A Strategy for assessing peak purity of pharmaceutical peptides in reversed-phase chromatography methods using two-dimensional liquid chromatography coupled to mass spectrometry. Part II: Development of second-dimension gradient conditions.

The importance of therapeutic peptides continues to increase in the marketplace for treating a range of diseases including diabetes and obesity. Quality control analyses for these pharmaceutical ingredients usually depends on reversed-phase liquid chromatography, and it is critically important to ensure that no impurities coelute with the target peptide at levels that would compromise the safety or effectiveness of the drug products. This can be challenging due to the broad range of properties of impurities that can be present on one hand (e.g., amino acid substitutions, chain cleavages, etc.), and the similarity of other impurities on the other hand (e.g., d-/l-isomers). Two-dimensional liquid chromatography (2D-LC) is a powerful analytical tool that is well suited to address this particular problem; the first dimension can be used to detect impurities over a broad range in properties, while the second dimension can be used to focus specifically on those species that might coelute with the target peptide in the first dimension. While hundreds of papers have been published on the use of 2D-LC for proteomics applications, there are very few papers that have focused on its use for characterisation of therapeutic peptides. This paper is the second in a two-part series. In Part I of the series, we studied several different column / mobile phase combinations that could be useful in 2D-LC separations of therapeutic peptides, with a focus on selectivity, peak shape, and complementarity to other combinations, particularly for isomeric peptides under mass spectrometry-friendly conditions (i.e., volatile buffers). In this second part in the series, we describe a strategy to derive second-dimension (2D) gradient conditions that both, ensure elution from the 2D column, and increase the likelihood of resolving peptides with very similar properties. We find that a two-step process yields conditions that place the target peptide in the middle of the 2D chromatogram. This process begins with two scouting gradient elution conditions in the second dimension of a 2D-LC system, followed by building and refining a retention model for the target peptide using a third separation. The process is shown to be generically useful by developing methods for four model peptides, and application to a sample of degraded model peptide to demonstrate its utility for resolving impurities in a real sample.

A strategy for assessing peak purity of pharmaceutical peptides in reversed-phase chromatography methods using two-dimensional liquid chromatography coupled to mass spectrometry. Part I: Selection of columns and mobile phases.

The current study describes the development of a 2D-LC-MS-based strategy for assessing main peak purity in the analysis of pharmaceutical peptides. The focus is on 2D-LC using reversed-phase (RP) separations in both dimensions, and particularly peptide isomer selectivity, since compounds with the same mass to charge ratio are not readily differentiated by mass spectrometry and therefore must be separated chromatographically. Initially, 30 column / mobile phase combinations were evaluated for both general separation performance (i.e., selectivity and peak shape) and isomer selectivity using forcibly degraded peptide samples and mixtures of synthetic diastereomers. A ranking of more than 300 UV and MS chromatograms suggests that when developing a new method, screening a set of four columns and four volatile mobile phases with differing characteristics should be adequate to both cover the selectivity space, and yield good separation performance. When 2D-LC-MS is to be used to evaluate peak purity for a new method, our results show that a second-dimension separation comprising a C8/C18 column possessing no ionic functionality, and an acetic acid / ammonium acetate mobile phase buffered at pH 5, provides good selectivity at 25 °C for peptide isomers with a MW <10 kDa. Retention data for 29 diverse peptides (1 < MW < 14 kDa, 3.7 < pI < 12.5) measured in this study using a variety of column and mobile phase conditions (i.e., 30 in total) are consistent with the classification of these various chromatographic conditions using the previously reported Peptide RPC Column Characterisation Protocol. For the investigated peptides trifluoroacetic acid was found to reduce selectivity differences between columns of diverse properties, probably due to its potential to form ion-pairs with peptides. Trifluoroacetic acid often improves peak shape for very large peptides (i.e. MW > 10 kDa). In the current dataset which also contain smaller peptides it received the highest ranking for 40% of the column and mobile phase combinations due to better selectivity and/or peak shape. The reported work here constitutes part one of a series of two papers. The second paper focuses on the use of retention modelling for rapid and accurate selection of the shallow gradients (i.e., << 1% ACN/min) required to obtain sufficient peptide isomer retention and separation in the second dimension. The overall results presented in this series of papers provides the guidance needed to develop a 2D-LC-MS method from start to finish for the analysis of main peak purity of therapeutic peptides.

