The most likely the pink color comes from your oxidized antioxidant. Polyphenols are commonly oxidized to brightly colored quinones. Use ascorbic acid as a control.  

It is Pinkish 

@yuri V Geletii 

So I should I take the reading while is white before turning pink?

Or I should take the absorbance reading when its pink? 

Hi,

I hope your procedure: ABTS 7 mM, Ammonium or potassium persulphate 2.4 mM, bothe are equal volume kept it 12 hours, each 1 mL mixed with methanol or ethanol. Dilute this mixture up to 0.706 at 734 nm.

Plant extract 1 mL, ABTS solution 1 mL = 2 mL 7 mints incubate it under dark condition at room temperature, Control: 1 mL solvent+ 1 mL ABTS, Reference standard Quercetin or ascorbic acid.

Result: From green to pinkish colour will turn depending upon your sample concentrations Ex: 50 ug/mL, 100 ug/mL, 200 ug/mL, 300 ug/mL  Check IC50 value of each sample by regression value of each samples. 

Dear Olivia, 

I do not know details of your experiment. There  are numerous antioxidant assays based on ABTS. I suspect you add too much antioxidant. You should have your solutions green after after adding the sample. Take a difference between absorbance readings in control (solution without antioxidant) and with antioxidant. Calibrate your assay with ascorbic acid. Important, the antioxidant activity depends on pH. Use buffered solutions. 

Hi  Arjun,

yes its something like that.  I did  at increasing concentration of the plant extract.

However, at 7 minutes, the higher concentration extract turn from green to colourless; after more than 7 minutes (maybe >15 minutes) it turn to pinkish.

So should I actually take the reading at 7 minutes or wait further?

by the way, is it possible to run this antioxidant test using HPLC?

After 7 minutes, filter the extract+abts solution with 0.45um filter and subject to HPLC?

or just stick to spectrophotometer?

Hi, 

I'm not sure about your technique for HPLC but if you want to know antioxidant activity compounds you can try HPTLC method, from HPTLC method which spots showing antioxidant activity those compounds you can identify through LC-MS. 

Procedure: 10 uL to 20 uL samples, standard 2 to 5 uL can spotted by HPTLC after spotted you can run with any solvent system 1. DCM: MeOH: W 70:30:4 or 2. T:EA:FA 80:20:2 or 3. EA:MeoH:W:FA 50:20:7:1.  prepare 0.4 mM DPPH solution spray on that TLC silica gel plate, purple backgrounds with yellow spots shows radical scavenging activity. Those spots can directly identify through LC-MS method. If you interested you can try this method.  

dear Olivia, if i remember correctly, 5 minutes incubation is enough for ABTS assay.... and don't forget to incubate in the dark......

Hello Li Yoke Ho. I used a methanolic extract and the same thing happened to me. Have you progressed with the absorbance readings? Did you reach any conclusions?