Characterization of a microparticulate strong anion-exchanger in the HPLC of acidic drugs

The use of Waters Spherisorb S5SAX for the HPLC of acidic compounds, including a number of non-steroidal anti-inflammatory drugs (NSAIDs), has been investigated. Adequate retention, separation, and peak efficiency and symmetry were obtained for most analytes on a 250 x 4.6 mm i.d. column using methanol containing ammonium perchlorate (10 mmol L(-1), pH 6.7 or pH 8.3) as eluent. The results of changes in (i) eluent pH (constant ionic strength); (ii) eluent ionic strength (constant pH); and (iii) adding water to the eluent (constant pH) were consistent with a retention mechanism dominated by ion-exchange with the bonded strong anion-exchange (SAX) moieties. However, there were some unexpected observations, including (i) a general decrease in retention at eluent pH values above 7.7; (ii) a marked increase in retention on adding 1% (v/v) water to the eluent; (iii) a subsequent marked decrease in retention on adding 5% (v/v) or more water; and (iv) decreased column activity with time. These observations may be due to (i) interaction between the charged SAX moieties and ionised surface silanols (with ionization increasing at higher eluent pH values) and (ii) influence of the solvation of silanols, analytes, SAX moiety, and counter-ion varying with both pH and water content. Nevertheless, the factors influencing separation of individual NSAIDs remain unclear especially as no relation between log k and pKa exists for these compounds. Hydrophobic interactions are unlikely to be important since basic and neutral compounds were hardly retained. Ease of accessibility of the counter-ion to the SAX moiety for analyte displacement may be a factor.     

Optimisation and characterisation of silica-based reversed-phase liquid chromatographic systems for the analysis of basic pharmaceuticals

Reversed-phase liquid chromatography using silica-based columns is successfully applied in many separations. However, also some drawbacks exist, i.e. the analysis of basic compounds is often hampered by ionic interaction of the basic analytes with residual silanols present on the silica surface, which results in asymmetrical peaks and irreproducible retention. In this review, options to optimise the LC analysis of basic pharmaceutical compounds are discussed, i.e. eluent optimisation (pH, silanol blockers) and stationary phase optimisation (development of new columns with minimised ionic interactions). The applicability of empirical based, thermodynamically based and test methods based on a retention model to characterise silica-based reversed phase stationary phases, as well as the influence of the eluent composition on the LC analysis of basic substances is described. Finally, the applicability of chemometrical techniques in column classification is shown.     

Reduction of silanophilic interactions in liquid chromatography with the use of ionic liquids

A suppression of silanophilic interactions by the selected ionic liquids added to the mobile phase in thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) is reported. Acetonitrile was used as the eluent, alone or with various concentrations of water and phosphoric buffer pH 3. Selectivity of the normal (NP) and the reversed (RP) stationary phase material was examined using a series of proton-acceptor basic drugs analytes. The ionic liquids studied appeared to significantly affect analyte retention in NP-TLC, RP-TLC and RP-HPLC systems tested. Consequently, the increased separation selectivity was attained. Due to ionic liquid additives to eluent even analytes could be chromatographed, which were not eluted from the silica-based stationary phase materials with 100% of acetonitrile in the mobile phase. Addition of ionic liquid already in very small concentration (0.5%, v/v) could reduce the amount of acetonitrile used during the optimization of basic analytes separations in TLC and HPLC systems. Moreover, the influence of temperature on the separation of basic analytes was demonstrated and considered in practical HPLC method development.     

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.     

