Reversed-Phase High-Performance Liquid Chromatography of Unsubstituted Aminobenzoic Acids

Abstract

High-performance liquid chromatographic (HPLC) characteristics of three position isomers of aminobenzoic acids (potential metabolites of important anesthetic drugs), were delineated with respect to their interactions with various mobile phases and stationary phases. HPLC with five hydrocarbonaceous phase, β-cyclodextrin silica (CDS), macrophase MP-1 polymer (MP), macroporous polystyrene/divinylbenzene (MPD), octadecylsilica (ODS), and propylphenylsilica (PPS), yielded results explicable in terms of substituent effects derived from the bifunctional amino– and carboxy groups. For cases where mobile phases contained sulfonates or quaternary ammonium salts both having longer chain alkyls, retention of analytes on all but CDS appeared to proceed predominantly via an ion-pairing mechanism. The extent of the corresponding counter-ion effects decreased in the order: MPD > ODS > PPS > MP, while the analyte retention order paralleled thier pH₂ values. On the other hand, an inverse relationship between the magnitude of capacity factors (k′) and pK₁ values of the title compounds was observed in experiments that produced retention data incompatible with ion-pair interaction rationales. The unique HPLC results obtained with the CDS phase are compared with those obtained with other phases.     

Retention Behavior of Benzodiazepines in Normal-Phase HPLC. Silica,Cyano, and Amino Phases Comparison

Abstract

The retention data for 16 benzodiazepines was evaluated on different normal-phase HPLC columns using the Soczewinski-Snyder model. The slopes and intercepts of the linear relationships between log k' and the mobile phase composition have been discussed in relation to solute and stationary phase characteristics. The good correlation between substituent chromatographic contribution and substituent electronic constant showed that the benzodiazepine molecule acts as a proton donor towards the adsorbent sites of the stationary phases investigated.     

High-performance liquid chromatographic separation of molecular species of neutral phospholipids.

Molecular species of neutral phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were resolved by reversed-phase high-performance liquid chromatography (HPLC) using mobile phases of acetonitrile-methanol-water containing tetraalkylammonium phosphates (TAAPs). Competitive interactions of TAAPs and analyte solutes with a reversed-phase HPLC column resulted in reduced retention of PC or PE with concomitant increase in detection sensitivity. The chromatographic data for PC and PE were distinctly different from those for negatively charged phospholipids where ion-pair retention mechanisms prevailed. While PC (or PE) components eluted at longer retention times with a larger size of TAAP, an increase in the TAAP concentration invariably caused a decrease in phospholipid retention times. Optimization of HPLC conditions by using high concentrations (25-100 mM) of tetramethylammonium phosphate in acetonitrile-methanol-water (70:22:8) facilitated elution of components with improved peak symmetry. HPLC separations of neutral phospholipids derived from animal sources were more complex than those from soybeans.     

Enantioselective HPLC separation of bioactive C5-chiral 2-pyrazolines on lux amylose-2 and lux cellulose-2: Comparative and mechanistic approaches

Stereoselective analytical HPLC separations have been developed for a series of biologically active chiral 2-pyrazolines (1-22) to be used in monitoring their resolution reactions or to custom semipreparative HPLC separations prior to biological assessment of both enantiomers. Polysaccharide-based chiral stationary phases (CSPs), namely, Lux amylose-2 and cellulose-2, have been used. Both normal (n-hexane/ethanol) and polar organic (ethanol, methanol, acetonitrile, or mixtures thereof) elution modes were very beneficial for the achievement of baseline separations. The impact of various chemical moieties embedded in the structures of 2-pyrazolines 1-22 and the adopted stationary phases on chiral recognition has been investigated. A case of reversed order of elution following alterations in either stationary phase or elution mode has been observed. Our findings recommend that normal elution mode can be used for optimizing semipreparative HPLC methods whereas polar organic mobile phases (such as acetonitrile and ethanol) are more suited to stereoselective reactions monitoring, routine quality control work, or for pharmacological and toxicological assays. These results settle the implementation of polysaccharide-based CSPs using different elution modes and declare the practicality of such CSPs in stereoselective HPLC.     

