The Initial Stage of the Development of Planar Chromatograms

The Initial Stage of the Development of Planar Chromatograms Agnieszka Pieniak, Mieczysław Sajewicz, Krzysztof Kaczmarski, and Teresa Kowalska* Key Words: Retention mechanism Initial stages of retention Impact of initial stages on Lateral analyte analyte interactions Densitometric determination of This paper was presented at the Symposium Planar Chromatography 2004, Visegrád, Hungary, May 23 25, 2004 Abstract The aim of the work discussed in this paper was to re-investigate the initial stage of the retention process in TLC, namely the moment of first contact between the analyte deposited at the origin and the mobile phase employed. It was our intention to assess the impact of dissolution of the analyte by the mobile phase on the overall mobility of the analyte in the chromatographic system. To accomplish this goal parallel experiments were performed with analyte samples applied to the stationary phase and chromatographed with and without drying (i.e. with and without evaporation of the solvent used to dissolve the analyte). As test analytes we selected two compounds (5-phenylpentanol and 2-phenylbutyric acid) able to participate in lateral interactions by hydrogen bonding and another four compounds (anthracene, phenanthrene, perylene, and tetralin) totally unable to self-associate. The results obtained are presented in two different ways, i.e. as the dependence of densitometrically measured concentration profiles and of retardation factors ( ) on the amount of analyte and on the mode of application (dry or wet) to the stationary phase layer. It was discovered that for both types of analyte (i.e. participating and not participating in lateral interactions) the process of dissolution at the origin is time-consuming and thus contributes to the overall retardation of migration of the analytes. A. Pieniak, M. Sajewicz, and T. Kowalska, Institute of Chemistry, Silesian University, 9 Szkolna Street, 40-006 Katowice, Poland; and K. Kaczmarski, Faculty of Chemistry, Technical University of Rzeszów, 2 W. Pola Street, 35-959 Rzeszów, Poland. 1 Introduction It is usually taken for granted that separations performed by planar and column liquid chromatography are physicochemically similar enough to be described and optimized within the same theoretical framework [1 4]. The main differences between these two modes which are most frequently perceived and consequently discussed are: (i) the open-bed or the closed-bed nature of the stationary phases [5, 6], and (ii) the two-dimensional effective diffusion in planar mode compared with onedimensional effective diffusion in column mode [7 9]. A crucial, although easily overlooked, difference between the two modes is, however, the manner of introduction of analyte samples in planar and column chromatography. In the former technique samples are usually applied to the stationary phase as the smallest possible volumes of the respective solutions and the solvent is then evaporated from the layer before initiation of the chromatographic separation. In the latter, samples are most frequently applied as solutions to the top of the solid bed and solvent is never removed from the start. Thus dissolution of the analyte by the mobile phase is an initial stage in the development of planar chromatograms which is totally absent from column chromatography. It seems that the initial stage of dissolution of the analyte samples in planar chromatography has not attracted enough attention from method theoreticians [2 4, 7 9], and has certainly not yet been scrutinized densitometrically. Hence it was the main goal of this study to investigate densitometrically the impact of analyte dissolution on the overall process of retention. In our experiment we used two different kinds of analyte those able to participate in lateral interactions (an alcohol and a monocarboxylic acid) and those lacking any functionality and hence practically unable to participate in hydrogen bonding (polycyclic hydrocarbons). Our attention also focused on the rela- Journal of Planar Chromatography VOL. 18. JANUARY/FEBRUARY 2005 13 DOI: 10.1556/JPC.18.2005.1.2

tionship between the amount of the analyte applied to the layer and its velocity of migration (expressed as a numerical value of the retardation factor, ). 2 Experimental 2.1 Analytes Able to Participate in Lateral Interactions As analytes able to self-associate we used 5-phenylpentanol and 2-phenylbutyric acid. Alcohol samples were dissolved in n- octane and acid samples in decalin (the concentrations are given in the table and figure legends). n-octane was used as mobile phase for the alcohol and decalin for the acid, i.e. the same as were used as solvents for the analytes. For both compounds the adsorbent used was microcrystalline cellulose (precoated cellulose powder TLC plates from Merck, Darmstadt, Germany; #1.05730). The volume of analyte samples applied to the plates was 1 µl, irrespective of analyte concentration. Development was performed in ascending mode in a Stahl-type open chromatographic chamber. The migration distance of the mobile phase front