Anal. Chem. 2006, 78, 4713-4721
Apparatus for Pressurized Planar Electrochromatography in a Completely Closed System Tadeusz H. Dzido,* Paweł W. Płocharz, and Piotr SÄ la¸ zak
Department of Physical Chemistry, Chair of Chemistry, Medical University, Staszica 6, 20-081 Lublin, Poland
Pressurized planar electrochromatography (PPEC) is the mode which offers much higher separation efficiency in comparison to conventional planar chromatography, including both higher performance and much higher speed of separation. In this paper, we present a new device for performing PPEC in which the whole area of the chromatographic plate is pressurized. Both electrodes (anode and cathode) are washed with the mobile phase during the experiment, which prevents gas bubbles from collection in the region of the electrodes. This device enables directly controlling the flow rate of the mobile phase during the electrochromatography process. Mobile phase control offers the possibility of researching the influence of various properties of the PPEC system on separation efficiency. One important relationship to investigate is plate height vs mobile phase flow rate. This relationship helps to choose the optimal value of the mobile phase flow rate during the separation process. Considerable difference in shape of this relationship is demonstrated for conventional planar chromatography plates and high performance planar chromatography plates. Examples of the influence of some properties of the separating system on flow rate of the mobile phase are demonstrated, such as the buffer concentration in the mobile phase, the pH value of the buffer solution of the mobile phase, the type of chromatographic plate, and the voltage applied to the electrodes. Planar electrochromatography (PEC) is the mode in which the mobile phase is driven into movement relative to the adsorbent layer (stationary phase) by electroosmotic effect, not by capillary action, as it is in conventional thin-layer chromatography (planar chromatography, TLC). Its history is relatively long; however, its development is still in the beginning stages. First suggestions about the possibility of electroosmotic effect application for planar chromatography were expressed about 60 years ago by Martin1 and Synge.2 More detailed information was published in 1974 by Pretorius et al;3 however, the experimental details in this paper were not completely presented. There were problems with repeating the experiments by others, and this was probably the (1) Martin, A. J. P.; Consden, R.; Gordon, A. H. J. Biochem. 1946, 40, 33-38. (2) Synge, R. L. M.; Mould, D. L. Analyst 1952, 77, 964-969. (3) Pretorius, V.; Hopkins, B. J.; Scheieke, J. D. J. Chromatogr. 1974, 99, 2330. 10.1021/ac060044b CCC: $33.50 Published on Web 05/20/2006
© 2006 American Chemical Society
reason the next publications on this mode didn’t appear until 1994.4 It must be mentioned that during this time, capillary electrochromatography with high separation efficiency was successfully developed, and it still fascinates many researchers. Poole and Wilson5 published a paper in 1997 in which they concluded, on the basis of only theoretical considerations, that it was the right time to implement it in laboratory practice. In the first half of the past decade, planar electrochromatography was performed in open systems with the chromatographic plate initially dry4,6,7 (an open system means that the stationary phase of the chromatographic plate is not directly covered by the lid of the chamber, and as a result, some volume of the vapor phase is present above the adsorbent layer). The results obtained show selectivity changes and some shortening of the development time relative to conventional TLC separations. However, considerable changes of these effects, especially regarding an increase in separation efficiency and reduction in development time, were obtained when the chromatographic plate was prewetted before inserting it into the planar electrochromatography device. These results were obtained by Howard et al.8 and by Nurok et al.9-11 The latter authors were the first to apply open reversed-phase systems in planar electrochromatography. The separation performance measured by theoretical plate height was several times higher in the PEC system in comparison to TLC, and the development time was several times shorter. The experiments were performed in a special device for PEC in which the chromatographic plate was almost vertically9 and horizontally11,12 mounted. Planar electrochromatography performed in open systems has two main disadvantages, i.e., evaporation of the mobile phase from the chromatographic plate and its excessive flux to the surface of the adsorbent layer.11 Nurok et al.11 proposed an application of a (4) (5) (6) (7) (8) (9) (10) (11) (12)
Pukl, M.; Prosek, M.; Kaiser, R. M. Chomatographia 1994, 38, 83-87. Poole, C. F.; Wilson, I. D. J. Planar Chromatogr. 1997, 10, 332-335. Malinowska, I. J. Planar Chromatogr. 2000, 13, 307-313. Malinowska, I.;Ro´z˘ yło, J. K.; Karson, A. J. Planar Chromatogr. 2002, 15, 418-424. Howard, A. G.; Shafik, T.; Moffat, F.; Wilson, I. D. J. Chromatogr., A 1999, 844, 333-340. Nurok, D.; Frost, M. C.; Chenoweth, D. M. J. Chromatogr., A 2000, 903, 211-217. Nurok, D.; Frost, M. C.; Pritchard, C. L.; Chenoweth, D. M. J. Planar Chromatogr. 1998, 11, 244-246. Nurok, D.; Koers, J. M.; Carmichael, M. A. J. Chromatogr., A 2003, 983, 247-253. Nurok, D.; Koers, J. M.; Carmichael, M. A.; Liao, W.; Dzido, T. H. J. Planar Chromatogr. 2002, 15, 320-323.
