C.A.M.G. Cramers

Molecular mechanism of retention in reversed-phase high-performance liquid chromatography and classification of modern stationary phases by using quantitative structure-retention relationships

Quantitative structure-retention relationships (QSRRs) were derived for logarithms of retention factors normalised to a hypothetical zero percent organic modifier eluent, log kw, determined on 18 reversed-phase high-performance liquid chromatography (RP-HPLC) columns for 25 carefully designed, structurally diverse test analytes. The study was aimed at elucidating molecular mechanism of retention and at finding an objective manner of quantitative comparison of retention properties and classification of modern stationary phases for RP-HPLC. Three QSRR approaches were employed: (i) relating log kw to logarithms of octanol-water partition coefficient (log P); (ii) describing log kw in terms of linear solvation-energy relationship-based parameters of Abraham; (iii) regressing log kw against simple structural descriptors acquired by calculation chemistry. All the approaches produced statistically significant and physically interpretable QSRRs. By means of QSRRs the stationary phase materials were classified according to the prevailing intermolecular interactions in the separation process. Hydrophobic properties of the columns tested were parametrized. Abilities of individual phases to provide contributions to the overall retention due to non-polar London-type intermolecular interactions were quantified. Measures of hydrogen-bond donor activity and dipolarity of stationary phases are proposed along with two other phase polarity parameters. The parameters proposed quantitatively characterize the RP-HPLC stationary phases and provide a rational explanation for the differences in retention patterns of individual columns observed when applying the conventional empirical testing methods.

Comparative study of test methods for reversed-phase columns for high-performance liquid chromatography

Column selection in reversed-phase liquid chromatography (RPLC) is still not a straightforward process. A number of tests to characterise and classify RPLC columns have been suggested. Several tests are already applied in laboratory practice, while others are under development. The results of the various tests, however, are not always qualified to describe the properties of columns for RPLC. In this study different tests for RPLC-columns are studied and compared, viz. the Engelhardt, Tanaka, Galushko and Walters tests. The column descriptors hydrophobicity and silanol activity are investigated in particular. The tests are studied using approximately 20 silica, alumina and polymer based C8- and C18-columns. Hydrophobicity data from the tests generally were good and interchangeable between the tests resulting in a column classification that is independent of the applied test. It appears that buffering of the eluent is mandatory for adequate testing of column silanol activity. In contrast with the high-quality hydrophobicity data, the silanol activity results of the various tests differ significantly. As a consequence column classification with respect to silanol activity depends considerably on the applied test method.

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.

Increased speed of analysis in isothermal and temperature-programmed capillary gas chromatography by reduction of the column inner diameter

It is shown theoretically that for an isothermal analysis the relationship of the retention time to the column diameter is . The exponent z is determined by the column pressure drop and varies between z  1 for high plate number columns and z  2 for situations of low pressure drop. Equations derived for temperature-programmed conditions also lead to the same conclusion. The validity of the expressions was confirmed by experiments with columns of 30 and 50 μm internal diameter, installed in standard chromatographic equipment. With these small-diameter glass and fused-silica columns coated with non-polar stationary phases, plate numbers between 105 and 106 were obtained. A temperature-programmed run typically can be accomplished within 8 min on an 8 m x 50 μm column having over 105 plates. Several examples of high-speed, high-resolution analysis of complex samples are given.

Increased speed of analysis in directly coupled gas chromatography-mass spectrometry systems : capillary columns at sub-atmospheric outlet pressures

Abstract A theoretical treatment of the optimum gas chromatographic conditions for open-tubular columns, operated at vacuum outlet pressures, is presented. Equations are given for the minimum plate height, the optimum linear gas velocity and the optimum inlet pressure. The maximum column efficiency was calculated to decrease by 12.5% at most, compared with atmospheric outlet conditions. The gain in speed of analysis obtained with vacuum outlet columns is dependent upon the nature of the carrier gas and increases strongly with lower (sub-atmospheric) optimum inlet pressures. The use of short and/or wide-bore columns can therefore be recommended. Experimental results indicate the validity of the theory, although no loss in efficiency has been observed. The ultimate gas chromatography-mass spectrometry coupling device thus appears to be no device at all: the end of the column need only be inserted into the ion source of a mass spectrometer. In addition to the gain in speed of analysis, the many problems caused by wall effects and dead volumes in interface lines are avoided by this method. Moreover, the gas chromatographic peaks are narrower and thus higher, lowering detection limits.

