Comparative evaluation of aromatic bonded phases for reversed-phase chromatography

Summary Retention factors for alkylbenzenes, polyaromatic hydrocarbons, steroids, amino acids, peptides, nucleotides, nucleosides were measured with phenylsilica, benzylsilica, phenethylsilica and octylsilica stationary phases using hydro-organic eluents containing methanol, acetonitrile or tetrahydrofuran. The retention behavior of the three arylsilicas was similar but distinctly different from that of octylsilica. The difference is attributed to the chemical properties of the organic ligates, i.e., of the alkyl- and aryl functions. The slight variations of retention behavior observed with arylsilicas on the other hand are believed to arise from differences in the pertiment phase ratios. The method used for interpretation of data is based on the analysis of the pertinent retention moduli, relative column retentivities and relative phase ratios. In addition, relationships between eluite structure and retention were examined. The results give insight into various phenomena underlying the retention process in reversed-phase chromatography.Part IV in the series on Stationary Phase Effects in Reversed-Phase Chromatography.     

A refined physico-chemical model of solute retention in reversed-phase high-performance liquid chromatography with ternary mobile phases

Summary In our previous publication we have introduced a new model of solute retention in RP-HPLC systems with ternary mobile phases of the B+AB₁+AB₂ type (B: acetonitrile or tetrahydrofuran; AB₁: methanol; AB₂: water). That model proposed no stoichiometric differentiation between acetonitrile and tetrahydrofuran, alternatively present in the solvent system; moreover, it made some very rough assumptions only as to the intermolecular interactions among the mobile phase constituents.This paper introduces a significant refinement to the already established retention model, which is based on the simple quantitative relationships between acetonitrile and tetrahydrofuran, and the remaining components of the ternary liquid system. The refined model is tested with same experimental data.     

Peak-splitting in reversed-phase,ion-pair high-performance liquid chromatography of sympathomimetic drugs and its probable mechanism

Summary In the determination of ephedrine using reversed-phase, ion-pair liquid chromatography, a chromatographically pure sample was observed to give three peaks under certain mobile phase conditions. The mobile phases which produced maximum peak splitting were determined for ephedrine and a number of other sympathomimetic drugs.A proposal that peak splitting was the result of the composite interplay of two discrete chromatographic mechanisms, was investigated. The results of analysis by GC/MS confirmed that each peak was due to ephedrine, however, only one of the three split peaks was found to contain ion pairs. It is postulated that peak splitting is a physical phenomenon on reversed-phase columns and the separation of these drugs by ion-pair HPLC is based on a mixed rather than a single mechanism.This study has also shown that errors can arise in ion-pair HPLC when multiple peaks are assumed to indicate heterogeneity. Interconvertible species of the same solute can give rise to these peaks.     

Preparation and retention characteristics of different phenyl phases for reversed-phase liquid chromatography

Summary Three different phenyl phases were prepared. The amount of organic moiety bound on silica support was determined from thermogravimetric curves of the modified silica gel. The specific surface areas of gels were obtained from nitrogen sorption measurement at –196°C. The effect on separation and selectivity of the different bonded-phenyl functional groups was studied. The selectivity of the phenyl-bonded silica gels was examined and compared with octadecyl (ODS) silica in liquid chromatography. Corresponding to the high surface concentration of functional groups, the capacity factors of solutes, normalized to unit surface area of the adsorbent, k/S_BET were found to decrease in the sequence phenylmethyl>diphenyl>triphenyl. Polar solutes are retained in greater extent on the phenyl phases than on the ODS phase.     

Effect of the type of ion-pairing reagent in reversed-phase ion-pair chromatography

Summary The retention of positively and negatively charged and neutral solutes was studied in an aqueous phosphate buffer eluent, with constant inorganic-counterion concentration, on ODS-Hypersil stationary phase in the presence of various sulfonic acid ion-pairing reagents. The adsorption isotherms of the ion-pairing reagents d-camphor-10-sulfonic acid, sodium cyclohexylsulfamic acid and sodium alkylsulfonates (butyl-, hexyl- and octyl-) were determined by the breakthrough method.Dedicated to Professor J. F. K. Huber on the occasion of his 60th birthday.     