was 15 cm. 2.2 Analytes Unable to Participate in Lateral Interactions As analytes unable to self-associate by hydrogen bonding we selected four polycyclic hydrocarbons, anthracene, phenanthrene, perylene, and tetralin; their chemical structures are shown in Table 1. The compounds were dissolved in toluene (the concentrations are given in the table and figure legends). These compounds were chromatographed on precoated silica gel 60 F 254 TLC plates (Merck #1.05715) with n-hexane as Table 1 The molecular structures of the polycyclic hydrocarbons investigated. Compound Tetralin Anthracene Phenanthrene Structure Figure 1 Comparison of concentration profiles for different amounts of 5-phenylpentanol (a) dried after application or (b) developed without drying (microcrystalline cellulose was used as stationary phase and n-octane as mobile phase; the volume of sample applied was always 1 µl). The mean velocity of migration of n-octane was 1.64 mm min 1. mobile phase. The other development conditions were exactly the same as used for the alcohol and acid. 2.3 Densitometric Evaluation of the Results The chromatograms obtained were evaluated by densitometry. Densitograms were obtained by use of the Desaga (Heidelberg, Germany) model CD 60 densitometer equipped with Windowscompatible ProQuant software. profiles were recorded in reflectance mode in UV light at λ = 260 nm (for alcohol and acid) or λ = 254 nm (for the four hydrocarbons); the dimensions of the rectangular light beam were 0.02 mm 0.4 mm. The densitograms obtained were relatively smooth and therefore needed no extra smoothing. 3 Results and Discussion Perylene 3.1 Analytes Able to Participate in Lateral Interactions Thin-layer chromatograms of 5-phenylpentanol and 2-phenylbutyric acid were developed in two ways which differed sub- 14 VOL. 18. JANUARY/FEBRUARY 2005 Journal of Planar Chromatography

distance from origin [cm] Figure 3 Comparison of peak profiles calculated for the two-layer (higher peak) and threelayer (lower peak) adsorption isotherms. Table 2 Comparison of values for different amounts of 5-phenylpentanol dried after application or developed without drying (microcrystalline cellulose was used as stationary phase and n-octane as mobile phase; the volume of sample applied was always 1 µl). The mean velocity of migration of n-octane was 1.64 mm min 1. Figure 2 Comparison of concentration profiles for different amounts of 2-phenylbutyric acid (a) dried after application or (b) developed without drying (microcrystalline cellulose was used as stationary phase and decalin as mobile phase; the volume of sample applied was always 1 µl). The mean velocity of migration of decalin was 0.72 mm min 1. stantially in the method of application of the analyte samples to the adsorbent layer. The first was the classical method in which the sample solvent was removed from the origin (with a hair dryer) before initiating the chromatographic separation. The second method consisted in developing the chromatograms without evaporation of the solvent from the origin. By comparing the results obtained, we demonstrated the impact of dissolution of the analytes in the mobile phase on their migration (expressed as values calculated taking into the account the maxima of the bands concentration profiles). These results are given in Figures 1 3 and Tables 2 and 3. It is apparent from the leading tails in the densitograms presented in Figures 1 and 2 that all the concentration profiles obtained for 5-phenylpentanol and 2-phenylbutyric acid are indicative of anti-langmuir adsorption isotherms, typical of compounds able to participate in lateral analyte analyte interactions [10, 11]. The impact of dissolving the dried analyte samples in the mobile phase before the beginning of the retention process can easily be deduced from the results given in Tables 2 and 3. Retardation factors ( ) for dried spots are substantially lower than for undried spots. In other words, the time-consuming 0.75 0.91 0.95 1.00 0.89 0.93 1.25 0.86 0.91 1.50 0.79 0.89 2.00 0.73 0.83 Table 3 Comparison of values for different amounts of 2-phenylbutyric acid dried after application or developed without drying (microcrystalline cellulose was used as stationary phase and decalin as mobile phase; the volume of sample applied was always 1 µl). The mean velocity of migration of decalin was 0.72 mm min 1. 0.9 0.88 0.92 1.2 0.85 0.89 1.5 0.83 0.87 1.8 0.81 0.85 process of dissolving the analytes at the origin contributes measurably to overall retardation during their migration. It is also interesting to compare differences between values for dried and the wet samples of the alcohol and the acid Journal of Planar Chromatography VOL. 18. JANUARY/FEBRUARY 2005 15