Analytical Chemistry, Vol. 78, No. 13, July 1, 2006 4713
mobile phase with an appropriate concentration of buffer to diminish the disadvantages produced by these effects. Another solution was presented in a paper by our group. We have proposed restricted feeding of the chromatographic plate with the mobile phase solution from the reservoir.13 The plate number obtained in these systems was several times larger than that in conventional TLC. This increase in plate number was enhanced not only by the flat flow profile of the mobile phase but additionally by evaporation of the mobile phase from the chromatographic plate. This effect was discussed by Nurok et al.12 The main disadvantage of the planar electrochromatography mode performed in an open system is the low repeatability of migration distance of the separated bands, despite the application of procedure and technical modifications mentioned above. The considerable step in the development of this mode has been described in the next paper by Nurok et al., which is concerned with planar electrochromatography in a closed system under pressure.14 The chromatographic plate was covered with Teflon foil and a ceramic sheet, which were pressed to the adsorbent layer by a special metal block using a hydraulic press. The authors named the mode “pressurized planar electrochromatography” (PPEC). The results obtained by this mode are very promising because of higher reproducibility of retention and a much faster separation process in comparison to planar electrochroamtography in open systems and conventional TLC systems. The authors reported 1-min separation using PPEC mode in a reversed phase system in comparison to a 24-min separation using conventional TLC. Similar results showing an increase in separation efficiency in the PPEC system when a specially modified horizontal DS chamber for TLC is used were reported by our group.15 The mobile phase used in the experiments was previously equilibrated with the stationary phase in a special chamber. The sample application onto the chromatographic plate followed the prewetting procedure of the plate. Application of the equilibrated solution to feed the chromatographic plate during the electrochromatography process was responsible for the repeatability increase of the retention data, in comparison to electrochromatography data obtained in open and closed systems. Another device for planar electrochromatography in a closed system was reported by Tate and Dorsey.16,17 The authors applied a special cover grid made of Kel-F pressed with a brass weight16 and with a hydraulic press17 to the TLC plate. These authors investigated voltage characteristics along the chromatographic plate during the planar electrochromatography process, and on the basis of the data obtained, they reached som conclusions about flow stability and equilibration in the systems investigated. Especially promising results were obtained when the chromatographic plate was pressed with a hydraulic press (69 bar). Then the plate equilibration proceeded much faster in comparison to the previous experiments, in which the chromatographic plate was under 1 bar pressure. These authors obtained a separation (13) Dzido, T. H.; Majewski, R.; Polak, B.; Gołkiewicz W.; Soczewin´ski, E. J. Planar Chromatogr. 2003, 16, 176-182. (14) 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. (15) Dzido, T. H.; Mro´z, J.; Jo´z´wiak, G. W. J. Planar Chromatogr. 2004, 17, 404-410. (16) Tate, P. A.; Dorsey, J. G. J. Chromatogr., A 2005, 1079, 317-327. (17) Tate, P. A.; Dorsey, J. G. J. Chromatogr., A 2006, 1103, 150-157.
