I am trying to calculate H2O2 in plant tissue based on this protocol:
Fresh leaves (0.5 g) were homogenized using a
cold mortar and pestle in precooled acetone (5 ml) and the
homogenate was centrifuged at 12,000g for 5 min. One
milliliter of the supernatant was mixed with 0.1 ml of 5%
Ti(SO4)2 and 0.2 ml of 19% ammonia. After a precipitate
was formed, the reaction mixture was centrifuged at
12,000g for 5 min. The resulting pellet was dissolved in
3 ml of 2 MH2SO4 and the absorbance was read at 415 nm
using a spectrophotometer. The H2O2 concentration was
calculated according to a standard curve of H2O2 that was
prepared by using H2O2 ranging from 0 to 10 lM.
But I do not have H2O2 to make a curve. Does it possible to use some formula?
Pls. if I understood your question right:
If your standard calibration curve[conc of H2O2 vs absorbance] IS ALSO AT 415 nm, all you have to do is to draw a straight line from the aborbance point (noted by you experimentally) on Y-axis and note the point where this straight line touches the curve. Then draw a perpendicular from this point on the curve on the X-axis and note the point on the horizontal X-axis and thus know the concentration. Supposing your standard curve is not at 415nm rather at 454(or 450)nm or 596( or590) nm[depeding upon the substances in which the conc of H2O2 is to be noted, people also use these two wave lengths] then note the absorbance of your substance at that wave length. It is simple case if your calibration curve is also drawn at 415 nm.
I don't think Manohar method will work for you, since you don't have a calibration curve to start with.
Unfortunately, you cannot quantify anything without a calibration curve. Especially in your case, where the measure is an indirect one.
In the protocol you reported, it is not even clear if the calibration was performed on a similar matrix or in a specific solvent.
Moreover, UV absorption spectra are very unspecific. We don't even know how many molecule might have an absorption at that wavelength and not be related to H2O2.
why do you not buy some H2O2 ????It is cheap and readily available - even over the counter at a drug store
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