns, B. Department of Mechanical Engineering, Indiana UniversitysPurdue University Indianapolis.
Figure 4. Migration distance versus soak time on high-performance plates conditioned at 160 °C for the following four compounds listed in order of increasing Rf: 17R-acetoxyprogesterone, 2′-acetonaphthone, benzanilide, and o-nitroaniline. PPEC was performed for 4.5 min using the standard conditions.
regular plates when the time axis is extended to 60 min. Equilibration is substantially faster for the high-performance plates, and the increase in retention when the soak time is increased from 5 to 10 s ranges from ∼10% for the lowest migrating solute to ∼30% for the highest migrating solute. This very fast equilibration is most probably related to the lower carbon load and more exposed silica of these plates as compared to the regular plates. For both types of plates, the migration distance diminishes when the soak time is extended to several hours. The solution in which the plate was soaked was not thermostated, and this could cause a small error. The regular plates do not exhibit dependence of spot shape on soak time. However, the best spot shape for the highperformance plates is obtained for soak times of 10 s or less. There is slight elongation along the axis of migration with a soak time of 1 min, and a substantial elongation when the soak time is extended to 10 min. Reproducibility. Both the regular and the high-performance plates used in this report were pre-equilibrated in the run buffer for 20 min before a separation in the following reproducibility study. The variation in soak time was within a few seconds, and any contribution to the overall error from this source is considered to be insignificant. The plates were conditioned at 120 °C, as the reproducibility studies were performed in the early phase of this project. Inconsistent results were obtained during the initial attempts with the current apparatus. A line of spots applied near the origin of the plate would result in an irregular and irreproducible “halfmoon” pattern in a PPEC run. This behavior was traced to mechanical imperfections in the open ball-and-socket joint. The joint was disassembled and reconstructed to the best tolerances available in our precision machine shop. The ball-and-socket were then lapped to a precise fit using a mild abrasive slurry. This procedure substantially improved the reproducibility of retention. The ball-and-socket joint is at the end of a movable piston rod in the hydraulic actuator. The rod is free to rotate in its cylinder with the expectation that it would self-align. It was found, even after machining and lapping, that the reproducibility of retention depended on the orientation of this rod from run to run. While Analytical Chemistry, Vol. 78, No. 8, April 15, 2006
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every attempt was made to use the same orientation for each run, an occasional outlier was observed. The technique is clearly very sensitive to even tiny misalignment in the apparatus. The orientation of the rod is adjusted before each run by rotation to its optimum position, which is identified by a painted witness mark. This procedure is necessary because there is slight rotation of the rod when the hydraulic pressure is released. This part of the apparatus is being redesigned in order to eliminate this behavior. It is necessary to clean the load-bearing contact surfaces in the current apparatus on a regular basis in order to obtain and sustain good reproducibility. Three sets of eight replicate separations of the standard mixture were performed on regular plates, and a single run of seven replicates was performed on high-performance plates. Standard conditions were used with an analysis time of 10 and 4 min, respectively, for the regular and the high-performance plates. Table 1 lists these results, together with a set of results obtained with our original apparatus.3 The replicates on the regular plates were performed, as a check, after interruptions to service the apparatus, such as when the pressure gauge was replaced. The second set of replicates for the regular plates contains an outlier, where the retentions of the four analytes were between 2.2 and 2.7 times the standard deviations from the mean. This outlier results in the somewhat higher relative standard deviations for this set of replicates. The relative standard deviations reported in this table are substantially smaller than those in our original publication. Effect of Pressure. Migration distance diminishes with increasing pressure when all other variables are kept constant. This is illustrated in Figure 5a for the regular plates and Figure 5b for the high-performance plates. Apart from the variation in pressure, PPEC was performed under standard conditions. The run time was 7.0 min for the regular plates and 4.5 min for the high-performance plates. Only the plot for the regular plates has a data point for the highest pressure used (196 atm) because the sorbent layer of the high-performance plates separates from the backing at this pressure. The diminution of migration distance with pressure is more substantial for the regular plates than for the high-performance plates. This is interpreted as due to a less uniform packing in the former plates. The diminution in migration distance with increasing pressure is most probably due to an increasing overlap of the electrical double layer as the channels between particles become smaller. This would lead to a lower velocity of electroosmotic flow. This effect is also discussed in the section on buffer concentration. The decrease in size of the channels also results in a diminution of electrical current and, consequently, in less Joule heating. This in turn allows a larger electric field to be applied to compensate for the decrease in migration velocity. Effect of Temperature. As would be expected, the temperature at which a separation is performed has a large influence on retention and the quality of separation. Figure 6 is a plot of retentions of four of the analytes on regular plates versus temperature. Analysis time was 10 min using the standard conditions. The slopes of the lines in the corresponding plot for the high-performance plates are somewhat smaller, indicating a lesser dependence of retention on temperature. 2828 Analytical Chemistry, Vol. 78, No. 8, April 15, 2006
Figure 5. (a) Migration distance versus pressure on regular plates conditioned at 160 °C for 20 min. PPEC was for 7 min. In other respects, the analyte mixture and operating conditions are as in Figure 4. (b) Migration distance versus pressure on high-performance plates conditioned at 160 °C for 20 min. PPEC was for 4.5 min. In other respects, the analyte mixture and operating conditions are as in Figure 4.