Effect of Temperature and Mobile Phase Composition on RP-HPLC Separation of Cephalosporins

Abstract

The effects of mobile phase composition and column temperature on resolution in reversed-phase high performance liquid chromatography (RP-HPLC) were used to separate the following cephalosporins: cefonicid, cefaclor, cephazolin, cefodizime, cephaloridine, cephamandole and cephalotin, from a single sample. The capacity factor (k') was described as a function of temperature and mobile phase composition. Semi-empirically estimated values of k' were determined using a small number of experimental data for different temperatures between 20°C and 60°C, and mobile phase compositions (acetate buffer/isopropanol). The capacity factor of each cephalosporin was observed to decrease with increasing temperature and the volume fraction (V) of isopropanol in the mobile phase; linear relationships were obtained for plots of In k' versus 1/T and log k' versus log V. The method developed, while simple, reveals the optimal isocratic elution conditions for column temperature and eluent concentration for the complete separation and rapid analysis of the cephalosporins studied.     

Chiral Separations: A Comparison of Hplc and TLC

Abstract

Comparisons are made for separations attained in normalphase HPLC and TLC involving N-carbobenzyloxy-glycyl-L-proline and (+/?)?10-camphorsulfonic acid as chiral mobile phase additives/chiral counter ions (CMA). Possible reasons for differences in stereoselectivity of β-cyclodextrin CMA and chiral stationary phases (CSP) are discussed. In addition, differences in solubility and proposed separation mechanisms for native cyclodextrins (CD) versus derivatized CD are discussed. Inherent differences in high performance liquid chromatographic (HPLC) and thin-layer chromatographic (TLC) methods are outlined. The pros and cons of using HPLC and TLC are considered.     

Molecular species analysis of phosphatidylcholine by reversed-phase ion-pair high-performance liquid chromatography

The retention behavior of molecular species of phosphatidylcholine (PC) is studied by reversed-phase (RP) ion-pair high-performance liquid chromatography (HPLC). Mobile phases contain tetraalkyl ammonium phosphates (TAAPs) in methano-acetonitrile-water. The stationary phase is alkyl-bonded silica. Competitive interactions of TAAPs, analyte solutes, and an RP-HPLC column result in reduced retention of PC molecular species. PC molecular species are eluted at longer retention times with a larger size of TAAP in the mobile phase, and an increase in the TAAP concentration invariably causes a decrease in PC molecular species retention times. There is a linear correlation between the logarithmic retention factors (k) of PC molecular species and the total number of carbon atoms of TAAP, and the logarithm of k values of PC molecular species can be approximated as a linear function of the logarithm of the counter-ion concentration. There is found to be no distinct dependence between k values of PC molecular species and the mobile phase pH.     

RP-HPLC Retention Data of New 2-Amino-2-Oxazolines. An Approach of Their Lipophilic Properties

Abstract

A RP-HPLC procedure has been developed for measuring the capacity factor k' of a series of new 5-substituted-2-amino-2-oxazolines. the chromatographic behaviour measured on a μBondapack C₁₈ column with methanol-aqueous buffer as mobile phase was related to the volume fraction of methanol φ. the log k' value extrapolated to 0% organic modifier in the eluent (log k'_w) was chosen as a measure of the solute lipophilicity. A good correlation was found between the slope S and the intercept value (log k'_w) of the log k' versus φ straight lines for all these structurally related 2-amino-2-oxazolines.

Some parameters related either to chromatographic conditions or to chemical structure were shown to influence the capacity factor. A study made at different pH values indicated that log k'_w increased with the basicity of the mobile phase.

The influence of the eluent pH on the capacity factor of 2-amino-2-oxazolines was related to their pKa's values determined by a potentiometric method.

Since log k' is considered as a valuable indice of the lipophilicity, the determined values will be used for quantitative structure-activity relationship studies of these new class of structurally related compounds     

Study of the Lipophilic Character of Xanthine and Adenosine Derivatives: II. Relationships Between Log K', RM and Log P Values

Abstract

The log k' values of a series of xanthine and adenosine derivatives were measured by means of a reversed-phase HPLC. The HPLC data were shown to be well correlated with previously reported R_M and R_MC18 values. The equations describing the relationships log k'/R_M and log k'/R_MC18 allowed the calculation of the log k' values of some compounds, which were not tested in the HPLC system. Since the relationship log k'/log P is very close to the previously described relationships R_M/log P and R_MC18/log P one can conclude that reversed-phase TLC and HPLC are very similar in describing the lipophilicity of the compounds.     

