Contributions to reversed-phase column selectivity. II. Cation exchange.

The contribution of cation exchange to solute retention for type-B alkylsilica columns (made from high-purity silica) has been examined in terms of the hydrophobic-subtraction (H-S) model of reversed-phase column selectivity. The relative importance of cation exchange in the separation of ionized bases by reversed-phase chromatography (RPC) varies with (a) column acidity (values of the column cation-exchange capacity C), (b) mobile-phase pH and buffer concentration, and (c) the nature of the buffer cation. The effects of each of these separation variables on cation retention were examined. The contribution of cation exchange (and other ionic interactions) to solute retention is represented in the H-S model by properties of the solute (κ') and column (C), respectively. Values of κ' for 87 solutes have been examined as a function of solute molecular structure, and values of C for 167 type-B alkylsilica columns have been related to various column properties: ligand length (e.g., C(8) vs. C(18)) and concentration (µmol/m(2)), pore diameter (nm), and end-capping. These results contribute to a more detailed picture of the retention of cationic solutes in RPC as a function of separation conditions. While previous work suggests that the ionization of type-B alkylsilica columns is generally negligible with mobile-phase pH<7 (as a result of which cation exchange then becomes insignificant), the present study provides evidence for cation exchange (and presumably silanol ionization) at a pH as low as 3 for most columns.

Choosing an equivalent replacement column for a reversed-phase liquid chromatographic assay procedure.

The column selectivity parameters (H, S*, A, B and C) described in the preceding paper [L.R. Snyder, A. Maule, A. Heebsch, R. Cuellar, S. Paulson, J. Carrano, L. Wrisley C.C. Chan, N. Pearson, J.W. Dolan, J.J. Gilroy, J. Chromatogr. A 1057 (2004) 49-57] can be used to compare columns in terms of selectivity. A detailed procedure for such column comparisons is presented here, and evaluated by its use in finding suitable replacement columns for 12 different routine separations performed in five different pharmaceutical analysis laboratories.

Bile metabolites of polycyclic aromatic hydrocarbons in three species of fish from the Severn Estuary.

Six metabolites of polycyclic aromatic hydrocarbons (PAHs) were identified and quantified from the bile of 31 common eels (Anguilla anguilla), 29 European flounders (Pleuronectes flesus), and 15 conger eels (Conger conger) collected from the Severn Estuary and Bristol Channel during 1997. The bile metabolites were deconjugated by enzymatic hydrolysis and separated by reverse-phase HPLC with fluorescence detection. The major metabolite present in all fish was 1-hydroxy pyrene (75-94% of all metabolites detected) with lower proportions of 1-hydroxy chrysene (2-15%) and 1-hydroxy phenanthrene (2-8%), and small amounts of three benzo[a]pyrene derivatives (<3%). Metabolite concentrations (normalized to biliverdin content) were significantly higher in common eels than in the other two species and tended to be higher in all species at the beginning of the year than at the end. The data confirm the importance of 1-hydroxy pyrene as the key PAH metabolite in fish bile and suggest that the common eel is an ideal species for monitoring PAHs in estuarine environments.

Analysis of estrogens in river water and effluents using solid-phase extraction and gas chromatography-negative chemical ionisation mass spectrometry of the pentafluorobenzoyl derivatives.

A procedure was developed for the analysis of estrogens in environmental water and effluents. Samples were extracted by passing through polymer-impregnated solid-phase extraction discs or C18 cartridges, followed by gas chromatography-negative chemical ionisation mass spectrometry of the pentafluorobenzoyl derivatives. The derivatives were stable and gave diagnostic negative molecular ions as the base peak for each of the major estrogens studied. The absolute recovery of estrogens spiked into clean groundwater using the disc procedure was 84-116% at the 10 ng l(-1) level (calculation not based on use of internal standards). Using doubly deuterated estradiol as internal standard added prior to extraction, the % relative standard deviation of estrogen extraction and analysis in spiked groundwater at the 10 ng(-1) level was 2.6-9.8%. Detection limits were 0.2 ng l(-1) or below for the major estrogens, based on a 2.5 litre sample. The most abundant estrogen was estrone, with concentrations over the range 6.4-29 ng l(-1) in effluents, and 0.2 to 17 ng l(-1) in water from the River Thames.

Towards the development of molecularly imprinted polymer based screen-printed sensors for metabolites of PAHs.

This paper describes the fabrication of a sensor for 1-hydroxypyrene (1-OHP) based on a screen-printed carbon electrode (SPCE) modified with a molecularly imprinted polymer (MIP); 1-OHP was chosen as a model metabolite of polyaromatic hydrocarbons (PAHs). It was shown that 1-OHP could be readily oxidised at a plain SPCE and the electrochemical mechanism was found to involve an ECE (electron transfer-chemical reaction-electron transfer) process. The MIP for 1-OHP was prepared using only divinylbenzene (DVB) and styrene as monomers and the binding was only based on hydrophobic interactions. Batch binding studies revealed that optimum uptake of 1-OHP by the MIP occurred from solutions containing 35% water in methanol. Selectivity of the binding sites in the MIP was examined by performing uptake studies in the same solution containing either phenol or 1-naphthol; the specific binding of 1-OHP was twenty times greater than the former and five times greater than the latter. Preliminary calibration studies were performed with the MIP-SPCE using a two-step approach; accumulation was carried out in 35% water in methanol followed by measurement in 50% methanol-0.025 mol dm(-3) phosphate buffer pH 12. This two-step non-competitive affinity assay gave encouraging results and indicated potential for use in pollution studies.

Effect of temperature and flow-rate on analysis of basic compounds in high-performance liquid chromatography using a reversed-phase column.

The peak shape and retention of some basic probes together with a neutral reference compound were investigated as a function of temperature and flow-rate using a reversed-phase HPLC column at both pH 3.0 and pH 7.0. The retention of bases often showed an anomalous increase with temperature; retention mechanisms are complex as shown by studies of the effect of buffer cation concentration on retention. Considerable improvements in column efficiency for bases may result from operation at elevated temperature. Improvements did not seem attributable either to incidental changes in the retention factor, or (in this particular study where low sample masses were utilised) to the influence of sample load. The optimum flow-rate for highest efficiency is generally lower for basic compounds than neutrals, and due to the steepness of the Van Deemter curves obtained, high flow-rates appear to be particularly detrimental in the chromatography of basic compounds.