Guilty by dissociation: Part A: Development of a rapid Ultra-High Performance Liquid Chromatography (UHPLC)-MS/MS methodology for the analysis of regioisomeric diphenidine-derived Novel Psychoactive Substances (NPS).

This study describes the first reported development of a rapid, generic gradient Ultra-High Performance Liquid Chromatography (UHPLC) methodology with targeted triple quadrupole MS/MS using electrospray positive ionisation to detect and unambiguously confirm the identity of 33 substituted 1, 2-diarylethamine (or diphenidine) derivatives in solid drug samples. The in-house synthesised library included a range of derivatives possessing either electron donating/withdrawing substituents, commonly included in combinatorial libraries, of varying size and lipophilicity on the phenyl ring. These test probes were used to investigate if their order of elution and that of their regioisomers were dependent on the position and type of the substituent on the phenyl ring. In addition, investigations into the retention mechanism of the diphenidines under reverse-phase UHPLC conditions were undertaken. Common adulterants found within seized bulk samples were assessed to prove that the methodology was specific, and the developed UHPLC-MS/MS (tG = 10 min) protocol was applied to confirm the identity of the psychoactive components within four seized bulk samples provided by law enforcement.

Guilty by dissociation: Part B: evaluation of Supercritical Fluid Chromatography (SFC-UV) for the analysis of regioisomeric diphenidine-derived Novel Psychoactive Substances (NPS).

Supercritical Fluid Chromatography (SFC-UV) employing a carbon dioxide (CO2) and 10 mM ammonium acetate in MeOH-water (95:5 v/v) gradient provides a rapid analysis (tG <10 min) of 31 novel, regioisomeric diphenidine-derived psychoactive substances, on a range of stationary phases of differing polarity. Medium to large selectivity differences between regioisomers, were observed on the acidic, neutral and basic SFC phases. For individual substituted ortho-, meta- and para-isomers, the same elution order was observed irrespective of the nature of the stationary phase. The acidic silica stationary phases yielded longer retention of the diphenidines via electrostatic attraction, whereas the basic phases resulted in shorter retention via electrostatic repulsion. SFC effected baseline separation of seven of the eight substituted groups of ortho-, meta- and para-diphenidines evaluated on a range of stationary phases. A simple silica phase achieved baseline separation of six of the regioisomeric substituted diphenidines. As the size of the halo-substituent increased, the resolution between ortho-/meta-isomers decreased, resulting in co-elution of the ortho- and meta-bromodiphenidines. Fluphenidines and chlorodiphenidines generated an elution order of meta- < ortho- < para- whereas an elution order switch was observed for the iodophenidines. This contrasted with RP-UHPLC where the elution order for the fluphenidines and iodophenidines was para- < ortho- < meta- and para- < meta- < ortho- respectively. An orthogonal elution order of diphenidines was demonstrated between the RP-UHPLC and SFC stationary phases due to the polarity differences between the separation modes. In general, hydrophilic compounds, which were poorly retained on a C18 reverse phase column, were well retained on SFC columns.

Investigation into reversed-phase chromatography peptide separation systems part V: Establishment of a screening strategy for development of methods for assessment of pharmaceutical peptides' purity.