Figure 6. Migration distance versus separation temperature on regular plates conditioned at 120 °C for 20 min. PPEC was for 10 min. In other respects, the analyte mixture and operating conditions are as in Figure 4.
The four-component mixture was separated for 4.5 min under standard conditions on high-performance plates at 4, 21, and 42 °C, and the highest efficiency was at 21 °C for the three fastest migrating analytes. There is a substantial increase in migration distance with increasing temperature, and this could influence efficiency. To eliminate this effect, the experiment was repeated
Figure 7. Number of theoretical plates for o-nitroaniline versus separation temperature on high-performance plates conditioned at 160 °C for 20 min. PPEC was performed under the standard conditions. Separation time was adjusted from 8 min at 3.0 °C to 3.0 min at 56.3 °C, such that the migration distance at each temperature was close to 49 mm (see text).
Figure 8. Thermocouple readout temperature versus time on regular plates conditioned at 120 °C for 20 min. PPEC was performed under standard conditions for 10 min. The upper plot (9) is for a separation without temperature control and the lower plot ([) is for a separation with temperature control.
over a larger temperature range with the analysis time at each temperature adjusted to give nearly the same migration distance. The analyte used was o-nitroaniline and the experimental migration distances ranged from 46.6 to 51.6 mm in a random distribution. The error introduced by the range of migration distances is small compared to the effect investigated. The plot in Figure 7 shows that, under the experimental conditions described above, there is an increase in efficiency with an increase in temperature from 3 °C to an estimated 26 °C followed by a substantial loss of efficiency with further increases in temperature. A possible interpretation is that at lower temperature band broadening is primarily due to resistance to mass transfer and at higher temperature is primarily due to diffusion, whereas at intermediate temperatures neither effect dominates. During a run, the temperature initially rises and then falls to an intermediate value. This is illustrated in Figure 8 for a separation on regular plates under standard conditions. The figure shows the temperature profiles of a run with and without cooling. After an initial rise of ∼2 °C, the block remains within a range of ∼0.7 °C with cooling and consistently rises in temperature when there is no cooling. The temperature profile is reasonably
Figure 9. Migration distance versus buffer concentration on regular plates conditioned at 120 °C for 20 min. PPEC was performed at 2 kV for 20 min. In other respects, the same conditions and analyte mixture was used as in Figure 4.
reproducible for a given set of conditions, but for obvious reasons, it depends on variables such as the buffer concentration and the applied voltage. Much of the drift would be eliminated with a closed-loop system using feedback that adjusted either the temperature or the flow of coolant through the cooling passages to achieve a more constant temperature of the sorbent layer. There is a surprisingly linear relationship between the current at the start of a separation and the initial temperature at which the run is performed. The correlation coefficient is 0.99 for separations performed at six different temperatures in the range 3-57 °C using standard operating conditions. This is a limited temperature range, and the correlation may not hold over a larger range. There is a steady decrease in current throughout a run with or without coolant circulation. Effect of Buffer Concentration. In this study the acetate buffer concentration was varied from 5.0 to 100 mM. Each run was performed for 20 min at 2 kV on regular plates and using the same pressure and mobile phase as under standard conditions. There is a linear increase in migration distance with increasing buffer concentration, as shown in Figure 9. The average temperature during a run increases from 24.7 °C for the 5 mM separation to 26.6 °C for the 100 mM separation. This increase in temperature is a minor contributor to migration distance as can be seen by inspection of the plot in Figure 6. Interpolation of the latter plot indicates that at a buffer concentration of 5 mM the migration distance for o-nitroaniline, the fastest migrating solute, would increase by ∼2 mm for a temperature increment of 2 °C, whereas there is an increase in migration distance of 12 mm over the same temperature range when the buffer concencentration is increased from 5 to 100 mM. This increase in migration distance is interpreted as being due to a diminishing overlap of the electrical double layer. The dependence of electroosmotic flow on double layer overlap in column electrochromatography was first suggested by Wan9,10 and has been used to interpret results in PEC at atmospheric pressure.11 Relationship between Efficiency and Migration Distance. In contrast to all other forms of planar chromatography, the (9) Wan, Q.-H. J. Chromatogr., A 1997, 782, 181-189. (10) Wan, Q.-H. Anal. Chem. 1997, 69, 361-363. (11) Nurok, D.; Koers, J. M.; Carmichael, M. A. J. Chromatogr. A 2003, 983, 247-253.
