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PubMed2001

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.

Chromatography, High Pressure Liquid↗
PubMed2002

Quantitative relationship between the retention of peptides on a reversed-phase alumina support and their physicochemical parameters.

The retention time of 16 peptides was determined on polyethylene-coated alumina stationary phase using mixtures of acetonitrile (MeCN) and acidic and basic phosphate buffers as mobile phases. The majority of peptides showed nonlinear retention behaviour, their retention decreased with increasing concentration of MeCN in the lower concentration range, reached a minimum and increased again with increasing concentration of MeCN in the higher concentration range. Quadratic equations described well the atypical retention behaviour of peptides, the significance level being always over 95%. Principal component analysis indicated that the steric parameters of peptides exerted the highest influence on their retention on polyethylene-coated alumina stationary phase.

Acetonitriles↗
PubMed2001

Determinants of protein retention characteristics on cation-exchange adsorbents.

There are currently a large number of commercially available strong and weak cation-exchange adsorbents for preparative protein purification, typically prepared by coupling charged ligands to a mechanically rigid porous bead. Because of the diverse chemical nature of the base matrix (carbohydrate, synthetic polymer, inorganic) and the coupling and ligand chemistry, cation-exchange adsorbents from different suppliers can differ substantially in chemical surface properties and physical structure. The differences in chemical properties can be in ionic capacity, hydrophobicity, the presence of hydrogen bond donors/acceptors, and the nature of the charged functional groups. In order to probe the effects of these factors on protein affinity, the isocratic retention of a set of model proteins was examined on a set of cation-exchange adsorbents to obtain a quantitative assessment of retention differences between adsorbents. Two adsorbent factors were found to be the dominant determinants of overall protein retention: the anion type and the adsorbent pore size distribution. Protein retention on strong cation-exchangers was found to be greater than that on corresponding weak cation-exchangers. Protein retention was increased on adsorbents with pore size distributions that include significant amounts of pore space with dimensions similar to those of the protein solute.

Adsorption↗
PubMed1988

Retention in reversed-phase liquid chromatography: solvatochromic investigation of homologous alcohol-water binary mobile phases.

The mechanism of retention in reversed-phase liquid chromatography (RPLC) has been further investigated using the ET(30) solvatochromic solvent polarity scale. The retention behavior of a variety of solutes was measured using a homologous series of normal alcohols as the organic modifiers in hydroorganic mobile phases. The results imply that a systematic change in the extent of solvation of the stationary phase occurs with respect to the size of the organic modifier. It was also found that a linear extrapolation of the log k' versus ET(30) plots for different mobile phases using methanol, ethanol and acetonitrile (but not n-propanol) as modifiers, intersected at approximately the ET(30) value of pure water. This intersection is further evidence that the ET(30) model of solute retention is a useful tool with which to study the mechanism of retention in RPLC. The extrapolated retention value in water, log k'w, from the ET(30) plots should then prove to be a more reliable means of estimating solute lipophilicity using RPLC than the percent organic modifier model.

Alcohols↗
PubMed1985

High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. I. Factors influencing retention, peak shape and detector response.

The use of silica columns together with non-aqueous ionic eluents provides a stable yet flexible system for the high-performance liquid chromatographic analysis of basic drugs. At constant ionic strength, eluent pH influences retention via ionisation of surface silanols and protonation of basic analytes, pKa values indicating the pH of maximum retention. At constant pH, retention is proportional to the reciprocal of the eluent ionic strength for fully protonated analytes and quaternary ammonium compounds. The addition of water up to 10% (v/v) has little effect on retention if the protonation of the analytes is unaffected. Thus, it is likely that retention is mediated primarily via cation exchange with surface silanols. However, additional factors must play a part with compounds such as morphine which give tailing peaks at acidic or neutral eluent pHs.

Chromatography, High Pressure Liquid↗
PubMed1987

Reversed-phase liquid chromatography of site-specific mutagenized staphylococcal nuclease. Gradient retention and correlations with amino acid-based predictive scales.

Comparison of the gradient reversed-phase chromatographic retentions of twelve Staphylococcal nuclease mutants and the naturally occurring protein showed that the chain location and the chemical nature of the substituted amino acid(s) were equally significant in determining the retention. Correlations between the retention times of these nuclease mutants and of previously published data for interleukin 2 mutants and insulin variants with nineteen amino acid-based predictive scales revealed retention time to be significantly correlated to several scales. The use of mutagenized proteins allowed a more sensitive analysis of the individual amino acid contributions to retention than can be achieved by utilizing a more diverse set of proteins.

Amino Acids↗
PubMed1987

Effect of positional environmental domains on the variation of high-performance liquid chromatographic peptide retention coefficients.