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performance equal to ∼8000 theoretical plates for an 8.5-cm migration distance of the test solute. Such a high value of the plate number has not been reported for PPEC system until now. In two papers14,15 dealing with pressurized PEC systems, the authors dealt with some small part of the chromatographic plate extending out of the pressurized area of the chromatographic plate. This feature can lead to some changes in the flow rate of the mobile phase during the separation process, which can influence lowering of repeatability of retention data. Flow rate of the mobile phase is a very important parameter used for optimization of separation in chromatographic systems. An appropriate flow rate yields an optimal value of plate height of the chromatographic system. However, it is well-known that flow velocity can vary during one run, especially in an open system of PEC, which additionally complicates the experiments and leads to poor reproducibility of the migration distance. Measurement of the flow velocity of the mobile phase in planar electrochromatography systems has not been accomplished until now. The influence of flow rate of the mobile phase on the retention of solutes was expressed as plots of migration distance of the solute vs time of electrochromatogram development and vs voltage in open and pressurized systems.14,18 The data indicate that the flow rate of the mobile phase is considerably more stable when using a device for pressurized planar electrochromatography than in a device operated in an open system. The investigation of the voltage profile along the chromatographic plate and migration distance of the test solute (rhodamine B) vs the time of electrochromatogram development performed by Tate and Dorsey17 gave additional evidence that the flow of the mobile phase is stable if the planar electrochromatography system is pressurized. In the present paper, we describe the new device for planar electrochromatography in a closed system in which the whole area of the chromatographic plate is pressurized, and direct measurement of the flow rate is controlled. EXPERIMENTAL SECTION Materials Used. Mobile-phase solutions were prepared by mixing acetonitrile (ACN) for gradient liquid chromatography (POCh, Glwice, Poland) with buffer solutions of an appropriate pH value (8:2). The buffer solution was prepared by mixing a solution of citric acid, 0.1 M (analytical grade, Merck, Darmstadt, Germany), and a solution of disodium hydrogen phosphate, 0.2 M (analytical grade, POCh, Glwice, Poland). The range of pH values applied in the experiments was from 2.2 to 6.0. The appropriate buffer concentration indicated in the text or captions of the figures was prepared by dilution with bidistilled water. The mixture of test dyes was composed of 1-[[2-methyl-4-[(2-methylphenyl)azo]phenyl]azo]-2-naphthalenol (Sudan IV), 1-(2-pyridylazo)2-naphthol, 4-nitroaniline (supplied by POCH, Gliwice, Poland), and 1-(4-chlorophenylazo)-azobenzene, 4-(diethylamino)-azobenzene, and 1-(4-hydroxyphenylazo)-2-naphthol (synthesized in Department of Inorganic and Analytical Chemistry, Medical University, Lublin, Poland). The concentration of mixture components in the methanol/acetone (8:2) solution ranged from 0.01 to 0.1%. Silcolease PC-263 PEX (A), Silcolease PC-615 (B), and platinum catalyst (C) were from Rhodia Silicones, France. The chromato(18) Nurok, D.; Koers, J. M.; Nyman, D. A.; Liao, W. J. Planar Chromatogr. 2001, 14, 409-414.
Table 1. The Plates Applied and Their Characteristics Based on Refs 19, 20a plate name
catalog no.
plate dimension cm
mean particle size µm
particle size distribution µm
layer thickness mm
coverage density of C18 ligands µmol/m2
TLC RP18 F254s HPTLC RP18 F254s LiChrospher RP18W F254s
115423.0001 113724.0001 105646.0001
10 × 20 10 × 10 10 × 20
10-12 5-6 6-8
5-20 4-8 3-5
0.25 0.2 0.2
2.6 2.6 0.5
a
Poly(acrylic acid) in an amount of 2% was applied as a binder of the adsorbent layer in all plates.20
graphic plates were supplied from Merck (Darmstadt, Germany). The applied plates characteristic is presented in Table 1. Conventional Planar Chromatography. Conventional development of planar chromatograms was performed with plates which were cut into 2- × 10-cm pieces. A horizontal developing DS chamber (type DS-II-5x 10) from Chromdes (Lublin, Poland) was used for chromatogram development. The sample application procedure and chromatogram development was previously described.13,15 Preparation of the Chromatographic Plate to PPEC. The plates were washed with methanol by dipping in the solvent for 1 min. After that, the plate was dried in air and, next, in an oven at 105 °C for 10 min and left in a desiccator for cooling. Margins of 3 mm width were formed on the whole peripheral area of the chromatographic plate using Silicon sealant solution. The sealant solution was prepared just before application by mixing of components A + B + C listed above in a 100:10:3 proportion (by weight). The solution of the sealant was laid (one time) on the adsorbent layer using a small paint-brush. Then the plate was inserted into the oven at 60-70 °C for 10 min to polymerize the sealant. After that, the plates were left in a desiccator and were used for experiments within 1 day. The next stage of the chromatographic plate preparation was the prewetting procedure of the stationary phase, which was performed by dipping the plate in the mobile phase solution for 1 min. Immediately after the prewetting procedure, the stationary phase on the chromatographic plate was covered with a glass plate (of the same length and width as the chromatographic plate) and held in place by laboratory clips. The covering glass plate was equipped with a 3-mm-diameter hole that was used for sample application. The sample application (0.2-µL volume with handoperated 5 µL microsyringe) was described in a previous paper.15 The diameter of the sample spot was in the range of 1.0-1.5 mm and was positioned 20 mm from the edge of the plate. Immediately after sample application, the plate was inserted into the device for planar electrochromatography. Device for Pressurized Planar Electrochromatography. A conceptual view of the device for planar electrochromatography is presented in Figure 1. It is composed of the chamber for PPEC with the chromatographic plate; mobile-phase reservoirs; calibrated micropipet (100 µL); and high-voltage DC power supply, 10 kV, 120 W (Spellman, USA) with an ammeter. All elements are shown in the Figure as a side view with the exception of the chamber for PPEC with the chromatographic plate, which is presented as a top view (the area enclosed by the dotted line). All elements, with exception of the mobile phase reservoirs (9′) and power supply with an ammeter, are inserted in a separate cabinet (presented in the Figure as the dashed rectangle) made of Plexiglas. The separate Plexiglas cabinet disabled the power
Figure 1. Conceptual view of the device for planar electrochromatography: (1) chromatographic plate, (2) body of the chamber, (3 through 4) Tarflen block, (5) channel for the mobile phase, (6′) anode, (6′′) cathode, (7) Teflon tube, (8) 0.1-mL micropipet, (9′, 9′′) reservoirs, (10) waste, (11) valve, and (12) high-voltage DC power supply with an ammeter. Dashed rectangle represents Plexiglas cabinet. All elements are shown as a side view with the exception of the chamber for PPEC with the chromatographic plate, which is enclosed in the dotted rectangle.
