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ISSN 1998-9539

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

Dmitry V. Belykh,a,@ Elena N. Zainullina,a and Ol’ga M. Startsevab

aInstitute of Chemistry of the Federal Research Centre “Komi Scientific Centre of the Ural Branch of the Russian Academy of Sciences”, 167000 Syktyvkar, Russia

bSyktyvkar State University, 167000 Syktyvkar, Russia

@Corresponding author e-mail: belykh-dv@mail.ru

 

A method for determining the mass fraction of chlorophyll a in the spirulina biomass — the feedstock for the preparation of chlorophyll a derivatives — using 1H 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 the formation of more 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 pheophytin a in the extract. The content of pheophytin a in the extract analyzed for calculating of the chlorophyll a mass fraction in the air-dry biomass of spirulina was determined by the internal standard method. The 4-nitrobenzaldehyde were used as 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 ω(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 boundary 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.