High-performance liquid chromatography

Analysis of pigments of photosynthetic organisms using high-performance liquid chromatography.

The method is used to determine the qualitative and quantitative composition of pigments in the photosynthetic membranes of a number of bacteria.

Pigment analysis is carried out on a Spherisorb ODS2 column 5 μm “Waters” (USA). As a carrier, it uses silicon dioxide with a C18 alkyl group attached, and a carbon content of 11.5%.
For the separation of the pigment mixture, the approach is applied with a gradually increasing gradient of the concentration of ethyl acetate. For this, the following solutions are used: Solution A: 23% Ethyl acetate + 77% solution C; Solution B: pure ethyl acetate; Solution C: acetonitrile: water 9: 1 (V).
At the beginning of the analysis, the column is equilibrated with a solution “C”. After applying a sample to the column for the first three minutes, solution “A” is passed through it, then this solution is linearly replaced by solution “B” for 37 minutes. And the last 3 minutes a clean solution “B” is passed through the column. After the analysis, the column is regenerated by washing with solution “C” for 20 minutes.
The pigment content is estimated from the area of the curve on the chromatogram in the 415-550 nm region, using the LC-solution program (“Shimadzu”, Japan) and known extinction coefficients.

As part of the school it is proposed to measure the content of pigments in photosynthetic membranes of Alc. minutissimum by reversed-phase high-performance liquid chromatography.

Contacts: Maxim Bolshakov. e-mail: lfbv22@gmail.com