supply if the cabinet lid was removed. An ammeter was included in the series with the supply to monitor current flow. The anode (the electrode on the left side of the Figure) was connected to the ground and cathode was connected to the DC power supply by a special cable supplied by Spellman. A photograph of the chamber is presented in Figure 2. Figure 2a and b demonstrate the chamber in open and closed configuration, respectively. In part a, the chromatographic plate in the chamber is shown with a separated component mixture. The total dimensions of the chamber in the closed configuration are 150 × 175 × 69 mm (width, length, thickness). A detailed view of the chamber for PPEC is demonstrated in Figure 3. Dashed lines A-A and B-B indicate the cutting planes of the chamber that are shown as the cross section in Figure 3b and the longitudinal section in Figure 3a, respectively. The cathode (6′′), electrode line out (23), the base (19) of the screws, the screws (20), and the nuts (21) are additionally shown in Figure 3b. The chromatographic plate (1) in the chamber is horizontally positioned with the adsorbent layer face-down. The chromatographic plate is pressed with the lid to the 0.2-mm-thick Tarflen (poly(tetrafluoroethylene)) foil (manufactured by Zaklady Azotowe, TarnowMoscice, Poland) (14) and 2-mm-thick VMQ silicon sheet (Larkis, Dopczyce, Poland) (13). The VMQ silicon sheet is placed on a 28-mm-thick Tarnoform (polyacetal) body (2) of the chamber. Tarnoform is manufactured by Zaklady Azotowe, TarnowAnalytical Chemistry, Vol. 78, No. 13, July 1, 2006
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Figure 2. The chamber for planar electrochromatgraphy: (a) with opened lid (2 × 10 cm chromatographic plate with separated sample mixture is placed inside the chamber) and (b) with closed lid.
Moscice, Poland. The Tarnoform body (2) of the chamber is situated on a 10-mm-thick plate (18) made of hardened tool steel NC11 (Polskie Huty Stali, Huta Katowice, Poland). The lid of the chamber is composed of the 10-mm-thick plate (16) of hardened tool steel NC11 and the Tarnoform body (15), which is 17 mm thick from the side adjacent to the chromatographic plate. The pressurizing force that acts on the chromatographic plate is regulated with two screws (20) and two nuts (22) using a dynamometric wrench (Belzer, Germany). Two Tarflen blocks (4) with troughs (3) and channels (5) for the mobile phase are situated on the left and right sides of the chamber beneath the chromatographic plate (Figure 3a). Two electrodes, an anode (6′) on the left side and a cathode (6′′) on the right side of the chamber presented in Figures 1 and 3a, are situated in horizontal parts of the channels in the Tarflen blocks (4). The Tarflen block on the left side of the chamber stands for the anode block, and on the right side, for the cathode block. The bottom of the Tarflen blocks is situated on a VMQ silicon spacer (13) of 2-mm thickness. The anode block is connected with Teflon tubing (7) to two mobilephase reservoirs (9′), and the cathode block, to one mobile phase reservoir (9′′) and a calibrated micropipet (8) of 100-µL volume (Figure 1). The level of the mobile phase solution can be changed by elevation of the mobile-phase reservoirs. Operation of the Device for Pressurized Planar Electrochromatography. After the prewetting and spotting procedures, the chromatographic plate was immediately inserted into the chamber, and the chamber was closed with its lid. The lid of the chamber was pressed to the chromatographic plate with two nuts and screws using a dynamometric wrench with a moment of force equal to 10 Nm, which is equal to ∼120 atm of pressure in the separating system. The channels and troughs of both Tarflen 4716 Analytical Chemistry, Vol. 78, No. 13, July 1, 2006
Figure 3. Scheme of the chamber for planar electrochromatography under pressure: (a) longitudinal section and (b) cross section. (1) Chromatographic plate, (2) body of the chamber, (3 through 4) Tarflen block, (5) channel for the mobile phase, (6′′) cathode, (7) Teflon tube, (13) silicon sheet, (14) Tarflen foil, (15) polyacetal base of the lid, (16) steel plate of the lid, (17) frame for chromatographic plate, (18) steel base plate, (19) base of the screw set, (20) screw, (21) nut, (22) glass plate, (23) electrode line out to power supply DC, and (24) mount of the lid.