CEC separation of insect oostatic peptides using a strong-cation-exchange stationary phase

The separation of several insect oostatic peptides (IOPs) was achieved by using CEC with a strong-cation-exchange (SCX) stationary phase in the fused-silica capillary column of 75 microm id. The effect of organic modifier, ionic strength, buffer pH, applied voltage, and temperature on peptides' resolution was evaluated. Baseline separation of the studied IOPs was achieved using a mobile phase containing 100 mM pH 2.3 sodium phosphate buffer/water/ACN (10:20:70 v/v/v). In order to reduce the analysis time, experiments were performed in the short side mode where the stationary phase was packed for 7 cm only. The selection of the experimental parameters strongly influenced the retention time, resolution, and retention factor. An acidic pH was selected in order to positively charge the analyzed peptides, the pI's of which are about 3 in water buffer solutions. A good selectivity and resolution was achieved at pH <2.8; at higher pH the three parameters decreased due to reduced or even zero charge of peptides. The increase in the ionic strength of the buffer present in the mobile phase caused a decrease in retention factor for all the studied compounds due to the decreased interaction between analytes and stationary phase. Raising the ACN concentration in the mobile phase in the range 40-80% v/v caused an increase in both retention factor, retention time, and resolution due to the hydrophilic interactions of IOPs with free silanols and sulfonic groups of the stationary phase.     

Retention behaviour of strong acid anions in ion-exclusion chromatography on sulfonate and carboxylate stationary phases

Some factors influencing the retention of strong-acid anions on ion-exclusion columns were investigated using columns with sulfonate and carboxylate functional groups. The nature of the functional group on the resin, the eluent pH and the eluent ionic strength all significantly affected the retention and separation of these analytes. Retention was observed for all strong-acid anions over the eluent pH range 2.2-5.7 and increased with both decreasing eluent pH and increasing eluent ionic strength. Some separation of strong-acid anions was possible when using a resin with carboxylate functional groups. It has also been demonstrated that strong-acid anions are poor markers of column void volume for ion-exclusion chromatography. A more accurate value was obtained using the neutral polymeric material dextran blue. When using eluents of low ionic strength, poor or fronted peak shapes were observed. A mechanism for these observations is proposed that relates the shape to ionic strength changes across the peak. A system peak was encountered under most experimental conditions. The properties of this peak are discussed and a cause for the system peak postulated.     

Reversed Phase Chromatography – the Mystery of Surface Silanols

The nature of surface silanols is reviewed and their influence on retention of basic solutes in reversed phase chromatography is demonstrated and evaluated. The influence of the type of organic modifier and/or pH of buffer on retention of basic analytes with silica based RP columns is evaluated, and means for characterization of RP columns are discussed. Attempts are described to determine the surface silanol concentration of RP columns by chromatographic methods. Surprisingly the experimentally measured silanol concentrations with bare silica and with RP are by one order of magnitude lower than discussed in literature. At pH 7.6 values of 0.12 µmol m^?2 have been measured for benzylamine with RP columns, corresponding to about 43% of the measurable silanols of plain silica. The break-through curves of amines let suggest that even at low pH values unprotonated species are present within the pores of the RP stationary phases and their interaction with the bonded alkyl groups contribute to retention.     

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.     

The application of retention indices using the alkylarylketone scale to the separation of the barbiturates by HPLC. I. The effect of the eluent

Summary In order to determine the applicability of retention indices, based on the alkylarylketone scale, as the basis of a reproducible method of reporting retentions, the separation of ten barbiturates and a set of column test compounds were examined on an octadecylsilyl bonded silica (ODS-Hypersil) column with methanol-buffer pH 8.5 as eluent. The effects on the capacity factors and retention indices, on changing the eluent composition, pH, ionic strength and temperature, showed that the retention indices of the barbiturates were much less susceptible to minor changes in the eluent than the capacity factors. For non-ionised compounds the retention indices were virtually independent of the experimental conditions. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Mixed-mode reversed-phase and ion-exchange separations of cationic analytes on polybutadiene-coated zirconia