The paper describes a simple and rapid reversed-phase UHPLC method development screening strategy for the purity determination of peptide-based pharmaceuticals. The protocol utilises five disparate column and six volatile or non-volatile mobile phases (i.e., 30 combinations). The method development strategy has been demonstrated to be highly effective in identifying conditions which generate complementary selectivity and good peak shape. Columns with varying degrees of charge (positive and negative), in addition to their differing hydrophobic character, were used in combination with mobile phases within the pH range of 2.3 to 5.1. The novel ion-pair / chaotropic reagent ammonium hexafluorophosphate at pH 2.3 was shown to be an extremely useful mobile phase additive in that it produced excellent complementary separation and good peak shape. Methanesulfonic acid was demonstrated to be a good alternative to the ubiquitously employed trifluoroacetic acid which failed to generate optimum separation for the peptides investigated highlighting the importance of screening disparate mobile phase additives. Both ammonium hexafluorophosphate and methanesulfonic acid were shown not to adversely affect the stability of C18 columns or demonstrated any irreversible adsorption / memory effects. No pH hysteresis effects were demonstrated with any of the stationary phases on mobile phase pH cycling. No major problems have been observed with the novel mobile phase additives ammonium hexafluorophosphate and methanesulfonic acid, however, it is recommended that they be used with caution until long-term routine use has been established.

Investigation into reversed-phase chromatography peptide separation systems Part IV: Characterisation of mobile phase selectivity differences.

The differentiation of mobile phase compositions between sub-classes which exhibit distinct chromatographic selectivity (i.e. termed characterisation) towards a range of peptide probes with diverse functionality and hence the possibility for multi-modal retention mechanisms has been undertaken. Due to the complexity of peptide retention mechanisms in given mobile phase conditions, no attempt has been made to explain these, instead mobile phases have simply been classified into distinct groups with an aim of identifying those yielding differing selectivities for use in strategic method development roadmaps for the analysis of peptide mixtures. The selectivity differences between nine synthetic peptides (fragments of [Ile27]-Bovine GLP-2) were used to assess how fifty-one RPC mobile phase compositions of differing pH (range 1.8 - 7.8), salt types, ionic strengths, ion-pair reagents and chaotropic / kosmotropic additives affected chromatographic selectivity on a new generation C18 stationary phase (Ascentis Express C18). The mobile phase compositions consisted of commonly used and novel UV or MS compatible additives. The chemometric tool of Principal Component Analysis (PCA) was used to visualise the differences in selectivity generated between the various mobile phases evaluated. The results highlight the importance of screening numerous mobile phases of differing pH, ion-pair reagents and ionic strength in order to maximise the probability of achieving separation of all the peptides of interest within a complex mixture. PCA permitted a ranking of the relative importance of the various mobile phase parameters evaluated. The concept of using this approach was proven in the analysis of a sample of Bovine GLP-2 (1-15) containing synthesis related impurities. Mobile phases with high ionic strength were demonstrated to be crucial for the generation of symmetrical peaks. The observations made on the C18 phase were compared on three additional stationary phases (i.e. alkyl amide, fluorophenyl and biphenyl), which had previously been shown to possess large selectivity differences towards these peptides, on a limited sub-set of mobile phases. With the exception of the ion-pair reagent, similar trends were obtained for the C18, fluorophenyl and biphenyl phases intimating the applicability of these findings to the vast majority of RPC columns (i.e. neutral or weakly polar in character) which are suitable for the analysis of peptides. The conclusions were not relevant for columns with a more disparate nature (i.e. containing a high degree of positive charge).

Investigation into reversed phase chromatography peptide separation systems part III: Establishing a column characterisation database.