blocks were filled with mobile-phase solution from the syringe, by adjusting the level of the mobile phase reservoirs, or both. The solution of the mobile phase in the anode block was allowed to flow during planar electrochromatography experiments by holding different levels of the mobile-phase solutions in both reservoirs. This flow was to prevent a collection of gas bubbles in the vicinity of the electrode during the electrochromatography experiments and to transport them (bubbles) to the mobile phase reservoir (not to the chromatographic plate). The arrows in Figure 1 show the direction of flow of the mobile phase in the channels of the anode block. Then the voltage of an appropriate value was switched on to create an electric field and generate an electroosmotic flow of the mobile phase in the adsorbent layer of the chromatographic plate. The arrow marked as EOF in Figure 1 indicates the direction of the electroosmotic flow within the chromatographic plate. The mobile phase from the cathode block was directed during the electrochromatography process to the calibrated micropipet (the tube connecting the mobile phase reservoir (9′′) with the cathode block was blocked during the electrochromatography process). This enabled us to perform the measurement of the flow rate of the mobile phase, which passed through the chromatographic plate during the electrochromatog-
Figure 4. Plots of the mobile phase volume passed through the chromatographic plate vs time of PPEC experiment for various voltages: [ 0.75, 0 1, 9 1.5, O 2.5, b 3.5, and * 4.5 kV. TLC RP18 F254s plate (Merck, Darmstadt), 80% acetonitrile in buffer (3.74 mM citric acid, 12.52 mM disodium hydrogen phosphate, pH ) 6).
raphy process. This measurement was realized by control of the distance migration of the meniscus of the mobile phase (or air bubble injected) in the calibrated micropipet. After a desired time of separation process necessary for performing the experiments, the voltage was switched off, and the chromatographic plate was taken out of the chamber and dried in air. In all experiments, the chamber for PPEC was inserted in the Plexiglas cabinet to prevent the operator from coming in contact with high voltage during the experiments. Recording of chromatograms was performed with a TLC 2010 diode array scanner (J&M, Aalen, Germany). All experiments were performed in triplicate, apart from the experiments applied for determination of repeatability of the migration distance. Then the number of experiments was equal to seven. RESULTS AND DISCUSSION In Figure 4, the relationships between the volume of the mobile phase passed through the chromatographic plate vs time are presented for various values of voltage applied to the electrodes. Very good correlation between the volume and time is observed in this Figure. The correlation coefficient, r, is 0.9980 or higher. The slope of these plots represents the flow rate of the mobile phase in the planar electrochromatography system; however, the values of flow rate plotted vs voltage applied to the electrodes do not show linear relationships in the whole voltage range investigated (Figure 5). For higher values of the voltage (3.5-7.0 kV), this relationship is distinctly steeper. The effect is probably caused by the temperature increase (Joule heating) in the system. Therefore, it can be concluded that no or a minor effect of temperature influence on the flow rate of the mobile phase is present for a lower voltage range (the slope of the relationship is constant). These observations are confirmed by a current profile during the experiments (Figure 6). The current is practically constant if the voltage applied for polarization of the chromatographic plate is in the range of 0.75-1.5 kV. If a higher voltage is applied, then a higher drop of current is observed. Two main effects are responsible for such relationships. One is concerned with lowering of the viscosity of the mobile phase, and the second one, with a change of the ζ potential of the mobile phasestationary phase interface if the temperature increases. These
Figure 5. Plot of mobile phase flow rate vs polarization voltage. TLC RP18 F254s plate (Merck, Darmstadt). 80% acetonitrile in buffer (3.74 mM citric acid, 12.52 mM disodium hydrogen phosphate; pH ) 6).
Figure 6. Plots of current vs time of experiment. TLC RP18 F254s plate (Merck, Darmstadt), 80% acetonitrile in buffer (3.74 mM citric acid, 12.52 mM disodium hydrogen phosphate, pH ) 6), applied voltage: * 0.75, 9 1, 0 1.5, b 2.5, O 3.5, × 4.5, and + 5.5 kV.
observations are in accordance with theoretical relationships, which can be predicted from the equations presented below. The basic Smoluchowski equation for electroosmotic flow is expressed as follows,
ueo )
0rζE η
(1)
where 0 is the permittivity of a vacuum, r is the dielectric constant, ζ is an electrokinetic (ζ) potential, E is the electric field strength, and η is the viscosity of the mobile phase. It means that the electroosmotic flow is directly proportional to the ζ potential of the mobile phase-stationary phase interface and inversely proportional to the viscosity of the mobile phase. The viscosity of the solution is exponentially decreased with temperature according to the relationship21
η ∝ exp (constant/RT)
(2)
where R is the universal gas constant and T is the absolute (19) http://chrombook.merck.de/chrombook/index.jsp?j)1. (20) Schultz, M. Merck, Darmstadt, Germany, personal communication. (21) Atkins, P. W.; de Paula, J. Elements of Physical Chemistry, 4th ed., Oxford University Press: Oxford, 2006, p 272.
