Improved separation of palladium species in biological matrices by using a combination of gel permeation chromatography and isotachophoresis

Abstract

The binding of palladium to high-molecular-mass compounds in palladium-treated lettuce is investigated as an example for a biological matrix. The total palladium concentration in lettuce leaves is 10.3 ng/g wet weight. After homogenization, high-molecular-mass compounds (> 10 kDa) are isolated by ultrafiltration. For separation of these palladium species a combination of preparative gel permeation chromatography (GPC) and preparative isotachophoresis (ITP) is used. Palladium is determined in separated fractions by using a highly sensitive total reflection X-ray fluorescence (TXRF) method after preconcentration. After GPC separation, four main fractions of palladium species are collected, each containing palladium in ng quantities (3-10 ng). Two of these fractions are further separated by ITP, yielding at least three main peaks per GPC fraction, each containing palladium in the range of 0.3-3 ng. These palladium containing peaks are characterized by high-performance size exclusion chromatography (HPSEC) and capillary isotachophoresis (cITP) in parallel. HPSEC enables the estimation of the molecular mass of six main palladium peaks, covering a molecular mass range of 69-200 kDa. It is also shown that the estimation of molecular mass after separation is more reliable than the respective estimation directly in the first GPC run. However, cITP reveals that each of the separated peaks is still a mixture of at least five different compounds.

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

“…In multistage separation schemes, this technique serves usually as finalizing polishing step, as reported for the mitochondrial N-acetylaspartate biosynthetic enzyme [213]. Also fragile metallo-proteins were purified by native electrophoresis with the Prep Cell [214,215]. Recently, structural characterization of nucleoprotein-RNA rings succeeded after continuous elution separation of different ring diameters [216].…”

“…The analysis of metals differs from the determination of other cofactors. Methods like atomic absorption spectroscopy or X-ray fluorescence provide the basis for the detection of a specific metal [214,215].…”

Recent progress in capillary ITP

“…The product (100 mg) could be separated during a 30-min run and valve isolations of the glycoforms from the ITP stack into four or six fractions were possible. ITP was used to investigate the homogeneity of fractions of palladium bound to high-molecular-mass compounds in palladium-treated lettuce after preparative gel permeation chromatography and ITP (LE: 10 mM HCl 1 Tris, pH 8.0; TE: 5 mM EACA 1 Tris, pH 8.9) [84]. A column coupling ITP system was used for direct analysis of cystine in urine without sample pretreatment with an LOD at the 0.1 mM level (LE: 10 mM HCl 1 BisTris, 0.2% MHEC, pH 8.9; TE: 5 mM glycine 1 BisTris, pH 8.9) [85].…”

Speciation of Palladium in Plants: Method Development for Investigating Metabolic Changes

“…But in the latter case no palladium species were directly analysed. The first results on the speciation of palladium in plant material have been published in the last three years (Alt et al 2002, Weber et al 2004, Lesniewska et al 2004.…”

“…One solution to this problem was the introduction of a TXRF based method for reductive preconcentration and ultratrace determination of palladium (Messerschmidt et al 2000), which allows a very selective determination of palladium with a detection limit in the low pg-range. This method was also the basis of the first investigations of palladium species in plants (Alt et al 2002, Weber et al 2004), which will be discussed in more detail in the following chapters.…”