The Peptide RPC Column Characterisation Protocol was applied to 38 stationary phases, varying in ligand chemistry, base silica, end capping and pore size, which are suitable for the analysis of peptides. The protocol at low and intermediate pH is based on measuring retention time differences between peptides of different functionality to calculate selectivity delta values. The characterisation was designed to explore increases / decreases in positive or negative charge (deamidation), steric effect (i.e. racemisation / switch in amino acid order), oxidation and addition / removal of aromatic moieties. The necessity of developing a characterisation protocol specifically for peptide analysis was highlighted by the fact that the small molecule databases (Snyder's Hydrophobic Subtraction Model and the extended Tanaka protocol) failed to correlate with the Peptide RPC Column Characterisation Protocol. Principal Component Analysis was used to demonstrate that the protocol could be used to identify columns with similar or dissimilar chromatographic selectivity for the purpose of selectivity back-up or method development columns respectively. This was validated using peptide fragments derived from the tryptic digest of bovine insulin and carbonic anhydrase. It was also demonstrated that the presence of positively charged functional groups on the stationary phase was advantageous as it yielded very different chromatographic selectivity and improved peak shape.

Investigation into reversed phase chromatography peptide separation systems part I: Development of a protocol for column characterisation.

A protocol was defined which utilised peptides as probes for the characterisation of reversed phase chromatography peptide separation systems. These peptide probes successfully distinguished between differing stationary phases through the probe's hydrophobic, electrostatic, hydrogen bonding and aromatic interactions with the stationary phase, in addition, to more subtle interactions such as the phase's ability to separate racemic or isomeric probes. The dominating forces responsible for the chromatographic selectivity of peptides appear to be hydrophobic as well as electrostatic and polar in nature. This highlights the need for other types of stationary phase ligands with possibly mixed mode functionalities / electrostatic / polar interactions for peptide separations rather than the hydrophobic ligands which dominate small molecule separations. Selectivity differences are observed between phases, but it appears that it is the accessibility differences between these phases which play a crucial role in peptide separations i.e. accessibility to silanols, the hydrophobic acetonitrile / ligand layer or a thin adsorbed water layer on the silica surface.

Investigation into reversed phase chromatography peptide separation systems part II: An evaluation of the robustness of a protocol for column characterisation.

The robustness of the Peptide Reversed Phase Chromatography (RPC) Column Characterisation Protocol was evaluated using reduced factorial design, to ascertain the degree of control required for parameters including temperature, flow rate, dwell volume, a systematic shift in the gradient, amount of formic acid in the aqueous and organic, pH of the ammonium formate and amount of acetonitrile (%MeCN) in the strong solvent, where a loss of MeCN resulted in an unacceptable variation. Mitigations have been introduced to ensure the integrity of the data to allow RPC columns to be characterised using peptides as probes, with the definitive protocol described. In addition, the instrument and column batch to batch variability were assessed with good reproducibility.

Chromatographic retention behaviour, modelling and optimization of a UHPLC-UV separation of the regioisomers of the Novel Psychoactive Substance (NPS) methoxphenidine (MXP).

A detailed investigation into the chromatographic retention behaviour and separation of the three regioisomers of the Novel Psychoactive Substance (NPS) methoxphenidine (i.e. 2-, 3- and 4-MXP isomers) has revealed the ionization state of the analyte and stationary phase, to be the controlling factor in dictating which retention mechanism is in operation. At low pH, poor separation and retention was observed. In contrast, at intermediate pH, enhanced retention and separation of the three MXP isomers was obtained; it appeared that there was a synergistic effect between the electrostatic and hydrophobic mechanisms. At high pH, the MXP isomers were retained by hydrophobic retention. Accurate retention time predictions (<0.5%) were achievable using non-linear retention models (3 × 3). This allowed the optimization of the gradient separation of the MXP isomers using a two-dimensional gradient and temperature design space. Prediction errors for peak width and resolution were, in most cases, lower than 5%. The use of linear models (2 × 2) still afforded retention time and resolution accuracies of <2.3 and 11% respectively. A rapid and highly sensitive LC-MS friendly method (i.e. Rsmin > 5 within 4 min) was predicted and verified. The developed methodology should be highly suitable for the rapid, specific and sensitive detection and control of MXP regioisomers.

Retention modelling in hydrophilic interaction chromatography.

