Comparison of different retention models in normal- and reversed-phase liquid chromatography with binary mobile phases

The dependence on mobile phase composition of the retention of selected test analytes in different normal- and reversed-phase chromatographic systems is studied. The aim of this study is to compare the performance of six valuable retention models reported in the literature with a new empirical equation, which is first introduced in this study. All of these models are compared for different thin-layer chromatographic and high-performance liquid chromatographic systems by use of three criteria: the sum of the squared differences between the experimental and theoretical data, approximation of the standard deviation, and the Fisher test.     

The retention of selenium chains in reversed-phase liquid chromatography

Summary Organic polyselenides in which the selenium chains are terminated at both ends by ethyl-thio, butyl-thio, phenylthio, or phenyl groups were prepared. Their chromatographic behavior in HPLC on a bonded octadecyl phase with entirely or mainly methanolic eluents was investigated and compared with the corresponding polysulphides. The retention increment per Se atom is greater than that for S atoms. The ratio of Se to S increments is equal to the ratio of surface area increments. The S−Se bond does not have an appreciable influence on retention. Bonded to phenyl, selenium increases retention to the same extent as sulphur does. Some of the RSSe_nSR solutes were identified by mass spectrometry and other techniques.     

The retention of some azoles in reversed-phase liquid chromatography

The influence of the structure-related physicochemical properties of derivatives of nitrogen-containing heterocyclic compounds on their retention under the conditions of reversed-phase liquid chromatography was studied. The dependence of the chromatographic retention of azole derivatives on the composition of the mobile phase was determined. The presence of azole protonated forms was substantiated by conductometric data.     

Matrix effect on the retention in reversed-phase liquid chromatography

Summary A polymer-based, reversed-phase column (VA-C18), prepared by grafting octadecyl chain onto vinyl alcohol copolymer gel, was investigated for its chromatographic characteristics. n-Alkanes and n-alkyl alcohols were found to be retained only by hydrophobic interaction between the solutes and the octadecyl chain. In the case of aromatic hydrocarbons, in addition to the hydrophobic interaction, - interaction between the solutes and the based material was elucidated to contribute to the retention. For aromatic tertiary amines which are known to strongly interact with the residual silanol group of the silica-based reversed-phase columns to produce broadened and skewed peakes, the VA-C18 column also retained these substrates strongly by the combination of hydrophobic, -, and ionic interactions. In this case, however, symmetrical peaks were observed. From these results, it was determined that in the case of VA-C18, the base material was found not to produce undesirable effect although the solutes interact with the base. Further conclusion obtained was that in reversed phase liquid chromatography, chromatographic properties of base matrix is highly responsible for the overall retention.     

Models of retention of adamantylamidrazones in reversed-phase high-performance liquid chromatography

Rules governing the chromatographic behavior of some amidrazones of the adamantane series were studied under the conditions of reversed-phase high-performance liquid chromatography. The characteristics of the retention of sorbates in elution by aqueous-acetonitrile phases with various compositions were calculated. Correlations between the structure and physicochemical characteristics of sorbate molecules and their retention were studied.     

Convergence of retention for small solutes in reversed-phase liquid chromatography

Summary It is shown theoretically that when the concentration of organic solvent in the mobile phase increases, or solute size decreases, log k values of small solutes in reversed-phase liquid chromatography (RPLC) will tend to have a minimum value called the convergence point. A theoretical model for evaluating the convergent coordinates of small solutes is presented by using a stoichiometric displacement model for retention (SMDR). The physical meaning of the coordinates of each kind of convergence are also elucidated. The convergence points have either two-dimensional coordinates with a common ordinate (the logarithm of the phase ratio of the column, log ) or threedimensional corrdinates with two common axes: — log and the logarithm of the molar concentration of the pure displacing agent in mobile phase, log a_D. The other axis relates to the nature of the solutes, such as carbon number of a homolog, van der Waal's surface area, hydrophobic fragment constant etc. for the latter and those and/or concentration axis for the former. The model was tested with published data and found to give a good fit.     

