processes dominating forced-flow thin-layer chromatography

Processes Dominating Forced-Flow Thin-Layer Chromatography

H. Kalasz

Department of Pharmacology, Semmelweis University of Medicine, Nagyv~rad tar 4, P. O. Box 370, Budapest VIII, Hungary

Key Words

Forced-flow TLC Overpressured TLC Dyestuffs

Summary

The effect of the vapor phase and other special in- fluences on thin-layer chromatography have been in- vestigated. Comparisons were made of the relationships of time vs. developing distance and flow rate vs. efficiency using a planar arrangement of the thin-layer. Covering the layer facilitates the reproducibility and of the migration front but the most effective optimization step for thin-layer chromatography is provided by forced- flow of the mobile phase, It is suggested that planar chromatography with a covered sorbent layer and using a pressurised solvent stream should be called forced-flow thin -layer chrom tatography.

Introduction

In the last quarter of the century, liquid chromatography showed an impressive progress which realized in the high- performance methods. This development has changed the conditions and parameters of chromatography and these well observable alternations were also mirrored in the nomenclature of the newly developed procedure: it is generally called as high-performance liquid (column) chromatography (HPLC) [ 1-4].

Similar phenomenon is also characteristic for forced-flow thin-layer chromatography (FF-TLC). The method has been called overpressured thin-layer chromatography (OPTLC) referring to the fact that the sorbent layer is totally closed by a membrane and an excess of pressure (an overpressure) is applied on it [5-7]. However, the membrane which closes the sorbent of thin-layer chromatography does not only serve for the elimination of the vapor phase, but forced- flow of the mobile phase can also be applied. In our earlier papers [6-8] the importance of the elimination of the vapor phase was detailed. Subsequently the advantages of the forced flow have been placed into the focus of the investigations [8-10]. In this paper, a comparison of forced- flow thin-layer chromatography with the classical thin-

628

layer chromatography and column liquid chromatography, will be given, emphasizing some special characteristics of the planar methods.

The generally used classifications of chromatography [11-12] distinguish between column and planar techn. iques. In liquid column chromatography (LCC) the sorbent has been prewashed and equilibrated with the mobile phase prior to chromatography. The sample is dissolved in the same solution as the eluent (mobile phase) and the sample solution is injected or applied by some way into the mobile phase before the column containing the stationary phase. In LCC, the basis of the separation and band spreading is the triple equilibrium between solvent-sorbent, solvent-solute and sorbent-solute as it is depicted in Fig. 1. LCC employs sensitive and specific detectors for monitoring the effluent, i.e., the sample components which are in solution and the separation can be evaluated quantitatively and qualitatively according to the measured values using flow-through cells and homogeneous phases [ 11]. Separation of several samples can be carried out consecutively.

In planar methods, thin-layer chromatography (TLC) be. came the most widely used technique. Several samples can be separated at the same time and the components of the separated samples can be detected by specific color reagents. The direct visual observation of both the separation proce~ and the results of chromatography helps to solve some special problems as the detection of spots with zero move- ments, multiple solvent fronts, etc. In TLC, the triple equil. ibrium of LCC is replaced by quintuple processes represen. ting the possible interactions between vapor-solvent-sorbent- solute as it is shown in Fig. 1. Moreover, the generally used techniques of TLC employ a dry stationary phase at the

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Factors and equil ibria dominating column liquid chromatography (left) and thin-layer chromatography (right).

Chromatographia Vol. 18, No. 11, November 1984 Originals

0009-5893/84/11 0628-05 $ 03.00/0 �9 1984 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH

beginning of the separation. The samples are spotted onto the dry sorbent and are dried on it, and the mobile phase also runs through the sorbent bed which was dry prior to the procedure. Using a multicomponent mobile phase ~veral solvent fronts can be observed, and even in a single- component volatile mobile phase, a diffuse solvent front is moving forward [6]. This means that the solvation of the stationary phase with the running solvent takes place paral- lely with the actual chromatographic processes and the constant evaporation and condensation of the solvent are ~perimposed on the development [6, 7], i.e., on TLC it- ~lf.