The retention and selectivity of the chromatographic separation of basic (cationic) analytes on a polybutadiene-coated zirconia (PBD-ZrO2) stationary phase have been studied in greater detail than in previous studies. These separations are strongly influenced by the chemistry of the accessible surface of zirconia. In the presence of buffers which contain hard Lewis bases (e.g., phosphate, fluoride, carboxylic acids) zirconia's surface becomes negatively charged due to adsorption of the buffer anion at the hard Lewis acid sites. Consequently, under most conditions (e.g., neutral pH), cationic analytes undergo both hydrophobic and cation-exchange interactions. This mixed-mode retention process generally leads to greater retention factors for cations relative to those on silica-based reversed phases despite the lower surface areas of the zirconia phase, but, more importantly, adsorption of hard Lewis bases can be used to control the chromatographic selectivity for cationic analytes on these zirconia-based stationary phases. In contrast to our prior work, here we show that when mixed-mode retention takes place, both retention and selectivity are easily adjusted by changing the type of hard Lewis base buffer anion, the type of buffer counter-ion (e.g., sodium, potassium, ammonium), the pH, and the ionic strength of the eluent as well as the type and amount of organic modifier.     

pH gradient high-performance liquid chromatography: theory and applications

pH gradient high-performance liquid chromatography (HPLC) is a method of reversed-phase high-performance liquid chromatography suitable for ionogenic substances. It consists in programmed increase during the chromatographic process of the eluting strength of eluent with respect to the analytes separated. On the analogy of the conventional organic modifier gradient reversed-phase HPLC, in the pH gradient approach the eluting strength of the mobile phase increases due to its changing pH: increasing in case of acids or decreasing in case of bases. At the same time the content of organic modifier remains constant. A theory of the pH gradient HPLC has been elaborated. The resulting mathematical model is easily manageable. Its ability to predict changes in retention and separation of analytes following the changes in chromatographic conditions is demonstrated. The pH gradient method is uniquely suitable to determine pKa values of analytes. An equation is presented allowing to calculate pKa values basing on appropriate retention data. The effects on pKa are discussed of the concentration of methanol in the mobile phase. The RP HPLC-derived pKa data correlate to the reference pKa values (w(w)pKa) but are not identical. That may be explained by the effects on the chromatographically determined pKa of the specific interactions of analytes with stationary phases. The proposed pH gradient RP HPLC procedure offers a fast and convenient means to get comparable acidity parameters for larger series of compounds, like drug candidates, also when the analytes are available only in minute amounts and/or as complex mixtures.     

Characterisation of reversed-phase stationary phases for the liquid chromatographic analysis of basic pharmaceuticals by thermodynamic data

This paper describes the characterisation of reversed-phase liquid chromatography (RPLC) columns using thermodynamic measurements. Retention versus 1/T data were used to construct Van't Hoff plots. The slope of these plots indicates the standard enthalpy of transfer of the analyte from the mobile to the stationary phase. The standard entropy can be calculated from the intercept. Van't Hoff plots were linear for the investigated RPLC columns, meaning that for basic analytes over the temperature range studied no changes in the retention mechanism occurred. Enthalpies and entropies of transfer of basic analytes from the mobile to the stationary phase revealed information about the types of interaction of protonated and neutral compounds with the stationary phases. However, a clear view using the present set of basic compounds on how these thermodynamic data may explain the observed substantial differences in peak symmetry cannot be given. It is considered that addition of N,N-dimethyloctylamine (DMOA) to the eluent will results in a dynamically coating of the stationary phase. Addition of DMOA to the eluent resulted for protonated basic compounds in a reduction of both enthalpy and entropy. In practice, with DMOA in the eluent symmetrical peaks were obtained. It is assumed that this is due to blocking residual silanols and/or ion exclusion effects.     

Suppression of deleterious effects of free silanols in liquid chromatography by imidazolium tetrafluoroborate ionic liquids

Silica-based stationary phases are commonly used in liquid chromatography, but their surface acidity causes known problems, especially when separating basic compounds. Deleterious effects of free silanols are not fully removed by standard prevention procedures consisting in adding alkylamines or other amino quenchers to the eluents. We found that ionic liquids of the imidazolium tetrafluoroborate class, added to mobile phases at concentrations of 0.5-1.5% (v/v), blocked silanols and provided excellent thin-layer chromatographic separations of strongly basic drugs which were otherwise not eluted, even with neat acetonitrile as the mobile phase. The silanol suppressing potency of imidazolium tetrafluoroborates was demonstrated to markedly exceed that of the standard mobile phase additives, like triethylamine, dimethyloctylamine and ammonia. The proposed new mobile phase additives were also demonstrated to provide reliable lipophilicity parameters of base drug analytes as determined by gradient mode of high-performance liquid chromatography. By applying the readily available and environmentally friendly imidazolium tetrafluoroborate ionic liquids, simple and efficient means of improvement of liquid chromatographic analysis of organic bases were elaborated.     

