Hello everybody
Recently, I've performed TPC assay and I have had some problems. Well, I started with 100 uL of extract that was filtered through a syringe filter 0.45 um. It was added 400 uL of Folin Ciocalteu and finally 2 ml of Na2Co3 (10%). After 30 min of stirring absorbance was measured at 725 nm.
The problem is:
At the moment of measurement, the blue solution had many little particles like bubbles and after a few minutes a white precipitate was deposited in the bottom of the tube (I attached the image). This made measurement difficult because of those particles moving around in the solution, therefore absorbance was not precise but changeable, irregular.
Some details of the procedure:
- Sodium carbonate was diluted in cold distilled water with less than 15 min of agitation.
- During the reaction time of 30 min, solution was stirred between 1000 - 1500 rpm.
I'd really appreciatte your advice. Thank you in advance.
Regards
The assay is not selective proteins, fats and any reducing agent present in the plant material would contribute and react with FCR reagent, for best practice try fractionate the biomolecules then you would observe more reliable results, running various controls would too help you to monitor the course of reaction and any unexpected behviour
Thank you both for replying
Dear Dr. Mushtaq
How do I fractionate biomolecules?
Read the part that describe the determination of total phenols in artcicle attached.
The Folin-Cio-
calteu method has first been tested on model compounds.
Methanol (0.5 mL) in the reaction mixture sometimes
induces the formation of a precipitate, as was observed
with gallic acid, ellagic acid, or (+)-catechin. It is thus
advisable to introduce the sample in a water solution when
this is possible. When compounds to be estimated are
poorly soluble in water but soluble in methanol, as with
ellagic acid, the color reaction is run in centrifugation tubes
and the precipitate eliminated before reading the absor-
bance. The reproducibility of the measurement is not
affected.
- Badges
- Science topic
- Similar topics
- Psychology