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Figure 7. Volume of the mobile phase passed through the RP18 F254s TLC plate vs time of experiment for various buffer concentrations: 9 no buffer; 0 1.34 mM citric acid, 2.32 mM disodium hydrogen phosphate; b 5.37 mM citric acid, 9.26 mM disodium hydrogen phosphate; O 10.74 mM citric acid, 18.52 mM disodium hydrogen phosphate. pH of all buffer solutions is 4.6, 80% acetonitrile.
temperature. The ζ potential can be related to the thickness, δ, of the electrical double layer and superficial charge density, σ, according to the equation
ζ)
σδ 0r
(3)
The thickness of the electrical double layer can be expressed by the equation
δ)
x
0rRT 2cF2
(4)
where c is the molar concentration of the buffer salt and F is the Faraday constant.22,23 Therefore, both terms, ζ potential and viscosity, lead to an increase in the electroosmotic flow if the temperature rises. However, as was reported in previous investigations, the impact of the ζ potential with a temperature increase to the electroosmotic flow is much greater than that of a viscosity decrease with a temperature increase in capillary electrochromatography systems.24,25 The flow rate of the mobile phase can also be regulated by buffer concentration in the mobile phase. Figure 7 demonstrates the influence of the buffer concentration on the volume of the mobile phase passed through the separating system. A higher buffer concentration leads to an increase in the mobile phase flow rate (slope of the relationship). This effect is consistent with previous data presented for capillary electrochromatography,26-29 (22) Knox, J. H.; Grant, I. H. Chromatographia 1991, 32, 317-328. (23) Hjerten, S. Chromatogr. Rev. 1967, 9, 122-219. (24) Wahlhagen, K.; Unger, K. K.; Hearn, M, T. W. J. Chromatogr., A 2000, 893, 401-409. (25) Cahours, X.; Morin, P.; Dreux, M. J. Chromatogr., A 1999, 845, 203216. (26) Szumski, M.; Buszewski, B. J. Chromatogr., A 2004, 1032, 141-148. (27) Wie, W.; Luo, G. A.; Hua, G. Y.; Yan, C. J. Chromatogr., A 1998, 817, 6574. (28) Wan, Q. H. J. Chromatogr., A 1997, 782, 181-189.
4718 Analytical Chemistry, Vol. 78, No. 13, July 1, 2006
Figure 8. The influence of pH value of buffer solution in the mobile phase on flow rate of the mobile phase. TLC RP18 F254s plate (Merck, Darmstadt). 80% acetonitrile in buffer; 2.5 kV.
planar electrochromatography in opened systems,18 and planar electrochromatography in closed systems.30 However, interpretation of the effect is more complicated due to the influence of the buffer concentration on the ζ potential and double layer thickness, which can overlap in the particulate stationary phase of the electrochromatography system. Different values of the flow rate are characteristic of various chromatographic plates used in our experiments. A higher flow rate (28.5 µL/min) is demonstrated by the system with LiChrospher RP18 W plates, and a lower (19.5 µL/min), with RP18 HPTLC plates (80% ACN; buffer concentration, 3.74 mM citric acid and 12.52 mM disodium hydrogen phosphate, pH 6; voltage, 2.5 kV). This effect correlates with coverage density of C18 ligands bonded to the silica surface (compare data in Table 1). A higher coverage density of the stationary phase (RP18 HPTLC) leads to a lower electroosmotic flow in comparison with the system with a stationary phase of lower coverage density (LiChrospher RP 18W). Similar relationships are known for the systems of capillary electrochromatography26 and were reported by Nurok et al. for pressurized systems of PEC.14 The other possibility for controlling electroosmotic flow is presented by use of buffer solutions having various pH values as components of the mobile phase. It is clearly seen that a higher pH value of the buffer solution leads to a higher flow rate of the mobile phase (Figure 8). The direction of this increase is wellknown from the data reported for the capillary electrochromatography.31 The explanation of the effect is concerned with the increase in dissociation of free silanols of the silica surface. This effect leads to a higher charge density of the electrical double layer in the stationary phase and mobile phase interface if the pH of the mobile phase increases. A characteristic feature of the planar electrochromatography system is higher performance in comparison to the conventional TLC reported in preceding papers.9-15,18 The performance of the chromatographic system is usually characterized by presentation of the plate number, N, or plate height, H. For column chromatography systems, it is convenient to characterize the performance (29) van den Bosch, S. E.; Heemstra, S.; Kraak, J. C.; Poppe, H. J. Chromatogr., A 1996, 755, 165-177. (30) Nurok, D. J. Chromatogr., A 2004, 1044, 83-96. (31) Keith, D.; Bartle, M.; Myers, P. J. Chromatogr., A 2001, 916, 3-23.