Comparison of the chromatography of octadecyl silane bonded silica and polybutadiene-coated zirconia phases based on a diverse set of cationic drugs

In this study, we compare the separation of basic drugs on several octadecyl silane bonded silica (ODS) phases and a polybutadiene-coated zirconia (PBD-ZrO2) phase. The retention characteristics were investigated in detail using a variety of cationic drugs as probe solutes. The ODS phases were selected to cover a relatively wide range in silanol activity and were studied with ammonium phosphate eluents at pH 3.0 and 6.0. Compared to any of the ODS phases, the PBD-ZrO2 phase showed very significant differences in selectivities towards these drugs. Due to the presence of both reversed-phase and ion-exchange interactions between the stationary phase and the basic analyte on ODS and PBD-ZrO2, mixed-mode retention takes place to some extent on both types of phases. However, very large differences in the relative contributions from ion-exchange and reversed-phase interactions on the two types of phases led to quite different selectivities. When phosphate is present in the eluent and adsorbs on the surface, the PBD-ZrO2 phase takes on a high negative charge over a wide pH range due to phosphate adsorption on its surface. On ODS phases, ion-exchange interactions result from the interactions between protonated basic compounds and ionized residual silanol groups. Since the pH of the eluent influences the charge state of the silanol groups, the ion-exchange interactions vary in strength depending on pH. At pH 6.0, the ion-exchange interactions are strong. However, at pH 3.0 the ion-exchange interactions on ODS are significantly smaller because the silanol groups are less dissociated at the lower pH. Thus, not only are the selectivities of the ODS and PBD-ZrO2 phases different but quite different trends in retention are observed on these two types of phases as the pH of the eluent is varied. More importantly, by using the large set of "real" basic analytes we show the extreme complexity of the chromatographic processes on the reversed stationary phases. Both the test condition and solute property influence the column performance. Therefore, use of only one or two probe solutes is not sufficient for column ranking.     

Separation of Proteins on Polar Bonded Phases by Hydrophobic Interaction Chromatography

Abstract

Hydrophobic interaction chromatography (HIC) with a polar bonded phase (“Acetamide”) developped for size exclusion chromatography (SEC) is described. Retention of proteins depends on the surface area of the stationary phase, the pH and ionic strength of the eluent. For efficient separation the pore diameter should be 25 nm or more. The surface area should be large to achieve retention even at low ionic strength. Separation is only possible with a gradient from high to low ionic strength. Gradient volumes of 10 empty column volumes with column lengths above 15 cm are recommended. Selectivity can be optimized via pH adjustment. The advantage of this column packing is its applicability for two different separation modes: SEC and HIC.     

High-pressure liquid chromatography of drugs: II. An evaluation of a microparticulate cation-exchange column

A microparticulate cation-exchange column has been evaluated for the chromatography of thirty compounds selected as representative of a wide variety of drug substances. Although the column exhibited a strong partition effect besides the expected ion-exchange mechanism, the retention of drugs could be predictably influenced by variation of the eluent ionic strength and organic solvent content.

For acidic drugs the column showed little selectivity (although a salting-out effect increased retention at high eluent ionic strengths), but for basic substances Partisil SCX may afford a useful separative medium offering reasonable chromatographic efficiency (HETP ≈ 0.1 mm). The column longevity, however, is at present questionable.     