Indirect detection in reversed-phase liquid chromatography: Ion-pair retention models for cations on polystyrene polymers

Summary The distribution equilibria of cationic compounds in reversed-phase chromatographic systems (ion-pair chromatography) have been studied on the basis of their effect on a detectable mobile phase component. The solid phase was a polystyrene-divinylbenzene copolymer and the detectable component, a quaternary ammonium ion, 1-methylpyridine. The solutes were mono- and divalent amines and quaternary ammonium ions. The cations can be retained by ion-pair adsorption and ion exchange. Expressions for the ion-pair retention of the solutes and the mobile phase cation (system peak) have been developed assuming Langmuir distribution of ion pairs to a solid phase with one kind of binding site. The validity of the expressions has been tested by evaluation of ion-pair distribution constants using non-linear curve fitting techniques. Good agreement for the constants of common ion pairs was obtained from different kinds of capacity ratio expressions. Ion exchange retention can appear beside ion-pair retention, and it has been observed in the pH range 1.6–6.1. The effect depends not only on cations in the mobile phase, but also on the nature of the buffering systems.     

Simple models for the effect of aliphatic alcohol additives on the retention in reversed-phase liquid chromatography

Four retention models for the effect of aliphatic alcohol additives on the retention of analytes in reversed-phase liquid chromatography have been developed following either a semi-thermodynamic treatment or an empirical approach. Their performance was tested using the experimental retention times of six non-polar analytes (alkylbenzenes) and ten o-phthalaldehyde derivatives of amino acids under different isocratic chromatographic runs when a small amount of ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol or 1-heptanol was added to methanol/water mixtures containing a constant amount of methanol. It was shown that for the structurally simple alkylbenzenes all the models can be adopted for retention prediction with good results. In contrast, just one out of four models, that with the fewest approximations, predicts satisfactorily the retention properties of amino acids derivatives. However, the most interesting feature is that this model can predict the effect of an alcohol-additive on the retention properties of solutes, even if this additive was not used in chromatographic runs done for the fitting procedure, provided that it belongs to the same homologous series of alkanols. This feature is also observed in all models described the retention of alkylbenzenes.     

Thermodynamic studies of pressure-induced retention of peptides in reversed-phase liquid chromatography

The pressure-induced retention of peptides on reversed-phase HPLC was studied by systematically changing organic solvent composition and temperature at both low (19 bar) and high (318 bar) pressures using a homologous series of hydrophobic poly-L-phenylalanine (n = 2-7) as the model compound. Based on van' t Hoff plots under different organic solvent compositions and pressures, the enthalpy change for the solute (deltaH) was determined. Moreover, both the enthalpy and entropy change for each phenylalanine residue (deltadeltaH and deltadeltaS), which corresponds to solute retention on a microenvironment along the depth of C18 chain, were also calculated by direct subtractions. Results indicate that under acetonitrile (ACN) compositions above 35%, the pressure caused deltadeltaS value to change from a negative to a positive value and both deltaH and deltadeltaH to change from a negative to a less negative value, all leading to a thermodynamic state closer to those under 35% acetonitrile composition. This implies that the pressure-induced retention observed in this study was an entropy-favored but enthalpy-unfavored process and was explained by pressure-induced desorption of solvent molecules that were associated with the stationary phase or with the peptide solute. Under 35% acetonitrile composition, however, it was found that neither deltadeltaH nor deltadeltaS value was significantly changed by the pressure. Whereas, both deltaH value and the intercept of van't Hoff plots under 35% acetonitrile composition were increased by pressure. This indicates that under low organic solvent composition, 35%, most of the acetonitrile molecules adsorbed on the surface of the stationary phase and only little solvent molecules were dissolved in the bulk stationary phase where the phenylalanine residues were partitioned. This study has provided new thermodynamic insights to the pressure-induced retention for peptides and proteins.     