An additional point is worth mentioning. The loading of the chromatographic system with the samples and the detection of the separated compounds are done in a heter 0genous phase system, so the real start of a TLC separation

when the mobile phase touches the sample and dissolves it. Similarly, the evaluation of the separation character- ~tics and the quantitative determination of the individual ~ample components are carried out by the reaction of the dried spots with the solution of the reagents; furthermore, photometric detection is also performed in the presence of the solid particles of the stationary phase bed [14].

Fig. 2 Set up for forced-flow thin-layer chromatography.

Experimental

Materials

TLC plates, 200 x 200 mm in size, precoated on glass with silica gel 60 F 254 were purchased from E. Merck (Darm- stadt, FRG). Solvents and chemicals were supplied by Reanal (Budapest, Hungary). Test Mixture II was a kind gift of Dr. J/inchen (Camag Inc. Muttenz, Switzerland).

Apparatus

Ihe chamber of Desaga (Heidelberg, FRG) was used as the ~t-up for conventional TLC. Saturation of the chamber was achieved by lining the glass tank with fftlter paper for 1 hour prior to development. The non-saturated chamber was n0t lined with filter paper. In a covered plate the vapor phase over the sorbent layer is eliminated when covered with a glass plate [6, 7]. The system for forced-flow thin- layer chromatography is detailed in several earlier publica- ~0ns [5-7] . The sorbent layer is sandwiched between its �9 pport and a plastic foil which is firmly pressed up to 1 mPa labout 10 atm). The mobile phase is supplied by a liquid chromatographic pump with adequate flow rate. The linear ~ape of the solvent front is arranged by a chanel in or over the sorbent and by bordering the thin-layer plate [9]. A h0me-made set up is demonstrated in Fig. 2 and the Chrom- pres l0 instrument of Labor MIM (Budapest, Hungary) is th0wn in Fig. 3.

Results

Fig. 4 shows the separation of the components of Test Mixture II on silica gel plates using, dichloromethane as the mobile phase and a conventional TLC chamber. The samples

Fig. 3

Chrompres 10 (Labor MIM, Budapest), equipment for thin-layer chromatography under pressure.

were applied in spots and lines as demonstrated in Figs. 4/a, 4/b and 4/c; the separations were performed in a non- saturated chamber on a non-covered plate, in a non-saturated chamber on a covered plate and in a saturated chamber on a non-covered plate (Figs. 4/a, 4/b and 4/c, respectively). The covered plate in a saturated chamber results in the same general picture as a covered plate in a non-saturated chamber (Fig. 4/b) and occupies middle position between the cases of saturated chamber - non-covered plate and non-saturated chamber - non-covered plate. The spots near to the solvent front are concentrated during the TLC procedure while this phenomenon is less pronounced for the components moving far from the front. The concentration of the spots is drastic in the case of the non-saturated chamber, while the saturated chamber gives only moderate concentration of the spots. Fig. 5 indicates the fact that the position of the spots after thin-layer chromatography is practically the same as their front line after frontal chromatography of the mixture consisting of the same running solvent and dye-components to be separated.

Fig. 6 demonstrates how the movement of the solvent front is slowing down in the classical TLC chamber. The front distance vs. time diagrams are given in saturated, non-

Chromatograph ia Vol. 18, No. 11, November 1984 Originals 629

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Comparison of elution and frontal chromatography of the dye mixture using dichloromethane as the mobile phase and silica gel as the stationary phase. The chromatograms were obtained by eluti0n development (left) and by frontal chromatography (right).

saturated and UM chamber (it is the covered plate) and using FF-TLC. The striking common characteristic of the diagrams characterising the runs in the classical chamber is that they show a declining form under each chamber condition used here. Saturation of the chamber slightly facilitates the front movement, unsaturation moderately decreases the flow velocity, while the covered plates eliminate the influence of saturation or non-saturation.

Fig. 7 shows the front distance vs. time diagrams in saturated and non-saturated chambers using an aqueous solvent for the development. In this case, there is no remarkable dif- ference between the running characteristics.

Fig. 8 gives the flow velocity vs. plate height characteristics in the case of phenylalanine and DOPA (dihydroxyphenyl- alanine) performing the separation on silica gel using chloroform-methanol-pH 6.5 buffer (7 : 5 : 1); and carrying out the chromatography on Fixon 50x 8 chromatoplates using citrate buffer at pH 3.6 (Fig. 9). In both systems, the curves exhibit a definite minimum although both the places and the values of the minima were different.

