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BecTHHK Cn6ry. Cep. 4. 2013. Ban. 2
J. Polanski, M. Sajewicz, M. Knas, A. Zywocinski, M. Weloe, T. Kowalska
TEMPERATURE EFFECT WITH MOLECULAR ROTORS IN THIN-LAYER CHROMATOGRAPHY
Introduction. Microcrystalline chirality of silica gel utilized as stationary phase in thin-layer chromatography (TLC) was demonstrated with use of the circular dichroism spectroscopy (CD) [1] and discussed in the context of the observed deviations of the migration route of the selected chiral analytes from linearity (e.g., [2-4]). In paper [5], a mini-review was provided, summarizing our initial findings referring to these deviations and pointing out to the two-dimensional (2D) effective diffusion, inherent of the chiral thin-layer chromatographic systems, as an important factor contributing to the reported phenomenon. At the same time, we felt that the explanation of this striking effect with the chirality of stationary phase and the 2D effective diffusion does not sufficiently enough cover the question.
In our more recent studies [6, 7], the deviation of the migration route of the selected (mostly chiral) analytes from linearity in the silica gel based thin-layer chromatographic systems was revisited and now the effect was concisely labeled as lateral relocation. This time, we focused our attention on molecular structure of chiral analytes apt to lateral relocation and we perceived that all these compounds bear an undeniable structural similarity to the propellers equipped with one, two, or even more freely rotating blades. On the one hand, lateral relocation poses a quantification problem with certain analytes (considerably invalidating densitometic detection and quantification thereof) yet on the other hand, it might prove profitable for nanotechnology, opening a new testing ground for molecular gears. Further, we have assumed that the propeller-like structures of the investigated analytes can be viewed as molecular rotors, hence in the planar chromatographic systems they can be subjected to the same Magnus force, which affects the rotating objects in the macroscopic systems and deviates the trajectories of their motion from linearity [6, 7].
Without answering the question, whether the analogy between the forces acting on the rotating objects on the molecular level and in the macroscopic world is physically justified, in this study we compare the magnitudes of lateral relocation for a selected set of propeller-like analytes at the two working temperatures (4 °C and 22 °C). The starting point of our considerations is the capillary forces, because the analytes move in the thin-layer chromatographic systems owing to the capillary action. The free-energy gain AEm of a solvent entering a capillary is given by the following empirical relationship [8]:
r
where y is the free surface tension; Vn denotes the molar volume of the solvent; r is the
Jaroslaw Polanski — professor, University of Silesia, Katowice, Poland.
Mieczyslaw Sajewicz — Dr., University of Silesia, Katowice, Poland.
Magdalena Knas — PhD student, University of Silesia, Katowice, Poland.
Andrzej Zywocinski — Dr., Institute of Physical Chemistry PAS, Warsaw, Poland.
Marcel Weloe — student, University of Mainz and University of Silesia, Katowice, Poland.
Teresa Kowalska — professor, University of Silesia, Katowice, Poland; e-mail: teresa.kowalska@us.edu.pl
© J. Polanski, M. Sajewicz, M.Knas, A. Zywocinski, M. Weloe, T. Kowalska, 2013
capillary radius. From Eq. (1), it follows that the free surface tension (y) is very important for capillary flow, and a higher tension leads to more efficient flow. It is also well known from the fundamentals of physics that the lower the temperature of a given liquid, the higher is the respective free surface tension. The consequence of this interdependence in the thin-layer chromatographic practice is that due to the more efficient capillary flow at lower temperatures, the development of the thin-layer chromatograms usually goes faster than at an ambient temperature [9-11]. Faster development of the chromatograms means faster flow of the mobile phase and hence, a stronger blow of the molecular propeller blades, which might result in stronger pronounced lateral relocation of the test analytes also.
However, the temperature-drop induced acceleration of the analyte's motion in the faster flowing mobile phase can be counterbalanced by the opposite effect, which is the stronger intermolecular forces acting between an analyte and stationary phase at a lower temperature than at an ambient one [9-11]. These stronger intermolecular interactions result in shortening of the analyte's migration route and a lower numerical value of the retention parameter (RF), which can apparently curb an extent of the lateral relocation effect.
