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.     

Retention of ionizable compounds in high-performance liquid chromatography. IX. Modelling retention in reversed-phase liquid chromatography as a function of pH and solvent composition with acetonitrile-water mobile phases

The influence of pH and solvent composition of acetonitrile-water mobile phases on the retention of acids and bases on a polymeric stationary phase is studied. Very good relationships between retention and mobile phase pH are obtained if the pH is measured in the proper pH scale. The fit of retention to pH for a particular solvent composition provides the pKa values of the equilibria between the different acid-base species and the retention parameters of these species at this solvent composition. Several models are tested that relate these parameters to solvent composition and properties in order to propose a general model to predict retention for any mobile phase pH and composition.     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.     

蛋白质在离子交换色谱上选择性和非吸附特性

对蛋白质在离子交换柱上选择民性和非吸附特性进行了研究。蛋白质在有机磷酸锆阳离子色谱柱上,其保留作用随流动相ｐＨ值在离子强度的增加而减小;蛋白质在强阳离子和强阴离子色谱柱上的保留作用,即是流动相中的ｐＨ值等于蛋白质的等当点,其净电荷为零。不册蛋白质仍有不同程度的保留,这主要是由于蛋白质的三维结构使电荷 密度的大小和分布的不均匀以及离子交换填料表面性质的影响。     

The Separation of Organic Analyte Cations on a Low-Capacity Cation Exchange Column Using Indirect UV Detection

Abstract

The retention of organic analyte cations on a low-capacity cation exchange column using indirect UV detection was studied. It was found that a combination of cation exchange/reversed-phase interactions affected the retention of organic analyte cations provided the analytes have both a cationic charge site and a hydrophobic center. The factors that influenced the organic analyte cation retention were: concentration of organic modifier, concentration of UV absorbing analyte, pH, and mobile phase ionic strength. Elution orders for several of the organic analytes studied on the low-capacity cation exchange column were different than those observed on silica-based strong cation exchange columns.     

Comparison of column properties in reversed-phase chromatography: monolithic, cholesterolic and mixed bonded stationary phases

A commercial Chromolith C18 column and two new stationary phases with mixed ligands bonded on the Kromasil silica gel support, SG-MIX and SG-Chol, were characterized using simple tests based on the retention of non-polar, basic and acidic compounds. Polar and methylene selectivity tests in acetonitrile-water and methanol-water mobile phases revealed lower hydrophobicities of the SG-MIX and SG-Chol columns in comparison to the Chromolith column. The columns were further characterized using new test criteria - gradient oligomer capacity and isomeric selectivity and peak symmetry of naphthalene di-sulphonic acids in aqueous mobile phases. The cholesterolic column shows greater gradient oligomer selectivity for the separation of oligoethylene glycol samples than the SG-MIX and the Chromolith columns. Increased retention and peak tailing, but decreased isomeric selectivity for naphthalene-di-sulphonic acids was observed with the SG-MIX column, because of interactions with various polar bonded groups.     

Comparison of peak shapes obtained with volatile (mass spectrometry-compatible) buffers and conventional buffers in reversed-phase high-performance liquid chromatography of bases on particulate and monolithic columns

Retention factor, column efficiency and asymmetry factor were recorded for nine basic compounds on a number of RP-HPLC columns using phosphate and a variety of (MS-compatible) volatile mobile phase buffers of acid and neutral pH, in order to assess any effects of the buffer on performance. With formic or acetic acid, some phases gave partial or complete solute exclusion effects (reduced or negative k) compared with results using phosphate buffers at low pH. Despite its possible suppression of mass spectrometer sensitivity, trifluoroacetic acid was useful in enhancing retention times of relatively hydrophilic protonated bases, due to ion-pair effects. Peak shape was relatively poor on some pure silica-based ODS phases at pH 7 compared with results at acid pH. At low pH and at pH 7, ammonium and potassium phosphate gave very similar k, but the former may be preferable due to its volatile cation. Improved peak shapes, attributed to superior silanol masking effects, were obtained with ammonium phosphate at pH 7, but not at acid pH. Ammonium acetate gave acceptable peak shape at pH 7, but due to very limited buffer capacity, poor results were obtained for solutes having a pKa close to the mobile phase pH. Due to its instability, ammonium hydrogen carbonate is not a viable alternative buffer at pH 7.     

