Peer Reviewed: Forced-Flow Techniques in Planar Chromatography

Force Techin

Planar chromatogra phy complements HPLC and, for certain separations, is the technique of choice.

David Nurok Indiana University—Purdue University Indi anapolis

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A N A LY T I C A L C H E M I S T R Y / O C T O B E R 1 , 2 0 0 0

eFlow

Pla Chromatogra Chromatog --

t has been more than 60 years since Nicolai Arkadevic Ismailov and Maria Schraiber of the Institute for Pharmaceutical Chemistry in Kharkov (Ukraine) first described thin layer chromatography (TLC) (1). This technique was performed by spreading finely ground aluminum oxide onto a glass plate, placing a sample in the center of the plate, and then introducing a solvent in a dropwise manner, resulting in a set of concentric solute zones. The technique is also called planar chromatography and we use the two names interchangeably in this article. Since that time, the technique has undergone a steady stream of innovations, one of which is overpressured layer chromatography (OPLC)—the major topic of this article. Yet, with techniques such as HPLC, is there any need for TLC, OPLC, and other forms of planar chromatography today? In fact, HPLC and planar chromatography should be considered complementary. HPLC is the technique of choice for many separations. It can be automated, handle a larger variety of detectors than planar chromatography because it generates a relatively high number of theoretical plates, and separate complex mixtures. On the other hand, conventional TLC and OPLC can separate multiple samples simultaneously on a single plate. Conventional TLC is an attractive alternative to HPLC for the quantitative analysis of multiple samples containing only 4–7 components; OPLC can handle 12–15 components. Both modes of planar chromatography work with a variety of postseparation visualizing reagents (2) and the mobile phase never interferes with detection because it is always removed first. Moreover, the separation bed is discarded

after each run, allowing samples that would contaminate the bed to be used without a cleanup step. Finally, soft sorbents, such as cellulose, which do not work with HPLC because of bed compaction, can be used in OPLC. There are also classes of compounds that are best determined by planar chromatography, such as toxic components in a mixture. Components are separated by planar chromatography, then the developed plate is dipped into a suspension of bioluminescent bacteria (3). The toxic compounds are present as dark spots on a luminescent background. For the above reasons, planar chromatography is widely used in industry and academia. It is popular in the pharmaceutical industry because substantial cleanup is not necessary, and, in contrast to HPLC, all components in a sample (even those that do not migrate) contribute to the resulting chromatogram. Planar chromatography’s ability to simultaneously separate multiple samples is exploited in screening for trace residues of compounds. Examples include veterinary drugs or anabolic steroids in meat, toxic contaminants in water, and drugs of abuse in biological fluids (3).

What is planar chromatography? TLC remains the best known of the planar chromatographic techniques. Silica gel is the most widely used stationary phase for TLC and is available as a conventional or as a high-performance (HPTLC) layer. The HPTLC layer is prepared from smaller particles with a narrower distribu-

