Reversed-phase Retention Research Articles

Reversed-Phase HPLC Retention of Deoxyribonucleosides as a Function of Mobile Phase Composition

Abstract Comparison of approaches describing retention of deoxynucleosides (dCyd, dUrd, dGuo, dThd, and dAdo) as a function of the mobile phase composition has been performed. The volume fraction (F) of organic modifier was changed from 0.05 to 0.30, and to 0.12 for methanol and acetonitrile, respectively. Linear regression has been carried out for the following equations: log k'=log k'w—S X F (1), log k'=H + Klog(1/F) (2), k'=A + B(1/F) (3), where k' is capacity factor, and log k'w, S, H, K, A and B are experimental coefficients. The best fits were obtained for the eqns. (1) and (3), and the poorest correlations were obtained for eqn. (2). The slope ratios Bmethanol/Bacetonitrile and Sacetonitrile/Smethanol are changed in comparatively narrow ranges with average values of 2.86 and 2.30, respectively. Unlike the slope S of eqn. (1), the slope B of eqn. (3) can characterize the properties of solute and organic modifier simultaneously. For each solute, the intercepts A calculated for acetonitrile and methan...

Use of canonical correlation analysis for the evaluation of chromatographic retention data

The retention characteristics of 38 nonionic surfactants having different hydrophobic moieties have been determined by reversed-phase thin-layer chromatography using alumina support and methanol as an organic modifier. The hydrophobicity ( R M values) of each surfactant decreased monotonously with increasing concentration of methanol, the linear correlation between the R M value and the concentration of methanol in the eluent was significant in each instance. Canonical correlation analysis was used to find the relationship between the intercept and slope values (related to the molecular hydrophobicity and specific hydrophobic surface area of surfactants) of the above linear correlation and the molecular substructures of surfactants. To compare the information from canonical correlation analysis and other multivariate mathematical—statistical methods, the same data matrix was evaluated by principal component analysis (PCA) and cluster analysis too. Both relationships in the canonical correlation analysis were significant, proving the significant influence of molecular substructures on the reversed-phase retention behaviour of nonionic surfactants. Significant linear correlations were found between the canonical variates and the first coordinate of the nonlinear map of principal component variables, but no correlation was found between the weighted canonical coefficients and the corresponding PCA parameters suggesting similar but not identical information content of the methods. The distribution of the chromatographic parameters and substructures were identical on the cluster dendrogram and on the two-dimensional nonlinear map of PCA loadings showing the similarity of these methods.

Pulsed electrochemical detection of alkanolamines separated by multimodal high-performance liquid chromatography

Pulsed electrochemical detection (PED) is applied to alkanolamines separated by high-performance liquid chromatography (HPLC). A multimodal HPLC column with both cation-exchange and reversed-phase retention modes is used with an acidic mobile phase to assure alkanolamines are in their cationic form. Baseline resolution of alkanolamines, including positional isomers, is possible. Detector response for a representative alkanolamine, tris(hydroxymethyl)aminomethane (Tris), is shown to be linear over a concentration range of more than three decades. The limit of detection for Tris is 20 n M (500 fmol in a 25-μl injection) and the standard deviation of the PED response for 10 μM Tris is better than 0.4%. HPLC-PED is demonstrated to permit the sensitive and precise determination of alkanolamines in a biological sample (blood) and a commercial formulation (shaving gel) with minimal sample preparation.

Selectivity due to conformational differences between helical and non-helical peptides in reversed-phase chromatography

The reversed-phase retention behaviour of two series of peptides, one non-helical and the other α-helical, was studied under various linear AB gradients in order to determine the effect of peptide conformation on selectivity of the separation. The non-helical series, designated X1, with the sequence Ac-XLGAKGAGVG-amide, exhibited negligible α-helical content in a hydrophobic medium; whereas, the amphipathic α-helical series, designated AX9, with the sequence Ac-EAEKAAKEXEKAAKEAEK-amide, exhibited high α-helical content in a hydrophobic medium. We have shown that plots of log k vs. φ (where k is the median capacity factor and φ is the median volume fraction of organic solvent) are very similar for any one peptide conformation, i.e., peptides from either the non-helical or amphipathic α-helical series exhibit similar S (solute parameter) values and the b (gradient steepness parameter) values are also similar for 17 different amino acid substitutions within each series of peptides. If mixtures of peptides from the two different series are separated using either increasing or decreasing gradient rates, large increases in resolution occur due to selectivity, which may be attributed to the difference in the log k vs. φ plots for each series of peptides. In addition, by using a polymer of an X1 peptide, which is 20 residues in length, it has been shown that the molecular mass difference between the X1 and the AX9 series of peptides is not sufficient to account for the selectivity difference. The S value of a non-amphipathic α-helical peptide further suggested that the difference in selectivity between the two series of peptides was due to differences in conformation. We believe that the peptide mixtures presented here providea a good model for studyingselectivity effects due to conformational differences between peptides, an important concern when attempting to develop rational approaches to the prediction and optimization of peptide separation protocols from primary sequence information alone.

