The Influence of the Specific Surface Area of Adsorbent upon the Optimization of the Process of Adsorption Thin-Layer Chromatography

Abstract

An important aspect of the influence of specific surface area of adsorbent on R_M values of substances obtained in the process of adsorption thin-layer chromatography has been presented in the paper. The experiments have been conducted with four adsorbents, each having different specific surface area, that is, from 50 to 500 m²/g and with the use of mixed binary solvent mobile phases of benzene-aliphatic alcohol type. It has been proved that R_M values of investigated substances of group B change regulary according to the specific surface area of adsorbent for individual concentrations of mobile phase. The relationship may be described by means of square trinominal. The parameters of trinominal may be tabulated and the differentiation of the parameters for particular substances is observable at the same time. The relationship in question may be utilized to calculate R_M, values of studied substances on any adsorbent with known specific surface area.     

The Role of the Specific Surface Area of an Adsorbent in the Optimization of Mixture Separation Conditions in Thin-Layer Chromatography

Abstract

The very important aspect of effect of the magnitude of specific surface area on R_M values obtained by using TLC method is presented. In experiments were performed on 4 adsorbents of different specific surface areas 50 – 500 m²/g and with mixed binary mobile phases.

It is shown that R_M values of chromatographed substances aromatic hydrocarbons are lineary dependente upon the specific surface area of adsorbents for each composition of mobile phase. This relationship can be described by a straight line, with the parameters a and b that can be tabularized. These lines can be used to calculate the R_M values of chromatographed substances for any adsorbent if is specific surface area is known. The illustrational comparison of experimentally obtained and theoretically predicted R_M values for different chromatographic system are presented.     

Comparison of Methods for Determination of the RM Values of Substances in Thin - Layer Chromatography by Using Binary Mobile Phase

Abstract

Methods of calculating the function R_M= f(?₁) of some aromatic substances from the data of experimental and theoretically calculated parameters have been compared. It has been shown that there is a possibility to calculate this function with high accuracy on the basis of the measured partition coefficients of the substances chromatographed in partition systems consisting of components of the mobile phase as well as parameters obtained theoretically. Thus obtained results were also compared with the functions R_{M 1.2} = f (?₁) obtained experimentally and a high agreement was reached. This in turn contributes to optimization of systems of thin-layer chromatography.     

Determination of the lipophilicity of some aniline derivatives by reversed-phase thin-layer chromatography. The effect of the organic phase in the eluent

Summary The lipophilicity of aniline and 36 ring-substituted aniline derivatives was determined by reversed-phase thin-layer chromatography using methanol, acetone and acetonitrile as the organic phase. The R_M value of each compound linearly decreased with increasing concentration of the organic solvent, Acetonitrile showed the highest and methanol the lowest solvent strength, however, the difference between the solvent strength of methanol and acetone was negligible. The site of the substitution considerably influenced the lipophilicity particularly in the case of –NO₂ groups. Good correlation was found between the R_M value extrapolated to zero organic phase concentration and the partition coefficient between n-octanol:water. The change in the R_M value caused by a 1% increase of the organic phase concentration also correlated with the partition coefficient, however, it was of secondary importance. The lipophilicity parameters determined by reversed-phase thin-layer chromatography correlated to a lesser extent with the calculated lipophilicity values of aniline derivatives.     

Possibility of calculating the Rm values in thin-layer chromatography using a ternary mobile phase

Summary On the basis of an equation derived it is possible to establish theoretically the R_M values for a ternary mobile phase. The R_M values of phenol and its derivatives, naphthalene and its derivatives and some polycyclic aromatic hydrocarbons were measured. A good agreement has been found between the calculated and the experimentally obtained R_M values. Analysis of the results showed some regularities of certain values obtained in binary systems when related to the ternary mobile phase.     

Chromatographic retention data of N-alkylamides of benzomorpholine-2-carboxylic acid in the study of structure-activity relationships

Summary The adrenolytic activity of N-alkylamides of benzomorpholine-2-carboxylic acid was correlated to their log k and R_M values determined by reversed-phase liquid chromatography and thin-layer chromatography. Mixtures of water with several organic modifiers were used as the mobile phase.     

Reversed-Phase Systems for the Separation of Coumarins and Furocoumarins by Thin-Layer and High-Performance Liquid Chromatography

Abstract

The retention behaviour of eleven derivatives of counarin was investigated by reversed-phase (RP) thin-layer and high performance liquid chromatography. The linear relationships between log k'or R_M values and the content of organic modifier in the aqueous mobile phase obtained for wide composition ranges indicate that the plots can be used to determine R_Mw and log k_w values by extrapolation to pure water. The effects of individual substituants on the retention and the correlation between TLC and HPLC data was analysed.     