O C T O B E R 1 , 2 0 0 0 / A N A LY T I C A L C H E M I S T R Y

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tion of sizes than the conventional The third approach is OPLC, (a) (b) layer. Typical HPTLC plates have which was first described by Ernö an average particle diameter of 4.5 Tyihák, Emil Mincsovics, and µm, with 80% of the particles in Huba Kalasz of the Research Instithe range 3.0–5.0 µm; the contute for Medicinal Plants in Budaventional layer average diameter is kalasz, Hungary (10). A TLC plate 10 µm, with 80% of the particles is covered by a sheet of flexible main the range 5.5–12.0 µm. The terial and subjected to a high exHPTLC layers have extremely ternal pressure. This allows the mo(c) (d) smooth surfaces and are well suitbile phase to be pumped through ed to quantitative analysis. Other the sorbent layer. The method is a commercially available sorbent hybrid between conventional TLC layers include aluminum oxide, and HPLC and incorporates sevcellulose, and various bonded eral features of each technique. phases. These choices allow anaApparatuses for OPLC are comlysts considerable flexibility in mercially available and in their designing a separation. third generation of design (11). Planar chromatography also FIGURE 1.Arrangements of TLC plates for - vari has its own list of sophisticated ous OPLC techniques. detection devices. The scanning densitometer is the most widely Arrows show direction of mobile-phase flow. The eluent-directing channel is the solid line. (a) Samples spotted for linear one-direcSome basic relationships used and allows a TLC separation tional OPLC. (b) Samples spotted for two-directional OPLC. (c) Plate A major disadvantage of classical to be presented as a series of peaks arrangement for long distance OPLC. (d) Samples spotted for cirTLC is the inverse relationship (4). In this arrangement, results cular OPLC. Mobile phase is introduced at the center of the plate. (Adapted with permission from Ref. 17.) between the velocity of solvent with a relative standard deviation migration and the distance miof 0.6%–1.5% in both precision grated by the solvent front, and accuracy are attainable (5). Densitometry is usually performed in either the UV–vis or k u= (1) fluorescent modes. Imaging detectors are also becoming 2Z f more widely used because of the speed of data acquisition in which u is the velocity of the front, k is the solvent ve(6). Other detection techniques include IR, Raman and locity constant, and Zf is the distance migrated by the front. photoacoustic spectroscopies, MS, flame ionization, and The value of k is given by radioactivity sensing (5, 7, 8). Although most TLC separations are still performed using (2) capillary-mediated flow, methods based on forcing the mo- k=2K0dp(g/h)cosu bile phase through the TLC layer are also available. One in which K0 is the permeability constant for the layer; dp is of the earliest is rotational planar chromatography (RPC), in which the TLC plate is rotated at a high angular velocity the average particle diameter; g and h are the surface tento generate a centrifugal force that moves the mobile phase sion and viscosity, respectively, of the mobile phase; and u is the contact angle. To obtain a high k, the ratio of g to through the layer in a circular profile. The method is now well established, and apparatuses for preparative or analyti- h should be high, and cosu should be close to unity (the cal separations are commercially available (4, 9). RPC shares mobile phase should wet the layer efficiently). A high value of k is not always possible, and it is particularly low when many of the advantages of other planar chromatographic performing reversed-phase chromatography with a nonpomethods, including simultaneous separation of multiple lar layer (e.g., bonded C18), which is poorly wetted by an samples and the removal of solvent before scanning the aqueous mobile phase. plate. However, RPC never reaches an overall mobile-phase By substituting Zf for column length, an equation for velocity that would give the highest separation efficiency, the linear velocity of an HPLC mobile phase (4) is modibecause the radial velocity of solvent migration diminishes fied to describe the migration velocity of the solvent front. from the center to the circumference of the plate. A different approach is to use electroosmotic flow to DPK 0d 2p (3) transport the mobile phase through the layer. Like other sep- u = hZ f aration methods that use electroosmotic flow, it could bein which D P is the pressure gradient from the inlet to the come a rapid and efficient way to separate multiple samples. This approach is discussed briefly at the end of this article. solvent front. Equation 3 is only an approximation be-

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cause, unlike HPLC, the solvent front is continually entering dry sorbent. The solvent pump is designed to deliver the mobile phase at a fixed rate, and D P increases with the migration distance to yield a constant solvent-migration velocity throughout a linear development. In OPLC, mobile-phase surface tension is irrelevant, and the ability to wet the layer is less important than in conventional TLC. To obtain high efficiency in either technique, a layer of small particles must be used. The dependence of velocity on particle diameter in conventional TLC severely limits the maximum obtainable solvent pathlength. In OPLC, the migration velocity ultimately depends on the magnitude of D P and hence on the properties of the solvent pump. In the commercial instrument, the pump can work with layers having particles as small as 3 µm in average diameter. The efficiency of any chromatographic separation depends on the linear velocity of the mobile phase. The inverse relationship between u and Zf in conventional TLC results in a velocity that is substantially less than optimum during most of the separation. This diminution in linear velocity results in a maximum development distance beyond which the drop in efficiency causes a significant loss of separation quality. This length is substantially shorter for an HPTLC plate than for a conventional one and, for this reason, HPTLC plates are always used with relatively short, solvent pathlengths, typically ~5 cm. In contrast, the migration velocity in OPLC is constant and close to optimal, and an HPTLC layer yields a higher efficiency than a conventional one, irrespective of the distance migrated. The time required for the mobile phase to cover the same distance in OPLC is typically 5- to 10-fold shorter than TLC, depending on the mobile phase’s surface tension, viscosity, and ability to wet the stationary layer. Separation time is further reduced because the number of theoretical plates required to achieve a separation is generated in a shorter distance, thanks to the near-optimal solvent flow rate.