Figure 9. Plate height vs flow rate of the mobile phase for PPEC systems with (O) RP18 F254s TLC and (b) RP18 F254s HPTLC plates. Test solute: 1-(4-hydroxyphenylazo)-2-naphthol. The mobile phase is as in Figure 4.
by presentation of the plate number of a column because each band leaves the column, so its migration distance is the same for all components being eluted from the column. However, in planar chromatography systems, all sample components are placed on the chromatographic plate after the separation procedure, and the plate number is the characteristic value of each component band. It is the main reason the plate number is not commonly used to characterize the performance of the planar chromatography system. The relationship between plate height, H, and flow rate (volumetric or linear flow rate) cannot be determined in conventional planar chromatography (with the exception of overpressure planar chromatography, OPLC, systems) due to the the migration of the mobile phase front through the chromatographic plate at various velocities according to the equation
Zf ) (κt)1/2
Figure 10. Separation of test mixture, (a) conventional planar chromatography with RP18 F254s TLC plate, (b) electrochromatography (PPEC) with RP18 F254s TLC plate (Merck, Darmstadt); mobile phase as in Figure 4, voltage 2.5 kV. Test mixture: (1) 1-[[2-methyl4-[(2-methylphenyl)azo]phenyl]azo]-2-naphthalenol, (2) 1-(2-pyridylazo)-2-naphthol, (3) 1-(4-chlorophenylazo)-2-naphthol, (4) 4-(diethyloamino)-azobenzene, (5) 1-(4-hydroxyphenylazo)-2-naphthol.
(5)
where Zf, κ, and t are the migration distance of solvent front, the velocity constant, and the time of migration of the solvent front, respectively. H vs the development distance is applied to characterize planar chromatography systems.32 The optimal migration distance of the mobile phase is equal to ∼7 and 4 cm for conventional TLC and HPTLC plates, respectively. A larger migration distance leads to an increase in the plate height of the chromatographic systems. These restrictions are not more valid for PPEC systems because the flow rate does not depend on the migration distance of the mobile phase but, rather, on the value of electric field applied. Our device enables one to measure relationships between plate height and the flow rate of the mobile phase. In Figure 9, the examples of this relationship are demonstrated. The results are presented for TLC and HPTLC plates. It can be observed that plate height was much lower when PPEC was performed with HPTLC plates, especially in the range of a higher flow rate. The explanation of the effect is concerned with the diameter of stationary phase particles and its distribution. Mean particle diameter of the stationary phase is equal to 10-12 µm and 5-6 µm for TLC and HPTLC plates, respectively; however, the distribution (4-8 µm) of the particles in HPTLC plate is much (32) Tyihak, E.; Mincsovics, E. J. Planar Chromatogr. 2002, 11, 137-176.
more narrow than that in TLC one (5-20 µm).19 A characteristic feature of the plot for the HPTLC plate is its much lower steepness in comparison to a TLC plate. Similar relationships were obtained for OPLC systems.32 The results indicate the possibility of performing an electroosmotically driven separation using a higher flow rate with HPTLC plates with no considerable loss of separation performance. The examples of application of our device to a separation of the components of an artificial mixture in conventional TLC and PPEC systems are presented in Figure 10. As is shown, the separation selectivity is different in the two systems investigated (conventional TLC, Figure 10a; PPEC, Figure 10b), despite the same chromatographic plate (RP18 TLC) being applied in the experiments. Improvement of separation performance is demonstrated for PPEC in comparison to TLC. Migration distance of the bands in the PPEC system is more than double that in the TLC system for the same (5 min) development time. As was discussed above, the front of the mobile phase in a conventional TLC system migrates with decreasing velocity if the developing distance increases. It means that the separation time using a conventional TLC system will be considerably longer than that in a PPEC system for the same migration distances when applying the conditions presented in this figure. This is the evidence that the main advantage of planar electrochromatography performed Analytical Chemistry, Vol. 78, No. 13, July 1, 2006
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Table 2. Repeatability of Migration Distance of the Solutes, Zs, and Plate Height, H, in Conventional TLC and PPEC Systems with 80% Acetonitrile in Buffera PPEC (RP18 F254s TLC plate)
conventional TLC (RP18 F254s TLC plate) substance 1-(2-pyridylazo)2-naphthol 1-(4-chorophenylazo)2-naphthol 4-(diethyloamino)-azobenzene 1-(4-hydroxyphenylazo)2-naphthol a
no. of experiments
Zs mm
RSD
H µm
RSD
7
13.43 18.60
0.026 0.026
74.79 66.13
0.051 0.053
PPEC (RP18 F254s HPTLC plate) Zs mm
RSD
H µm
RSD
0.073
8.88
0.078
63.37
0.036
17.26
0.074
14.29
0.053
57.01
0.036
28.57 40.83
0.084 0.070
20.89 34.86
0.049 0.041
48.33 37.10
0.044 0.050
Zs mm
RSD
15.54
H µm
61.52 47.68
RSD
0.058 0.081
3.74 mM citric acid, 12.52 mM disodium hydrogen phosphate; pH ) 6. Development time 5 min, 2.5 kV (PPEC).
