Separation of Tetracycline Antibiotics by Hydrophilic Interaction Chromatography Using an Amino-Propyl Stationary Phase

In this work the potential of hydrophilic interaction chromatography (HILIC) is explored for the analysis of tetracycline antibiotics. The choice of the polar stationary phase is first discussed and it is demonstrated that aminopropyl stationary phases lead to higher efficiencies and peak symmetry than bare silica ones. The influence of the composition of the mobile phase is studied next : the concentration of the weaker solvent (acetonitrile), the nature and concentration of the more polar solvent (water or methanol), pH, the nature and ionic strength of the buffer. It is shown that high efficiencies are reached only with a citrate buffer that impairs the interactions with the residual silanol groups whatever the mobile phase pH is. We demonstrate that the citrate buffer strongly interacts with the cationic moiety of the aminopropyl stationary phase and thus reduces the accessibility of silanols. The separation of oxytetracycline, tetracycline and chlortetracycline is achieved in a few minutes at pH 3.5 or 5, with no peak tailing as usually observed in reversed phase liquid chromatography with an opposite elution order when compared with reversed phase liquid chromatography.     

Determination of diethanolamine or <Emphasis Type="Italic">N</Emphasis>-methyldiethanolamine in high ammonium concentration matrices by capillary electrophoresis with indirect UV detection: application to the analysis of refinery process waters

Alkanolamines such as diethanolamine (DEA) and N-methyldiethanolamine (MDEA) are used in desulfurization processes in crude oil refineries. These compounds may be found in process waters following an accidental contamination. The analysis of alkanolamines in refinery process waters is very difficult due to the high ammonium concentration of the samples. This paper describes a method for the determination of DEA in high ammonium concentration refinery process waters by using capillary electrophoresis (CE) with indirect UV detection. The same method can be used for the determination of MDEA. Best results were achieved with a background electrolyte (BGE) comprising 10 mM histidine adjusted to pH 5.0 with acetic acid. The development of this electrolyte and the analytical performances are discussed. The quantification was performed by using internal standardization, by which triethanolamine (TEA) was used as internal standard. A matrix effect due to the high ammonium content has been highlighted and standard addition was therefore used. The developed method was characterized in terms of repeatability of migration times and corrected peak areas, linearity, and accuracy. Limits of detection (LODs) and quantification (LOQs) obtained were 0.2 and 0.7 ppm, respectively. The CE method was applied to the determination of DEA or MDEA in refinery process waters spiked with known amounts of analytes and it gave excellent results, since uncertainties obtained were 8 and 5%, respectively.     

Comparison of tryptophan interactions to free and grafted BSA protein

The binding of d- and l-tryptophan molecules to bovine serum albumin (BSA) protein has been studied using liquid chromatography and ultrafiltration in the pH range from 7 to 11. A hydrophobic interaction between tryptophan and BSA has been observed at pH 7.0 on BSA grafted chromatographic column. However, this interaction is negligible at higher pH for which the interaction to the stereospecific site was predominant. For both grafted and free proteins, the complexation mechanism was a competitive binding of d- and l-enantiomers on a single site. The apparent complexation constants for both d- and l-tryptophan show a maximum in the pH range 9-10. The variations of the apparent complexation constants versus pH were the result of the protonation of both the amino acid and a single site of the protein assuming that the complexation occurs between the zwitter-ionic amino acid form and the unprotonated BSA site. The apparent pK(BSA) is slightly shifted from 8.3 for grafted BSA protein to 9.4 for free BSA protein. This shift is presumably as a result of the different protein conformation.