Apparatuses for OPLC To run OPLC, a TLC layer is covered by a flexible sheet, which is forced into close contact with the sorbent surface by application of a suitable pressure. In the commercial design, a poly(tetrafluoroethylene) cover sheet and a pressurized water bag supply the external pressure. Other designs have used gas pressure or a hydraulic press to supply the pressure (12–14). Apparatuses for OPLC have been commercially available for about 15 years. The original apparatus was bulky, difficult to handle, and had a tendency to develop leaks and other maintenance problems. Third-generation equipment appears to be much improved and more user-friendly and allows the use of a stepwise elution gradient (11). The pre-

pared TLC plate is placed in a holder in a cassette-type apparatus, which is inserted through a slot into the chromatograph. External pressures of up to 50 bar can be used, although a lower external pressure must be used with soft sorbents (e.g., 15 bar with cellulose) to avoid layer compaction. A secondary advantage of using a higher external pressure is increased efficiency, even when the flow rate is held constant. The improvement in efficiency is caused by the flexible membrane forming a better seal with the layer surface and a slight compression of the layer that results in reduced interstitial porosity (15, 16). The reduced plate height is a dimensionless number that can be used to compare different chromatographic systems. In OPLC, the minimum values of the reduced plate height are 2.1–3.5 (3, 11) and depend on the properties of the sorbent layer and the operating conditions. The corresponding range for a good HPLC column is ~2.0–2.5 (3), which suggests that, under optimum conditions, OPLC is comparable in efficiency with HPLC. At higher reduced mobile-phase velocities, however, the reduced plate height for OPLC is substantially larger than for HPLC.

Mobile-phase delivery In the linear mode, the mobile phase enters the stationary layer from a narrow eluent-directing channel in the flexible cover that contacts the TLC plate surface. For some applications, the channel is scratched directly into the sorbent layer. The channel and direction of mobile-phase flow are illustrated in Figure 1a. The pump delivers the mobile phase to the center of this channel, which must be filled very rapidly to obtain a linear solvent front. This is achieved by bringing the pump to the operating pressure with the inlet valve closed. Chromatography begins by opening the valve, which lets the mobile phase flash into the trough. It is necessary to seal the edges of the TLC plate before performing a separation in the linear mode to prevent the mobile phase from running off the plate. About 2 mm of the layer is scraped from the plate’s edges, and the exposed backing and 2 mm of the sorbent layer are coated with a suitable polymer. It is important that this be done carefully. An uneven solvent front can result if any of the original layer is left under the polymer or if cracks develop in the polymer surface. Plates with sealed edges are commercially available.

On-line and off-line modes OPLC may be performed on-line by allowing the mobile phase to flow directly from the apparatus to an HPLC de-


