I've been , using spectrophotometer to measure chlorophyll content and following Equations were used (µg/ml or mg/l). These equations are for 1 cm path length.
[Cha] =12.64 A664 - 2.99 A647
[Chb] = -5.6 A664 + 23.26 A647
Since I have plenty of samples, I want to use microplate reader to measure the Chlorophyl content in DMF.
how to convert the absorbance of 200µL of sample in a microplate into a 1-cm path length? What is the modification I want to do for those equation?
You will need to know the path length in that type of plate using 200µl volume and use that to convert your OD readings to a path length of 1cm. It should be possible to retrieve that info (path length vs volume) from the manufacturer website.
It would be really important to be confident about the reproducibility of the volumes between wells, and about the homogeneity of the microtiter plate itself (also between wells). If you are confident the engineering of the plate is good, and the volumes are consistent, and you have access to a normal 1cm-path cuvette and spectrophotometer, then just measure one of your samples both ways, and calculate a conversion factor, either for the absorbances or for the chlorophyll concentrations. The factors should be the same, and would be the same as the ratio [1/path length of one of your wells in cm], but you don't need to measure it, you can calculate it indirectly. Contrary to a cuvette, the path length for your microtiter plate would vary depending of what volume you had filled the wells with. If you don't have enough sample to fill the cuvette, then dilute the sample and take that dilution into account. You can of course do this dual reading for several samples and calculate an average, more accurate.
I would be interested to know where did you find a microtiter plate resistant to DMF. My email is [email protected]
there is lillte profit for several reasons. Plate readers use filters that have got bandwidth different from the ones described for specrophotometers and band interference modifies the apparent coefficient of absorption, chlorophyll samples require a quick lap between extraction and reading for stability to phaeophytin degradation, some brand is resistant to methanol but for other solvents you might use very expensive glass coated ones, but again cleaning would be a problem. rfes in http://www.labhut.com/products/microplates-and-seals.html?material=1552 and dr Warren method for polystryrene plates and methanol https://www.researchgate.net/publication/233274740_Rapid_Measurement_of_Chlorophylls_with_a_Microplate_Reader
Article Rapid Measurement of Chlorophylls with a Microplate Reader
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