I'm going to measure chlorophyll spectrophotometrically. Does anyone have a tried-and-tested, detailed protocol?
You would need to add more details... What kind of sample? Range of concentrations? Chlorophylls are usually measured in visible spectra (different types of chlorophylls absorb at different wavelengths: the most common ones are around 400 nm and 500 nm if I remember correctly). And ratios of absorbances (types of chlorophylls) are commonly used too.
You can try spectrophotometric method with mentioned formulas:
- Chlorophyll a (mg/g FW) = [(0.0127 (OD663) - 0.00269 (OD645)] × 100
- Chlorophyll b (mg/g FW) = [(0.0229 (OD645)- 0.00468 (OD663)] × 100
- Total chlorophyll content = [0.0202 (OD645) + 0.00802 (OD663)] × 100
- Chlorophyll a ratio b = Chlorophyll a / Chlorophyll b
Reference paper: Morphogenic and biochemical variations under different spectral lights in callus cultures of Artemisia absinthium L.
Article Morphogenic and biochemical variations under different spect...
Yes, lot of methods are used. But DMSO based method is easiest and less time consuming.
Usually, I use 80% aceton for extraction of chlorophyll quantification.
Article Determination of Accurate Extinction Coefficients and Simult...
Chl a = 12.25*(A663.6 - A750) - 2.55 *(A646.6 - A750) Chl b = 20.31*(A646.6 - A750) - 4.91 *(A663.6 -A750) Chls a + b = 17.76*(A646.6 - A750) + 7.34 *(A663.6 - A750)
(ug / mL)
Here, I show the usual calculation for 80% aceton extraction case but the coefficients differ in different solvents. I'm not sure the coefficients for DMSO extraction. Does anyone know those for DMSO and its ref. article?
- Badges
- Science method