I am conducting research on a physiological basis of rice under high temperature stress.
I already have started to measure chlorophyll content. I weighed 100mg of leaf sample and put it into test tube. Then I added 5ml of DMSO to it. I boiled this mixture in a water bath at 70°C for 30 minutes. I allowed the mixture to cool for about 4 hours. Finally, I measured the solution extract in a spectrophotometer at wave length 645 and 663nm.
Theoretically, I expected to get a reading of absorbance value at a range of less than one. But practically, when I measured, I observed that the reading of absorbance value ranged between 1 and 2.5A.
Please help me with this. What mistake did I made with the procedures? If the absorbance values are greater than 1, is it an error?
Thank you Philip Morris. I used method given by Hiscox and Israelstam (1979). Are you familiar with this method?
I follow the Wellburn (paper titled 'Spectral determination of chlorophylls a and b as well as carotenoids using various solvents with spectrophotometers with different resolution' published in 1994 J Plant Physiology vol 144 pp 301-313), using 20 mL of 80% v/v acetone as extractant for every 20 mg dried leaf sample of rice seedling. Wellburn recommended formulae to compute for the chlorophyll concentration, these formulae were derived from earlier reports and the results were comparable with those obtained following other methods.
Thank you Evangelina S Ella. My advisor directed me to decrease the weight of sample from 100mg to 25mg. the results showed some comparison.
Dear Hilali SALEH Hilali ,
You can estimate chlorophyll a, b and c by both methods but you must notice that each methods have a specific formula to estimate the type of chlorohyll.
Also, you must estimate at three different OD so, you must check the equation for each method
with best luck
Khaled
You may please check the assay mixture you are putting into the cuvette. It must be a clear and homogenous mixture (a solution in other words). If it is not a solution then minute particles of the ground leaf samples may had been included in the extracted mixture. However, even if it is a solution and absorbance is still very high then you need to dilute the solution taking note of the dilution factor necessary in your computation. From my experience, absorbances greater than 1.7 may not follow Beer-Lambert's Law anymore and so the functionality between the amount of the absorbing component and absorbance becomes curvilinear and no longer linear.
Thank you to all, Khaled, i used to measure absorbance at 663 nm; 645 nm and 480 nm. The method was Hiscox and Israelstam 1979 with minor modification ie (I reduced the weight from 100 mg up to 25 mg of leaves sample).
cha=12.19 (OD 663) -3.45 (OD 645)xV/(1000xW)
chb=21.9 (OD 645) -5.32 (OD 663xV/(1000xW)
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