Characterization of several stationary phases prepared by thermal immobilization of poly(methyltetradecylsiloxane) onto silica surfaces

Variations of a thermal immobilization procedure using poly(methyltetradecilsiloxane) and silica produced fourteen stationary phases with carbon contents of 4-18%. The stationary phases were chromatographically evaluated with the Engelhardt, SRM 870 and Tanaka tests. Classifications using USP and Euerby procedures indicate that the new immobilized phases are different from most commercial phases although there was some similarity with phases that have high ion-exchange interactions. The retention mechanism involved in the separation of basic solutes on several of the new stationary phases was studied by varying pH, type of Lewis base and the ionic strength of the eluent. The separations are strongly influenced by the chemistry of the accessible free silanols. The stationary phases present good selectivity at intermediate pH where the basic analytes were protonated, suggesting use of intermediate pH for these separations. Stability tests show that the stationary phases have poor stability at very high pH, even at 23°C, but good stability in acidic mobile phases, even at 75°C, as expected for an immobilized polymer stationary phase.     

Effect of temperature on the chromatographic retention of ionizable compounds. III. Modeling retention of pharmaceuticals as a function of eluent pH and column temperature in RPLC

We propose a general simple equation for accurately predicting the retention factors of ionizable compounds upon simultaneous changes in mobile phase pH and column temperature at a given hydroorganic solvent composition. Only four independent experiments provide the input data: retention factors measured in two pH buffered mobile phases at extreme acidic and basic pH values (e. g., at least +/- 2 pH units far from the analyte pK(a)) and at two column temperatures. The equations, derived from the basic thermodynamics of the acid-base equilibria, additionally require the knowledge of the solute pK(a )and enthalpies of acid-base dissociation of both the solute and the buffer components in the hydroorganic solvent mixture. The performance of the predictive model is corroborated with the comparison between theoretical and experimental retention factors of several weak acids and bases of important pharmacological activity, in mobile phases containing different buffer solutions prepared in 25% w/w ACN in water and at several temperatures.     

Effect of the density of the C(18) surface coverage on the adsorption mechanism of a cationic compound and on the silanol activity of the stationary phase in reversed phase liquid chromatography

RPLC columns with different surface coverages (a C(1) endcapped column with a bonding density of 3.92 micromol/m(2) and four C(18)-bonded, endcapped columns, with octadecyl chain densities of 0.42, 1.01, 2.03, and 3.15 micromol/m(2)) were used to investigate the effects of the density of the surface coverage of RPLC columns on the adsorption mechanism of a cationic compound, amitriptyline chloride, and on the silanol activity of these columns. The mobile phases used were acetonitrile-water (30/70, v/v) solutions, buffered at either pH 2.7 or pH 6.9. At pH 2.7, the residual silanol groups are not ionized. At pH 6.9, some of these groups are ionized and these surface anions can strongly interact with the cationic compound. The adsorption isotherms were measured by frontal analysis (FA) at pH 2.7 and by frontal analysis by characteristic points (FACP) at pH 6.9, because the very high retention observed at neutral pH made FA measurements excessively long and poorly accurate. The adsorption energy distributions (AEDs) were calculated when possible, according to the expectation-maximization (EM) algorithm. A bimodal and a trimodal energy distribution were found for all the columns at pH 2.7 and 6.9, respectively. The third site measured at pH 6.9 was attributed to the strong ion-exchange interactions between the ionized silanol groups and the amitriptylinium cation. The contribution of the ionized silanol groups to the overall retention is maximum for the phases with intermediary bonding densities (1.01 and 2.03 micromol/m(2)). The peak tailing is most pronounced for the lowest (C(1) column) and the highest (3.15 micromol/m(2)) surface coverages.     

单柱离子色谱法测定一价阳离子的流动相研究

对单柱离子色谱法测定一价阳离子的流动相进行了系统研究，阐述了一价阳离子的保留行为和电导检测行为与流动相之间的关系，分别对无机酸（硝酸）、有机酸（柠檬酸）和芳香碱（苯胺）为流动相测定一价阳离子进行了讨论，其中有机酸和无机酸是较为适宜的流动相。     

Retention of ionizable compounds on high-performance liquid chromatography: VII. Characterization of the retention of ionic solutes in a C18 column by mass spectrometry with electrospray ionization

The elution of ions from a C₁₈ column with mobile phases containing methanol (60%, v/v) and aqueous buffers is studied by mass spectrometry. It is demonstrated that the anions are excluded from the stationary phase by the ionized silanols. However, the ionized silanols interact strongly with cations, which are retained in the column. These cations are later eluted from the column by ion exchange with the cations present in the pH buffered mobile phase. The size of the ions, the mobile phase cation concentration and the mobile phase pH are the main parameters that affect elution of the retained cations. It is also demonstrated that there are at least two different types of ionizable silanols, with different acidities, that contribute to the retention of cations. An estimate of the pK_a values of these two groups of silanols in 60% methanol is given.     