HPLC-SEC: a new approach to characterise complex wastewater effluents

This work investigates the use of HPLC-SEC to characterise dissolved organic matter (DOM) of complex wastewater effluents. A silica-based column, sodium acetate eluent and multiple detections were employed: UV-254 absorbance for humictype, and tryptophan-like (Ex/Em = 270/355) and tyrosine-like (Ex/Em = 270/310) fluorescence for protein type compounds. Effects of eluent pH, eluent ionic strength and injection volume on separation efficiency were tested. Humic-type and protein-type fractions were clearly differentiated and eluted within and out of calibration range. Eluent ionic strength had the greatest influence on global resolution; the lowest eluent concentration of 0.01 M produced the best separation for all wastewater effluents tested at any detection. UV-254 absorbance was higher at neutral and basic eluent pH while tryptophan-like fluorescence depended on the sample composition rather than on the eluent pH or ionic strength. Tyrosine-like fluorescence decreased significantly with the increase of eluent ionic strength. Accurate molecular weight measurements could not be done, the separation being influenced by secondary interactions, but could be approximated using separate calibrations with sodium salts of polystyrene-sulfonates and protein standards. The results show that this method is suitable for determining DOM in wastewater at low eluent concentrations (up to 0.03 M), at neutral or slightly basic pH.     

Retention pattern profiling of fungal metabolites on mixed-mode reversed-phase/weak anion exchange stationary phases in comparison to reversed-phase and weak anion exchange separation materials by liquid chromatography-electrospray ionisation-tandem mass spectrometry

Retention properties of 79 fungal metabolites (including neutral, acidic, basic, and amphoteric compounds) were evaluated on distinct mixed-mode reversed-phase/weak anion exchange (RP/WAX)-type stationary phases by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) in gradient as well as in isocratic elution mode. The RP/WAX separation materials were prepared by functionalising thiol-modified silica with N-(10-undecenoyl)-3-aminoquinuclidine and N-(10-undecenoyl)-3-alpha-aminotropane, respectively. To evaluate complementarity in chromatographic selectivity the physico-chemically heterogeneous solute set was analysed also on a RP phase (C(18)), an amino-type WAX phase, and a commercially available RP/WAX-like mixed-mode phase. Analytes may interact with the RP/WAX ligands via (attractive/repulsive) ionic, RP-like hydrophobic, as well as hydrophilic (HILIC) retention mechanisms. Individual interactive increments were found to be basically controlled by the nature and amount of organic modifier, pH value of eluent, and ionic strength of buffer additives. It could be demonstrated that RP/WAX columns offer the potential to separate compounds by exploiting a combination of various chromatographic interaction modes, which is not accessible with conventional RP and WAX columns. Such multi-modal properties increase both versatility and degrees of freedom for adjustment of chromatographic selectivity. For example, highly polar mycotoxins such as moniliformin were well retained on RP/WAX-type phases without compromising RP-selectivity for neutral (e.g. aflatoxins) and most basic solutes (e.g. epimer separation of ergot alkaloids) under fully MS-compatible conditions like a hydro-organic eluent with acetonitrile as organic modifier and an acetic acid/ammonium acetate buffer. Flexibility of the employed mixed-mode separation materials may be of value particularly for LC-ESI-MS/MS-based bioanalytics involving analytes with widely varying physico-chemical properties or applications prone to matrix effects.     

Retention behaviour of acidic, neutral and basic drugs on a CN column using phosphate buffers in the mobile phase

The retention behaviour of a set of sixteen more or less drugs on a CN column with methanol-phosphate buffer as eluent was studied. The influence of the volume percentage of methanol, pH and the ionic strength on the capacity factors (k') of the drugs was determined using an experimental design consisting of three pH values (3, 5 and 7), four ionic strengths (0.025, 0.05, 0.075 and 0.1) and three volume percentages of methanol (10, 30 and 50%). The selected drugs were basic, acidic and neutral compounds with different polarity properties. The number of carbon atoms, considered here as reflecting the hydrophobic characteristics of the solutes, varied from 7 to 25. The influence on the retention of drugs on a CN column of the different parameters studied was evaluated. The volume percentage of organic modifier and the pH are the most important factors. A change in ionic strength is important only when large molecules are chromatographed. As the interaction between pH, ionic strength and volume percentage of methanol is small, optimization of the three parameters separately is possible.