Effect of mobile phase composition on the retention of selected alkaloids in reversed-phase liquid chromatography with chaotropic salts

Sodium hexafluorophosphate, perchlorate and trifluoroacetate were applied as ion-ion interaction reagents in reversed-phase liquid chromatography. The separation of chosen alkaloids was performed by changing the kind of the organic modifier (methanol, acetonitrile, tetrahydrofuran), concentration of the ion-ion-interaction reagents and the concentration of phosphate buffer at constant pH (2.7) in the mobile phase. Obtained results were analyzed in connection to a dynamic ion-exchange model of retention and ion-ion interaction effects. The perturbation method was applied to test proposed retention theories. The formation of ion-complexes controlling the retention in chaotropic systems was confirmed. On the basis of the relationships of capacity factors (k) versus salt concentrations derived experimentally, absolute increases in capacity factors, the desolvation parameters and the limiting retention factors were calculated and compared for all the investigated compounds in eluent systems studied. The selectivity of the proposed mobile phases was compared on the basis of the separation of alkaloid mixture.     

Prediction of peptide retention time in reversed-phase high-performance liquid chromatography.

Peptide retention in reversed-phase chromatography depends mainly on the amino acid composition of peptides and can therefore be predicted by summing the relative hydrophobic contributions of each constitutive amino acid residue. The prediction is correct for small peptides but overestimates the retention times of peptides larger than 10-15 residues. A new prediction model is proposed in which the contribution to peptide retention of each amino acid residue is not a constant but a decreasing function of peptide length. From the retention times of 104 peptides, the parameters of decreasing functions were estimated by a non-linear multiple regression analysis. The contribution to peptide retention of charged, polar and non-polar residues appears to be differently affected by peptide length. The secondary structure of most peptides during reversed-phase high-performance liquid chromatography could be responsible for this. The high correlation between the predicted and observed retention times of peptides which were not used to establish the model indicates a good predictive accuracy of the new model.     

An abnormal temperature dependence of alkylpyrazines' retention in reversed-phase liquid chromatography

Retention behaviors of pyrazine and alkylpyrazines on various stationary phases in reversed-phase liquid chromatography were examined. An abnormal temperature effect on the retention of alkylpyrazines with a mobile phase consisting of acetonitrile and water was observed when changing the column temperature. On the other hand, no similar trend was found with a methanol-water mobile phase. For all the columns investigated in this work, the above tendency to the temperature-dependence was consistently observed, suggesting that the abnormal temperature effect on the retention of alkylpyrazines could be mainly induced by an acetonitrile-based mobile phase.     

Comparison of retention of aromatic hydrocarbons with polar groups in binary reversed-phase high-performance liquid chromatography systems

The retention of aromatic hydrocarbons with polar groups has been correlated as log k1 versus log k2 for reversed-phase high-performance liquid chromatography systems with different binary aqueous mobile phases containing methanol, acetonitrile or tetrahydrofuran as modifiers. Distinct changes in separation selectivity have been observed between tetrahydrofuran and acetonitrile or methanol systems. Methanol and acetonitrile systems show lower diversity of separation selectivity. The changes in retention and selectivity of aromatic hydrocarbons with various polar groups between any two chromatographic systems with binary aqueous eluents (tetrahydrofuran vs. acetonitrile, tetrahydrofuran vs. methanol and methanol vs. acetonitrile) have been interpreted in terms of molecular interactions of the solute with especially one component of the stationary phase region, i.e. extracted modifier, and stationary phase ordering. The ordering of the stationary phase region caused by modifier type influences the chromatographic selectivity of solutes with different molecular shape.     

反相液相色谱中溶质保留与溶剂化结构参数间的定量关系

本文研究了等度条件下反相液相色谱中溶质保留与溶剂化结构参数间的定量关系;考察了反相液相色谱保留值变化规律式lnk'=a+cC~b中参数a.c 与溶剂化结构参数间的定量关系,结果表明能较好地预测等度和不同冲洗剂组成下溶质的保留值,将乙腈-水和甲醇-水冲洗剂下的a.c值定量关联,表明在考虑溶质的氢键作用参数后,其相关性有所提高.     

