I have been trying to optimize H202 Radical scavenging activity antioxidant assay, I have tried with Ruch et al, 1989. As per the protocol principle, H202 + antioxidant => H20 + 02 , this implies , as the antioxidant (Sample) concentration increases the OD values should decrease. But my sample OD values decrease. In most of the references, the blank solution and control solution were not clearly mentioned, can any one suggest a clear protocol?
Hello. The blank is usually the solvent used for sample preparation.
Hi Mr. Govindrajan
Blank is usually the solvent in which you prepare your sample. Standards used are the standard antioxidants like Butylated Hydroxy anisole BHA, alpha- tocopherol, Ascorbic acid, Butylated hydroxy toluene BHT, quercetin etc. In your case with the hydrogen peroxide radical scavenging activity, you can use BHA, alpha tocopherol, ascorbic acid (Vit. C) as standards. Pls find attached some papers for ready reference.
Gud Luck
regards
Hello
In hydrogen peroxide radical scavenging activity the reference (Ruch et al, 1989) which you are using if fine. Just take phosphate buffer as a blank without hydrogen peroxide in it. Repeat your protocol I am sure you will get it right :)
I have also tried the same protocol. However my OD values are increasing with increasing the concentration of plant extract. Please suggest me how to proceed?
My students are using DPPH and hydrogen per oxide as radicals for citrus juice.
PBS is used as a blank. We are facing the same problem OD is increased with increase in concentration.
Dear Karthivashan,
As per to the principle of this assay the absorbance should get decrease. But in ur case I think molarity of H2O2 is responsible for contrary results. Also do prepare the H2O2 solution freshly.
Hello, the best blank for this assay is to take hydrogen peroxide and you can also take the phosphate buffer for the same, according to your convenience.
Hello, i agreed with all of you, when i repeated the same assay in triplicate, i also faced the same problem, but the solution for this, prepare the solution freshely as Ms. ankita told in her answere as well as to keep in mind which protocol you are following as in many papers, they prepared their sample in phosphate buffer, insted of preparing milli Q.
For each concentration, you should make blank sample according to some published papers but its showed, increase in O.D of blank also. So, its better to take the average of all the blank O.D & proceed further.
Thanking you
firstly when for screening purpose it gives good result but on repetition with increase of conc.absorbance increases why this happen
Dear karthivashan,
I think what you are trying to say is that the OD of your sample is increasing with its concentration while it is supposed to decrease if it scavenges peroxide, the reason for this might be that your compound itself is U.V active and hence the absorbance seems to increase with the increase in its concentration. You could try to determine the OD of your compound corresponding to the concentration range you have used in experiment without addition of peroxide there by difference between the OD with Peroxide and Without peroxide will correspond to the actual OD of peroxide which will decline if your compound scavenges peroxide. This will work only if all the other conditions are kept identical.
This is the same situation i facing right now...OD value increase with increasing in sample concentrations... i already used phosphate buffer as a blank...
I have taken phosphate buffer as blank,solutionof hydrogen peroxide prepared in buffer as control and sample+buffer +H2O2 AS SAMPLE
Having the same challenge. My sample OD is increasing with increasing concentration. I will consider the method suggested by Rishi Chinmay Thakkar it makes good sense.
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