Comparison of Octadecyl-Bonded Alumina and Other Stationary Phases for Lipophilicity Estimation by High Performance Liquid Chromatography

Abstract

A recently developed octadecyl-bonded alumina stationary phase (ODA) was evaluated for determining the lipophilicities of organic compounds by high performance liquid chromatography. Using a column packed with this material and mobile phases consisting of methanol and aqueous buffers, the correlation between the octanol-water partition coefficients (log P's) of compounds of various chemical classes and the logarithms of their chromatographic capacity factors (log k's) was found to be superior to that obtained using columns packed with octadecylsilica, poly butadiene-coated alumina or octadecyl-derivanzed polystyrene-divinylbenzene copolymer. In contrast to results obtained with other columns, phenols and other hydrogen-bonding compounds did not need to be treated as a separate data set on the ODA column to obtain good correlations between log k's and log P's. The resistance of ODA to degradation by alkaline solvents allowed the use of a basic mobile phase (pH > 10) for suppressing ionization and determining the lipophilicities of organic bases which could not be evaluated within the stable pH range of ODS (pH 2–8). The log P's of five basic pharmaceutical compounds determined in this manner were found to be significantly higher than previously reported values. Evidence is presented which indicates that the previously reported log P values of these compounds are inaccurate, due to improper correction for ionization.     

Hydrophobic Interaction Chromatography of t-RNA's and Proteins

Abstract

Various microparticulate siliceous bonded stationary phases having weakly hydrophobic ligates were developed for HPLC of proteins and t-RNA's by hydrophobic interaction chromatography (HIC). It was confirmed that optimal separation of different types of biopolymers can be obtained by using a set of stationary phases having appropriate hydrophobic properties. Thus, the separation of t-RNA's is best carried out on stationary phases which are more hydrophobic than those optimal for HIC of proteins. Plots of log k' of both proteins and t-RNA's against the salt molality in the eluent yielded straight lines at sufficiently high salt concentrations in the eluent. The limiting slopes represent the hydrophobic interaction parameter for the particular chromatographic system and can serve as measures of the hydrophobic character of either the biopolymer or the stationary phase. Stationary phases with covalently bound polyether chains at the surface were found to be most suitable for HIC of proteins and t-RNA's.     

Reversed-Phase Systems for the Separation of Coumarins and Furocoumarins by Thin-Layer and High-Performance Liquid Chromatography

Abstract

The retention behaviour of eleven derivatives of counarin was investigated by reversed-phase (RP) thin-layer and high performance liquid chromatography. The linear relationships between log k'or R_M values and the content of organic modifier in the aqueous mobile phase obtained for wide composition ranges indicate that the plots can be used to determine R_Mw and log k_w values by extrapolation to pure water. The effects of individual substituants on the retention and the correlation between TLC and HPLC data was analysed.     

HPLC Phospholipid Separation Mechanisms on Silica,Amino, and Diol Columns

Abstract

The HPLC separation of phospholipid mixtures was investigated on silica, amino, and diol columns, using mobile phases consisting of acetonitrile, methanol, and phosphoric or trifluoroacetic acids in varying proportions. An explanation of the mechanism of these separations is given with regard to the individual column types.     

Improved High-Performance Liquid Chromatography of Phospholipids

Abstract

The reversed-phase HPLC of phosphatidylcholine and di- and triglycerides is presented with particular emphasis on improving the efficiency of the phospholipid separations so that they are comparable to those attained for di- and triglycerides. Elevated column temperatures (60°C) and addition of strong mineral acids or ion-pairing agents to the mobile phase were found useful in accomplishing this goal.     