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tector or off-line when the plate is dried mm slit cut in the first. The mobile phase (a) (b) after development, and the densitometry then passes from the second to the third 5 is performed separately. The on-line mode plate as illustrated in Figure 1c. The new5 is limited to a single sample, whereas the est commercial equipment allows a maxi4 off-line mode can be used to separate mum of three plates to be stacked, but 4 multiple samples in a single run using a older models could handle five plates, 1 2 1 8 20 3 20-cm TLC plate. which provides a solvent migration dis3 2 7 The on-line mode may be viewed as tance of at least 65 cm. Plates with a flex8 3 7 a form of HPLC that uses a planar “colible backing are used. Under the high umn”. Figure 2 shows the separation of external pressure used, the backing from 6 a mixture of furocoumarin isomers by the upper plate is sufficiently flexible to 6 OPLC and HPLC using the same moconform to the layer surface of the lower bile phase (18). The OPLC separation plate, which allows OPLC to be performed. 40 5 10 20 is similar to that of an HPLC column Figure 3b shows the separation of a of moderate efficiency. The analysis time mixture of dyes using this technique with Time (min) Time (min) of ~1 h could be substantially reduced a 34-cm development distance. The gap FIGURE 2.Furocoumarin isomers indicates the position of the slit. The sepwith a higher flow rate, which is possible separated by OPLC and HPLC. aration takes ~27 min, but the analysis with an external pressure of 50 bar in the latest equipment. time per sample is substantially less be(a) OPLC and (b) HPLC, using silica as a stationary phase and chloroform:n-hexane OPLC is more commonly performed cause several samples can be separated :diethyl ether:ethyl acetate:water (58.5:40: in the linear off-line mode, which allows simultaneously. The number of samples 0.9:0.6:0.04). 1, iso-bergapten; 2, angelicin; for a separation path of up to 18 cm. Sam- 3, psoralen; 4, bergapten; 5, pimpinellin; 6, separated is less than with a single plate, sphondin; 7, xanthotoxin; and 8, iso-pimpples are spotted 10-mm apart, therefore, because the longer separation distance up to 18 samples can be spotted in a sin- inellin. Lengths for OPLC layer and HPLC results in larger spots. column are 18 cm and 10 cm, respectively. gle run with an unused border on each Off-line OPLC may also be performed External pressure for OPLC is 25 bar. (Adapted with permission from Ref. 18.) side of the spotting line. Figure 3a shows in the circular mode, in which the mobile the separation of toxic drugs using this phase is pumped onto the center of the mode (19). Alternatively, the mobile phase TLC plate. This results in a radial flow can be introduced in the middle of the plate, as illustrated profile that, on geometric grounds, is advantageous when in Figure 1b. This is referred to as the two-directional mode separating low-Rf solutes (21). Samples are spotted in a raand is only suitable for samples containing five to eight com- dial pattern around the center of the plate, as illustrated ponents because the separation path is limited to 8 cm. The in Figure 1d. The low-Rf advantage is at its best when the “spotting circle” has a small radius. Although the low-Rf shorter pathlength results in a smaller spot size after development, and, therefore, samples can be spotted 5 mm apart. advantage is lost with a large spotting circle, the greater radius allows more samples to be spotted. This allows 35 samples to be spotted for each of the two In studies at the Research Institute for Medicinal Plants, directions, for a total of 70 samples in a single run. Szabolcs Nyiredy and colleagues used circular OPLC to The maximum migration distance of 18 cm can be exscreen a large number of plant extracts for three ergot alkatended by using long-distance OPLC (20). In this techloids. Silica gel HPTLC plates with a flexible backing were nique, TLC plates are stacked and the mobile phase from used, and on each plate 72 samples were spotted 3 mm the first plate is directed to the second plate through a 0.1-

(a)

0.2

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FIGURE 3.Separations using off-line OPLC.

(b)

0.4

0.6 Rf

0.8

1.0


Rf

0.5

(a) Toxic drugs separated on a silica layer with an 18-cm pathlength using a mobile phase of trichloroethylene:2butanone:n-butanol:acetic acid:water (17:8:25:6:4). From top to bottom, compounds are flecainide, norverapamil, verapamil, acebutolol, ethylmorphine, aminophenazone, correlation standards, chlorpromazine, atenolol, trimipramine, chloroquine, clozapine, caffeine, and metoclopramide. (Adapted with permission from Ref. 19.) (b) A mixture of dyes separated on a silica layer with a 34-cm pathlength by long-distance OPLC using toluene as the mobile phase. (Adapted with permission from Ref. 20.)

