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For the first time, stepwise pH gradient thin-layer chromatograms of biologically active substances with controlled developing solvent velocity are presented and described in the paper. Change in buffer pH of the mobile phase solution influences retardation, selectivity, and shape of the separated substances' spots. The conducted research has confirmed that the mobile phase's pH gradient could be an essential factor to optimize the conditions of the separation of substances in reversed-phase high-performance thin-layer chromatography. The reproducibility of the gradient retardation factor values of separated substance zones is satisfactory.

Fast and reliable methods for the determination of hydrophobicity and acidity are desired in pre-clinical drug development phases to eliminate compounds with poor pharmacokinetic properties. Reversed-phase high-performance liquid chromatography (RP HPLC) coupled with time-of-flight mass spectrometry (RP HPLC-ESI-TOF-MS) is a convenient technique for that purpose. In this work we determined the chromatographic measure of hydrophobicity (logkw) and dissociation constant (pKa) simultaneously for a large and diverse group of 161 drugs. Retention times were determined by means of RP HPLC-ESI-TOF-MS for a series of pH and organic modifier gradients. We were able to measure retention times for 140 out of 161 (87%) compounds. For those analytes logkw and pKa parameters were calculated and compared with literature and ACD Labs-calculated data. The determined chromatographic measure of hydrophobicity and dissociation constant was closely related to literature and theoretically calculated values. Applied methodology achieved the medium-throughput screening rate of 100 compounds per day and proved to be a simple, fast and reliable approach of assessing important physicochemical properties of drugs. This technique has certain limitations as it is not applicable for very hydrophilic analytes (logP

Binding to melanin is considered to be a reason for several adverse effects of drugs and should be known to reduce the failure rate due to inappropriate pharmacokinetics in search for better pharmaceuticals. A new, reliable and convenient method of determination of affinity of drugs and drug candidates to melanin has been proposed employing magnetic beads. For that aim the reaction conditions to effectively covalently immobilize melanin on surface of superparamagnetic beads have been determined. Binding efficiency of melanin towards antipsychotic and other basic drugs has been determined and compared to that obtained in the affinity HPLC systems employing aminopropylsilica stationary phases with immobilized melanin. The magnetic beads method provided melanin binding data correlating well with the ability of agents to evoke extrapyramidal symptoms. Quantitative structure-property relationships have been derived describing the melanin binding efficiency in terms of structural descriptors of drugs from calculation chemistry. Thus, an approach has been proposed to evaluate a priori melanin binding potency of drug candidates based solely on their chemical formula.

The purpose of this work was to propose a theoretical model of the combined pH/organic modifier gradient in reversed-phase high-performance liquid chromatography (RP HPLC) with special emphasis on its applicability to polyprotic analytes. The model was developed and approximated to be useful for a data set comprising organic modifier gradients obtained at different pH changes and different gradient durations. It was evaluated regarding its ability to describe experimental data. The chromatographic pKa and lipophilicity parameter, log k w , were obtained by fitting to the proposed model and comparing to the literature values.

Silica-based stationary phases are commonly used in liquid chromatography, but their surface acidity causes known problems, especially when separating basic compounds. Deleterious effects of free silanols are not fully removed by standard prevention procedures consisting in adding alkylamines or other amino quenchers to the eluents. We found that ionic liquids of the imidazolium tetrafluoroborate class, added to mobile phases at concentrations of 0.5-1.5% (v/v), blocked silanols and provided excellent thin-layer chromatographic separations of strongly basic drugs which were otherwise not eluted, even with neat acetonitrile as the mobile phase. The silanol suppressing potency of imidazolium tetrafluoroborates was demonstrated to markedly exceed that of the standard mobile phase additives, like triethylamine, dimethyloctylamine and ammonia. The proposed new mobile phase additives were also demonstrated to provide reliable lipophilicity parameters of base drug analytes as determined by gradient mode of high-performance liquid chromatography. By applying the readily available and environmentally friendly imidazolium tetrafluoroborate ionic liquids, simple and efficient means of improvement of liquid chromatographic analysis of organic bases were elaborated.