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Figure 10. Number of theoretical plates for o-nitroaniline versus migration distance on high-performance plates conditioned at 160 °C for 20 min. PPEC was under standard conditions for between 1.0 and 7.5 min, depending on distance migrated. The upper plot (9) is for the high-performance plates and the lower plot (() is for the regular plates.
Figure 12. Separation of a nine-component mixture in 2 min. PPEC was performed at 9 kV and 63 atm using the standard mobile phase on a high-performance layer. Analytes listed in order of increasing Rf: 4-cholesten-3-one, 4-androsten-17β-ol-3-one acetate, 17R-acetoxyprogesterone, androstenedione, 4-pregen-11R-ol-3,20-dione, benzanilide, o- nitroaniline, hydrocortisone alcohol, and benzamide. 4-Cholesten-3-one does not migrate and is used to mark the starting position.
Figure 11. Height equivalent of a theoretical plate (HETP) for o-nitroaniline versus migration distance. The same raw data were used as for Figure 10. The upper plot (() is for the regular plates and the lower plot (9) is for the high-performance plates.
efficiency of PPEC, as measured by the number of theoretical plates, increases with increasing migration distance. To illustrate this, o-nitroaniline was allowed to migrate for various time periods using standard conditions. Analysis time ranged from 2.5 to 17.5 min for the regular plates and from 1.0 to 7.5 min for the highperformance plates. Migration velocity is substantially higher on the high-performance plates. During the longest run for each type of layer, the o-nitroaniline migrated 56.3 mm on the regular layer and 69.3 mm on the high-performance layer. The results of the study are presented in two ways. Figure 10 shows an increase in the number of theoretical plates for both the high-performance and the regular layer with increasing migration distance. At higher migration distances, this increase in nearly linear for the highperformance plates and this strongly suggests that still higher efficiency will be obtained with a longer migration path than is possible with the current apparatus. Figure 11 shows that the HETP initially diminishes with increasing migration distance and then reaches a plateau value. The HETP value at 69.3 mm corresponds to 94 000 theoretical plates/m for the high-performance plates, and there have been a few separations exhibiting efficiencies of between 100 000 and 112 000 theoretical plates/m. 2830 Analytical Chemistry, Vol. 78, No. 8, April 15, 2006
Two such examples are for the fifth peak in Figure 7a of ref 3 and for the seventh peak of Figure 12. In both cases, the peak is for o-nitroaniline. While this may be a coincidence, it is always the faster migrating solutes that exhibit the high efficiencies. It is not possible to routinely obtain efficiencies of over 100 000 theoretical plates with the current apparatus. The initial spot width makes a substantial contribution to the width of the final spot, with the result that the apparent efficiency of the layer is distorted, especially for solutes with a short migration distance. As an illustration, the initial peak width at halfheight represents 38.5 and 24.8% of the final peak width at halfheight for the o-nitroaniline peak at 7.6 and 69.3 mm, respectively. A “real” efficiency can be calculated according to the method of Kaiser7 if it is assumed that the initial spot has zero width and that the amount of band broadening is independent of the initial width. This calculation is further explained in the section on Calculations Used. For the high-performance plates, the data used in Figures 10 and 11 yield “real” efficiencies of 433 theoretical plates at 7.6 mm and 11 516 theoretical plates at 69.3 mm, as compared to values of 163 and 6520 theoretical plates, respectively, where the initial spot width at half-height has an average experimental value of 0.52 mm. While it is impossible to have an initial spot of zero width, such a calculation illustrates the importance of a very small initial spot. One approach to obtaining such a spot would be to use the Fenimore contact spotter. Fenimore12 reported that this device yields spots with diameters (12) Fenimore, D. C.; Meyer, C. J. J. Chromatogr. 1979, 186, 555-564.