Conventional Conven TLC is an is TL alternative

apart along a spotting circle with a radius of 3.5 cm. The mobile phase—acetonitrile:ethanol:toluene (85:10:5)—was allowed to migrate 9.3 cm from the plate’s center. Thus, the distance from the spotting circle to the final position of the solvent front was 5.8 cm, which is adequate for separating the alkaloids. The sample throughput was increased by stacking five plates. The stacking concept is similar to that used in long-distance OPLC, except the mobile phase is delivered to all plates simultaneously through a narrow channel drilled through the center of the top four plates. This allows the simultaneous separation of 360 samples. The technique seems well suited for the screening of reaction mixtures in combinatorial chemistry. OPLC can also be performed in the two-dimensional mode (22, 23). The sample mixture is spotted near the corner of the TLC plate, and the first development is performed with a suitable mobile phase. The plate is removed from the apparatus, allowed to dry, rotated 90°, and reinserted into the apparatus. A second development is then performed with a mobile phase of different selectivity. The difference in selectivity between the two developments can be improved by using a commercially available dual-phase plate. The first development is performed on a silica layer in the normal-phase mode, followed by the second development on a bonded C18 layer in the reversed-phase mode.

Mobile phase There are several approaches to selecting an optimum mobile phase for TLC (4, 24), and any of these can be used as a scouting technique to find a satisfactory solution for OPLC. Pre-equilibration between mobile and stationary phases does not occur in OPLC, and, for this reason, an unsaturated development chamber (i.e., one in which essentially no pre-equilibration occurs) must be used when performing the scouting. There is also a statistical method of mobile-phase selection that was developed specifically for OPLC, which predicts, with 95% confidence, the best mobile phase for separating a sample containing a specified number of compounds (25). Data are available only for steroids, but the approach could be applied to other classes of compounds. One of the most useful scouting approaches is the Prisma technique, which is a structured trial-and-error method (26). The first step is to perform TLC in an unsaturated chamber with a set of solvents that span a range of selectivities and in which all components of the sample mixture have Rf values between 0.2 and 0.8. If any of the Rf values are outside this range, the mobile-phase strength is adjusted by

to

HPLC

adding a weak solvent, such as hexane, or strong solvent, such as acetic acid, to bring all the values into this range. If none of the mobile phases yields a satisfactory separation of the sample mixture, the three best phases are mixed in a set of defined proportions (e.g., 1:1:1, 1:8:1), and each of these are evaluated. If none of these yields a satisfactory separation, one of the three “best” mobile phases is discarded and the next best mobile phase is used instead. Ideally, the mobile phase should contain one component that does not contribute to solute migration. This solvent can be used for a pre-run to prevent the “disturbing effect”, which is discussed in the next section.

Some necessary precautions Solvent demixing is a phenomenon that occurs with polar layers such as silica gel or alumina and with a mobile phase consisting of two or more solvents of substantially different polarity. The TLC layer will have an affinity for the most polar of these solvents, and the mobile phase becomes depleted in this component. The greatest depletion occurs in the region behind the solvent front, where there is a resultant drop in solvent strength. Significant demixing causes a sharp transition between the depleted and nondepleted zones and results in an invisible secondary front, which is known as the b front. Solutes with somewhat higher values of Rf than the b front are unable to cross the front and, therefore, migrate with the front. Such solutes cannot be separated from each other. A very sharp peak is obtained when scanning a spot that migrates with the b front. Demixing can be prevented, or significantly reduced, in conventional TLC by allowing the layer to pre-equilibrate with the mobile-phase vapor before chromatography. There are several specially designed chambers for accomplishing this. In principle, pre-equilibration for OPLC can be achieved by pumping gas saturated with mobile-phase vapor across the plate’s surface before commencing chromatography. This option is not available with current apparatuses, and demixing is always a possibility when performing OPLC with multicomponent mobile phases on polar layers. Demixing is usually not a problem in the reversed-phase mode and is never a problem in either chromatographic mode when the mobile phase consists of a single solvent. Even when demixing occurs, it does not affect the quality of