Discussion

The chromatographic separation of samples using LCC is accompanied with peak broadening or band spreading if the development is done by isocratic elution, i.e., the com- position of the eluent is not changed during the chromatographic process. However, the stationary phase - mobile phase ratio is kept constant by the simple fact, that the stationary phase is thoroughly equilibrated with the mobile phase.

In TLC two different influencing factors have to be taken into consideration. The first is the effect of the vapor phase which is realized in the constant evaporation and conden-

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Front distance - t ime diagram of running of chloroform on a silica gel plate in various systems including FF-TLC.

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630 Chromatograpma Vol. 18, No. 11, November 1984 Originals

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Flow velocity (u} vs. eff iciency (HETP, H) relationship on Fix ion 50 X 8 chromatoplates using citrate buf fer as the mobile phase, for phenylalanine and DOPA.

sation of the volatile solvent component(s) from and to the mobile phase running on the stationary phase bed. This effect can mostly be eliminated by covering the sorbent. However, the behavior of the sample components is rather influenced by those processes which play a role near to the solvent front and which determine the shape and thickness of the diffuse solvent front [6, 9] of the volatile mobile phase. It was published by Vernin [15] and also discussed by us [9] that the solvent - sorbent ratio is sharply changed near to the front line. Although the covering of the sorbent makes the experiments reproducible [6], the fact that a gradient in the sorbent-solvent ratio exists remains characteristic for all planar chromatographic systems operated with a sorbent which had been dry prior the chromatographic van. In these cases the solvation of the stationary phase takes place at and near the solvent front. This solvatation procedure constructs a particular series of the solvent- s0rbent ratios around the front, and the components (some constituents of the dye-mixture here) located near to the front line behave according to the rules of this gradient in the solvent-sorbent ratio.

The solvation procedure can be well observed if a volatile solvent is used for the development of silica gel plates as the gradually darkening zones are moving forward. This is assumed to be the reason for the spots definitely concentrat- ing at positions near to the solvent front. In our previous experiments [16], the possibility of displacement actions was excluded; thus the observed phenomenon is assumed as the consequence of frontal chromatography of the volatile and rather apolar organic solvents on the silica gel with diffuse or diminished-arch step. This phenomenon helps the separation and improves the efficiency of TLC. Fortunately, the covering of the stationary phase does not eliminate this beneficial effect; in other words, FF-TLC keeps the advantages of both frontal chromatography of the mobile phase and improved efficiency.

At the same time, FF-TLC gives the possibility for a controlled and constant supply of the mobile phase. Thus, the

method provides an unique tool for the optimization of the flow rate in the case on TLC. In our earlier publication [7] optimal flow rate was found for the separation of amino acids on Fixion 50 x 8 chromatoplates. Recently, Hauck and Jost confirmed our finding that in planar chromatographic systems there is an optimum mobile phase flow velocity. They showed it on HPTLC plates [10] and their results indicated a velocity of about 0.20-0.25mm/sec for minimum plate height while our results pointed out 0.12 mm/sec, using Fixion 50 x 8. Fig. 8 presented here gave the optimal flow velocity as 0.16 mm/sec with TLC silica gel plates.

The plate height values of the individual components are different but every value is low enough to clearly demonst- rate the advantageous effect of planar chromatography at tile optimal flow rate for the efficiency of the separation. At the same time, the declining form of front distance vs. time diagrams indicates the long-lasting procedure of TLC over a certain but short development distance. This distance is very limited in the case of HPTLC plates but moderately short lengths can also be reached in a brief period on any kind of support and stationary phase.

Several efforts have been published [17-21] to realize a controlled and/or fast development. Among them, special- ly arranged chambers [ 17-18] and the use of pumps are the most important. However, only the simultaneous closing of the stationary phase bed and th e application of a pump for the forced-flow of the mobile phase can provide the necessary conditions for the reproducibility of the chromatographic separation and optimization of the efficiency [5-10]. Several types of systems were constructed or predicted for this purpose including chambers for circular [5], linear [6-10] and two-dimensional [22, 23] developments. As the forced-flow of the developing solvent system is the basic characteristic and requirement for fast and effective separation, in order to differentiate this method from any other type of TLC, it is proper to use it as the proper term describing the procedure.