Reversed-Phase Separation of the Major Deoxyribonucleosides and Their Mononucleotides Using Tetrabutylammontum Hetaerons

Abstract

Simultaneous separation of the four major deoxyribonucleosides and their monophosphate nucleotides was achieved using tetrabutyl ammonium phosphate hetaerons with a reversed-phase (C8) packing material. Baseline resolution for all eight solutes was achieved within 48 minutes, using a 7.5% methanol mobile phase, 2.0 mM in TBA, buffered with 50 mM phosphate at pH 4.8. The effect of methanol and TBA concentrations upon the retention of neutral and anionic solutes was studied in detail. It was determined that changes in solute k' with increasing methanol could be explained by essentially independent phenomena. These are: 1) a decrease in the partition coefficient of the TBA cation with increasing organic concentration, resulting in lower surface charge densities, and 2) a decrease in the hydrophobic interactions of the solutes with the reversed-phase HPLC. The overall effect was a log-linear decrease in k' with increasing methanol concentration. An empirical equation was derived for the above model which was found to be helpful in determining the optimal separation conditions for the nucleosides and nucleotides.     

Liquid Chromatographic Behavior of Selected Aza-arenes and Potential Metabolites

Abstract

Reversed phase HPLC was applied to analyses of selected aza-arenes and potential metabolites (5,6-benzoquinoline, 5,6-benzoquinoline-N-oxide, and the N-methyliodide salt of 5,6-benzoquinoline). Naphthalene and anthracene were employed as reference materials. Water-methanol and ammonium phosphate-methanol mobile phases were used with several commercial octadecyl reversed phase columns and a column using laboratory synthesized ODS stationary phase. Chromatographic behavior of the hydrocarbon reference compounds were excellent on all packings with either mobile phase. Benzoquinoline and derived materials were more difficult analytical subjects; water-methanol mobile phases proved unsuitable giving very long retention times with unacceptable efficiencies. Ammonium phosphate containing mobile phases were more appropriate for the nitrogen containing materials especially when used with end capped or polymeric end capped stationary phases.     

Studies on Steroids. CLXXVIII. Separation of Estrogen Glucuronides by High-Performance Liquid Chromatography

Abstract

Separation of monoglucuronides of estrone, estradiol, estriol and 16-epiestriol by high-performance liquid chromatography on a reversed-phase column has been carried out. The effects of pH and salt concentration of a mobile phase on the k' value were investigated with a TSK GEL LS-410 ODS-SIL column. Isomeric monoglucuronides of estriol and 16-epiestriol were distinctly separated on this column when 0.7% disodium hydrogen phosphate (pH 3.0)/tetrahydrofuran was used as a mobile phase.     

Assessment of Mobile Phase Flow Resistance of Fused Microplatelet Alumina-Based Stationary Phases for Reversed Phase High Performance Liquid Chromatography

Abstract

The mobile phase flow resistances (φ's) of several stationary phases consisting of surface-modified Unisphere^TM fused microplatelet alumina particles were determined and compared with corresponding values obtained from stationary phases based upon more conventional spherical and angular silica and alumina particles. Although the φ value obtained for a fused-microplatelet alumina-based phase was slightly lower than the value obtained on a spherical alumina-based phase with similar particle dimensions and surface modification, factors other than particle shape were found to have a substantial effect on mobile phase flow resistance. Larger φ values were obtained with phases with smaller average particle diameters. These were attributed to the presence of greater numbers of microparticles with diameters less than 5 microns in such phases. Larger φ values obtained for wide pore (21 nm) and octyl-bonded alumina-based phases over narrow pore (11 nm) and octadecyl-bonded phases were attributed to the former's ablities to entrap larger volumes of stagnant mobile phase within their pores.     

Solvent Effects in Reversed Phase Liquid Chromatooraphy for High and Low Loadings of Octadecylsilane on Microparticle Silica. I

Abstract

The selectivity of two ODS bonded phase packings, one with a high carbon load, the other with a low carbon load, were investigated. Many organic solvent/water mobile phases were used with three sample mixtures of varying polarity. When the separations were normalized, trends in the solubility parameters of the various classes of organic solvents were noted. Analyzing the separation factors and considering possible mobile phase, solute, and bonded phase interactions furnished some insight into possible separation/selectivity mechanisms.     

Application of Iso-Selective Gradient Elution for the Separation of Selected Phthalates

Abstract

In this paper, the overlapping resolution mapping scheme was applied to the gradient HPLC separation of selected phthalates. The procedure employs a minimum of seven experiments for the determination of the optimum mobile phase gradient for the desired separation. In the present work, solvent systems for iso-selective multisolvent gradient elution were used. The overlapping resolution mapping procedure was found to be a rapid and systematic approach for optimizing HPLC separations.