hi Mirella, follow the attached protocol and you will be fine. I think you will need to use colour blanks too. Also if there is pulp present you will need to remove this via centrifugation to prevent any issues. 

i used microgram concentration for my positive control...... and remember to incubate in the dark..... all the best

Hello Mr. Naim,

I am sort of confused about the outlined way of preparing the standards in the attached protocol. Could you break it down please? I am employing ascorbic acid to prepare standards, and propyl gallate as positive control.

Thanks in advance for the help,

Mirella

Another concern,reading though the protocol, is the use of eppendorfs. Won't it be better using cuvettes straightaway?

Regards

Mirella 

there is absolutely no issue in using eppendorfs. I normally use glass tubes.

Firstly make ascorbic acid stock 1 (10mM) : Add 0.0176g Ascorbic Acid Powder to 10ml methanol or ethanol (ethanol is safer as it's a lot less toxic) or water or ethanol.

Make stock 2 (5mM): add 500 microliters stock 1 to 500 microliters water or ethanol.

Make stock 3 (250uM): Add 50 microliters stock 2 to 950 microliters water or ethanol.

Use stock 3 to make standard curve.

Standard curve

Ascorbic Acid Concentration  (uM)          microliters stock 3        microliters water

0                                                                      0                                 100

5                                                                      2                                 98

10                                                                    4                                 96

25                                                                    10                               90

50                                                                    20                               80

125                                                                  50                               50

250                                                                  100                             0

This should give you an excellent standard curve.

Thanks Mr O' Sullivan. Another question: what parameters should I use to plot the standard curve please (that is on the axes)? Do I use the absorbance of my set up positive control(propyl gallate) in determining DPPH % inhibition?

hi. Ascorbic Acid concentration goes on the x axis (horizontal), while the absorbance at 515nm is on the y-axis. See attached graph. The positive control can be compared to the ascorbic acid curve.

Thanks alot Mr O'Sullivan. I will surely work with the given protocol tomorrow in the lab. Certainly, I'll get back to you on my results.

Please do. I look forward to hearing your progress 

Some time it gives too dark colour, thus can't be measure. You can dilute your sample and than proceed with measurement. After you can multiply the dilution-fector during calculation.  

the antioxdant activity of orange juice, the same protocol for antioxidant free radical from plant extract, but we used the concentration as  microgramms/ml  and used the volume in the case of juice for exemple equivalent trolox/ l of juice. We can consulted the reference attached file .

we can used the reference attached file

Hello Mr O'Sullivan,

I carried out the given protocol with minor changes: it was a SUCCESS! Attached hereby is the obtained standard curve. Check it to confirm. My sample falls within the range of prepared concentrations. Thanks alot.

Another question: I analysed my two samples (orange and orange juice) at different dilutions(ranging from undiluted to 1:500) with DPPH. How can I calculate the antioxidant activity, so to plot a graph of antioxidant activity versus sample dilution please?

Dear Mr O'Sullivan,

I wish to plot a graph of % DPPH inhibition versus sample concentration. Looking at the needed formula, I wonder if the absorbance of 0 mM, as seen in your protocol, can be used as control to calculate % RDSA please.

Earnestly awaiting your response.Thanks.

can you please out the standard curve into Excel as I can't open it here on this computer.

here are answers to your other questions:

1. antioxidant activity/ DPPH scavenging activity formula= ((1-abs of sample/ abs of control))*100. do this for each dilution.

2. Ascorbic Acid Equivalents (µM). Basically what you need is the equation of the line from the Ascorbic Acid standard curve. Lets say the equation is -0.0005x + 0.681. The formula= (Abs sample-0.681)/-0.0005) which will give Ascorbic Acid Equivalents (µM).

Hello Mr. O'Sullivan,

Attached hereby is a photoshot of my graph": hope it suffices.

Based on the answers you gave me, the ascorb acid equivalent serves for what, as pertaining to plotting a graph, please?

Could you kindly tell me in details the kind of graphs as I can plot for antioxidant activity analysis please? Sketches are most welcome.

Thank you Sir.

The Ascorbic Acid standard curve is excellent.

Above I have given you answers to you questions.

1. You don't require a graph to determine antioxidant activity. Just use the abs you go like so:

antioxidant activity/ DPPH scavenging activity formula= ((1-abs of sample/ abs of control))*100. do this for each dilution.

2. Ascorbic Acid Equivalents (µM). Basically what you need is the equation of the line from the Ascorbic Acid standard curve which you just sent me. Lets say the equation which is -0.0006x + 0.5908. The formula= (Abs sample-0.5908)/-0.0006) which will give Ascorbic Acid Equivalents (µM).

dear Mirella Ejiugwo

follow attached journal

Ok Mr. O'Sullivan,

Does the control refer to my set up positive control (being BHT) ?

Dear All,I'd appreciate if any of you could refer me to a website or a source of documentation on antioxidant activity, particularly vitamin C, in orange and orange juice. Thanks a lot.

Hello Mr. O'Sullivan,

Based on theprotocol you sent me, do I have to calculate %FRSA for each sample dilution, or ...?

%FRSA???

% FRSA = Free radical scavenging activity.Called also, 

Use this calculation as this is all you need: antioxidant activity/ DPPH scavenging activity formula= ((1-abs of sample/ abs of control))*100. do this for each dilution.

Hi Mr. O'Sullivan, 

Happy Saint Patricks Day. Yes the reference is: Brand-Williams, W., Cuvelier, M.E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT-Food Science and Technology, 28, 25-30.

Hello Mr. O'Sullivan,

Same to you and thanks a lot. Can I send my thesis to you to have an overview today please? I'd really need your evaluation as soon as possible. I've to hand it in tomorrow morning.

Yes send it on. What time tomorrow? 

11 a.m. please. Should I forward to you on this platformas an aattachment ?

Yes please 

I updated it most recently. Same some errors.

I could only skim through it but it looks great. well done :)