after freshly preparing of FRAP reagent i add 3 ml of the reagent to 0.1 ml of solution. but when i tried to take the reading in spectrometer the absorbance value kept increasing for about one hour before reaching little stable stage.
what could be the problem???
" i add 3 ml of the reagent to 0.1 ml of solution." What solution? Of antioxidant?
If yes, then the products from antioxidant may strongly absorb light around 600 nm. Look at the spectral changes in the range 400-900 nm.
Omar Alajil
Hi! You could try this way:
At the dark, you must mixture:
90ul of sample or standard (I use trolox)
270ul of deionized water
2.7mL of FRAP reagent (freshly prepared)
Incubation: water-bath 37°C for 30 minutes. After this, read at 595nm.
FRAP REAGENT:
2.5 mL of 10mM TPTZ (in HCl 40mM)
2.5 mL of 20mM ferric chloride
25 mL acetate buffer 0.3M pH 3.6
Dear Omar Alajil
Yes, FRAP assay is used to determine kinetic property of electron transfer. The assay based on electron transfer capacity of test substance within time period.
Based on the potential of antioxidant (electron donating) capacity rate of absorbance will increase.
Kindly refer
Article Antioxidant and free radical scavenging potential of crude s...
Article Purification and characterization of fucose-containing sulph...
Manoj Saravana Guru Mohan Ram, you did not respond to my and Omar's suggestions. Instead, you describe what is the FRAP assay. Do you assume that we need your explanation of FRAP?
Hey, try this method
1. Add 0.9ml of HPLC grade water to 0.1ml of sample solution.
2. Then add 2ml of FRAP reagent to it and mix well.
3. Incubate it for 30mins at 34 degree Celsius
4. Read the Abs. at 593nm
*FRAP reagent prepared by mixing the following solutions accordingly in the ratio of 10:1:1
300mM Acetate Buffer
10mM TPTZ in 40mM HCl
20mM ferric chloride hexahydrate
Banti Chakraborty thanks for your valuable suggestion. i will try the solution you mentioned
however the method i followed is similar to the method you mentioned except i didn't use HPLC grade water.
Yurii V Geletii & Manoj Saravana Guru Mohan Ram & Amanda da Costa Gomes
thank you all for valuable suggestions and contributions
Amanda da Costa Gomes your methods looks interesting . thanks for your contribution .
i did keep the sample in the dark after adding the 0.1 ml of sample but i didn't think to add the sample to reagent in the dark.
Manoj Saravana Guru Mohan Ram thanks for the helpful answer and attachment of highly valuable articles
Yurii V Geletii thanks for answer and interest
and as you said the solution is the sample extract (antioxidant), yes
moreover
actually in FRAP method the procedure is to use use wavelength 593 , i didnt understand the need to check absorbance at 400 to 900.
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