(Tables 2 and 3). For 5-phenylpentanol the greater the concentration of the analyte sample (i.e. the larger the amount of alcohol chromatographed) the more rapid was the growth in differences between values (i.e. values) for dried and wet samples. The behavior of 2-phenylbutyric acid was substantially different. For this compound differences between values obtained for different amounts of sample were relatively stable (within experimental error). These phenomena and the different behavior of the alcohol and acid can most probably be explained on the basis of the different tendency of alcohols and acids to self-associate by hydrogen bonding. 3.1.1 Physicochemical Explanation of the Phenomena Observed It is well known that alcohols self-associate by hydrogen bonding, forming linear associative n-mers with an average number (n) of monomer molecules which grows with increasing concentration of the alcohol in a sample. The velocity of migration of the higher n-mers obviously decreases, which is reflected in a rapid decrease of values. This is especially well pronounced for the dried alcohol samples. It seems readily understandable that for alcohol samples applied to stationary phase without drying the average associative n-mers quasi-automatically consist of a smaller number of the monomers than samples which are dried. Hence the difference between the values (i.e. ) grows rapidly with increasing concentration of the alcohol samples spotted (Table 2). Carboxylic acids self-associate to produce associative multimers of substantially different structure. In experiments in the gaseous, liquid, and solid states it has been confirmed that the acids predominantly form cyclic dimers and that this regularity is barely affected by the concentration of the carboxylic acid solution. This seems the main reason why values are relatively stable and almost unaffected by increasing the concentration of the acid samples spotted (Table 3). 3.1.2 Computer Modeling of the Different Band Widths One other observation worthy of note is that the widths of the peak profiles depend on the mode of application of the analytes to the stationary phase (either with or without drying before start of chromatography). The example of 5-phenylpentanol is more persuasive than that of 2-phenylbutyric acid. In Figure 1 it is clearly apparent that the chromatographic bands obtained from the dried alcohol sample spots are considerably wider than for those developed wet. It should be noted that alcohol was dissolved in n-octane and the acid in decalin. Dissolution of the alcohol in n-octane is faster than that of the acid in decalin, so it seems that the dissolution process plays a minor role in the substantial broadening of the chromatographic bands of the alcohol otherwise more pronounced broadening should be observed for the acid. The phenomenon of substantial band broadening for the dried alcohol spot can be explained by assuming (as was done earlier) that the alcohol forms longer n-mers when dried than when undried. This means that the dried alcohol can form more adsorbed layers on the adsorbent surface than the undried alcohol. Simulated peak profiles calculated for two-layer (Eq. 1) and three-layer (Eq. 2) isotherm models are presented in Figure 3 [12]: (1) Eqs (1) or (2) were coupled with the equilibrium dispersive (ED) model [13]: Although the ED model does not take into the account the crosswise diffusion which always occurs in TLC, the lengthwise spot profiles are qualitatively identical with those calculated from the ED model. For illustrative explanation we used the simple ED model, which was solved for the values w = 0.146 cm min 1, F = 0.25, D a = 0.025 cm 2 min 1, q s = 1 mol L 1, K = 0.05 L mol 1, and K d = 20 L mol 1 ; the initial concentration was 2 mol L 1 and the sample application time was 1 min. From the band profiles presented in Figure 3 it is clearly apparent that the greater the number of adsorption layers the wider the peak observed so the value measured at the maximum of the concentration profile should be lower. The pattern presented in Figure 3 is very similar to that for 5-phenylpentanol developed in the two ways discussed above. An analogous pattern can also be obtained by assuming that the adsorbent surface can bind the monomer alcohol molecules and the dimers, trimers, etc. From the discussion above it can be concluded that the decrease in the retardation factor ( ) for the dried alcohol spot is because of dissolution and more efficient self-association (higher n value of self-associated n-meric aggregates) of the analyte compared with development of the wet spot. 3.2 Analytes Unable to Participate in Lateral Interactions Table 4 Comparison of values for different amounts of anthracene and phenanthrene dried after application or developed without drying (silica gel was used as stationary phase and n-hexane as mobile phase; the volume of sample applied was always 1 µl). The mean velocity of migration of n-hexane was 2.44 mm min 1. Anthracene Phenanthrene Anthracene Phenanthrene 0.050 0.45 0.51 0.075 0.45 0.51 0.100 0.43 0.46 0.150 0.43 0.46 0.200 0.43 0.46 (2) (3) 16 VOL. 18. JANUARY/FEBRUARY 2005 Journal of Planar Chromatography