The retention behaviour of acidic, basic and quaternary ammonium salts and polar neutral analytes has been evaluated on acidic, basic and neutral hydrophilic interaction chromatography (HILIC) stationary phases as a function of HILIC operating parameters such as MeCN content, buffer concentration, pH and temperature. Numerous empirical HILIC retention models (existing and newly developed ones) have been assessed for their ability to describe retention as a function of the HILIC operating parameters investigated. Retention models have been incorporated into a commercially available retention modelling programme (i.e. ACD/LC simulator) and their accuracy of retention prediction assessed. The applicability of HILIC modelling using these equations has been demonstrated in the two-dimensional isocratic (i.e. buffer concentration versus MeCN content modelling) and one-dimensional gradient separations for a range of analytes of differing physico-chemical properties on the three stationary phases. The accuracy of retention and peak width prediction was observed to be comparable to that reported in reversed-phase chromatography (RPC) retention modelling. Intriguingly, our results have confirmed that the use of gradient modelling to predict HILIC isocratic conditions and vice versa is not reliable. A relative ranking of the importance of the retention and selectivity of HILIC operating parameters has been determined using statistical approaches. For retention, the order of importance was observed to be organic content > stationary phase > temperature ≈ mobile phase pH (i.e. pH 3-6 which mainly effects the ionization of the analyte) ≈ buffer concentration. For selectivity, the nature of the stationary phase > mobile phase pH > buffer concentration > temperature > organic content.

Chromatographic and spectroscopic analysis of the components present in the phenanthridinium trypanocidal agent isometamidium.

The chromatographic isolation and characterisation of the four compounds present in the quaternary phenanthridine veterinary trypanocidal agent, isometamidium chloride hydrochloride (ISM), is reported. The isolated compounds were unambiguously characterised using spectroscopic (NMR, UV, IR and MS) methods as 3-amino-8-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium (1a) and related isomers, 8-amino-3-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium, 3,-8-diamino-7-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium and 3,-8-bis[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium. During the course of this study, it was realised that the nature of the solvent used in the NMR study was critical as in DMSO-d6 the quaternary group in the compounds was reduced to dihydro forms (e.g. 2a).

The influence of stationary phase on pressure-induced retention, selectivity and resolution changes in RP-LC.

This paper reports the influence of a diverse range of stationary phases and differing mobile phase modifiers on pressure-induced retention changes in reversed-phase liquid chromatography (RP-LC). The practical implications of these effects in the Tanaka column characterization using ultra high-performance liquid chromatography (UHPLC) conditions, and implications for HPLC to UHPLC translations in order to increase productivity and resolution are investigated. The stationary and mobile phase combinations responded to a similar degree to elevated pressure; hence, the authors believe that reliable column characterization parameters should be obtainable when UHPLC format columns are evaluated using the Tanaka approach. Analytes exhibited differing pressure-induced retention changes even for only modest increases in pressure (i.e. ΔP(total) 85 bar as shown when one transfers from a 3- to 2-µm particle). The degree of pressure-induced retention changes correlated with the analyte's molar volume and refractivity. The hydrophobicity of the analytes, as measured by logD, only exhibited a weak correlation. Hence, translating a RP-LC methodology from large to smaller particle size material of the same type may result in an increased or decreased selectivity and hence resolution between two analytes depending on their differing response to the pressure-induced retention changes. This potentially has a major impact on LC method development/optimization strategies and LC method translations.

An appraisal of the chemical and thermal stability of silica based reversed-phase liquid chromatographic stationary phases employed within the pharmaceutical environment.

