The coupling of size-exclusion HPLC with ICP-MS in bioinorganic analysis

Abstract

T he detection of a metal compound in a sample is the prerequisite of any further action concerning its identification, characterisation and the role in biochemistry. The actual problem can be reduced to proving that the analytical signal detected by the spectrometer is due to the presence of a metal compound and not to that of a simple ion. This can be done by preceding the detection by a separation technique that would differentiate between the free metal or metalloid ion and the same element bound with a larger, usually macromolecular structure.The detection of the presence of metal complexes with biological macromolecules has commonly been realised by ultrafiltration using a filter with a small cut-off molecular mass, usually 500 − 5 000 Da. The concentration of the element of interest was determined in the initial sample and in the fi l t rat e, u s u a l ly by graphite furnace AAS; the metal retained was considered to be bound to a macromolecular species. A refinement of this technique by the successive u l t ra fi l t ration through membranes with molecular we i g h t cut-offs of 500, 5 000 and 30 000 Da has widely been used to study the distribution of metal-species as a function of the molecular weight [1][2][3][4]. The method allows only a ro u g h speciation and is time consuming.An improvement in terms of the resolution can be obtained by the use of size-exclusion chromatography which also seems to be less cumbersome and faster allowing the on-line detection to be used. Because the resolution of SEC is insufficient for the discrimination of the small aminoacid heterogeneities, the coupling SEC-ICP-MS is the most popular technique for the first screening of an unknown sample in view of the presence of macromolecular species of elements. SEC using a short guard column with ICP-MS detection allows the rapid quantification of the bound metal fraction by comparison of the chromatographic signal with that, obtained in parallel, by flow-injection ICP-MS analysis of the sample. This paper presents the technique, reviews its applications and shows some recently established potential in the fields of the plant, animal and clinical biochemistry. Size-exclusion chromatography in bioinorganic analysisSize-exclusion chromatography (SEC) is based on the molecular sieve effect and enables species to be sep a rat e d according to their size, and to a lesser extent, shape. The average time a substance spends in the pores is determined by its size which for a given shape, can usually be related directly to its molecular weight. The following conditions have to be taken into consideration for separation of biological constituents containing metals: interactions between metals and bu ffe rs and ch ro m at ographic support mat e ri a l should be minimized, and the pH of the eluent should be weakly alkaline to prevent the metals from dissociating. PackingSeparation by SEC should be independent of the analyte's charge but in practice the stationary phase surface displays ch a rged pro p e rties so that a mixed mode sep a ra...

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