Separation and Determination of Iron-Containing Proteins via Liquid Chromatography−Particle Beam/Hollow Cathode−Optical Emission Spectroscopy

Abstract

Presented here is a method for the quantitative determination of iron-containing metalloproteins. Four iron-containing metalloproteins (transferrin, myoglobin, hemoglobin, and cytochrome c) were separated by high-performance liquid chromatography (HPLC) and determined through particle beam/hollow cathode-optical emission spectroscopy (PB/HC-OES) by the Fe (I) 371.9 nm optical emission. Parametric optimization of sample introduction, nebulization, and hollow cathode source conditions is performed for the suite of Fe-metalloproteins. Response curves for the Fe (I) emission were obtained under optimized conditions with detection limits for triplicate injections occurring on the nanogram level for iron ( approximately 24 ng) with variability of <7% RSD over the concentration range of 0.1-100 microg/mL iron in the metalloproteins. Response curves for S (I) emission yielded similar analytical characteristics. Optical emission detection of the liquid chromatography separations of the iron-containing metalloproteins demonstrates the feasibility of the PB/HC-OES system as a simple element-specific detector for liquid chromatography. The retention times of the four analytes are similar to those determined by UV absorbance (216 nm), demonstrating the ability of the PB interface to preserve the chromatographic integrity of the separation. Additionally, empirical formula calculations based on Fe (I) and S (I) emission response ratios provide a much higher level of specificity than single-element protein determination.