Mobile phase pH and temperature are major factors in determining retention, selectivity and chromatographic performance of ionizable compounds. This imposes a requirement that stationary phases must ideally be stable in both acidic and basic conditions coupled with good thermal stability, in order to be able to chromatograph these compounds in either their ionized or ion-suppressed modes. The development of a range of new high and/or low pH stable silica based RPLC stationary phases (including sub-2 µm fully porous and sub-3 µm fused core-shell materials), which are specially designed for the analysis of ionizable compounds and their chemical and thermal stability is reviewed. The ability to utilize both pH and temperature as selectivity variables allows the chromatographer to exploit a much wider method development design space including previously prohibited alkaline conditions. This greatly increases the probability of satisfying the desired chromatographic selectivity and performance criteria.

A comparison of the chromatographic properties of silica gel and silicon hydride modified silica gels.

The retention properties of a silica gel column and a type C silica (silicon hydride) column for bases, sugars and polar acids were compared in hydrophilic interaction chromatography (HILIC) mode with formic acid or ammonium acetate as aqueous phase modifiers. The type C silica column was much more retentive for a series of model bases than the silica gel column and, surprisingly, retention of bases increased on the type C silica column when, the higher pH, ammonium acetate containing mobile phase was used. The retention of sugars was greater on the type C silica column than on the silica gel column and also increased on the type C silica column with increased pH suggesting either a silanophilic mechanism of retention or some unknown mechanism. Three type C silica based columns, type C silica, cogent diamond hydride and a ß-pinene modified column, which it was hoped might exert some additional stereochemical discrimination, were tested for metabolomic profiling of urine. In general the unmodified type C silica column gave the strongest retention of the many polar metabolites in urine and could provide a useful complement to established HILIC methods for metabolomic profiling.

Validation of the extended Tanaka column characterization protocol by multivariate analysis of chromatographic retention of low-molecular-weight analytes on reversed phase columns using methanol and acetonitrile as organic modifiers.

The validity of the extended Tanaka column characterization procedure against the retention behavior of 101 analytes of widely differing properties chromatographed on five differing stationary phase chemistries has been established using a chemometric technique called principal component analysis (PCA). It was concluded that the simple and conveniently determined column characterization parameters covered the same space in the PCA loading plot as the retention times for the 101 differing analytes. This confirms that the ten column characterization parameters of the extended Tanaka protocol encode the same information as the retention times of the 101 analytes. Significant selectivity differences were observed between stationary phases and the mobile-phase modifiers - MeOH and MeCN. PCA contribution plots served as a convenient way to highlight specific selectivity differences between stationary phases. logD values exhibited a poor correlation with retention indicating that retention in RP-LC is not solely dictated by the analyte's hydrophobicity. The use of MeOH was found to generate greater selectivity differences with the five stationary phases than when MeCN is used.

Chromatographic retention behaviour, modelling and separation optimisation of the quaternary ammonium salt isometamidium chloride and related compounds on a range of reversed-phase liquid chromatographic stationary phases.

This paper describes the reversed-phase liquid chromatographic behaviour of the trypanocidal quaternary ammonium salt isometamidium chloride and its related compounds on a range of liquid chromatographic phases possessing alkyl and phenyl ligands on the same inert silica. In a parallel study with various extended polar selectivity phases which possessed different hydrophobic/silanophilic (hydrogen bonding) activity ratios, the chromatographic retention/selectivities of the quaternary ammonium salts was shown to be due to a co-operative mechanism between hydrophobic and silanophilic interactions. The highly aromatic and planar isometamidium compounds were found to be substantially retained on stationary phases containing aromatic functionality via strong π-π interactions. The chemometric approach of principal component analysis was used to characterise the chromatographic behaviour of the isometamidium compounds on the differing phases and to help identify the dominant retention mechanism(s). Two-dimensional (temperature/gradient) retention modelling was employed to develop and optimise a rapid liquid chromatography method for the separation of the six quaternary ammonium salts within 2.5 min which would be suitable for bioanalysis using liquid chromatography-mass spectrometry. This is the first reported systematic study of the relationship between stationary phase chemistries and retention/selectivity for a group of quaternary ammonium salts.

