The procedure for a given analytical method is strictly defined. So I don't think there are any possible exceptions to the procedure. Can you logically explain why you want to extend the time?

In many publications, the measurement system is monitored

for 30 minutes, but some authors also use a shorter incubation time (from 3 to 18

minutes). I haven't come across a long time. Some compounds, e.g. catechins, react fairly quickly with DPPH and the results may be different for different times.

Mirosław Grzesik I have eight samples with six dilutions for each so it may take more than thirty minutes if I wanted to test them all at the same time.

Aws Bunyan, So you have a reason to reduce the incubation time. However, who will guarantee you that you will get reliable results in this way. You can only check it experimentally. Preliminary tests are often necessary.

Hello Aws Bunyan!

I tested seven samples sequentially with about 8 minutes interval of addition of DPPH and a small break after 4 samples.

Sample-1+DPPH --> 8 min ---> Sample-2+DPPH --> 8 min ---> Sample-3+DPPH --> 8 min ---> Sample-4+DPPH --> BREAK --> Absorbance after 30 min from Sample-1 & same for the remainders

You can go this way too.

Hi Aws Bunyan

* DPPH reacts with antioxidants by mixed SET and HAT mechanisms, the proportion of which changes with the type of antioxidant.

* The behavior of DPPH reaction (Kinetic behavior) depends on the type of Antioxidant:

* Antioxidants can react with DPPH by very rapid electron transfer and by slow hydrogen atom transfer (see Figure in attachment). Compounds reacting rapidly (dominant electron transfer) are ascorbic acid and simple phenols with no ring adducts; reactions slow with the addition of acid groups or other side chains to aromatic rings. Small compounds with fast initial rates show a linear increase at low concentrations, but the linear reaction range shrinks, and saturation concentrations decrease with the addition of bulky ring adducts and multiple rings (see Figure in attachment). Molecules with complex structures more readily get in the way of each other and impede access to DPPH at low concentrations and they strongly block reaction at high concentrations.

It means that Steric Accessibility is a major determinant of the reaction of DPPH with antioxidants. Thus, small molecules that have better access to the radical site have higher apparent Antioxidant Activity with this test.

* In 2000, Sanchez-Moreno and co-workers classified the kinetic behavior of the antioxidant compound as follows:

• 30 min (slow).

You can note that the reaction times between DPPH and antioxidants range from 4 minutes to many hours, or even days. However, it is accepted in most of the papers to take 30 minutes as an average reaction time of this test.