Quantitative structure-retention relationships (QSRR) have been employed in studying the molecular mechanism of chromatographic separations under pressure- (HPLC) and electro-driven (CEC) conditions. Logarithms of retention factors corresponding to zero percent of organic modifier in aqueous eluent, log k(w), were determined on eight reversed-phase stationary phases under both HPLC and CEC conditions at similar eluent flow velocities. QSRR equations describing log k(w) in terms of linear solvation energy relationship (LSER) parameters of analytes, in terms of simple structural descriptors acquired by calculation chemistry, and in terms of logarithms of n-octanol-water partition coefficients, were derived. Parameters of corresponding QSRR equations for individual stationary phases were compared for both HPLC andCEC modes and the resulting similarities and differences in retention mechanisms were discussed. It has been concluded that at least in the case of regular neutral analytes the specific inputs to separation mechanism due to the electric field in CEC are of secondary importance.

Previous studies have shown that four experimental runs, where both temperature T and gradient time tG are varied, can be used for the reliable prediction of separation as a function of these two variables (two-dimensional optimization). Computer simulation (e.g., DryLab) can then be used to predict "optimized" conditions for maximum sample resolution using either isocratic or gradient elution. Samples that contain a large number of components (e.g., n>15-20) present a greater challenge. Resolution for these more complex samples is often quite sensitive to small changes in T or tG in turn requiring greater accuracy in predictions that result from computer simulation. In the present study of several samples, we have examined computer simulation errors that can arise from inexact expressions for retention time as a function of T, tG or isocratic %B. Resulting conclusions are applicable to both complex and simpler samples, in either one- or two-dimensional optimization. Means to anticipate and minimize the impact of these predictive errors are examined.

Retention mechanism, separation possibilities and physicochemical properties of a laboratory-synthesized packing material for high-performance liquid chromatography (HPLC) containing cholesterol moiety immobilized on silica matrix were studied. In order to exhibit the original properties of the new phase, two referential materials – an octadecylsilica and an acylaminopropylsilica phase – were prepared starting from the same silica adsorbent. The chromatographic behavior of the three columns was examined with special regard to retention and selectivity in the reversed-phase HPLC mode employing a variety of mobile phase compositions. Special attention has been paid to possible specific applications of the phases and to temperature effects on retention and selectivity of selected polycyclic aromatic hydrocarbons and other analytes of highly diversified structure.

A high-performance liquid chromatography method for the direct separation of enantiomers of a homologous series of 1,4-disubstituted piperazine derivatives, using a chiral cellulose tris(4-methylbenzoate) stationary phase and a hexane-propan-2-ol eluent, is described. An atypical relationship was found between enantioselectivity and the carbon number of the alkyl substituent with a maximum corresponding to four-five carbon atoms in the substituent. The optimum enantioselectivity observed suggests the existence of a limited size interaction site on the stationary phase and an input to retention due to the steric exclusion of individual solutes.

A number of RP–HPLC systems have been characterized by the linear free energy relationship: (i) log SP=c+r.R 2 +s.π H 2 +a.∑α H 2 +b.∑β 2 +v.V x Here, SP is either log k′ or log kw for a series of solutes in a given system, where k′ is the capacity factor and kw is the capacity factor extrapolated to l00% water, and the solute descriptors are, R2 an excess molar refraction, π2H the dipolarity/polarizability, ∑α2H and ∑β2 the overall or effective hydrogen-bond acidity and basicity, and Vx the McGowan characteristic volume. Comparison of the coefficients in Eq. (1) with those for water-solvent partitions confirms that the modified electrostatically coated C18 phase of Pagliara et al. (J. Liq. Chromatogr., 18 (1995) 1721) can be used to obtain solute lipophilicities, as log Poct. For RP–HPLC systems based on poly(styrene–divinylbenzene), the coefficients in Eq. (i) are nearer those for the correlation of water–alkane partition coefficients, as log Palk, than for the correlation of log Poct, suggesting that the RP–HPLC systems with poly(styrene–divinylbenzene) phases could be used as a rapid method for determination of solute lipophilicity, as log Palk or as log Pcyc, where the latter is the water–cyclohexane partition coefficient. Eq. (i) has also been applied to RP–HPLC log k′ values obtained with an immobilized artificial membrane (IAM) phase. A good regression equation was obtained, but the coefficients in this equation are substantially different to those for regressions with log Poct, log Palk, or log Pcyc as the dependent variable. On the other hand, log k′ values from the RP–HPLC system of Miyake al. [J. Chromatogr., 389 (1987) 47], consisting of silica gel coated with dipalmitoyl phosphatidyl choline as a stationary phase, with aqueous acetonitrile mobile phases, yielded coefficients in Eq. (i) very similar to those for log Poct.