Table 5 Comparison of values for different amounts of tetralin and perylene dried after application or developed without drying (silica gel was used as stationary phase and n-hexane as mobile phase; the volume of sample applied was always 1 µl). The mean velocity of migration of n-hexane was 2.44 mm min 1. Tetralin Perylene Tetralin Perylene Saturated solution 0.21 0.26 1.0 0.61 0.65 1.5 0.62 0.65 2.0 0.63 0.65 Figure 5 Comparison of concentration profiles for different concentrations of phenanthrene (a) dried after application or (b) developed without drying (silica gel was used as stationary phase and n-hexane as mobile phase; the volume of sample applied was always 1 µl). The mean velocity of migration of n-hexane was 2.44 mm min 1. Figure 4 Comparison of concentration profiles for different amounts of anthracene (a) dried after application and (b) developed without drying (silica gel was used as stationary phase and n-hexane as mobile phase; the volume of sample applied was always 1 µl). The mean velocity of migration of n-hexane was 2.44 mm min 1. The results obtained for analytes unable to participate in lateral interactions are substantially different from those presented in Section 3.1. These results are summarized in Tables 4 and 5 and in Figures 4 7. As is apparent from comparison of the results obtained after spotting hydrocarbon solutions of different concentration, the Figure 6 Comparison of the concentration profiles of perylene dried after application (- - - -) or developed without drying ( ) for saturated solutions of the analyte in toluene (silica gel was used as stationary phase and n-hexane as mobile phase; for both samples the volume applied was 1 µl). The mean velocity of migration of n-hexane was 2.44 mm min 1. values remain almost constant (within the error limits), irrespective of the amounts of analytes analyzed (Figures 4 7). This observation is equally valid for chromatograms obtained from applied spots initially dry or wet. There is, however, a sub- Journal of Planar Chromatography VOL. 18. JANUARY/FEBRUARY 2005 17

symmetrical Gaussian-type shapes, irrespective of whether or not the spots were dried. For perylene (developed either dried or wet; Figure 6) a weak shift from the Gaussian-type concentration profile to the Langmuir-type profile is apparent; for tetralin (Figure 7) the concentration profiles are indisputably of the Langmuir type. Whether Gaussian or Langmuir, the concentration profiles of the hydrocarbons discussed in this paper give evidence of the absence of lateral interactions in these chromatographic systems, which is because of the absence of the functional groups from the molecular structures of these compounds. Acknowledgment The authors wish to thank Merck KGaA (Darmstadt, Germany) for supplying the test compounds and the precoated cellulose plates used in this study. References Figure 7 Comparison of concentration profiles for different amounts of tetralin (a) dried after application or (b) developed without drying (silica gel was used as stationary phase and n-hexane as mobile phase; the volume of sample applied was always 1 µl). The mean velocity of migration of n-hexane was 2.44 mm min 1. stantial difference between values obtained from these two kinds of application, analogous to that reported in the preceding section. Chromatograms obtained from initially dried analyte spots furnish lower values than those obtained from analyte spots developed while still wet (Tables 4 and 5). It can also be concluded that for analytes unable to self-associate by hydrogen bonding the impact of dissolution of the analyte in the mobile phase measurably affects (i.e. reduces) the overall mobility of the test sample in the chromatographic system. On comparing the shapes of the concentration profiles obtained from anthracene, phenanthrene, perylene, and tetralin one must admit the differences are minor and barely perceptible. The profiles of anthracene and phenanthrene (Figures 4 and 5) have [1] C.F. Poole, A Contemporary View of the Kinetic Theory of Planar Chromatography. In: Sz. Nyiredy (Ed.) Planar Chromatography. A Retrospective View for the Third Millennium, Springer, Budapest, 2001, pp. 13, 14, 24. [2] G. Guiochon and A. Siouffi, J. Chromatogr. Sci. 16 (1978) 470 481. [3] A.M. Siouffi, F. Bressolle, and G. Guiochon, J. Chromatogr. 209 (1981) 129 147. [4] G. Guiochon and A.M. Siouffi, J. Chromatogr. 245 (1982) 1 20. [5] F. Geiss, J. Planar Chromatogr. 1 (1988) 102 115. [6] Sz. Nyiredy, Zs. Fatér, L. Botz, and O. Sticher, J. Planar Chromatogr. 5 (1992) 308. [7] B.G. Belenky, V.V. Nesterov, E.S. Gankina, and M.M. Smirnov, J. Chromatogr. 31 (1967) 360 368. [8] B.G. Belenky, V.V. Nesterov, E.S. Gankina, and M.M. Smirnov, Zh. Fiz. Khim. 42 (1968) 1484 1489. [9] J.M. Mierzejewski, Chem. Anal. (Warsaw) 20 (1975) 77 89. [10] K. Kaczmarski, M. Sajewicz, A. Pieniak, R. Piêtka, and T. Kowalska, J. Liq. Chromatogr. Related Technol. 27 (2004) 1967 1980. [11] M. Sajewicz, A. Pieniak, R. Piêtka, K. Kaczmarski, and T. Kowalska, J. Liq. Chromatogr. Related Technol. 27 (2004) 2019 2030. [12] K. Kaczmarski, W. Prus, C. Dobosz, P. Bojda, and T. Kowalska, J. Liq. Chromatogr. Related Technol. 25 (2002) 1469 1482. [13] G. Guiochon, S.G. Shirazi, and A.M. Katti, Fundamentals of Preparative and Nonlinear Chromatography, Academic Press, Boston, MA, 1994. Ms received: March 22, 2004 Accepted by SN: December 13, 2004 18 VOL. 18. JANUARY/FEBRUARY 2005 Journal of Planar Chromatography