Retention Behavior of Poly(L-Tryptophan)S and Poly(D,L-Tryptophan)S in Reversed-Phase Liquid Chromatography

Abstract The reversed-phase liquid chromatographic retention behavior of poly(l-tryptophan)s and poly(d,l-tryptophan)s whose molecular weight ranged from 5.4 kD to 37.25 kD is examined using a C-8 chemically bonded stationary phase and binary mobile phases of tetrahydrofuran (THF)-water and ternary mobile phases of THF-water-methanol. The retention of poly(l-tryptophan)s is compared with that of poly(d,l-tryptophan)s. Linear-Solvent-Strength (LSS) model is examined in describing the retention behavior. The effect of adding a third solvent to the binary mobile phase is also discussed.

Reversed-phase liquid chromatography as a useful probe of hydrophobic interactions involved in protein folding and protein stability

We have evaluated the potential of reversed-phase liquid chromatography (RPLC) as a probe of hydrophobic interactions involved in protein folding and stability. Our approach was to apply RPLC to a de novo designed model protein system, namely a two-stranded α-helical coiled coil. It was shown that the reversed-phase retention behaviour of various synthetic analogues of monomeric α-helices and dimeric coiled-coil structures correlated well with their stability in solution, as monitored by circular dichroism during guanidine hydrochloride and temperature denaturation studies. In addition, an explanation is offered as to why amphipathic coiled coils, an important structural motif in many biological systems, are more stable at low pH compared to physiological pH values. The results of this study suggest that not only may RPLC prove to be a useful and rapid complementary technique for understanding protein interactions, but also the de novo designed coiled-coil model described here is an excellent model system for such studies.

Statistical evaluation of molecular descriptors and quantitative structure-property relationship studies of retinoids.

Various molecular descriptors, including connectivity indices, sums of the intrinsic state values, electrotopological state indices, topological equivalence indices, kappa indices, normalized Bonchev-Trinajstic and Shannon information indices, Wiener and Platt's F numbers and molecular weight, were calculated for 73 retinoids, whose structures and properties were taken from the literature. A novel methodology using statistical analyses (cluster, factor and stepwise regression) in selecting relevant molecular descriptors for quantitative structure-property relationship (QSPR) studies has been developed. The analyses were used in correlating molecular structure with affinity, pharmacokinetic properties and reversed-phase retention of retinoids.

Reversed-phase retention characteristics of some bioactive heterocyclic compounds

The lipophilicities and specific hydrophobic surface areas of sixteen bioactive heterocyclic compounds were determined by reversed-phase thin-layer chromatography using various eluent systems. The data were evaluated by principal component analysis (PCA), separately calculated from the correlation and covariance matrices, and by cluster analysis. The ratio of the variances explained by the first PC component was high and was very similar for both PCA methods, suggesting that the eluents have common elution characteristics. The lipophilicities and specific hydrophobic surface areas of the compounds are well separated on the two-dimensional non-linear maps of PC variables, indicating the different information contents of the two physico-chemical parameters. The information contents of cluster analysis and two-dimensional non-linear mapping techniques were found to be similar but not identical.

Reversed-Phase Retention Behavior of Fluorescence Labeled Phospholipids in Ammonium Acetate Buffers

Abstract Subcomponents of fluorescent derivatives of phosphatidylethanolamine (PE) and phosphatidylserine (PS) were resolved by reversed-phase high-performance liquid chromatography (HPLC) with mobile phases containing acetonitrile, methanol, water, and ammonium acetate. The fluorescence labeled phospholipids (PL) include N-(rhodamine B sulfonyl)-PE, N- (7-nitrobenz-2-oxa-l, 3-diazol-4-yl) -PE, N-(5-fluoresceinthiocarbamoyl) (FL)-PE, N-(1-pyrenesulfonyl)-PE, and N-(5-dimethylaminonaphthalene-l-sulfcnyl)-PE. Among the compounds studied, FL-PE exhibited the highest degree of selectivity for component resolution. The HPLC behavior of the five PE derivatives was examined under variable concentrations of ammonium acetate. Capacity factors of the PL subcomponents increased with increasing concentrations of the acetate buffers. Incorporation of triethylamine into the mobile phase alleviated peak broadening and improved detection sensitivity of polar PL (FL-PE and PS). Fatty acid structures of molecular species i...

