Destructive method is used for measurement of chlorophyll content in plant tissues. Dimethyl sulfoxide solvent (DMSO) is used for chlorophyll extraction from five leaf disc in dark accodring to method by Hiscox and Israelstam (1979). Absorbance of extracts is read by Beckman gratin  spectrophotometer (model U-1100, ltitach, Ctd, tokyo, Japan) at 645 and 663 nm. Chlorophyll a (mg/cm-2) Chlorophyll  b (mg/cm-2) and total chlorophyll content (mg/cm-2) is calculated from absorbance at 663 nm and 645 nm according to Arnonʹs (1949) equations: Chlorophyll a = (ml solvent)[(0.0127 Absorbance 663) - (0.00269 Absorbance 645)]/Leaf area (cm²) , chlorophyll b = (ml solvent) [(0.0229 ×Absorbance 645) - (0.00468 Absorbance 663)]/Leaf area(cm²) , Total chlorophyll content = (ml solvent)(0.0202 ×Absorbance 645) + (0.00802 Absorbance 663)]/ Leaf area(cm²)

Ravi Kant sir.. i truly appreciate your efforts, but the method is already known to me along with the equations and I too make use of Hiscox method, but I am curious to know about the stability of compounds employing DMSO over Acetone. Acetone being liquid at room temperature is not as efficient as DMSO I suppose since the melting point of DMSO is 18 or 190C so the samples are frozen at a normal temperature of 15-200C prevalent in a temperate region in Shimla for almost half of year.

I highly recommend to use DMF for the extraction. I requires very few plant tissue and could be stored in 4 C for several weeks. Be careful, DMF dissolves plastic.

Dear B. Sharma,

It would be better to store y oursamples before extraction (for example, -70, or -18 if -70 is not possible). Chlorophylls are very labile at room temperature and the light when in solution. With the same protocol of extraction you can measure Chl a, b and carotenoids.

Regards.

We have used pure acetone for the extraction and quantification of photosynthetic pigments and after extraction we keep the extract at -20º C for months...without symptoms of degradation

That's an interesting concept Sir. Thank you