While attempting to derive a set of high-performance liquid chromatographic amino acid retention coefficients from a set of 298 related peptide analogues of a 13-amino acid peptide, we found that each position within the peptide would require a different set of retention coefficients to accurately predict peptide retention times. Furthermore, our results show that peptides having the same amino composition but slightly different sequences can have very different retention times. We believe that individual sequence domains and the resulting differences in the solid phase-mobile phase interactions must be taken into account for the accurate prediction of peptide retention times.

Amino Acids↗
PubMed1985

Liquid chromatographic retention behavior of organometallic compounds and ligands with amine-, octadecylsilica- and beta-cyclodextrin-bonded phase columns.

Effects of solvent composition and ligand variation on the retention of organometallic compounds have been studied using an amino, an octadecylsilica (ODS) and a beta-cyclodextrin (beta-CD) bonded phase column in either a normal-phase or a reversed-phase mode. The retention behavior for the organometallic compounds with the amino column can be rationalized using the displacement model. The "apparent" molecular areas are greater for compounds capable of strong hydrogen bonding. The retention in the ODS column roughly follows an argument based on the expected solubility behavior while mixed retention mechanisms are involved for the solubility behavior while mixed retention mechanisms are involved for the beta-CD column, i.e. both inclusion process and solubility or solvophobic interactions are possibly operative.

Amines↗
PubMed2002

Supercoiled circular DNA and protein retention in non-equilibrium chromatography temperature and velocity dependence: testimony of a transition.

Non-equilibrium chromatography (NEC) is a chromatographic mode for the rapid separation of polymers. The retention behavior of various proteins (human, chicken, bovine serum albumin) and supercoiled circular double-stranded DNA (plasmids) was investigated using a phosphate buffer as a mobile phase at different velocities and column temperatures with a C1 column with very low-packing particle diameter as a stationary phase. It was shown that the two factors (temperature and velocity) constituted important parameters in the retention mechanism of plasmids and proteins in NEC. The protein was retained more than the plasmid. At all the temperatures (5, 10, 15, 20, 25 degrees C) the plasmid retention increased over the entire flow-rate range (0.02-1.8 ml/min). For the protein, the retention curve presented a decrease in the relative retention time until a critical value of the mobile phase flow-rate, followed by an increase. The transition between the two well known NEC methods, slalom chromatography and hydrodynamic chromatography was clearly visualized for proteins at the lowest temperature, but did not appear for plasmids due to their strong compact structure.

Chromatography↗
PubMed2002

New equations describing the combined effect of pH and organic modifier concentration on the retention in reversed-phase liquid chromatography.

Six equations that express the combined effect of mobile phase pH and organic modifier content on sample retention in reversed-phase liquid chromatography (RPLC) are developed based on either the adsorption or the partition model for retention. The equations are tested against five retention data sets taken from literature. In the tests two pH scales are used, w(w)pH and s(s)pH. It is shown that a new seven-parameter equation works more satisfactorily, because it exhibits good numerical behavior, gives low values of the sum of squares of residuals and represents the experimental retention surfaces successfully. In addition, the danger of overfitting, which leads to the prediction of physically meaningless retention surfaces, is minimized by using the proposed new seven-parameter equation. Finally, the possibility of obtaining reliable pK values of weak acids or bases chromatographically by means of the derived equations is also considered and discussed.

Chromatography, Liquid↗
PubMed2003

Retention mechanism of fatty alcohol ethoxylates in reversed-phase liquid chromatography.

Fatty alcohol ethoxylates (FAEs) are widely used nonionic surfactants that have distributions in both alkyl and poly(ethylene oxide) (PEO) chain length. Generally, two-dimensional liquid chromatography technique is required for the complete characterization of both distributions. By selecting a proper stationary and mobile phase condition, however, we can obtain fully resolved chromatograms of a FAE sample (Brij 30) with respect to both alkyl and PEO chain length by using a single reversed-phase C18 column and aqueous acetonitrile mobile phase. FAEs show a peculiar reversed-phase liquid chromatography (RPLC) retention behavior with an aqueous-organic mobile phase, the retention mechanism of which has not been fully elucidated. For a fixed alkyl chain length, FAEs with higher-molecular-mass PEO block elutes first and the van't Hoff plot of the retention factor shows a curvature. The unique retention behavior can be understood from the opposite thermodynamic characteristics associated with RPLC retention of PEO block and alkyl chain: the sorption process of PEO to the non-polar stationary phase shows deltaH(o) > 0 and deltaS(o) > 0 while the alkyl chain shows deltaH(o) < 0 and deltaS(o) < 0 in contrast. The relative magnitude of the two contributions can change the elution order of the FAE. Therefore the often found, inverted elution order of FAEs (the early elution of FAEs with longer PEO block) is due to the positive enthalpic interaction of PEO blocks, which is a characteristic of the hydrophobic interaction. And the curvature of the van't Hoff plots was analyzed assuming the temperature dependent thermodynamic variables.