Influence of various salts on the reversed-phase retention of some dansylated amino acids in TLC

The retention of 14 dansylated amino acid derivatives was determined using aqueous LiCl, NaCl, KCl, RbCl and CsCl solutions as eluents in reversed-phase thinlayer chromatography. The salts exerted typical salting-out effects, the retention of each dansylated amino acid increased with increasing concentration of salt in the eluent. This effect has been tentatively explained by the suppression of the dissociation of the polar groups in the solute molecules resulting in increased apparent lipophilicity. The correlation between the increased retention of dansyl amino acids and the salt concentration was found to be linear. The hydrated radii and energy of hydration of cations as well as the hydrophobicity of free amino acids and the pK value of the α-amino groups simultaneously influenced the retention.

Semi-automated chromatographic procedure for the isolation of acetylated N-terminal fragments from protein digests

Several published procedures have been combined to develop a general strategy for the specific identification and isolation of the acetylated-N-terminal fragment from all other proteolytic fragments. This ruse can be divided into four steps: (i) succinylation of the substrate to block lysine NH 2 groups; (ii) enzymatic digestion of the modified protein; (iii) automated phenylisothiocyanate derivatization of the protease derived fragments to block newly generated “free” N-termini; and (iv) reversed-phase high-performance liquid chromatography with on-line photodiode array spectroscopy. The individual phenylthiocarbamyl-peptide species exhibit an increased reversed phase retention time and a greater UV (210–297 nm) profile compared to the corresponding control (-phenylisothiocyanate) digest. The N-terminal acetylated fragment shows neither a retention time shift nor an augmented UV profile. To validate each process step, synthetic peptides and acetylated-N-terminal proteins of known sequence were used as test samples. The desired fragment was isolated from three proteins and positively identified by electrospray mass spectrometry and amino acid composition, Proteins with other N-terminal blocking groups should be amenable to this procedure.

Lipophilicity determination of some monoamine oxidase inhibitors: the effect of methanol and ammonium chloride

The lipophilicity ( R M value) of seventeen monoamine oxidase inhibitory drugs was determined by reversed-phase thin-layer chromatography, and the effect of salt concentration on the reversed-phase retention was studied by adding ammonium chloride to the eluent. Each drug exhibited regular retention behaviour, its R M value linearly decreasing with increasing concentration of methanol in the eluent. Ammonium chloride decreased the retention: the effect was higher at lower salt concentrations, which indicates that the phenomenon is of saturation character. The influence of ammonium chloride depended on the concentration of methanol (on the dielectric constant of the eluent) suggesting that methanol suppresses the dissociation of ammonium chloride resulting in a modified salting-in effect.

Chromatographic separation and molecular modelling of triazines with respect to their inhibition of the growth of L1210/R71 cells

The potential anti-cancer activity of triazines was characterized by the inhibition of the growth of L1210/R71 cells. The retention times for fifteen triazine derivatives were measured by high-performance liquid chromatography on octyl silica and silica gel columns. The slope and intercept values of the plot of the logarithmic capacity factor versus acetonitrile concentration were calculated from the reversed-phase retention measurements. The adsorption properties of the compounds were characterized by the retention data obtained on silica gel columns using high and low concentrations of ammonium salts in the hydro-organic mobile phase. The non-polar, non-polar unsaturated and polar surface areas, the surface energies, the dipole moments and the Van der Waals radii of the molecules were calculated from their chemical structures after energy minimization on the basis of molecular mechanics. Correlation analysis of these parameters showed that the inhibitory effect is dependent on the polar and non-polar surface areas of the molecules. The reversed-phase slope showed a significant correlation with the difference between the accessible and the total non-polar surface areas of the compounds, whereas the intercept values correlated with the non-polar accessible surface area. The adsorption properties of the triazines on silica gel cannot be described by the molecular parameters investigated here.

Simple validatory tools for judging the predictive performance of parafac and three‐way PLS

AbstractThe methods PARAFAC and three‐way PLS are compared with respect to their ability to predict reversed‐phase retention values. Special attention is paid to simple validatory tools, the meaning and use of which are explained.

Molecular Modeling for Mobile Phase Optimization in RP-HPLC

Abstract The applicability of molecular parameters calculated on the bases of molecular mechanics have been investigated for the prediction of reversed-phase retention behavior of structurally unrelated series of drug molecules. Non-polar, non-polar unsaturated and polar surface areas, surface energies, dipole moments, van der Waals radii and hydrophobicity values expressed by the logarithm of the octanol/water partition coefficients have been calculated from the molecular structure. The reversed-phase retention behavior was described by the slope and the intercept of the straight lines obtained by plotting the log k1 values against the acetonitrile concentration of the mobile phase. The acetonitrile concentration (OP%0) which was needed for the log k1 = 0 retention was also calculated from the slope and intercept values. Step-wise linear regression analyses have been applied for revealing the correlations between the investigated parameters. The slope values could be described by the difference of the no...