Eleven new chemically bonded silica stationary phase materials were synthesized and characterized physicochemically in search for the separation systems suitable for analysis of natural nucleosides and nucleotides. A set of 11 fundamental nucleosides and cyclic nucleotides was analyzed in 15 reversed-phase high-performance liquid chromatographic (RP-HPLC) systems. The HPLC systems comprised additionally 4 reference columns of different chemical structure. For the test solutes 10 structural descriptors were derived by molecular modelling. Sets of retention parameters and of structural descriptors were subjected to chemometric analysis by the method of principal component analysis (PCA). It was demonstrated that of the total number of 11 newly synthesized stationary phase materials 8 hydrocarbon-bonded silica phases had separation properties regarding nucleosides and nucleotides similar to a standard octylsilica phase. The phases which comprised aromatic fragments in their ligands did not differ significantly from the aliphatic hydrocarbon-silica materials. The new phases comprising methylimidazole and to a lesser extent pyridine and cyano moiety, had specific properties regarding the separation of nucleosides and cyclic nucleotides. It has been demonstrated that PCA of structural descriptors of solutes obtained by molecular modelling facilitates identification of structural features which determine retention in individual HPLC systems.

There are numerous measures of hydrophobicity employed in the quantitative structure-bioactivity relationship studies. Individual bioactivity parameters may appear best predicted by the specific hydrophobicity parameters. Introduction of an original reversed-phase material, the immobilized artificial membranes (IAMs) opened new perspectives for bioactivity predictions in the case of the hydrophobicity-driven processes. Comparative studies of performance of the HPLC-derived hydrophobicity measures demonstrate the advantages of the IAM-derived hydrophobicity parameters in predicting the human skin permeation by non-ionizable agents as exemplified by steroid hormones. On the other hand, the skin permeation data of agents ionized at physiological pH appeared less dependent of the retention on IAMs. The IAM columns increase the means of characterization of various aspects of hydrophobicity. Their advantages over the slow equilibrium methods of hydrophobicity determination are the simplicity of operation and the suitability of the generated retention measures for predicting specific bioactivity parameters.

Quantitative structure (reversed-phase)-retention relationships (QSRRs) derived by means of various statistical procedures are reviewed from the viewpoint of identifying retention affecting structural factors and understanding the mechanism of separations. A brief summary of the theoretical background of QSRRs is followed by presentation of reversed-phase high-performance liquid chromatographic (RP-HPLC) separation theories of relevance to the reported QSRRs. Hydrophobicity parameters derived by RP-HPLC are discussed in detail in relation to liquid-liquid partition coefficients with special emphasis on properties of new reversed-phase materials. Reported QSRR equations are critically reviewed, bearing in mind their statistical significance and physical meaning. Information on retention mechanism, as extracted by factorial methods of data analysis, is briefly analysed. It is concluded that QSRRs employing molecular descriptors expected to model fundamental intermolecular interactions and the QSRRs employing solvatochromic parameters are of similar potency for retention prediction and provide rationalization of the RP-HPLC retention mechanism. QSRR studies are demonstrated to be of value in the search for new reliable and precise descriptors of the structures of solutes of relevance to their properties, including properties other than chromatographic, e.g., bioactivity.