Ultra‐high performance separation of basic compounds on reversed‐phase columns packed with fully/superficially porous silica and hybrid particles by using ultraviolet transparent hydrophobic cationic additives

The use of the tetrabutylammonium additive was investigated in the ultra‐high performance reversed‐phase liquid chromatographic elution of basic molecules of pharmaceutical interest. When added to the mobile phase at low pH, the hydrophobic tetrabutylammonium cation interacts with the octadecyl chains and with the residual silanols, thus imparting a positive charge to the stationary phase, modulating retention and improving peak shape of protonated basic solutes. Two sources of additive were tested: a mixture of tetrabutylammonium hydroxide/trifluoroacetic acid and tetrabutylammonium hydrogen sulfate. Retention and peak shape of 11 basic pharmaceutical compounds were evaluated on commercially available ultra‐fast columns packed with octadecyl stationary phases (Ascentis Express C18 2.0 µm, Acquity BEH C18 1.7 µm, Titan C18 1.9 µm). All columns benefit from the use of additive, especially tetrabutylammonium hydrogen sulfate, providing very symmetric peaks with reasonable retention times. Focusing on the probe compounds amitriptyline and sertraline, efficiency and asymmetry values were investigated at increasing retention factor. The trend is very different to that obtained in reversed‐phase conditions and the effect lies in the complex molecular interaction mechanisms based on hydrophobic and ion exchange interactions as well as electrostatic repulsion.     

Duplicating the Retention of Cationic Analytes Obtained with Ammonium Formate Mobile Phases when Switching to UV Transparent Mobile Phase Additives in RP-LC

This study explored feasibility of utilizing sodium phosphate and mixtures of sodium phosphate and sodium perchlorate salts in mobile phases as UV transparent alternatives to the ammonium formate salts commonly used in LC–MS mobile phases. Chromatography experiments were run at pH 3.5 in 25% acetonitrile mobile phase, using several model cationic analytes to evaluate cation retention on two different C18 columns as the type or amount of salt was varied. For both columns, phosphate consistently showed less cation retention than formate. In other respects, the two columns showed very different behavior. The study suggests that it is feasible to use UV transparent mobile phase additives to provide comparable cation retention of formate mobile phases, but that the exact composition needed for optimal retention agreement is column dependent.     

A study of retention and overloading of basic compounds with mixed-mode reversed-phase/cation-exchange columns in high performance liquid chromatography

The retention and overload of bases were studied on two new mixed-mode, silica based phases possessing ionic carboxylate functionalities of different acidity embedded within a hydrophobic ligand (SiELC Primesep). At low pH, good peak shapes were obtained for small solute mass, suggesting that the mere presence of a mixed-mode hydrophobic/ionic retention mechanism is not responsible for the poor peak shape that can occur on conventional reversed-phases with ionised silanols. Somewhat inferior, but still acceptable peak shape for bases was obtained on a column containing a mixture of discrete ion exchange and reversed-phase particles (Hypersil Duet). In both types of column, the ionic sites favourably increased the capacity for ionised bases, reducing considerably the deterioration of peak shape with load observed with conventional RP columns. The combined ionic and reversed-phase interaction can give strong retention of bases under certain conditions, necessitating careful choice of stationary and mobile phase.     

Prediction of retention behaviour and evaluation of pka values of peptides and quinolones in liquid chromatography.

The present paper examines the effect of the solute ionisation on the retention behaviour in liquid chromatography of a series of peptide and quinolone compounds of biological interest, using acetonitrile-water media as mobile phases and a polymeric-based stationary phase. Polymeric columns with polystyrene-divinylbenzene (PS-DVB) polymer show advantages over silica-based reversed-phase packings since the former are stable in a wide pH range. (s)(s)pKa values have been evaluated using chromatographic data in acetonitrile-water mixtures with acetonitrile percentages of 30, 35, 40 and 50% (v/v) for quinolones and 12.5 and 20% (v/v) for peptides. The quinolones show great retention on PS-DVB phase stationary. It was thus necessary to work with a higher acetonitrile content in the mobile phase than for the less retained peptides. The pH values were measured in the hydroorganic mixtures, used as mobile phases, instead of in water and account was taken of the effect of activity coefficients. The derived equations permit the chromatographic determination of (s)(s)pKa. values of the peptides and quinolones in acetonitrile-water mixtures by fitting it to the experimental data in a nonlinear least-square procedure and also permit the prediction of the effect of (s)(s)pH on their chromatographic behaviour. We have also compared the obtained (s)(s)pKa values with those previously obtained in acetonitrile-water mixtures from potentiometric measurements.     

