Dear Poonam

As you are performing your activity with coloured extract. Try to use one blank solution of your extract for each reading.

Then subtract each OD with Blank Extract OD. Then you get real absorbance.

Can you let me know what blank solution you are using?

Sir I am using phosphate buffer as a blank.

Dear Poonam

As you are performing your activity with coloured extract. Try to use one blank solution of your extract for each reading.

Then subtract each OD with Blank Extract OD. Then you get real absorbance.

I haven't performed this method by Ruch et al 1989. By the way, may I know what component are you following through in this O. D.  measurement following Ruch et al method. If the O. D. you are measuring is due to it is hydrogen peroxide itself then the higher the hydrogen peroxide scavenging capacity of the sample the less will be the sample's level of hydrogen peroxide and thereby less O. D. will be observed. However, if the component you are following through is a substance that accumulates when hydrogen peroxide decreases then O. D should increase.

Hello Ma'am,

According to the methodology with slight modification following constituents were used for this assay.

1 ml plant extract( different concentrations) + 0.6 ml h2o2 solution + 2.4 ml phosphate buffer.

and phosphate buffer was also used as blank

Poonam ,

Will explain me the procedure of yours

There are two ways to directly measure H2O2 in the sample: (i)  by directly measuring H2O2 level at the wavelength by which H2O2 maximally at  the wavelength of 230nm and compute the level based on the molar absorbance  (or extinction) coefficient of H2O2 (0.04mM-1cm-1 or 1.0 unit change in absorbance for every 0.025mM or (ii) by indirectly measuring the level of the substance that reacts with H2O2 (like NADH that absorbs maximally at 340 nm) and in the presence of NADH peroxidase to catalyze the reaction to convert H2O2 to other product like H2O and oxidized NADH in the form of NAD+. In Ruch et al 1989 H2O2 was measured directly with the extract sample and without this extract, and the difference in OD where OD without sample (control) that they had designated as Ac =  amount of total H2O2 initially present minus the OD with sample designated as As = amount of total H2O2 left / remaining after H2O2 scavenging by the sample (and if sample has H2O2 scavenging capability, H2O2 levels with sample derived  or calculated from As, definitely, be less than without sample calculated from Ac and so with the OD getting less (i.e., As < Ac, making the value of Ac-As positive), that is as the extract concentration increases, more H2O2  is scavenged and less H2O2 remains. Therefore the change in OD between the assay without sample (Ac) and with sample As in Ac-As increases. It is then expected to observe an increasing order in OD change (OD without sample, Ac, minus OD with sample As, i.e. Ac-As) with increasing extract concentration. Note that it is the change in OD (without sample - with sample) that has to be considered here not simply OD.

Mam,

even i have tried the same ie Ruch et al method .Mine absorbance value also increases as conc increases why it is so not able to understand .

how to calculate BLANK EXTRACT OD

Hello Poonam,

Can you tell one thing whether you have taken ascorbic acid as standard then wt about absorbance value 

WHETHER IT WAS INCREASING ALONG WITH CONCENTRATION.

Hi Poonam,

I suggest you to follow the following protocol. I have tried many assay, and find that most of the protocol has major drawback of high background. Please see the attached paper.

Hi,

please, how do you know what concentrate of standard to use? I am following  the method of Ruch et al, 1989. and using the ascorbic acid as the standard.

Than you

Could you please share the protocol Martina

increasing trend of OD may be due to accumulation of extract. The absorbance of (Phosphate buffer + extract) only may be subtracted from the absorbance of PB+ extract + H2O2. It may give actual scavenging pattern of H2O2.

Good evening doctor @Debanjan Mukhopadhyay. I have a question about the reference that you attached to your answer. Can ferrous ammonium sulphate be substituted by any other ferrous salt in this assay? Ferrous chloride and Ferrous sulphate are the only available salts to me.

Hi Amira, ferrous sulphate you can try, but ferrous chloride is often oxidised to feric chloride within even container.So I will not recommend that. Hope this will work.

Thanks

Thank you doctor @ Debanjan Mukhopadhyay for your answer. I have another question for you please, it is well known that phenolic compounds have the ability to chelate metal ions, how do the results of this assay ensure that the decrease in color is due to hydrogen peroxide scavenging and not to chelation of ferrous ions if the tested extract contained phenolic compounds

For this particular reason, in the protocol section of the paper, I mentioned about keeping appropriate blank where no hydrogen peroxide should be added. You should always keep a blank to check whether your compound by itself has any colour or not. I have tested several compounds in the paper such as gallic acid. Which at high concentration has this property. Have a details look on the paper.

Thanks

@ Debanjan Mukhopadhyay Thank you very much. You helped me a lot.

Standard protocol is available in literature.

pleasedly consult recent journals for protocols