Comparison of two principal component analysis methods to evaluate reversed-phase retention data

The retention of twelve 2-nitro-4-cyanophenyl esters showing marked herbicidal activity was determined in 23 reversed-phase thin-layer chromatographic systems. The retention data set was evaluated by principal component analysis (PCA). To assess the effect of the information loss caused by normalization, PCA was separately carried out on the covariance (method A) and on the correlation matrix (method B). The ratio of the variances explained was very similar for both methods, however, the PC loadings and the coordinates of the two-dimensional nonlinear maps showed poor correlation. The distribution of the 2-nitro-4-cyanophenyl esters and that of chromatographic systems showed differences on the two-dimensional nonlinear maps of PC loadings and PC variables, however, the general trend was similar independently of the application of method A or B. The findings indicate that the application of the correlation matrix as basis for the PCA calculations may lead to slightly distorted results that strongly advocates the use of covariance matrix in PCA.

Linear solvation energy relationships in the study of the solvatochromic properties and liquid chromatographic retention behaviour of benzodiazepines

The solvatochromic behaviour of a series of benzodiazepines was studied in order to characterize their molecular properties [polarity/polarizability and hydrogen bonding (HB) acidity and basicity]. Such experimental parameters, compared with those calculated according to Kamlet and co-workers, were employed in the study of the chromatographic retention behaviour by means of linear solvation energy relationships. Various high-performance liquid and thin-layer chromatographic systems were analysed for testing the confidence of this methodology in the study of different retentions in reversed-phase (RP) and normal-phase (NP) chromatographic systems. The most important parameters influencing benzodiazepine retention were established as size and HB basicity for RP retention and polarity and HB acidity or basicity for NP retention. For this series of solutes the present method also appears suitable for predicting retention in the studied systems and for selecting the optimum chromatographic conditions for analytical purposes.

Effects of peptide hydrophobicity and charge state on molecular ion yields in plasma desorption mass spectrometry

Plasma desorption mass spectra were obtained for a series of peptides, grouped in four mass ranges having approximately 9, 20, 30, and 40 amino acid residues. Within each group, the individual peptides differed in hydrophobicity, charge state, and retention time, as measured on a reversed-phase HPLC column. Comparison of the molecular ion intensities in the positive ion mass spectra of peptides from each group showed a strong dependence upon hydrophobicity and no correlation with charge state. Plasma desorption mass spectra of mixtures of all the peptides within each mass range generally resulted in the desorption of a single residue and suppression of the ion signal from other components. In most cases, this could be correlated with hydrophobicity, as calculated from the Bull and Breese index; however, a better correlation existed when the results were compared with reversed-phase retention times. In general the spectra of mixtures were not influenced by charge state (except in the absence of hydrophobic peptides), as the same component in each peptide mixture produced the most abundant ions in both positive and negative ion spectra.

Liquid Chromatographic and Spectral Analysis of the 17-Hydroxy Anabolic Steroids

The liquid chromatographic properties of various 17-hydroxy anabolic steroids are examined under reversed-phase conditions. These anabolic steroids are now listed as controlled drugs in many states due to their abuse potential in athletics, body building, and other areas. These nonesterified steroids are separated on a C18 stationary phase with a 70% methanol in water mobile phase. In a few cases, two compounds display very similar retention properties. However, dual-wavelength detection at 254 and 280 nm allows for their differentiation. Reversed-phase retention parallels steroid lipophilicity based on hydroxyl and methyl group substituents. Also, those steroids containing a dienone substructure are more polar than steroids containing an enone moiety.

Prediction of the high-performance liquid chromatographic retention behaviour of some benzodiazepine derivatives by thin-layer chromatography

The reversed-phase retention behaviour of eighteen benzodiazepine derivatives was studied using high-performance liquid chromarography (HPLC) and thin-layer chromatography (TLC). In each instance the retention decreased with increasing concentration of the organic mobile phase in the eluent, that is, no anomalous effect was observed. In reversed-phase TLC the R M value of benzodiazepine derivatives depended linearly on the organic phase concentration and logarithmically on the buffer concentration in the eluent. A highly significant correlation was found between the HPLC log k 0 value and the reversed-phase TLC parameters, suggesting that TLC can be used for predicting the HPLC retention behaviour of benzodiazepine derivatives. As the TLC parameters explained about 75% of the total variance, the predictive power of TLC is limited.