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separation, provided that all the solutes in a is the zeta potential, E is the magnitude of the electric field, and h is the viscosity of mixture have migration distances behind the mobile phase. In principle, the electric the b front. Thus, it is usually possible to field can be selected to yield an optimal find a set of experimental conditions such that demixing causes no problems. flow velocity, which is independent of the The “disturbing effect” is another cause distance traveled by the mobile phase and, of a secondary front in OPLC. The front under most conditions, the particle diameappears as a very irregular wavy line with ter. The expected plug-flow profile should the layer in the “disturbed zone” in front have a uniform cross-sectional velocity and of the line appearing to be only partially yield a higher efficiency than the laminar wetted by the mobile phase. Solutes that profile in a pressure-driven system. These migrate with the secondary front or in the combined properties should yield a fast and disturbed zone often have irregular shapes, efficient system when working with very which result in poor quantitation by densmall particles and a high electric field. sitometry. It is believed that the disturbing In 1974, the first report on using electroeffect is related to the mobile phase’s disosmotic flow in both column and planar 0 2 4 placement of adsorbed gas in the stationary chromatography was published (28). A Migration distance (cm) layer (27). Although much of the displaced mixture of four steroids was separated in gas escapes ahead of the solvent front, some 4 min compared with 60 min by TLC. In FIGURE 4.Separation of a is dissolved in the mobile phase or is presspite of impressive improvement in performent as tiny bubbles trapped behind the pri- mixture of dyes on 18a C ance, it was more than 20 years before TLC layer using electroosmoticusing electroosmotic flow was described mary solvent front. There is a pressure graflow. dient behind the primary front, with the again, and this lengthy hiatus may be due pressure behind the secondary front being to the unfortunate paucity of experimental Applied voltage is 2.0 kV. The mobile sufficient for the gas to completely dissolve. phase is 80% aqueous ethanol containing detail in the earlier report. This is in contrast 1.0 mM N-[tris(hydroxymethyl)methyl]-3The secondary front occurs between the to CE and column electrochromatography aminopropanesulfonic acid buffer. (Adaptzone of fully dissolved and partially diswhere the attractive features of electroossolved gas. The waviness of this line may motic flow have been well exploited. be related to irregularities in the stationary layer. The speed of the technique has been confirmed for The disturbing effect can be prevented by using a mobile normal-phase chromatography. Six pyrimidines were sepaphase that incorporates a solvent (e.g., hexane for most rated on a silica layer with acetonitrile as the mobile phase. normal-phase separations) that is too weak to cause solute The analysis time was 12 times faster than with conventionmigration. The pure, weak solvent is used for a pre-run imal TLC (29). Separation in the reversed-phase mode is mediately before the analytical separation, preventing a subabout two to three times faster, depending on the mobile sequent manifestation of the disturbing effect. phase (30). Finally, it is important that the external pressure be subFigure 4 shows the separation of a mixture of dyes on stantially higher than the pressure used to drive the mobile a bonded C18 layer using the electroosmotic technique. The separation is more efficient than for the same system phase through the layer. If this condition is not met, the pressurized membrane will not form a good seal on the plate with conventional TLC. There is poor power dissipation in a TLC layer. Therefore, it is expected that the separasurface. This will lead to a wall effect and lower efficiency. tion quality will improve when the thermal contribution to band broadening is reduced by external cooling, suggesting that this could be a promising technique in the future.

TLC using electroosmotic flow

We thank Szabolcs Nyiredy and Emil Mincsovics for their contributions.

Applying an electric field across a wet TLC layer containing both ionized silanol groups and mobile ions results in an electroosmotic flow due to the presence of an electrical double layer. If the TLC layer is treated as a bundle of capillary tubes, the flow velocity is predicted by u = «zE 4ph

(1)

(2)

(4)

in which « is the dielectric constant of the mobile phase, z

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References


(3)

Ismailov, N. A.; Schraiber, Farmatsiya M. S.(Sofia) 1938,1 (as referenced in Kirchner,Thin-Layer J. G; Chromatography, 2nd ed.; Wiley: New York, 1978). Jork, H.; Funk, W.; Fischer, W.; Wimmer, Thin-Layer H. Chromatography: Reagents and Detection Methods, Vols. 1a and 1b; VCH: Weinheim, Germany, 1990. Poole, C. J.F. Chromatogr., A 1999,856, 399—427.