Fatty Alcohols↗
PubMed2003

Analysis and critical comparison of the reversed-phase and ion-exchange contributions to retention on polybutadiene coated zirconia and octadecyl silane bonded silica phases.

The two major modes of retention of basic compounds in reversed-phase liquid chromatography on both octadecyl silane bonded silica-based (ODS) and polybutadiene coated zirconia (PBD-ZrO2) materials are hydrophobic and ion-exchange (Coulombic) interactions. Although the influence of reversed-phase and Coulombic interactions on the chromatography of organic cations is qualitatively well recognized, the quantitative relationship between hydrophobic and ion-exchange interactions remains unclear. In this work, the retention mechanisms on both of the above types of phases were probed by studying the retention of a homologous series of p-alkylbenzylamines as a function of the ammonium concentration in the eluent. The various columns tested were studied in terms of plots of retention factor vs. the inverse of the displacingcation concentration. The analysis of such plots as well as plots of log k' vs. number of methylene groups in the solutes and plots of log k' vs. log[NH4+] clearly shows that at least two types of sites--a pure reversed-phase site and a "hydrophobically-assisted ion-exchange site" similar to the type of site described by Neue [J. Chromatogr. A 925 (2001) 49] are needed to explain the observations. In addition, we have found a quantitative measure of the relative amount of reversed-phase and ion-exchange interaction on a given solute has on a given stationary phase which allows unambiguous classification of columns. It is now clear that ion-exchange contributions to retention on PBD-ZrO2, sometimes exceeding 90%, are even more important than previously thought and relative to hydrophobic interaction much more significant on PBD-ZrO2 than on ODS type-B silicas.

Buffers↗
PubMed1998

Prediction of retention times of peptide nucleic acids during reversed-phase high-performance liquid chromatography.

Peptide nucleic acids (PNAs) are synthetic biopolymers consisting of nucleobase side chains attached to an amino ethyl glycine backbone. At present this family of compounds enjoys a well deserved popularity in biomedical research, due to a number of favorable biological and chemical properties of PNAs compared to conventional oligonucleotides. PNAs are basically peptides, and are synthesized, purified and analyzed by traditional peptide chemistry, chromatography and mass spectrometry techniques. In the current report, we analyzed factors that influence the elution behavior of 29 PNAs on reversed-phase high-performance liquid chromatography using a water-acetonitrile-trifluoroacetic acid gradient elution system on C18 columns. We found that increasing the temperature from 25 degrees C to 55 degrees C resulted in improved peak shape and resolution. The retention times of the PNA analogs were dependent upon the length of the polymers with longer PNAs eluting later. Mixtures of PNAs with length, originating from inefficient monomer coupling during the polymer assembly, could be separated by single chromatographic runs. The retention time also depended upon the cytosine, thymine, adenine and guanine contact of the polymers. These differences in the contribution to the retention times could be explained by simple hydrophobicity features of the monomer side chains at pH 1.8. Based on all data, a linear equation was generated which predicted the retention time of any synthetic PNA based on composition and length. Comparison of the predicted and observed retention times showed a remarkable reliability of the algorithm.

Chromatography, High Pressure Liquid↗
PubMed1999

Effect of eluent composition on retention behavior of anions in ion chromatography on anion-exchangers modified with heparin.

Effect of eluent composition on retention behavior of inorganic anions have been investigated in ion chromatography using anion-exchangers modified with heparin. Both cation and anion of the eluent affected the retention of analyte anions and unusual retention behavior was observed on the modified stationary phase. The retention time of anions decreased with decreasing eluent concentration when sodium sulfate, magnesium sulfate and chlorides of alkali metals were used as the eluent, whereas it increased with decreasing eluent concentration when aluminum sulfate, copper sulfate and sulfuric acid were used as the eluent. The retention of nitrate increased in the order of Li+, Na+, K+, Rb+ and Cs+ when their chlorides were used as the eluent. When sodium perchlorate and chlorides of alkaline-earth metals were used as the eluent, the eluent should include heparin. Otherwise, the modifier was partially bled from the column.

Anions↗
PubMed1999

Semi-empirical topological method for the prediction of the chromatographic retention of cis- and trans-alkene isomers and alkanes.