Indirect detection in reversed-phase liquid chromatography: Ion-pair retention models for ionic compounds on μ Bondapak phenyl

Summary Retention models for ionic compounds in reversed-phase ion-pair chromatographic systems have been developed on the basis of the capacity ratios of solutes and ionic mobile phase components using indirect detection. The binding to the adsorbent, μBondapak Phenyl, has been expressed by ion-pair distribution constants and tested in the retention models by non-linear regression. All the ionic compounds followed adsorption models of the Langmuir type comprising two kinds of binding sites with widely different adsorption capacities and binding constants. The results indicate certain differences between the two sites regarding the binding of cationic and anionic components, respectively.     

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 microm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.     

Analysis of selected ionic liquid cations by ion exchange chromatography and reversed-phase high performance liquid chromatography

The chromatographic behavior of 8 ionic liquids - 7 homologues of 1-alkyl-3-methylimidazolium and 4-methyl-N-butylpyridinium - has been investigated with a strong cation exchange adsorbent. In particular, the dependence of the retention properties of these solutes on mobile phase composition, pH, and buffer concentration was evaluated with the aim of optimizing and improving the selectivity and retention of solute separation. While using the SCX stationary phase, several interactions occurred with varying strengths, depending on the mobile phase composition. Cation exchange, nonspecific hydrophobic interactions, and adsorption chromatography behavior were observed. Reversed phase chromatography occurred at low concentrations of acetonitrile, electrostatic and adsorption interactions at higher organic modifier concentrations. Elevated buffer concentrations lowered the retention factors without affecting the selectivity of ionic liquids. Obtained results were further compared to the chromatographic behaviour of ionic liquids in the reversed phase system. All analyzed ionic liquids follow reversed-phase behavior while being separated. Much lower selectivity in the range of highly hydrophilic compounds is obtained. This suggests preferred use of ion chromatography for separation and analysis of compounds below 4 carbon atoms in the alkyl side chain.     

Modeling the effect of solvation on solute retention in reversed-phase liquid chromatography

The retention of a homologous series of alkylbenzenes was determined on octyl and octadecyl reversed-phase columns in several polar organic liquids. Free energies of transfer were calculated by the SM5.0R classical solvation model for each organic liquid tested and for several alkanes. The relationships between the measured retention factors and the calculated free energies of transfer were then investigated. Although the natural logarithms of the retention factor and the calculated free energies of transfer were linearly correlated, the obtained free energies of transfer of the solutes did not completely explain the retention behavior of the solutes. Nonetheless, even in these pure organic liquids, the energetics of RPLC retention behaved very similarly to those of partitioning.     

Factors influencing retention and resolution of substituted alkylbenzenes in reversed-phase liquid chromatography

Summary The retention data of isomeric xylenes, ethyltoluenes and diethylbenzenes, and of mesitylene, benzene, toluene and ethylbenzene were obtained on a reversedphase column using methanol-water and ethanol-water mobile phases at four different temperatures. This database was used to relate the dependence of solute retention and resolution on the polarity of the mobile phase, solute dipole moment, and column temperature. The additivity of the free energy of the transfer of solute molecules or solute segments from the stationary phase to the mobile phase, was examined for the isomeric compounds. For this, the logarithm of the net retention volume was substituted for the free energy. Deviations from the additivity of free energies indicate that the separation of isomeric substituted alkylbenzenes is governed by their differential interactions with both the polar mobile phase and nonpolar stationary phase. Among the disubstituted alkylbenzenes,ortho-isomers favor the mobile phase more andpara-isomers tend to prefer the stationary phase more. Themeta-isomers are found to follow the additivity rule closely. These trends are amplified as the polarity of the mobile phase is increased indicating that these isomers are resolved better in water-rich mobile phases. These findings are substantiated by analogous results from gas-liquid chromatographic retention data, estimation of dipole moment effects, and examination of the entropic and enthalpic contributions to the net retention volume.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.