Retention behaviour of metal complexes with 5-sulphoquinoline-8-ol in reversed-phase ion-pair liquid chromatography

Summary The reversed-phase ion-pair liquid chromatographic behaviour of several metal complexes with 5-sulphoquinoline-8-ol (HQS) on an octadecyl-modified silica column was investigated by using a fluorescence detector. The aluminum complex gave two peaks on the chromatograms when eluents containing phosphate buffer were used. The effects of the pH of the eluent, HQS concentration, the nature of anions in the eluent, and the column temperature on the retention behaviour of the aluminum complex are discussed on the basis of the equilibria and the kinetics of the complexation of the aluminum ion with HQS and anions in the eluent. The equilibrium constant for the interconversion reaction between the two Al(III)-HQS species which correspond to the two peaks observed and the second stepwise formation constant for the Zn(II)-HQS complex were evaluated from the HPLC data obtained.     

Study on retention behavior of peropyrene-type polycyclic aromatic hydroccrbons with various bonded stationary phases in reversed-phase liquid chromatography

Summary The retention behavior of 15 peropyrene-type polycyclic aromatic hydrocarbons was investigated on various bonded stationary phases in reversed-phase liquid chromatography. On diphenyl and naphthylethyl bonded phases, high correlations were obtained between the molecular polarizability of solutes and their retention. However, very low or no correlations were found on various octadecyl bonded phases. These facts are discussed by using the electrostatic interaction concept between the solutes and the stationary phase. We conclude that these observations are due to two reasons: the difference in the degree of planarity of polycyclic aromatic hydrocarbons and the high ability of planarity recognition of octadecyl bonded phases.     

Retention behaviour of an homologous series of oligodeoxythymidilic acids using reversed-phase ion-pair chromatography

Summary The retention behaviour of an homologous series of phosphorylated oligodeoxythymidylic acid (pd(T)_5–18) oligonucleotides was studied using reversed-phase ion-pair chromatography with isocratic elution conditions. The effects of temperature, pH, eluent ionic strength, percentage organic modifier, concentration and alkyl chain length of the ion-pairing reagent were investigated. The retention behaviour was generally explicable by current theoretical models of ion-pair chromatography. However, the marked effect of mobile phase pH on the retention of the oligonucleotides was unexpected, and this was ascribed to the presence of ionisable residual silanols on the surface of the reversed-phase packing material.     

Application of reversed-phase and ion-pair HPLC for investigation of nucleotide metabolism in erythrocytes,reticulocytes and tumour cells

Summary The determination of nucleotides, nucleosides, and nucleobases was carried out in cells of different metabolic complexity: in mature and immature red blood cells, in Ehrlich ascites tumour cells from different proliferation stages, and in other tumour cells. The maturation of reticulocytes to erythrocytes is accompanied by loss of organelles and energy-requiring processes as well as the switch from aerobic to anaerobic ATP production. The profile of purine nucleotides, nucleosides, bases, and pyridine dinucleotides, by reversed-phae HPLC, shows large concentration changes during the maturation of red blood cells. The concentrations of purine mono and triphosphates are two to four times greater in reticulocytes in comparison with erythrocytes; the difference in the concentrations of nucleosides and nucleobases between reticulocytes and erythrocytes is even greater. Application of ion-pair HPLC showed that the Ehrlich ascites cells loose major portions of purine mono-, diand triphosphates between the 7th and 11th day after inoculation. Fast growing solid sarcoma tumours of rats (MV 202 Ner) contain higher amounts of nucleotides than slowly growing tumours of identical cell type.     

Preparation and evaluation of n-octadecylphosphonic acid-modified magnesia-zirconia stationary phases for reversed-phase liquid chromatography