A new index is proposed for the prediction of the chromatographic retention of the cis- and trans-n-alkene isomers and alkanes. This index is based on the hypothesis that the chromatographic retention of the molecule is due to the interaction of each carbon atom with the stationary phase, and consequently the index is reduced by its neighbours' steric effects. The topological values are obtained by a numerical approximation considering the general behaviour of the chromatographic retention of the compounds. The simple linear regressions between the chromatographic retention and the index proposed for all branched alkanes and also isomers of the studied straight-chain C5 and C14 alkenes (1-ene, cis- and trans-2-, 3-, 4-, 5-, 6- and 7-enes) is very good (the correlation coefficient is r = 0.9999), and the elution sequence is correct for most of them. The models have a high predictive ability, as established by cross-validation values (r2cv). Thus, this new method, different from those already existent, can be used as complementary tool for the elucidation of the molecular structure, or prediction of the chromatographic retention of the cis- and trans-alkene isomers and branched alkanes. It could be extended with success, in the future, to the other types of compounds.

Alkanes↗
PubMed1999

Prediction of protein retention at gradient elution conditions in ion-exchange chromatography.

This work presents a prediction procedure for protein retention in ion-exchange chromatography, where two linear gradient experiments of different length give the protein retention time at other linear gradients. The procedure predicts the retention time of early and late eluting proteins with similar precision and predictions by extrapolation deviate approximately 3% or less from the experimental retention times. By using the ionic strength, this procedure predicts protein retention times obtained with divalent ions in the eluent more accurately than a well-established procedure that uses the protein co-ion concentration.

Chromatography, Ion Exchange↗
PubMed1999

Chromatographic evaluation of structure selective and enantioselective retention of amines and acids on cellobiohydrolase I wild type and its mutant D214N.

The mechanisms of structure selective and enantioselective retentions of amines and acids on two chiral stationary phases based on wild type cellobiohydrolase I (CBH I) and its mutant D214N have been investigated. All the amino alcohols tested had an enantioselective site that overlaps with the catalytically active site of CBH I, whereas the enantioselectivity of prilocaine was not affected by the mutation. The hydroxyl group of the amino alcohols did not seem to be an important contributor to the total binding strength whereas a bromo substituent in the aromatic ring promotes a high enantioselectivity (alpha=7.05). Interestingly, the chiral recognition site of the acid warfarin overlaps with the binding site of the amino alcohols. Di-p-toluoyltartaric acid and dibenzoyltartaric acid were strongly retained probably due to electrostatic attraction, but no enantioselectivity was observed. The difference in retention characteristics for the amino alcohols on the two stationary phases was strongly pH-dependent. A change in elution order of different amino alcohols occurred when changing the pH from 5.0 to 7.0. The difference between the two phases was lower at low pH. The retention times could also be affected by ionic strength and by use of cellobiose as a mobile phase additive but no indication of ion-pair retention of the amines was observed, when adding hexanesulphonate as counter ion to the mobile phase. The temperature dependence of the retention of the enantiomers of propranolol at pH 7.0 on the mutant D214N was similar to what was earlier observed on the wild type CBH I at lower pH.

Acids↗
PubMed1993

Effect of the alpha-amino group on peptide retention behaviour in reversed-phase chromatography. Determination of the pK(a) values of the alpha-amino group of 19 different N-terminal amino acid residues.

We have examined the contribution of the alpha-amino group to retention behaviour for peptides in reversed-phase chromatography using two series of peptide analogues, one containing an N alpha-acetylated terminal and the other containing an alpha-amino group (non-acetylated). The effect of the alpha-amino group, at pH 2, on the hydrophobicity of the side-chain of the N-terminal residue was obtained by referencing the retention time of the acetylated or non-acetylated peptide to the retention time of a glycine analogue. It was shown that the presence of an alpha-amino group could decrease or increase the hydrophobicity of the side-chain of the N-terminal residue with respect to the hydrophobicity of the side-chain in the absence of an alpha-amino group. The effect was also shown to be sequence dependent, with respect to the N-terminal residue. Increasing pH was shown to increase retention time dramatically for the non-acetylated analogues, through the deprotonation of the alpha-amino group. By separating pairs of acetylated/non-acetylated analogues over the pH range 2-9, it was possible to determine the pK(a) of the alpha-amino group, where it was shown that the pK(a) was dependent on two probable factors: (1) the inherent hydrophobicity of the stationary phase; and (2) the amino acid substituted in the N-terminal position. Interestingly, the pK(a) values determined were very similar to that found in proteins. It was also possible to determine the pK(a) values of some of the substituted amino acids containing ionizable side-chains. This study shows that, in order to understand fully the retention behaviour of peptides containing an alpha-amino group in reversed-phase chromatography, one must incorporate an alpha-amino group contribution and its effect on the hydrophobicity of the side-chain of the N-terminal residue.

Acetylation↗