(4) (5) (6) (7)

(8) (9)

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(13) (14) (15) (16) (17)

(18) (19) (20) (21) (22) (23) (24) (25) (26) (27)

(28)

Poole, C. F.; Poole,Chromatography S. K.; Today; Elsevier: Amsterdam, 1991. Sherma, J. Anal. Chem. 1998,70, 7 R—26 R. Liang, Y.; Baker, M. E.; Yeager, B. T.; Denton, Anal. M. B. Chem. 1996,68, 3885—3891. Mukherjee, K. D., Applications of Flame Ionization De tectors in Thin-Layer Chromatography. Handbook In of ThinLayer Chromatography; 2nd ed.; Sherma, J.; Fried, B., Eds.; Marcel Dekker: New York, 1996; 361—372. Fried, B.; Sherma, Thin-Layer J. Chromatography, 4thed.; Marcel Dekker: New York, 1999; pp 249—267. Nyiredy, Sz.; Meszaros, S. Y.; Dallenbach-Tolke, K; Nyiredy-Mikita, K.; Sticher, J. PlanarO. Chromatogr. 1988,1, 54—60. Tyih k, E.; Mincsovics, E.; Kalasz, J. Chromatogr. H. 1979,174, 75—81. Mincsovics, E., et J. AOAC al. Int. 1999,82, 587—598. Kaiser, R. E.; Rieder, Planar R.Chromatography, I. Vol. 1; Kaiser, R. E., Ed.; Heuthig: Heidelberg, Germany,1986; pp 165—191. Witkiewicz, Z.; Mazurek, M.; Bladek, J. Planar Chromatogr. J. 1993,6, 407—411. Flodberg, G.; Roeraade, J. Planar J. Chromatogr. 1995,8, 10—13. Tyih k, E.; Mincsovics, E.; Siouffi, J. Planar A. Chromatogr. M. 1990,3, 121—125. Botz, L.; Wehrli, E.; Sticher, J. Planar Chromatogr. O. 1993,6, 13—20. Mincsovics, E.; Ferenczi—Fodor, K.; Tyih k, - E. Overpres sured Layer Chromatography. Handbook In of Thin-Layer Chromatography, 2nd ed.; Sherma, J.; Fried, B., Eds.; Marcel Dekker: New York, 1996; pp 171—203. Zogg, G. C.; Nyiredy, Sz.; J. Sticher, Planar Chromatogr. O. 1988,1, 351—354. Ojanper , I; Goebel, K.; Vuori, J. Liq. Chromatogr. E. & Rel. Technol. 1999,22, 161—171. Botz, L.; Nyiredy, Sz.; Sticher, J. Planar Chromatogr. O. 1991,4, 115—122. Geiss, F. Fundamentals of Thin Layer Chromatography; Huthig: Heidelberg, Germany, 1987. Habibi-Goudarzi, S.; Ruterbories, K. J.; Steinbrunner, J. E.; Nurok, J.D. Planar Chromatogr. 1988,1, 161—167. Nagy-Tur k, A.; V gh, J. Chromatogr., Z. A 1994,668, 501—507. Nurok, D. Chem. Rev. 1989,89, 363—375. Nurok, D.; Kleyle, R. M.; McCain, C. L.; Risley, D. S.; Ruterbories, K. Anal. J.Chem. 1997,69, 1398—1405. Dallenbach-Toelke, K.; Nyiredy, Sz.; Meier, M.; Sticher, O. J. Chromatogr. 1986,365, 63—72. Nyiredy, Sz.; Meszaros, S. Y.; Dallenbach-Tolke, K; Nyiredy-Mikita, K.; Sticher, J. High Resolut. O. Chromatogr. 1987,10, 352—356. Pretorius, Victor.; Hopkins, B. J.; Schieke, J. ChroJ. D. matogr. 1974,99, 23—30.


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Peer Reviewed: Forced-Flow Techniques in Planar Chromatography

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