Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases for Liquid Chromatography. Part II: Chemical and Thermal Stability.

In the first part of this review, stationary phases (silica, hybrid silica, hydride silica and non-silica stationary phases) were characterized and compared with respect to selectivity, efficiency, resolution, solvent consumption and analysis time. The present review focuses on the thermal and chemical stability of stationary phases. Stationary phases of high chemical and thermal stability are required for separations that are carried over a wide pH and/or temperature range.

Silica, hybrid silica, hydride silica and non-silica stationary phases for liquid chromatography.

Free silanols on the surface of silica are the "villains", which are responsible for detrimental interactions of those compounds and the stationary phase (i.e., bad peak shape, low efficiency) as well as low thermal and chemical stability. For these reasons, we began this review describing new silica and hybrid silica stationary phases, which have reduced and/or shielded silanols. At present, in liquid chromatography for the majority of analyses, reversed-phase liquid chromatography is the separation mode of choice. However, the needs for increased selectivity and increased retention of hydrophilic bases have substantially increased the interest in hydrophilic interaction chromatography (HILIC). Therefore, stationary phases and this mode of separation are discussed. Then, non-silica stationary phases (i.e., zirconium oxide, titanium oxide, alumina and porous graphitized carbon), which afford increased thermal and chemical stability and also selectivity different from those obtained with silica and hybrid silica, are discussed. In addition, the use of these materials in HILIC is also reviewed.

How to select equivalent and complimentary reversed phase liquid chromatography columns from column characterization databases.

Three RP-LC column characterization protocols [Tanaka et al. (1989), Snyder et al. (PQRI, 2002), and NIST SRM 870 (2000)] were evaluated using both Euclidian distance and Principal Components Analysis to evaluate effectiveness at identifying equivalent columns. These databases utilize specific chromatographic properties such as hydrophobicity, hydrogen bonding, shape/steric selectivity, and ion exchange capacity of stationary phases. The chromatographic parameters of each test were shown to be uncorrelated. Despite this, the three protocols were equally successful in identifying similar and/or dissimilar stationary phases. The veracity of the results has been supported by some real life pharmaceutical separations. The use of Principal Component Analysis to identify similar/dissimilar phases appears to have some limitations in terms of loss of information. In contrast, the use of Euclidian distances is a much more convenient and reliable approach. The use of auto scaled data is favoured over the use of weighted factors as the former data transformation is less affected by the addition or removal of columns from the database. The use of these free databases and their corresponding software tools shown to be valid for identifying similar columns with equivalent chromatographic selectivity and retention as a "backup column". In addition, dissimilar columns with complimentary chromatographic selectivity can be identified for method development screening strategies.

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.

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.

Effects of pH and temperature on the chromatographic performance and stability of immobilized poly(methyloctylsiloxane) stationary phases.

The effects of mobile phase pH, temperature, buffer type and buffer concentration on the selectivity and stability of four stationary phases, with different PMOS loadings, prepared by the thermal immobilization of poly(methyloctylsiloxane) on to silica (PMOS-SiO2), were evaluated with both hydrophobic and hydrophilic basic solutes. These solutes show longer retention times at near neutral pH, where both the silanols and the basic solutes are partially ionized, and shorter retention times in more alkaline pH, where the silanols are mostly ionized and the basic solutes are not ionized. Increases in temperature and buffer concentration also result in shorter retention times. These PMOS-SiO2 stationary phases are quite stable at low pH and are also stable at ambient temperature (23 °C) using pH 7 phosphate. The PMOS-SiO2 stationary phases are more stable at higher pH using triethylamine (pH 11) and borate (pH 10) buffers than with phosphate and carbonate buffers. Temperature increases stationary phase degradation, while buffer concentration has a minimal effect on stationary phase degradation, indicating that these PMOS-SiO2 stationary phases have stabilities similar to the equivalent chemically bonded phases.

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.

Selectivity of some basic solutes on a poly(methyltetradecylsiloxane)-silica stationary phase.

Complex analyses of polar compounds, especially basic ones, require more selective stationary phases. The present paper describes a stationary phase prepared by thermal immobilization of poly(methyltetradecylsiloxane) onto chromatographic silica (PMTDS-SiO(2)). This stationary phase presents hydrophobic and ion-exchange interactions that confer both high retention and unique selectivities for basic solutes. The influence of ion-exchange interactions is confirmed by the increase in retention factors of basic solutes when the mobile-phase pH changes from acidic to neutral and by the decrease in retention factors when the mobile-phase pH changes from neutral to alkaline. The ion-exchange properties of the stationary phase are enriched in neutral mobile phase (pH 7-7.5) using soft Lewis bases such as tricine and tris as buffers but are suppressed in both acidic (pH 2.5-6) and highly alkaline mobile phases (pH≤10). Increasing both temperature and flow rate permits more rapid separations while maintaining the selectivity. The stability of the stationary phase is evaluated with acid, neutral and alkaline mobile phases.

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.

Chromatographic evaluation using basic solutes of the silanol activity of stationary phases based on poly(methyloctylsiloxane) immobilized onto silica.

The chromatographic behaviors of some basic solutes were evaluated on stationary phases based on poly(methyloctylsiloxane) immobilized onto silica (PMOS-SiO(2)). The test solutes present both hydrophobic and hydrophilic properties. Evaluations of the pH effect used 80:20 v/v methanol/buffered mobile phase over the pH range of 5-11.5 with inorganic buffers such as borate, carbonate and phosphate and with organic buffers such as citrate, tricine and triethylamine. Evaluations in acidic mobile phases used 50:50 v/v and 30:70 v/v methanol/buffer (pH 2.5; 20 mmol/L) mobile phases. The buffer concentration effect used 65:35 v/v methanol/phosphate (pH 7; 20 and 100 mmol/L) mobile phases. The results are compared with those obtained with two chemically bonded stationary phases. The immobilized phases show greater contributions from an ion-exchange mechanism than do the commercial phases. The results indicate that the silanol activity of PMOS-SiO(2) stationary phases can be adequately evaluated by using appropriate basic probes and mobile phases having different pH, using different buffers.