Theoretical determination of optimum composition of the binary mobile phase for distribution of substances in thin-layer chromatography

The measure of the distribution of a mixture of substances in TLC with a binary mobile phase is expressed as ΔRM which represents the difference between the R_M values of substances i and j on use of the binary solvent 1 + 2 as the mobile phase. The possibility of determining its maximum value at an optimum composition of the binary mobile phase is demonstrated in this paper. This value can calculated from experimental and theoretical functions R_M1,2 = f(Φ₁) of substance i and j. More simply, ΔRM can be calculated theoretically from easily measurable adsorption parameters of excess adsorption isotherms and from chromatographic parameters obtained for pure solvents. The ΔRM value calculated theoretically can be utilized in a pilot technique for determination of the optimum composition of the mobile phase in gradient liquid chromatography.     

The Correlations between Capacity Factors k′ and RM Values in Liquid Column and Thin-Layer Chromatography

Abstract

The purpose of this studie being carried out is to define some regularities between Thin-layer and Liquid Column Chromatography; i.e. between experimental obtained and theotetical calculated R_M-values and experimental obtained logk-values for some non-active organic substances in mixed binary solvents systems. It was studied the kind these correlations change with changing mobile phase compositions. The present paper is a certain aspect of new studies on possibilities of the use Thin-layer Chromato-graphy as a pilot technique for Liquid Column Chromatography (1,2). In a study on the optimization of chromatographic process it is necessary to define the correlations between R_M-values which characterize the retention in TLC and k′-values which characterize the retention in in LCC.

In the paper the changes of these correlations with the changes of mobile phase compositions are studied. The conformity between theoretical calculated and experimental obtained R_M-values of chromatographed substances for differemt mobile phase compositions are investigated. A good agreement between theoretical and experimental data makes possible to foresee on the basis of the correlations between log k′- and R_M-values the retention in column chromatography.     

Thin-layer adsorption chromatography with mixed mobile phases. 1. Characterization of chromatographic systems showing energetic heterogeneity of adsorbent surfaces with regard to admolecules of solvents and solutes

Thin-layer adsorption chromatography with a multicomponent mobile phase is discussed. A new equation for predicting R_M-values in TLC with mixed mobile phases using the R_M-ValUeS obtained for pure solvents is proposed. This equation takes into account effects of energetic heterogeneity of the adsorbent surface with regard to adsorbed molecules of solvents and solutes. Experimental verification of this equation is presented for R_M-data obtained by TLC using a binary mobile phase.     

Retention data from reverse‐phase high‐performance thin‐layer chromatography in characterization of some bis‐salicylic acid derivatives

The chromatographic behaviour of salicylic acid derivatives was investigated using reversed‐phase high performance thin‐layer chromatography (RP HPTLC) with methanol–water and dioxane–water binary mixtures as mobile phase in order to establish relationships between chromatographic data and selected physico‐chemical parameters that are related to ADME (absorption, distribution, metabolism and elimination). Some of the investigated compounds were screened for antioxidant activity. Examination of chromatographic behaviour revealed a linear correlation between R_M values and the volume fraction of mobile phase modifier. Obtained R_M⁰ values were correlated with lipophilicity, solubility, human intestinal absorption, plasma‐protein binding, and blood–brain barrier data. The comparison among chromatographic data obtained by two mobile phase was performed with a statistical technique, principle component analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Lipophilicity determination of some photosynthetic pigments by reversed-phase thin-layer chromatography. The effect of support and the organic phase in the eluent

Summary The lipophilicity of some photosynthetic pigments was determined by reversed-phase thin-layer chromatography using silica, alumina, cellulose, polyamid and diatomaceous earth as supports and acetone and ethanol as organic mobile phase. The R_M value of each compound linearly decreased with increasing concentration of the organic modifier. The supports exerted high impact on the separation: no acceptable separation was achieved on cellulose, polyamid, alumina and diatomaceous earth supports while silica supports produced the best separation. The retention of photosynthetic pigments depended on the origin of silica and on the type of organic mobile phase.     

Lipophilicity study of eight cephalosporins by reversed‐phase thin‐layer chromatographic method

The lipophilicity (R_M0) and specific hydrophobic surface area for the representatives of four generation cephalosporins have been determined by reversed‐phase thin‐layer chromatography, and the effect of different mobile‐phase modifiers (such as methanol, acetonitrile, acetone, 1,4‐dioxane and 2‐propanol) on the retention has been studied. The compounds studied showed typical retention behavior; their R_M values decreased linearly with increasing concentration of the organic modifier in the eluent. The linear correlations between the volume fraction of the organic solvent and the R_M values over a limited range were established for each solute, resulting in high values of correlation coefficients (>0.95 in most cases). R_M values were determined by various concentrations of organic modifier, and the correlation obtained was extrapolated to 0% of organic modifier. Chromatographically established logP (R_M0) parameters were compared with computationally calculated partition coefficients values (AClogP, ALOGP, KOWWIN, ALOGPs, XLOGP2, MLOGP and XLOGP3) and experimental octanol–water logP values (measured by the shake flask method). The received results demonstrate that RP‐TLC may be a good alternative technique for analytics in describing the lipophilic nature of investigated cephalosporins as well as the activity. Copyright © 2015 John Wiley & Sons, Ltd.     