(measured with an optical micrometer scale) between 0.1 and 0.3 mm, depending on the original concentration of the analyte. The width at half-height would be somewhat smaller than these values. This simple device is no longer sold, but other devices that are commercially available may possibly result in spot sizes smaller than those reported here. Calculation of efficiency, using an initial spot width at halfheight of 0.2 mm, predicts 285 and 8993 theoretical plates for migration distances of 7.6 and 69.3 mm, respectively. This would represent an increase in the number of theoretical plates of about 75 and 38%, respectively, above the corresponding experimental values. Thermal-Transfer Effects. A sheet of aluminum nitride ceramic is positioned between the sheet of Teflon covering the sorbent layer and the face of the movable die block that applies pressure to the sorbent layer in all of these studies. A sheet of this material is also positioned between the glass support of the TLC plate and the fixed die block. This ceramic was selected because it is an electrical insulator with good thermal conduction properties. While Teflon is also a good electrical insulator, it has poor thermal conduction properties. The effect of Teflon’s poor thermal conductivity can be minimized by working with a very thin sheet of the material, such as that used for covering the sorbent layer. To illustrate this, a single run was performed without the aluminum nitride ceramic sheet between the 0.01-in.-thick Teflon cover sheet and the die block. The resulting separation exhibited small differences in retention, as compared to a separation with the nitride sheet in place, but the overall quality of separation was very similar. All other separations in this report were with the nitride sheet in place. There is a significant drawback to working without the nitride sheet, because a pinhole leak in the Teflon would cause the apparatus to short circuit catastrophically. It would, however, be practical to work without the nitride sheet, if a thin sheet of insulating material of sufficient mechanical strength covered the sorbent layer. The low thermal conductivity of the material could be compensated by an appropriate adjustment in the temperature of the water circulating through the die blocks. Back-to-Back Plates. The sample throughput of the apparatus can be doubled by using two plates that are back-to-back. Such a separation was performed using a separate electrode for each TLC plate, and the separations on each plate were similar. The temperature was controlled only on the sorbent face, as the two glass backing plates were in contact. This could be rectified by placing a block with channels for a circulating liquid between the plates. This configuration, together with the ability of PPEC to separate a two-dimensional array of samples,3 should provide a substantial advantage for high-throughput separations as the method is refined in the future.
Some Conclusions. PPEC is a fast and efficient form of planar chromatography. An example of this is illustrated in Figure 12, which shows a 2-min separation of a nine-component mixture. The current study was performed with sorbent layers designed for conventional TLC, and it is reasonable to assume that layers designed specifically for PPEC would yield improved performance. Such layers would consist of smaller particles than those of the high-performance plates used in the current study. The latter plates consist of smaller particles (∼7 µm3) than the regular plates, which have a particle size of ∼11 µm, yet yield substantially higher electroosmotic flow. Sorbent layers for PPEC might have particle sizes in the order of 3 µm, a size that does not present problems in HPLC. To utilize the benefits of such plates, the initial spot size needs to be substantially smaller than that used in the current study. The efficiency of the system, as measured by the number of theoretical plates, increases with increasing migration distance. Higher efficiencies than those in this report should be attainable with an apparatus that allows a migration path longer than that of the current apparatus. Solutes exhibit a higher migration velocity at elevated temperature, but also exhibit poorer efficiency due to greater band broadening, which is ascribed to increased diffusion. This could be overcome by working at a larger electric field, which would result in a higher mobile-phase velocity. The current apparatus is limited to a maximum applied voltage of ∼10 kV. Above this value, high-voltage breakdown leading to a short circuit occurs. Another problem is that this apparatus is extremely sensitive to mechanical alignment, and this sometimes results in poor separations. A more robust instrument is being designed. ACKNOWLEDGMENT The research was supported by a grant from the Eli Lilly and Company Foundation. The following are also thanked: Merck KGaA for the gift of the high-performance plates and EMD Chemicals, Inc. for the gift of the regular plates; Emil Mincsovics for the gift of the silicone rubber that was used for sealing the edges of the TLC plates; Stellar Industries Corp. for the gift of the aluminum nitride ceramic; Steve White of CAD & Graphics Design for preparing Figure 3; Jim Zimmerman for designing the electrical connections to the apparatus; Sivakumar Krishnan, Shahid Osman, and Benjamin Burns for performing the finite element analysis; Jennifer Hovis for providing laboratory space while the apparatus was tested at Purdue University in West Lafayette, and Don Risley for the gift of solutes. Received for review December 21, 2005. Accepted February 16, 2006. AC052262V
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