The aim of this study is to gain a practical insight in the temperature dependence of lateral relocation with the selected propeller-like analytes. The lateral relocation effects observed at the lower working temperature (i. e., at 4 C) are going to be interpreted as an interplay between the opposite stimuli of the accelerated mobile phase flow and the stronger analyte — stationary phase intermolecular forces.
Experimental.
Test analytes. The following four commercially available test analytes were used in our experiment: (S)-2-phenylpropionic acid, S(+)-naproxen, and ruthenium(III) acetylacetonate (Sigma-Aldrich, St. Louis, MO, USA; cat. nos 279900-1, 28478-5 and 282766, respectively); and 4-aminobenzoic acid (Acros Organics, New Jersey, USA, cat. no. 146212500). The four non-commercial ferro compounds, i. e., (S)-4-nonanoyloxy-4'-(2-methylbutyloxycar-bonyl)biphenyl (S-ferro A), (S)-4-[4-(1-methylheptyloxycarbonyl)phenyl]-4'-[6-(cyanoeta-noyloxy)pentyloxy]-biphenyl-4-carboxylate (S-ferro C5), (S)-4-[4-(1-methylheptyloxycarbo-nyl)phenyl]-4'-[6-(cyanoetanoyloxy)hexyloxy]biphenyl-4-carboxylate (S-ferro C), and (S)-4-(3-methyl-2-chloropentanoyloxy)-4'-heptyloxybiphenyl (S-ferro D), were synthesized in the Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland. Chemical structures of the test analytes are given in Table 1. All the organic solvents used in the experiments were of the analytical purity grade, and water was double distilled and de-ionized (with use of the Elix Advantage model Millipore system, Molsheim, France).
Stationary phases. Thin-layer chromatographic experiments were performed on the commercial glass plates (20 x 20 cm2) precoated with the 0.25 mm thick silica gel 60 F254 layer (Merck; cat. no. 1.05715.0001). Before use, each plate was carefully washed by pre-development with methanol + water, 9 : 1 (v/v), and then dried at ambient temperature for 3 h. Then one part of the washed and dried plates was impregnated with the solution of L-arginine (Merck, cat. no. 1.01542.0100) in methanol by conventional dipping for 2 s. Concentration of L-arginine was equal to 3 • 10_1 mol/L and it was calculated in such a way as to deposit 0.5 g L-arginine per 50 g of the dry silica gel layer. The impregnated chromatographic plates were again dried at ambient temperature for 3 h. The other part of the washed and dried plates was left non-impregnated. Finally, the preconditioned adsorbent layers of the two types were ready for the experiments. Details concerning stationary phases used for each individual test analyte are given in Table 1.
Mobile phases. The one-directional (1D) development of the test compounds was carried out at 4 ± 1 °C and 22 ± 1 °C for the distance of 15 cm, using the mobile phases listed
Table 1
The investigated test analytes, their chemical structures, numbers of the respective propeller blades, the employed stationary and mobile phases, and the individual scanning wavelengths, k
Molecular No. X, nm
Test analyte structure in (2) propeller blades Stationary phase Mobile phase (v/v)
(S')-2-Phenylpropionic acid 1 1 silica gel impregnated with L-arginine Acetonitrile (ACN) + methanol (MeOH) + water (H20), 5:1:0.75 210
S-(+)-Naproxen 2 1 silica gel impregnated with L-arginine ACN + MeOH + H20, 5:1:1.5 210
Ruthenium(III) acetylacetonate (optically inactive) 3 3 silica gel impregnated with L-arginine Ethyl acetate (EtAc) 280
S-Ferro A A 2 silica gel n-Hexane (HA) + 2-propanol (PrOH), 8:2 280
S-Ferro C5 5 2 silica gel HA + 2-PrOH, 7:3 280
S-Ferro C 6 2 silica gel HA + 2-PrOH, 8:2 310
S-Ferro D 7 2 silica gel HA + 2-PrOH, 8:2 260
4-Aminobenzoic acid 8 2 silica gel HA + 2-PrOH, 8:2 290
in Table 1. In the case of the plates impregnated with L-arginine, the mobile phases have contained an additional amount of 0.5 % (v/v) glacial acetic acid to fix pH < 5.
