Design of a flow injection method for chlorophyll determination in in vitro plants.

A flow injection (FIA) method was designed for the determination of chlorophylls a and b in small in vitro Dieffenbachia maculata "Sublime" plants. In the first step, the pigments from spinach leaves were separated, purified by solvent extraction and freeze-dried, to obtain standards for the FIA optimization. The sample extraction procedure was optimized. Four solvents were tested: diethyl ether, methanol, acetone and ethanol. The ethanol 96% was the optimal solvent for FIA purposes. It allows to the efficient extraction of the pigments and water can be used as carrier. The best FIA conditions found for the quasi-simultaneous quantification of chlorophylls a and b were a flow rate of 10.84mLmin(-1), a sample injection volume of 1.45mL and a reactor length of 63cm. The detection was performed with the automatic wavelength scanning Cintra 10e spectrometer, at 649 and 665nm. The results obtained by the FIA method were compared to those obtained by the Arnon method. A deviation less than 5% was found between results for both methods. The concentration (mgg(-1)) of chlorophylls a and b during three periods of the plants (in vitro, acclimatization, and adult) was determined to evaluate the whole in vitro procedure. It was found an increment of both pigment concentrations since the in vitro step till the adult stage, while the chlorophylls a to b ratio decreases. The designed method is suitable especially for the determination of the pigments at low concentrations in small samples with appropriate analytical quality.