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Eight commercially available sub-2 microm octadecyl silane columns (C18 columns) have been characterised by the Tanaka protocol. The columns can be grouped into two groups that display large differences in selectivity and peak shape due to differences in hydrophobicity, degree of surface coverage and silanol activity. Measurements of particle size distributions were made using automated microscopy and electrical sensing zone measurements. Only a weak correlation could be found between efficiency and particle size. Large differences in column backpressure were observed. These differences are not related to particle size distribution. A more likely explanation is differences in packing density. In order to take full advantage of 100-150 mm columns packed with sub-2 microm particles, it is often necessary to employ not only an elevated pressure but also an elevated temperature. A comparison between columns packed with sub-2, 3 and 5 microm versions of the same packing indicates potential method transferability problems for several of the columns due to selectivity differences. Currently, the best alternative for fast high-resolution LC is the use of sub-2 microm particles in combination with elevated pressure and temperature. However, as shown in this study additional efforts are needed to improve transferability as well as column performance.

In this study, a porous mixed-mode n-alkyl methacrylate-based monolith has been used in the separation of therapeutic peptides. While the sulfonic acid (SCX) moiety derived from 2-acrylamido-2-methyl-1-propanesulfonic acid supports the generation of a stable electroosmotic flow (EOF) at both acidic and basic pH values, the butyl ligands provide the nonpolar sites for chromatographic resolution. The performance of the monolith was evaluated regarding the influence of pH on chromatographic resolution of peptides. The suitability of the butylmethacrylate/SCX monolith for the analysis of therapeutic peptides containing basic centres, for example arginine, at moderately high pH 9.5 and the stability to repeat injections of a mixture of peptides was demonstrated. Separations with efficiencies as high as 5.0 x 10(5) plates/m were obtained and the migration behaviour of the peptides at both low (2.8) and high (9.5) pH values could be rationalised based on their charge, molecular mass/shape and relative hydrophobicities.

A range of ten perfluorophenyl and perfluoroalkyl stationary phases has been evalua- ted using standard chromatographic tests and probes. Principal Component Analysis of the data has indicated that the phases can be divided into distinct groupings. Extending the dataset to include standard alkyl and phenyl phases provided further data interpretation to support the orthogonal selectivity claims made for perfluorinated phases. The analysis of a range of basic analytes showed an unusual extended retention of hydrophilic basic analytes with perfluorophases. Furthermore, a non- linear relationship between the amount of organic modifier and the logarithm of the retention factor was observed, for the hydrophilic bases, which could not be modelled with LC prediction softwares. This was in sharp contrast to the alkyl and phenyl pha- ses examined. Basic analyte retention on perfluoroalkyl phases could be modelled adequately for the lipophilic bases. Exploration of the retention mechanism of these perfluoro phases indicated that silanol interactions were important in retention and selectivity. Using a rapid, isocratic, high organic modifier methodology, it was possible to analyse a mixture containing a lipophilic steroid, hydrophilic base and an internal standard in a 4 minutes with a perfluorophenyl phase. This had previously only been achievable with an alkyl phase under gradient elution conditions.

A range of C18 bonded silica stationary phases, two specifically designed for capillary electrochromatography (CEC) use, have been evaluated for their performance in the CEC analysis of basic analytes of differing physico-chemical properties. The performance of the phases towards basic analytes will be discussed in terms of various CEC parameters and by Principal Component Analysis (PCA) of five LC chromatographic parameters – hydrophobic and steric selectivity, hydrogen bonding and ion-exchange capacities at pH 7.6 and 2.7. The CEC phases, which the PCA highlighted as possessing high non-acidic silanol activity, were shown to give substantially enhanced EOF at pH 7.8 and did not exhibit the phenomenon of “drying-out” at pH values below 6.1 which was pronounced with hydrophobic LC phases. The Hypersil CEC Basic C18 phase was found to provide superior performance in analysing the basic analytes compared to the Hypersil CEC C18 phase, this was attributed by PCA to its non-acidic base silica. Through the use of experimentally derived electrophoretic mobility values, LC retention factor, and electrosmotic flow values in CEC, it was possible to predict the CEC retention behaviour of ten basic analytes to an accuracy of ± 6%.

Silica stationary phases of varying purity have been compared and evaluated for the LC separation of pharmaceutically relevant basic compounds of differing molecular weight, log P, and pK a using aqueous/organic mobile phases. The least pure silica gave the best separations. All phases showed a linear relationship between In k and volume fraction of ACN (30-70%) or MeOH (15-35%). The investigations confirmed that all the unbonded phases examined possessed a hydrophobic/adsorption and ion-exchange character. The use of silica with MS friendly mobile phases, and high volume fractions of ACN, has been shown to be applicable to LC/MS analysis of basic analytes.

The capillary electrochromatographic separation of six substituted barbiturates in the ion-suppressed mode was investigated on commercially available C18, C8, and phenyl-bonded packing materials. The effect on the retention behavior of mobile phase composition, buffer pH, buffer ionic strength, and temperature was studied. Successful chromatography of the barbiturates was achieved with all three packing materials over a pH range from 2.3 to 7.8. It was notable that a significant electroosmotic flow (EOF) was generated with a pH 2.3 mobile phase for all the packing materials, suggesting that capillary electrochromatography (CEC) in the ion suppression mode can be applied to a wide range of weakly acidic compounds of pharmaceutical interest. The C18, C8, and phenyl-bonded packing materials gave similar selectivities and retention times, and approximately linear relationships between percentage organic modifier in the mobile phase or temperature versus ln k were observed. Increasing the buffer ionic strength reduced the EOF but did not result in changes in selectivity. The use of methanol compared with acetonitrile had no effect on selectivity but resulted in a large increase in retention of the analytes. The experimental investigations have shown that the CEC separation of a series of weakly acidic compounds of pharmaceutical relevance can be achieved readily using a range of commercially available packed capillaries.

The operational parameters and factors which control the performance of capillary electrochromatography (CEC) using commercially available CEC instrumentation are evaluated and discussed. The CEC of neutral or ion-suppressed acidic analytes shows marked advantages such as increased column efficiency and reduced analysis times compared to conventional high-performance liquid chromatography (HPLC). Development of the CEC method using C-18 stationary phases follows similar guidelines to that of HPLC and, due to the high efficiencies obtained, the time required for method development can be substantially reduced. The technique of CEC is extremely attractive for the analysis of pharmaceuticals due to its excellent quantitative characteristics: good repeatability, reproducibility, wide UV detector linearity, and excellent detection limits. The analysis of acidic and neutral analytes by CEC using reverse-phase material is highly successful. However, in order to analyze highly basic analytes a strong cation exchange stationary phase is needed. This phase can produce staggering and as yet unexplained “apparent” efficiencies of over 40×106 plates per meter; however, to-date these results are highly non-reproducible. The application of CEC to the pharmaceutical industry has been demonstrated in the analysis of a wide range of structurally diverse pharmaceutical compounds using capillaries packed with reverse-phase materials. © 1997 John Wiley & Sons, Inc. J Micro Sep9: 373–387, 1997

Methylthio activating groups have been found to improve the yields in six different isoquinoline syntheses: in four cases the improvement was from zero, in the unactivated system, to between 54 and 94%.