Three n-octadecylphosphonic acid-modified magnesia-zirconia reversed stationary phases (C₁₈PZM) are prepared via the strong Lewis base interactions between organophosphonate and magnesia-zirconia composite. And two of them are end-capped by using trimethylchlorosilane as end-capping agent in different procedures. Stability studies at extreme high pH conditions (pH 9-12) show that both the non-endcapped and endcapped columns are quite stable at pH 12 mobile phase. The reversed-phase liquid chromatographic behavior of three C₁₈PZM stationary phases are comparatively investigated in detail using a variety of basic compounds as probes. The retention of basic compounds on the three phases is studied over a wide range of pHs. And the possible retention mechanisms of basic compounds on the three stationary phases are discussed. The results show that the basic solutes retain by a hydrophobic and cation-exchange interaction mixed mechanism on three stationary phases when they are operated in eluents at pH values near to the pK_a of the Brönsted conjugate acid form of the analyte, suggesting that inherent zirconol groups on ZM are not expected to interact with bases via cation-exchange interaction at lower pH. Nonetheless, the non-endcapped phase differs markedly from the edncapped ones in retention and selectivity of basic solutes using eluents at pH 4.1, implying a complex retention mechanism at this pH. The cation-exchange sites under such conditions are more likely due to the adsorbed Lewis base anionic buffer constituents (acetate) on accessible ZM surface sites than the chemisorbed phosphonate. Although the three phases exhibit very similar chromatographic behavior with eluents at pH 10.1, and show in general satisfactory separation of basic compounds and alkaloids studied, the performance for a specific analyte, however, differs largely from column to column.     

Rapid analysis of opium alkaloids by ion-pair high performance liquid chromatography

A new procedure for the rapid analysis of four major opium alkaloids is described using ion-pair, reversedphase high performance liquid chromatography and recently developed radial compression column technology. The solvents were aqueous 10mM potassium perchlorate with 5.0mM n-butylamine (pH 3.0) and pure acetonitrile. A gradient was run for five minutes from 10% to 70% acetonitrile and held at that level for two minutes. Flow rates were 3 ml/min. Two column packing materials were compared with this solvent system: standard C-18 packing and a fully-end-capped C-18 material. Results obtained with the fully-end-capped C-18 packing and the two solvent ion-pair gradient showed elution times under six minutes, with greater sensitivity (<50 nanograms) than previous systems.     

Selectivity studies on branched and linear alkyl bonded stationary phases in reversed-phase liquid chromatography

Summary Reversed-phase liquid chromatographic retention characteristics for the sixteen acyclic C₁−C₅ N-alkylbenzamide congeners were measured on various branched and linear, alkyl bonded hydrocarbon stationary phases. Retention factors, k′, were determined in acetonitrile-water mobile phase compositions on ethyl, n-octyl, n-dodecyl, n-octadecyl, 1-ethyladamantyl, 4-butyloctyl, and 2,4,4-trimethylpentyl stationary phases. Statistical analysis of the two main effects investigated — type of stationary phase and percentage of organic modifier (acetonitrile) in the mobile phase — described greater than ninety percent of the variability in the data for most of the comparisons. Selectivity effects due to variation in the mobile phase dominated the results.     

Determination of tylosin and related macrolides in fermentation broth by gradient elution ion-pair liquid chromatography

Summary A gradient elution ion-pair HPLC assay was developed for the analysis of tylosin and related macrolides in fermentation broth. The effect of the ion-pair agent concentration and identity was studied to optimize the reproducibility of the separation. The assay compares favorably with the biological assay and is sufficiently durable for the analysis of many samples per column.     

Computer-assisted prediction of small peptide sequencing in reversed-phase liquid chromatography

Summary A computer-assisted prediction system for small peptide sequencing has been constructed based on the retention prediction approach in reversed-phase liquid chromatography. The system has the basic function to predict retention of small peptides at any chromatographic conditions and this function can be useful for the prediction of sequencing. The outline of the system construction and the performance is discussed.     

Column selectivity in reversed-phase liquid chromatography. VII. Cyanopropyl columns

Eleven cyanopropyl ("cyano") columns were characterized by means of a relationship developed originally for alkyl-silica columns. Compared to type-B alkyl-silica columns (i.e., made from pure silica), cyano columns are much less hydrophobic (smaller H), less sterically restricted (smaller S*), and have lower hydrogen-bond acidity (smaller A). Because sample retention is generally much weaker on cyano versus other columns (e.g., C8, C18), a change to a cyano column usually requires a significantly weaker mobile phase in order to maintain comparable values of k for both columns. For this reason, practical comparisons of selectivity between cyano and other columns (i.e., involving different mobile phases for each column) must take into account possible changes in separation due to the change in mobile phase, as well as change in the column.     

On-column endcapping and derivatization in reversed-phase high-performance liquid chromatography

Summary On-column endcapping and derivatization or regeneration of C₈ and C₁₈ reversed-phase HPLC columns with newly introduced reagents were studied. These treatments can increase column life expectancy by restoring retention times and original chromatographic characteristics of the columns. This is illustrated by examples. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984