Thin-layer chromatography (TLC). In the thin-layer chromatographic experiments, we used the solutions of the test analytes in the organic solvents at the concentration equal to 1.00 mg/mL. With one compound (S-ferro C5), the lower concentration of 0.2 mg/mL was used to obtain the optimum experimental conditions. Each sample was applied to the plate 1.5 cm above the lower edge in the aliquot of 2 ^L/spot. Application of the samples was made with use of the AS 30 model autosampler (Desaga, Heidelberg, Germany), equipped with the Windows-compatible ProQuant software. Five samples in an equal distance of 1.5 cm, or three samples in an equal distance of 2.5 cm from one another were applied per one plate, and then the chromatograms were developed in the 1D mode. After the development, the plates were dried at ambient temperature for 3 h, and each plate was densitometrically scanned in the 1-mm intervals, in the direction of the development. Upon these parallel scans, the 2D chromatographic bands were constructed. Densitometric scanning was performed in the ultraviolet (UV) light from the mercury lamp (in the reflectance mode) at an appropriate wavelength, carefully chosen for each individual test analyte and given in Table 1 (to provide maximum sensitivity for each analysis). Maxima of the concentration profiles were used for calculation of the respective RF values and for calculation of the lateral relocation effects (understood as a difference between the maximum of the concentration profile and the trajectory running perpendicularly to the lower edge of the chromatographic plate and starting at the sample spotting site). Each chromatographic experiment was performed at least in triplicate (and in most cases, there were between seven and ten repetitions).
Results and discussion. The results obtained in this study are summarized in Table 2 and illustrated by Fig. 1 and 2. The results given in Table 2 indicate that with the propeller-shaped molecules, lateral relocation appears at both investigated temperatures (i. e., at 4 °C and 22 °C). The purposely employed test compound (4-aminobenzoic acid) shows no lateral relocation effect, independent of the working temperature applied. This finding additionally emphasizes the fact that lateral relocation phenomena are inherent of the chiral and/or propeller-like analytes. With each chiral and/or propeller-like compound, the maximum magnitude of lateral relocation observed at the lower temperature (4 °C) was lower, or barely the same, as at 22 °C. In most cases, the development times and the RF values were also lower at the lower temperature, as extensively discussed in the classical papers on the subject [9-11]. The less pronounced lateral relocations and the lower RF values observed at the lower temperature can be explained with stronger intermolecular forces between the test compounds and stationary phase at the lower temperature than at an ambient one. Moreover, it has to be admitted that no definite directional trends with lateral relocation of the investigated compounds were observed (as indicated in Table 2, and Fig. 1 and 2). At the moment, it seems rather difficult to explain this directional randomness of the observed lateral relocations. One significant reason could be the different handedness of the enantiomeric excess with silica gel from one batch of the bulk product (and consequently, from one chromatographic plate) to another. Although the industrial precipitation of silica gel for TLC is not stereospecific, the precipitate is not strictly racemic either, but scalemic (as demonstrated in paper [1]), possibly with random predominance of the left-handed, or the right-handed microcrystalline form, depending on the employed precipitate batch. Another reason can be the molecular structure of certain propeller-like analytes which induces directional randomness of lateral relocation. Finally, one can anticipate a combination of the random enantiomeric excess of silica gel and specificity of
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Fig. 1. 2D chromatograms obtained at 22 C and derived from the respective densitometric scans for (S)-2-phenylpropionic acid (a); S-(+)-naproxen (b); ruthenium(III) acetylacetonate (c); S-ferro A (d); S-ferro C5 (e); S-ferro C (f); S-ferro D (g) and 4-aminobenzoic acid (h): emphasizing lateral relocations of these test analytes and the lack of lateral relocation for last test analyte; lateral relocation is purposely emphasized by the black line, i. e., the trajectory running perpendicularly to the lower edge of the chromatographic plate and starting at the sample spotting site
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Fig. 2. A comparison of the temperature-dependent handedness and magnitude of lateral relocation for S-(+)-naproxen (a); S-ferro C (b), and S-ferro D (c): lateral relocation is purposely emphasized by the black line, i. e., the trajectory running perpendicularly to the lower edge of the chromatographic plate and starting at the sample spotting site
a
a given molecular structure with a given propeller-resembling analyte as jointly responsible for the directional randomness of lateral relocation.
