ISOLATION AND PURIFICATION OF CHLOROPHYLL-A AND CHLOROPHYLL-B FOR THE DETERMINATION OF STABLE CARBON AND NITROGEN ISOTOPE COMPOSITIONS

A method is described to isolate and purify chlorophyll pigments from plant tissues for the determination of stable carbon and nitrogen isotope compositions. Chlorphylls are initially isolated by solvent precipitation, followed by preparative C18 reverse-phase high-performance liquid chromatography (HPLC) for final purification. The purity of the isolated pigments (chlorophyll a and chlorophyll b) was assessed by analytical HPLC, absorption spectroscopy, elemental analysis, and H-1 NMR spectroscopy. A step-by-step evaluation of the separation indicates that molecular and isotopic integrity of the pigments are preserved during purification. Measurement of stable isotope composition across the HPLC peak illustrated the necessity to collect the entire peak in order to maintain isotopic integrity. Multiple stable isotope compositions (delta-C-13, delta-N-15) of individual molecular markers (i.e., pigments) are a powerful new tool to identify the sources of complex organic matter mixtures in the bio- and geospheres.