1H NMR Quantitative Determination of Chlorophyll α in the Spirulina Biomass

A method for determining the mass fraction of chlorophyll a in the spirulina biomass - the feedstock for the preparation of chlorophyll a derivatives - using H-1 NMR spectroscopy without preliminary separation of the extract components is proposed (the extract was obtained by exhaustive extraction with a mixture of chloroform and ethanol). Because of the relative lability of chlorophyll a for its determination the demetallation was performed under mild conditions (action on the extract by 10 % hydrochloric acid) with formation ofmore stable pheophytin a, which served as a derivate for the determination of chlorophyll a. The chlorophyll a mass fraction in the studied raw materials was calculated based on the amount of pheophvtin a in the extract. The content of pheophytin a in the extract analyzedfbr calculating of the chlorophyll a mass fraction in the air-dry biomass of spirulina was determined by the internal standard method. The 4-nitrobenzaldehyde was used as an internal standard. The ratio of the amount of pheophytin a and 4-nitrobenzaldehyde added to the extract was determined by the signal integrated intensities of protons of the internal standard and protons in the meso-positions of the pheophytin a molecule. It was shown that, to increase the accuracy of determination, preliminary removal of a part of the lipid components by extraction with hexane is useful. The mass fraction of chlorophyll a in the raw material studied was co(chl) = 0.81 +/- 0.05 %. The result obtained corresponds to the data on the preparative yields of pheophytin a and methylpheophorbide a obtained from the same spirulina sample (the yields of these compounds can be used to judge the lower boundaty of the content of chlorophyll a in the raw material). The proposed technique can be adapted for other types of raw materials containing chlorophyll a or its derivatives.