Adsorption TLC with binary mobile phases

Summary An equation describing R_M values in TLC with a binary mobile phase has been derived and examined by using TLC data. Two different methods have been proposed to determine the chromatographic parameters characterizing energetic heterogeneity of the adsorption system and solute-solvent interactions. These methods differ in the estimation of the mole fractions of solvents in the surface phase. In method I an analytical equation for evaluating these mole fractions is assumeda priori. Method II utilized the excess adsorption isotherm measured for the mobile phae-adsorbent system.     

A simple molecular model for adsorption chromatography. VII. Relationship between the RM value and the composition for phenols in systems of the type (cyclohexane+polar solvent)/silica

Summary The effect of the composition of binary developing solvents on R_M values of phenols with one, two or three hydroxyl groups was investigated. In most cases linear R_M vs. (log X_S) relationship were obtained for wide composition ranges (X_S — mole fraction of polar solvent; diluting solvent: cyclohexane), the slope of the lines being related to the molecular mechanism of adsorption. The slopes of some derivatives of quinoline, aniline and phenols are compared and the effects of the molecular structure of the solute and the eluen strength of solvent are discussed. The experimental data for phenols are interpreted in terms of competitive solvation in the mobile phase by electron donor solvents; practical rules concerning optimization of TLC systems for the analysis of the compounds investigated are formulated.     

Isolation and characterization of methoxylated flavones in the flowers of Primula veris by liquid chromatography and mass spectrometry

Characterization of six flavones, which were named substances G1, G2, G3, G4, G5 and G6 according to their R_F values in normal-phase thin-layer chromatography, is reported. The pure flavones were purified after maceration with methanol by normal-phase solid-phase extraction, normal-phase medium-pressure liquid chromatography, normal-phase preparative thin-layer chromatography and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected fractions of several isolation steps were analyzed by normal-phase (NP) and RP-HPLC. Detection and identification of the substances G was accomplished by UV detection at 213–216 nm, diode array UV detection, or fluorescence detection (λ_ex=330 nm; λ_em=440 nm). The molecular mass, the elementary composition, and the structure of the six components was determined by electron-impact high-resolution mass spectrometry (EI-HRMS). Substance G4 was identified as 3′,4′,5′-trimethoxyflavone. The substances G1–G6 were shown to be mono-, di- tri- and pentamethoxyflavones. HPLC–electrospray ionization tandem mass spectrometry (ESI-MS–MS) of the flavones was carried out employing a 150×2 mm I.D. column packed with a 3 μm/100 Å octadecylsilica stationary phase and a mobile phase comprising 1.0% acetic acid in water–acetonitrile (50:50). Comparative RP-HPLC–ESI-MS of the raw methanol extract and the isolated substances G1–G6 proved that the isolated compounds were pure and were not artifacts. Finally, RP-HPLC–ESI-MS–MS was used to identify substances G1–G6 in phytopharmaceutical drugs.     

Spot and Mobile-Phase Front Anomalies in Planar (Thin-Layer) Chromatography

The advantages and variations of thin-layer chromatography are described in detail. The displacement mode of development is generated in a relatively short distance, and the fully developed displacement train concentrates sample components even from an elongated spot. Two-dimensional TLC using both elution and displacement thin-layer chromatography enables excellent separation of spots; results from 2D TLC cannot, however, be derived directly from two one-dimensional runs. Secondary mobile-phase fronts occur because of the dry state of stationary phase, which absorbs the mobile phase. Occasional problems with the reproducibility of R_F values arise because of adsorption of the mobile phase components by the dry stationary phase and evaporation at the solvent front. The use of 2D TLC in pharmacognosy enables separation of the components and also helps in the planning of preparative-scale separations by classical column chromatography and/or HPLC.     

Optimization of Solvent Composition for High Performance Thin-Layer Chromatography

Abstract

The methodology of mixture experiments has been applied to optimize the multicomponent solvent composition for high performance thin-layer chromatography (HPTLC). In this study, a mixture of five solvents for the mobile phase was maximized for the separation of twelve structurally related B(a)P metabolites on normal phase HPTLC plates. Volume fractions (rather than absolute quantities) of the mobile phase solvents were constrained by upper bounds by considering solvent strength. These restrictions defined an experimental region that is an irregular polyhedron formed from a truncated 4-dimensional simplex. The optimization of the expected separation response over the experimental region was based on a second-order Scheffé polynomial estimated from twenty-five experimental runs. Six functions for measuring separation based on R_f values were evaluated. A new response function is proposed that overcomes the difficulties of previously published criteria.     

Computer-aided optimization of stepwise gradient and multiple-development thin-layer chromatography

Summary Equations for the final R_F values for mobile phase gradient development and gradient multiple development are presented. Computer simulations of gradient development for both modes of thin-layer chromatography are discussed for the preliminary optimization of gradient programs.