Preparation and characterization of monolithic polymer columns for capillary electrochromatography.

A series of micro-monolithic columns with different porosities were prepared for capillary electrochromatography (CEC) by in-situ copolymerization of butyl methacrylate, ethylene glycol dimethacrylate, and 2-acrylamido-2-methyl-1-propane-sulfonic acid in the presence of a porogen in fused-silica capillaries of 100 microm I.D. Different column porosities were obtained by changing the ratios of monomers to porogenic solvents. Columns were investigated and evaluated under both pressure-driven (high-performance liquid chromatography, HPLC) and electro-driven (capillary electrochromatography, CEC) conditions. Each column exhibited different efficiency and dependency on flow velocity under electro-driven conditions. Abnormally broad peaks for some relatively bulky molecules were observed. Possible explanations are discussed. The differences in column efficiency and retention behavior between the two eluent-driven modes were studied in detail. In addition, other column properties, such as morphology, porosity, stability and reproducibility, were extensively tested.

Evaluation of time-of-flight mass spectrometric detection for fast gas chromatography.

Separations below 1 s of a mixture of organic compounds ranging from C5 to C8 have been performed to investigate the performance of a time-of-flight mass spectrometer in fast gas chromatography. The gaseous samples were focussed on a cold trap, and then injected after thermal desorption to obtain the required narrow input band-widths. Also, to obtain a very fast separation, a short narrow bore column was used, operated at above-optimum inlet pressures. With this system, it was possible to identify ten compounds within 500 ms, showing peak-widths (2.354sigma) as narrow as 12 ms. The spectral acquisition rate used for these analyses was 500 Hz. The quality of the recorded spectra and the comparison with library spectra was very high. Deconvolution algorithms offer the possibility of identifying overlapping peaks. It is shown that the spectral scan speed of the time-of-flight mass spectrometer is high enough for very fast separations.

Factors determining flow rate in chromatographic columns

SummaryIt is shown that the flow in chromatography is nearly always laminar in nature. Starting from the Darcy equation, expressions are given for the flow rate in both gas and liquid chromatography columns. The concepts of specific permeability, chromatographic permeability and column resistance factor are discussed for packed as well as open tubular columns. The experimental determination of all these factoers is demonstrated. The influence of the shape and pore volume of porous and non-porous supports on the column resistance factor and the chromatographic permeability is discussed.

The separation of herbicides by micellar electrokinetic capillary chromatography

SummaryA method for the separation of a number of herbicides consisting of chlorophenoxy acids by micellar electrokinetic capillary chromatography (MECC) was developed. Sodium dodecyl sulphate (SDS), Brij 35, cetyltrimethylammonium bromide (CTAB) and methanol were introduced into the buffers to investigate their effects on the separation of the herbicides. SDS combined with Brij 35 as the micellar agent was found to provide the best overall separation of these components.

Possibilities and limitations of steam distillation—extraction as a pre-concentration technique for trace analysis of organics by capillary gas chromatography

The quantitative performance of steam distillation—extraction was investigated for different types of organic substances at concentrations ranging from tens of parts per million to parts per billion (parts per 105 to 109). A theoretical model is introduced that describes the recovery of different classes of organic compounds as a function of the process time. The effect of variations of some process factors predicted theoretically are in good agreement with the experimental results. A 100 % recovery is obtained within 20 min for most substances. Enrichment factors between 50 and 200 and recoveries corresponding to a standard deviation of 10% can be achieved in many applications.