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.     

Evaluating the surface charge of C18 stationary phases

The surface charge of four C18 stationary phases was investigated by measuring the flow induced streaming potential, a well known electrokinetic property of charged surfaces. Three of the stationary phases (Symmetry, Gemini, and Xterra-MS) had significantly positive streaming potentials at both pH 3 and 4.5. The fourth (Zorbax-SB) appeared to be essentially neutral at pH 3 and became negative at pH 4.5. Apparent zeta potentials ranged from approximately +16 to -4 mV. The retention behavior was also investigated using chloride as model anion and glycinamide (in its protonated form) as model cation. When the retention factor (k) of glycinamide was subtracted from k of chloride anion, the resulting delta k values showed very similar trends as apparent zeta potential values, suggesting that the simple chromatographic method could be used to estimate zeta potential values, or that the zeta potential values could be useful for ranking columns according to ion exchange or exclusion behavior. The anion exchange capacity of the Symmetry and Gemini columns was also estimated, using a published chromatographic procedure, and the results suggest about 2 microEq. capacity per gram of packing.     

A dual-column HPLC method for the simultaneous determination of DHPG (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine) and its mono and diesters in biological samples

A convenient method for the simultaneous determination of various DHPG species present in biological samples is presented. This method utilizes a cation exchange column (25 cm X 4.6 mm i.d.) coupled in series to a short reversed-phase column (5 cm X 4.6 mm i.d.). The mobile phase consists of methanol:0.005M ammonium phosphate buffer, pH 2.5. There is a large polarity difference between DHPG and its esters due to the non-polar side chain of the ester moiety. The simultaneous determination of the diesters, monoesters, and DHPG in these samples using only the cation exchange or the reversed-phase column is not possible without time-consuming gradient elution. In the reversed-phase mode alone, the esters are highly retained relative to DHPG, whereas the esters are only slightly retained on a cation exchange column and are insensitive to changes in pH and ionic strength of the mobile phase. However, a combination of these two columns provides interesting selectivity for these compounds and offers a unique way of controlling the retention times of these species relative to each other. The retention time of esters can be selectively altered (with respect to DHPG) by changing the composition of methanol in the mobile phase. In contrast, the retention time of DHPG is controlled by changing the buffer strength and pH of the mobile phase.     

Enhanced‐fluidity liquid chromatography using mixed‐mode hydrophilic interaction liquid chromatography/strong cation‐exchange retention mechanisms

The potential of enhanced‐fluidity liquid chromatography, a subcritical chromatography technique, in mixed‐mode hydrophilic interaction/strong cation‐exchange separations is explored, using amino acids as analytes. The enhanced‐fluidity liquid mobile phases were prepared by adding liquefied CO₂ to methanol/water mixtures, which increases the diffusivity and decreases the viscosity of the mixture. The addition of CO₂ to methanol/water mixtures resulted in increased retention of the more polar amino acids. The “optimized” chromatographic performance (achieving baseline resolution of all amino acids in the shortest amount of time) of these methanol/water/CO₂ mixtures was compared to traditional acetonitrile/water and methanol/water liquid chromatography mobile phases. Methanol/water/CO₂ mixtures offered higher efficiencies and resolution of the ten amino acids relative to the methanol/water mobile phase, and decreased the required isocratic separation time by a factor of two relative to the acetonitrile/water mobile phase. Large differences in selectivity were also observed between the enhanced‐fluidity and traditional liquid mobile phases. A retention mechanism study was completed, that revealed the enhanced‐fluidity mobile phase separation was governed by a mixed‐mode retention mechanism of hydrophilic interaction/strong cation‐exchange. On the other hand, separations with acetonitrile/water and methanol/water mobile phases were strongly governed by only one retention mechanism, either hydrophilic interaction or strong cation exchange, respectively.