Chromatographic retention behaviour of monosubstituted benzene derivatives on porous graphitic carbon and octadecyl-bonded silica studied using molecular modelling and quantitative structure-retention relationships.

The retention behaviour of a series of 28 monosubstituted benzenes, representing a diverse range of functional groups and substituent shape, were investigated using porous graphitic carbon (PGC) and octadecyl-bonded silica (ODS) stationary phases. For the majority of analytes retention on PGC was greater than on ODS, and in most cases this effect occurred at both pH 2.5 and 7.0. The main trends observed on PGC (in comparison with ODS) were: (i) similar or reduced retention of low polarity molecules such as the hydrocarbon and halogenated analytes; (ii) increased retention of conjugated analytes with extended planarity; (iii) increased retention of polar and charged species; and (iv) substantial increases in retention for selected polar and negatively charged analytes, including some ionised and unionised acid analytes. Poor retention of positively charged analytes was observed on both stationary phases. Molecular modelling studies have explored the geometry of π-π stacking interactions in retention on PGC and have highlighted the strong retention of large conjugated analytes, with extended planar conformations, which can interact with the graphite surface with cofacial geometry. Quantitative structure-retention relationships showed the importance of hydrophobic (π) and electronic factors (e.g. mean polarisability and LUMO energy) in retention on PGC, whilst retention on ODS was correlated to hydrophobicity (logP and π).

Comparison of classical chromatographic tests with a chromatographic test applied to stationary phases prepared by thermal immobilization of poly(methyloctylsiloxane) onto silica.

Stationary-phase evaluation in reversed-phase liquid chromatography (RP-LC) is not a straightforward process. A number of tests to characterize and classify stationary phases have been suggested. The results of these various tests, however, do not always describe the real properties of the stationary phase. This study critically compares several tests for RP-LC stationary phases, including the Engelhardt, Tanaka, and SRM 870 tests, as well as an in-house test, with emphasis on the stationary-phase descriptors of hydrophobicity and silanol activity. The stationary phases were prepared by thermal immobilization of poly(methyloctylsiloxane) onto silica. Hydrophobicity data from the tests were generally good and interchangeable between the several tests. In contrast, the silanol activity results of the various tests differ significantly. As a consequence, stationary phase classification with respect to silanol activity depends considerably on the test method applied. A new classification method for silanol activity is proposed.

Characterization of a mixed-mode reversed-phase/cation-exchange stationary phase prepared by thermal immobilization of poly(dimethylsiloxane) onto the surface of silica.

A novel stationary phase prepared by the thermal immobilization of poly(dimethylsiloxane) onto the surface of silica (PDMS-SiO(2)) has been described, evaluated and compared with 229 commercially available RP-LC stationary phases using the Tanaka column classification protocol. The phase exhibited many unique chromatographic properties and, based on the phases in the database, was most similar to the fluoroalkylated phases (aside from the obvious lack of fluoro selectivity imposed by the C-F dipole). The phase exhibited classic reversed-phase behaviour in acid mobile phase conditions and mixed-mode reversed-phase/cation-exchange retention behaviour in neutral mobile phase conditions. The phase exhibited acceptable stability at both low and intermediate pH, conditions which should impart optimum chromatographic selectivity to the phase. Retention of basic analytes was shown to occur by a "three site model" as proposed by Neue. This new PDMS-SiO(2) stationary phase is extremely interesting in that the dominancy of its hydrophobic and ion-exchange interactions can be controlled by the influence of mobile phase pH, buffer type and concentration. The PDMS-SiO(2) stationary phase may provide a complementary tool to reversed-phase and HILIC stationary phases. The present results highlight the fact that the type of buffer, its concentration and pH can not only affect peak shape but also retention, selectivity and hence chromatographic resolution. Therefore, in method development and optimization strategies it is suggested that more emphasis should be given to the evaluation of these mobile phase operating parameters especially when basic solutes are involved.