A multiwavelength approach to the selection of absorbance ratios for the assessment of chromatographic peak purity.

The routine use of diode-array detectors (DAD), based on the linear photodiode array device, has transformed the practice of UV-vis detection in liquid chromatography (LC). Multiwavelength detection is widely employed to generate absorbance ratios as a relatively non-specific method for characterizing peak purity in LC. If several wavelength pairs are selected the selectivity of the absorbance ratio method and its sensitivity to an interfering impurity can be increased, however, these attributes still depend on the selection of suitable pairs of wavelengths. This paper presents a novel approach to the selection of absorbance ratios for the assessment of peak purity in LC, utilizing a matrix derived from all the spectral data collected. As with single absorbance ratios, the absorbance ratio matrix (ARM) generated (containing all possible finite absorbance ratios) is characteristic for the analyte and independent of the analyte concentration. Moreover, the ARM technique eliminates the need to select "appropriate wavelength pairs", for sensitive discrimination of small spectral differences, when used for peak purity assessment. The ARM is found to give comparably high sensitivity to the presence of co-eluting species, as compared with the use of the wavelength pair selected on the basis of the conventional optimization criteria.

Post-column continuous-flow analysis combined with reversed-phase liquid chromatography and computer-aided detection for the characterisation of peptides.

Peptide mapping is a key technique for structural identification of new proteins or the products of recombinant gene technology. The recognition of oligopeptides, separated by reversed-phase liquid chromatography, is limited by the conventional reliance on the correlation of retention times with standards, supported by dual-wavelength chromatograms. It has been reported that the recognition of phenolic compounds can be achieved by a novel technique, based on computer-aided photodiode-array detection of the pH-shifted solutes after post-column continuous-flow analysis. This work describes how the generation of the pH-shifted difference spectra for dipeptides, containing a tyrosyl residue, may be used to enhance peak recognition, when used in conjunction with absorbance ratios.

Determination of salicylazosulphapyridine, sulphapyridine and its metabolites in serum and urine.

Methods for the determination of salicylazosulphapyridine (Salazopyrin(R)), sulphapyridine, N-acetylsulphapyridine, sulphapyridine-O-glucuronide and N-acetylsulphapyridine-O-glucuronide in serum and urine have been developed. In one method samples were diluted with methanol to precipitate proteins present and thereafter injected directly onto the liquid chromatographic column. The sulphapyridine-O-glucuronide could not be determined by this method. For the determination of all the main sulphapyridine metabolites the sulphapyridine aglycones were formed after treatment with beta-glucuronidase. These sulphapyridine compounds were then extracted with isobutylmethyl ketone at pH 5, and re-extracted to a phosphate buffer at pH 13 prior to injection. Separation and retention of all compounds was affected both by pH and concentration of methanol in the mobile phase. The proposed method made determinations down to 1 mug ml(-1) possible, which was found to be sufficient. Comparisons were made with spectrophotometric methods.

Selective extraction of organic compounds as ion-pairs and adducts.

Selective and easily regulated systems for extraction of organic compounds as ion-pairs and/or adducts are presented. The effect of different kinds of hydrophobic agents that give adducts in the organic phase are demonstrated: mesitylene for nitrophenols, ethyl acetate and diethyl ether for hexestrol (diphenol), lipophilic alcohols for organic ammonium ion-pairs, dibenzo-18-crown-6 for ion-pairs of primary ammonium ions, HDEHP for hydrophilic aminophenols (adrenaline, isoproterenol, synephrine). It is shown that the extraction selectivity decreases with increasing content of the complexing agent in the adduct. The influence of the hydrogen-bonding character of the counter-ion and the organic solvent on the selectivity of ion-pair extractions is demonstrated with ammonium compounds (nortriptyline, amitriptyline and N-methylainitriptyline) and inorganic anions. Highly hydrophilic anionic compounds (e.g., glucuronides, cholic acid derivatives) can be extracted into chloroform as ion-pairs with large quaternary alkylammonium ions. The extraction efficiency of the cation increases with the number of methylene groups to a limit which is due to co-extraction of other sample components (e.g., buffer anions).

Two-phase titration of organic ammonium ions with lauryl sulphate and methyl yellow as indicator.

Conditions for determination of quaternary ammonium compounds and amines by two-phase titration have been calculated on the basis of extraction constants for the ion-pairs between the titrant (lauryl sulphate), the indicator (Methyl Yellow) and the sample. Methods for determination of extraction constants are presented. The precision and selectivity of the method are demonstrated by titrations of ammonium ions with different structures and degrees of substitution. The results are in agreement with the theoretical calculations.