Chromatographia Vol. 18, No. 11, November 1984 Originals 631

Conclusions

There are several factors governing thin-layer chromatographic separation. Among them, the role of the vapor phase and frontal chromatography of the mobile phase components can be emphasized. This latter procedure has an advantageous effect on the spots moving near to the solvent front. The method of performing chromatography on a totally closed stationary phase bed seems to be typically thin-layer chromatography. The controlled flow of the mobile phase permits fast development and optimum efficiency, i.e., generally smaller plate heights in comparison with liquid column chromatography, as well as effective separation and fast development as compared to classical thin-layer chromatography. At the same time, FF-TLC maintains the main advantages of TLC as the chromatographic method in which several samples can be analized and color reagents can also be used.

References

[11 Cs. Horv~th (Ed.) "High Performance Liquid Chromato- graphy - Advances and Perspectives", Vols. 1, 2 and 3. Acad. Press, New York, 1980 and 1983.

[2] B. L. Karger, in: J. J. Kirkland (Ed.) "Modern Practice of Liquid Chromatography", Wiley, New York, 1971; p. 3.

[3] L. R. Snyder, J. J. Kirkland "Introduction to Modern Liquid Chromatography". Wiley, New York, 2nd ed. 1979.

[4] A. Zlatkis, R. E. Kaiser (Eds.) "HPTLC - High Performance Thin-Layer Chromatography", Elsevier, Amsterdam, 1977; p. 9.

[5 ] 1t. Kaldsz, J. Nagy, E. Mincsovies, E. Tyih6k, J. Liquid Chr0- matogr., 3, 845 (1980).

[61 H. Kal6sz, E. Tyiht~k, E. Mincsovics, in: A. Frigerio, M. McCamish (Eds) "Recent Developments in Chromatography and Electrophoresis, 10". Elsevier, Amsterdam, 1980; p. 289.

[71 H. Kaldsz, J. Nagy, J. Liquid Chromatogr. 4, 985 (1981). [8] H. Kaldsz, J. Nagy, J. Knoll, in: A. Chodera (Ed) "Abstracts

of Presentations on 7th Congress of Polish PharmacologiM Society". Poznan, Poland, 1980; p. 223.

I91 H. Kaldsz, in: H. Kaldsz, L. S. Ettre (Eds.) "Chromatography, the State of the Art". Akad6miai Kiad6, Budapest, in press.

[10] H.R. Hauck, W. Jost, J. Chromatogr. 262, 113 (1983). [ l l l L.S. Ettre J. Chromatogr. 220, 29 (1981). I12] L. Szepesy, "Gdzkromatogr~fia" ("Gas Chromatography")

Miiszaki K6nyvkiad6, Budapest, 1970; p. 13. [131 T. Ddvdnyi, Acta Biochem., Biophys. 11, 1 (1976). I14] L. Leisztner, in: E. Tyihdk (Ed.) "A rdtegkromatogr~fia

zsebk6nyve" (Handbook of Thin-Layer Chromatography). Miiszaki K6nyvkiad6, Budapest, 1979; p. 124.

[15] G. Vernin, "La Chromatographie en Couche Mince", Dun0d, Paris, 1970.

[16] 1-1. Kaldsz, unpublished results. [17] s A. Perry, T. H. Jupille, L. J. Glunz, Anal. Chem. 47, 65

(1975). [18] H. Determann, "Gelchchromatographie". Springer, Berlin,

1967; p. 57. [19] M. Brenner, A. Niederwieser, Experimentia, 17, 237 (1961). [20] E. Soczewinski, J. Chromatogr. 138,443 (1977). [21] E. Soczewinski, T. Wawrynowicz, Chromatographia 11,466

(1981). [22] G. Guiochon, L. A. Beaver, M. F. Gonnard, A. M. Siouf~,

M. Zakaria, J. Chromatogr. 255,415 (1983). I23] G. Guiochon, M. F. Gonnard, M. Zakarla, L. A. Beaver, A. M.

Siouffi, Chromatographia 17, 121 (1983).

Received: June 6, 1984 Accepted: July 25, 1984 A

632 Chromatographia Vol. 18, No. 11, November 1984 Originals

processes dominating forced-flow thin-layer chromatography

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