In Fig. 1, we present the 2D chromatograms valid for the temperature of 22 °C and derived from the respective densitometric scans for the seven chiral compounds and one non-chiral compound, in this study used as the test sample (see Table 1). For graphical demonstration, we chose the higher working temperature (22 °C) with the better pronounced deviation of the analytes' migration tracks from linearity. Lateral relocation of the analytes is purposely emphasized by the black line, i. e., the trajectory running perpendicularly to the lower edge of the chromatographic plate and starting at the sample spotting site.
In Fig. 2, we compare the temperature dependence of lateral relocation with the three out of seven chiral test analytes, i. e., with 5-(+)-naproxen, 5-ferro C, and 5-ferro D. In the first two cases, lateral relocation was at the two investigated temperatures left-handed, and in the last case (5-ferro D) it was left-handed at 4 °C and right-handed at 22 °C. Let us remind that from the data presented in Table 2 it is evident that even at one and the same temperature, the test analytes could demonstrate either the left-handed, or the right-handed lateral relocation, depending on the experiment performed.
Table 2
The investigated test analytes, working temperatures, the obtained retardation factor (Rf) values, handedness, and maximum magnitudes of lateral relocation, mm
Test analyte Working temperature, °C Development time, h RF Handedness of lateral relocation Maximum magnitude of lateral relocation, mm
(S)-2-Phenylpropionic acid 4 0.40 0.64 right 2.0
22 1.30 0.65 right 2.0
S-(+)-Naproxen 4 0.55 0.82 left 3.0
22 1.00 0.90 left 3.0
Ruthenium (III) acetylacetonate (optically inactive) 4 0.55 0.81 right 2.0
22 1.25 0.81 right 3.0
S-Ferro A 4 1.15 0.87 left right 2.0 2.0
22 1.40 0.96 left 4.0
S-Ferro C5 4 1.10 0.72 left 3.0
22 1.15 0.76 right 5.0
S-Ferro C 4 1.00 0.61 left right 3.0 3.0
22 1.40 0.71 left 3.0
S-Ferro D 4 1.10 0.89 left 3.0
22 1.45 0.98 right 5.0
4-Aminobenzoic acid 4 1.15 0.19 none 0
22 1.30 0.39 none 0
Conclusions.
— Development of the chromatograms was faster at 4 °C than at 22 °C, which has a straightforward physicochemical explanation.
— Lateral relocation of the selected analytes was observed at the two working temperatures (i. e., at 4 C and 22 °C).
— Magnitudes of lateral relocation and the respective RF values were lower at 4 °C than at 22 C, which can be explained by stronger intermolecular forces between the analyte molecules and the adsorbent (silica gel) at the lower temperature than at an ambient one.
— The directional randomness of lateral relocation was established with six chiral test analytes and one non-chiral test analyte, which at this stage is difficult to interpret and certainly deserves an additional reflection and study. One reason can be the random handedness of the enantiomeric excess with different batches of the silica gel precipitate used as stationary phase. Another reason can be the molecular structure of certain analytes, which induces directional randomness of lateral relocations. Finally, one can anticipate a combination of the random enantiomeric excess of silica gel and specificity of a given molecular structure with a given analyte as jointly responsible for the directional randomness of lateral relocation.
Ms Magdalena Knas is the scholarship recipient within the framework of the "DoktoRIS Scholarship Programme for the Innovative Silesia", subsidized by the European Social Fund of the European Union.
References
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Статья поступила в редакцию 10 сентября 2012 г.
