Trolox slightly dissolved in water but its disslove in ethanol, methanol, DMSO. 

You can try heating the solution to dissolve Trolox more.

Trolox is a week acid insoluble in water. Add some NaOH to adjust pH to neutral

Thanks everybody, but I think Trolox is sensitive with light and temperature and when I mix it with solution like ethanol, methanol, DMSO or add some NaOH how can I control absorbance of these content?

Trolox is not light sensitive, do not raise pH > 8, keep a stock solution in a fridge. Trolox does not absorb light in a vis range

Thank you very very much, but can I ask one more question?

If I keep Trolox in a fridge, what temperature I need to set and the shelf life of the solution?

Thank you again.

I do not know. I have always used the freshly prepared Trolox stock solutions and kept in a fridge not longer that 6-8 hours. 

Hello, I know this post is from 2 years ago but the matter is relevant to me, so I would like to try to ask a question.

I'm doing my master's degree thesis and I encountered the same difficulties.

My plant extracts (leaves) were obtain with methanol-water (50/50) as solvent, and now I wanted to perform the ABTS assay on those extracts but when I'm building the Trolox standart curve the results were strange.

I dissolved Trolox in Methanol/water as well and it didn't dissolve, even after 3 hours mixing.

I tried to dissolve Trolox with methanol and it did dissolve well but then when I read the standart Trolox curve on the spectrophotometer, the values were very low maybe because I dissolved Trolox only in methanol and not in the same solvent as used for the extracts.

Is there any advice to make the Trolox solution with this type of solvent used?

Thanks you.

You can try to firstly dissolve your trolox in methanol and add the water after it dissolve.

that's a good idea, indeed. I'll try to do that, thank you!

As I have already commented, Trolox is a week acid is insoluble in water. Add some NaOH to adjust pH to neutral and you will dissolve Trolox. This is trivial. Trolox carboxylic group has pKa 3.9. The solubility of Trolox at pH 7.2 is 3 mg/mL (12 mM), which is high enough for antioxidant assays.The ABTS antioxidant assay should be used under physiological conditions (100% water, pH 7.4). Don't create additional problems by adding alcohols, which also scavenge radicals. I personally worked with Trolox in aqueous solutions and never had problems.

Hi,

Find the file attached herewith. It helps you a lot.

Kind regards,

Tilahun

Hello,

Here is the SOP for preparation of Trolox solution. I hope it will help you.

SOP of ABTS measurement.

B. Standard preparation

1. Prepare 1.5 mMTrolox stock solution, weight 0.0038 gTrolox into 10 volumetric flask and fill uptomark with 90% methanol and store in freezer (at -20oC for up to 2 days) or at -80oC for more than 6 months

2. Trolox standards (final concentration 0-1.44 mM) depending on the concentration of the sample) in methanol. This concentration of standards can be manipulated based on the concentration expected in the sample.

Reference

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26, 1231–1237. doi:10.1016/S0891-5849(98)00315-3

Thank you all for you help! I'm sure it will help me a lot with my analysis!

Just one more question if I may:

In most of the methos for ABTS assay, the required potassium persulfate solution concentration is 140mM but in others is 2.45mM.

Is there any advice about which one is the best for this assay?

Thank you very much once again!

Wish you a great weekend!

As pr the protocol developed by Re et al.(1999), I suggest you to prepare 2.45 2.45mM.

Good luck!

thank you all for your help!

The assays are improving!

Wish you all a great weekend!

Vera: for ABTS assay, you can prepare a 140mM potassium persulfate stock solution, and then add 89 uL of the stock solution to 5 mL of 7mM ABTS solution for the preparation of 2.45mM ABTS radical solution. 140mM and 2.45mM are concentrations of different solutions.

Dear All

I know my question might seem a little bit strange but since you provided great answers to previous questions I decided to ask that here. I'm doing DPPH antioxidant activity test and calculated RSA% and IC50 of my extracts but since most of the articles have reported the results based on Trolox equivalent I need to convert my results into that unit to be able to make comparisons. the problem is that I don't know how to convert %RSA to trolox equivalent. I appreciate if you could help me out.

Dear Tilahun Abera, I just asked a question related with storing temperature. As I can see, you say that trolox can be stored at -20ºC just up to 2 days. I tried to find an explanation in the reference, but I couldn´t. Is it that Trolox is that unstable? Thank you in advance!

Yurii V Geletii how much NaOH should be used? You say NaOH will not affect the ABTS/DPPH assay results?

I would advise to use 100 mM Phosphate buffer at pH 7.2. pH affects the results of all antioxidant assays. Therefor use the same buffer to evaluate antioxidant activity. pH 7.2-7.4 is a physiological value.

Hi all, I am conducting a research on the total phenol content by folin and total antioxidant capacity by dpph and cuprac, I am using NaOH extraction solution in my extracts,

My first question is about folin, dpph and cuprac method' s standarts preparation ,

Must standarts( gallic acid, trolox etc.) be prepared by extraction solution ( naoh for me)? They are generally prepared by methanol or ethanol?

Second question, I will perform analysis of phenolic acids by Hplc, Must my standarts that I will use in Hplc be prepared extraction solution naoh for me ? Briefly, Must the standarts be prepared what you use in your extraction procedure? is it true?