Figure 11. Electrochromatogram of test mixture, separation time 45 s, voltage 7.5 kV, RP18 F254s HPTLC plate (Merck, Darmstadt), 80% acetonitrile in buffer (3.74 mM citric acid, 12.52 mM disodium hydrogen phosphate; pH ) 6). Test mixture: (1) 1-[[2-methyl-4-[(2methylphenyl)azo]phenyl]-2-naphthalenol, (2) 1-(2-pyridylazo)-2naphthol, (3) 1-(4-chlorophenylazo)-2-naphthol, (4) 4-(diethyloamino)azobenzene, (5) 1-(4-hydroxyphenylazo)-2-naphthol.
in a pressurized system is a much shorter time of separation in comparison to conventional planar chromatography. As was mentioned above, a 1-min separation is possible with the pressurized planar electrochromatography mode.14 A similarly short (45 s) separation was obtained when using our device with a voltage of 7.5 kV (Figure 11). Comparable separation in conventional TLC takes ∼15 min. Inspection of Figures 10 and 11 and Table 2 suggests that performance of the PPEC systems is not as high as has been shown in previous papers.14,17 This effect may be due to the spotting mode and the pressure applied. Spotting of the sample on the prewetted plate may introduce some dispersion of the starting spot in comparison to the starting spot obtained on a dry layer. The pressure applied to the chromatographic plate was ∼120 atm, which was much higher than that for the systems of higher performance reported in previous papers. Higher pressure may lead to some changes in the structure of the adsorbent layer which is responsible for the performance of the PPEC system. On the other hand, our preliminary experiments demonstrated that higher pressure applied in the device leads to a more uniform migration distance of the solutes spotted side by side on the start line of the chromatographic plate. 4720
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In Table 2, the repeatability of migration distance of the test solutes is presented for conventional TLC and PPEC systems. The data indicate that lower, but comparable, repeatability was obtained for the PPEC system with RP18 HPTLC plates relative to conventional planar chromatography with RP18 TLC plates; however, the repeatability obtained in PPEC systems with RP18 HPTLC plates was somewhat higher than with RP18 TLC plates. It must be mentioned that repeatability of migration distances obtained in the experiments presented in our previous paper15 was somewhat better, despite application of the chamber with a small area of the chromatographic plate exposed to vapor phase. The experiments were performed with RP8 HPTLC plates that were under lower pressure (half of the applied pressure in this paper), lower polarization voltage (2 kV), and lower buffer concentration in the mobile phase in comparison to the system presented in this paper. Each of these variables can influence the repeatability of retention. However, the last two parameters can be especially responsible for lower Joule heating in the PPEC system, which can lead to higher repeatability of migration distances, in comparison to the data demonstrated in the present paper. It seems that more systematic investigation should be performed to explain the repeatability variations. Parameters such as electric field strength, mobile phase composition, buffer concentration, mode of sample application, equilibration of the electrochromatography system, and pressure exerted to the chromatographic plate should be considered to investigate this effect. CONCLUSIONS The device for planar electrochromatography presented in this paper enables researchers to perform a control of flow rate of the mobile phase passing through a separation system. This feature can be easily applied to investigation of a PPEC system concerned with optimization of separation conditions, especially including the properties which influence the flow velocity of the mobile phase. The results in this paper demonstrate that planar electrochromatography performed in a closed and pressurized system is a promising method for analytical separation. Its features, such as higher separation performance and shorter separation time, are very attractive for application in laboratory practice. However, the method is still in the development stage, and more effort should
be invested in optimization of elements of the separating system and constructional details of the device to make it more convenient for the operator. The problems that should be of interest in future investigations are concerned with sample application on the plate, production of the stationary phases dedicated to this method, temperature control of the PPEC system to avoid or minimize the problems concerned with Joule heating, and investigation of parameters influencing repeatability of the migration distance and bandwidth.
Note Added after ASAP Publication. The paper was posted on 5/20/06. A typographical error in eq 5 was noted and corrected. The paper was reposted on 5/25/06.
Received for review January 7, 2006. Accepted March 24, 2006. AC060044B
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