This is a summary of a UV-VIS Chlorophyll analysis and the Fixed multi-wavelength method that is preprogrammed in the INSIGHT software.
Although the method is specific for INSIGHT software and the Evolution 200 Instrument series, this summary can be used as a guide for performing this analysis on other Thermo Fisher Scientific UV-VIS instruments. However, instruments with a Spectral Bandwidth of 2NM or less will provide the highest resolution and most accurate results.
- Evolution 200 Series
- INSIGHT software
The chlorophyll method that is preprogrammed into the INSIGHT software is based on the Wisconsin State Lab of Hygiene, Environmental Science Section Method ESS 150.1, which also references Standard Methods for the Examination of Water and Wastewater, 17th Ed. pp. 10-31-10-39, 1989. Please refer to these methods for details.
If you open the INSIGHT method and click on Settings, then click on the Instrument Tab, you will see that the instrument is measuring the absorbance at 665 nm, 663 nm, 645 nm, 630 nm, and 750 nm. It then subtracts the 750 nm value from all of the other absorbance readings. The 750 nm subtraction is a correction for the turbidity of the sample. The INSIGHT software does not calculate the final concentrations because the chlorophyll method has optional ways to calculate the data.
Algal cells are concentrated by filtering a known volume of water through a membrane filter (47 mm, 5.0 μm pore size). The pigments are extracted from the concentrated algal sample in an aqueous solution of acetone. The chlorophyll a concentration is determined spectrophotometrically by measuring the absorbance (optical density - OD) of the extract at various wavelengths. The resulting absorbance measurements are then applied to a standard equation.
Pheophytin, a natural degradation product of chlorophyll, has an absorption peak in the same spectral region as chlorophyll a. It may be necessary to make a correction when pheophytin concentration becomes significantly high.
Corrected chlorophyll a refers to the method with the pheophytin correction (acidification method). Uncorrected chlorophyll a refers to the method without the pheophytin correction (Trichromatic method).
Calculate the uncorrected chlorophyll a concentration by inserting the turbidity corrected absorbance values in the following equation.
Uncorrected Chlorophyll a (μg/L) = [11.64 (Abs663) -2.16 (Abs645) +0.10 (Abs630) * E(F)] / [V(L)]
F = Dilution Factor (i.e., if the 663 Abs is >0.99 with the 1 cm cell, dilute, re-analyze and insert the dilution factor in the equation);
E = The volume of acetone used for the extraction (mL);
V = The volume of water filtered (L); and
L = The cell path length (cm).