An improvement in affinity capillary electrophoresis (ACE) was achieved by covalently linking bovine serum albumin (BSA) to a high-molecular- mass dextran ( M r 2 000 000) using cyanogen bromide. The efficiency of the binding reaction was greater than 99%, measured through quantitative separation of the protein and protein—dextran polymer network mixture on a bare capillary using capillary electrophoresis (CE). Baseline separation of leucovorin (LV) enantiomers was obtained under 9 min using a linear polyacrylamide-coated capillary (with an effective length of 20 cm) filled with the BSA—dextran polymer network. The present work offers several significant advantages over other approaches to ligand immobilization, and can be generally applied to a wide variety of ligand—substrate systems.

For a set of 22 drugs known to modify physiological effects of endogenous histamine, high-performance liquid chromatographic (HPLC) retention data were determined employing two reversed-phase columns, seven compositions of methanol—buffer eluent and three pH values (acidic, neutral and alkaline) of the buffer. Logarithms of capacity factors, normalized to 100% buffer eluent (log k ′ w ), were determined by extrapolation of the respective data in five HPLC systems studied. Chemometric analysis of the 5 × 22 matrix of log k ′ w data obtained in five HPLC systems for 22 drug solutes allowed the extraction of two main principal components which accounted for 96% of total data variance. The distribution of individual drugs on the plane determined by the two first principal component axes forms the patterns, which are in excellent agreement with the established pharmacological classification of the structurally diverse compounds studied. It was demonstrated that systematic information extracted by chemometric analysis of behaviour of solutes in diverse HPLC systems has direct relevance to the pharmacological properties of the solutes. The approach developed here should facilitate the preselection of drug candidates, at the same time reducing the costs and the use of laboratory animals.

Quantitative structure-retention relationships (QSRRS) were compared for a set of 1,4-benzodiazepine (BDZ) test solutes analyzed by high-performance liquid affinity chromatography (HPLAC), reversed-phase (RP) and adsorption (normal-phase, NP) high-performance liquid chromatography (HPLC). The HPLAC data reflected the enantioselective retention on a human serum albumin-based chiral stationary phase (HSA-CSP); RP-HPLC data were determined on a specially deactivated hydrocarbon-bonded silica material; NP-HPLC was performed on a graphitized carbon stationary phase. Molecular descriptors reflecting additive-constitutive properties of the solutes as well as their geometry and electron charge distribution were generated using molecular modelling software. The QSRR equations derived for each chromatographic mode involved different sets of molecular descriptors. Analysis of the physical meaning of the individual descriptors allowed for interpretation of separation mechanisms at molecular (submolecular) level and rationalization of the observed separation patterns. For HPLAC the contributions by hydrophobic, steric and electrostatic factors were quantified and accounted for the differential retention of enantiomers. Retention in RP-HPLC was demonstrated to be a net effect of both non-specific (dispersive) and directional (electrostatic) intermolecular interactions between solute and molecules of both stationary and mobile phases. QSRR equations derived for NP-HPLC proved the predominance of specific dipolar and charge-transfer attractive interactions. The QSRR-based models obtained appear a reliable and convincing proof for involvement of distinctive mechanisms in different HPLC systems.

High-performance liquid chromatographic retention data derived for a set of non-congeneric aromatic solutes on a graphitic carbon stationary phase with hexane as the eluent were analysed. The capacity factors were quantitatively related to structural information extracted from nineteen molecular descriptors of solutes by multivariate analysis. The descriptors considered included molecular weight, molecular refractivity, topological indices, information content indices, quantum chemical indices and structural indices based on electron charge distribution within a molecule. The first three principal components obtained by factor analysis of nineteen structural descriptors appeared meaningful for the description of retention. Most important for retention were structural features reflecting abilities of solutes to participate in intermolecular interactions of the electron pair donor-acceptor and dipole-induced dipole type. The structural factor related to molecular size appeared less important for retention but decisive for the prediction of boiling points. Multivariate analysis of structural descriptors was demonstrated to provide systematic information useful for the explanation of the mechanism of a chromatographic process.

Twelve non-homologous solutes with a wide range of functional groups were chromatographed on a series of well-characterized stationary phases with a range of methanol-water mobile phases. Retention correlations were found for quantum mechanically calculated molecular parameters. These parameters, the total molecular energy, and the maximal excess electronic charge difference, were found to be better descriptors of retention than the dipole moment or fragmental constants. The implication of these results in terms of retention mechanisms on reversed-phase materials are discussed.

The reversed-phase high-performance liquid chromatographic (HPLC) method of hydrophobicity evaluation based on polybutadiene-coated alumina columns was applied in quantitative structure-activity relationship (QSAR) studies of a group of pharmacologically active azole derivatives. The solutes (weak organic bases) were chromatographed at pH 7.30 and 11.5. The HPLC data obtained under alkaline conditions correlated well with hydrophobicity parameter calculated for non-ionized forms of the compounds. The same chromatographic data were demonstrated to provide the most significant information on the biological activity of the compounds under study. The polymer-coated alumina stationary phases developed by Schomburg and co-workers, which can be used over a wide range of eluent pH values and which show no tendency to undergo the specific interactions with solutes typical of silica-based materials, offer unique advantages from the point of view of QSAR applications.

The rational bases, experimental techniques and conditions required for the chromatographic determination of the structural data of importance for studies on quantitative relationships between chemical structure and biological activity of drugs (QSAR) are reviewed. Practical applications of the information gathered from various chromatographic modes in correlation with bioactivity data are discussed.

Accurate definition and measurement of the thermodynamic dead-volume, V m in liquid chromatography is essential for the correct evaluation of capacity ratios. Many different recipies for determining V m have been suggested. We propose that V m be defined as the total volume of all eluent components within the column bed. It is shown that V m so defined, is given by V m  V A* x A + V B* x B + … where V A* etc. are the elution volumes of isotopically labelled eluent components A etc., and x A etc. are the volume fractions of A etc. in the eluent fed to the column. For an ( N + 1) component eluent there will be ( N + 1) such peaks. If a mixture of same eluent components but with different composition is injected into the column, N solvent disturbance peaks will be obtained which, in general, will not coincide with the peaks for labelled eluent components. The cases of binary and ternary mixtures are examined in detail and the transition from peaks due to trace components into solvent disturbance peaks is explored and clarified. The treatment is generalised to ( N + 1) component mixtures and leads to important results relating to vacancy chromatography. Experimental data are presented for binary and ternary eluents which provide practical validation of the above equation. For binary eluents, A + B, the same data allow calculation of partition isotherms for A and B between bulk eluent and space within the column bed, while the elution volumes of the solvent disturbance peaks in A + B give the gradient of the isotherm. This theoretical connection is accurately confirmed by our experimental data and by that of previous investigators [R.M. McCormick and B.L. Karger, Anal. Chem. , 52 (1980) 2249]. On the basis of our theory and experimental data, a critique is given of the various methods currently claimed to give values for V m .

A chromatrographic polarity parameter, I – b · MR, has been calculated, where I is a gas-liquid chromatographic retention index, MR is the molar refractivity of the compound analysed and b is the stationary phase polarizability coefficient. To determine b , retention data are obtained on two phases of different polarities. The polarity parameter has been shown to correlate well with quantum chemically calculated dipole moments in the case of phenols without space-consuming substituents.

For a structurally diverse set of primary, secondary and tertiary heterocyclic amines, correlations were found between the Kovats retention indices obtained on the methyl phenyl silicone phase OV-101 and quantum chemically calculated parameters. The total energy calculated by CNDO/2 molecular orbital method was chosen as a measure of a solute's ability to participate in dispersive interactions with the stationary phase. As a measure of the solute's ability to undergo polar interactions with the stationary phase, a parameter was proposed which reflects the highest local intermolecular dipole moment. It is defined as the largest difference in electron charges between two atoms. A two-parameter regression equation was derived which describes satisfactorily the retention of structurally different polar solutes on a relatively non-polar stationary phase. Some evidence is also provided that charge-transfer complexes are formed during the separations process.