In this article we will discuss about the estimation and micro estimation of citric acid.
Estimation of Citric Acid:
Titrable Acidity Method:
1. Fermented broth.
2. 0.1 (N) NaOH.
3. 1.0(N) Succinic acid.
5. Titration set.
1. Standardise NaOH using 1(N) succinic acid.
2. Take in two separate 100 ml conical flasks 2 ml each of fermented broth and non-fermented medium (as control).
3. Titrate both, using phenolphthalin as indicator, end point is colourless to pink.
1 ml of 0.1 (N) NaOH = 7.0 mg of citric acid
From this calculate the yield of citric acid in g/100 ml as follows:
Normality of NaOH using 1 (N) succinic acid = 0.07(N)
Volume of NaOH required = X ml
Micro Estimation of Citric Acid:
1. Stock standard citric acid solution in 1 (N) NH2S04 50 µg/ml.
2. 10% trichloro acetic acid.
3. H2S04 18(N)/50%.
4. KBr-1 (M) 11.9% aq. solution.
5. H202 -6%.
6. Petroleum ether.
7. KMn04 aqueous solution 5%.
8. Thiourea solution- 2 g Borax in 100 ml 4% thiourea solution pH 9.2-9.5.
9. Oil bath 120°.
10. Sample (fermented broth).
1. Take different quantities of standard solution in the range of 10-100 µg in tubes after deproteinising with 10% trichloroacetic acid.
2. Take 1 ml of sample, add water to make up the volume to 3 ml.
3. Add 1 ml of 50% concentrated H2S04 in all tubes.
4. Place tubes in an oil bath at 120°C and boil until volume reduces to 1 ml and cool the tubes at room temperature.
5. Add 5 drops of KBr to each tube and then 10 drops of 5% KMn04 and allow to stand for 10 minutes. Colour of KMn04 to be maintained (by further addition).
6. Place tubes in ice water and add 6% H202 drop by drop shaking constantly till solution becomes just colourless.
7. Add 2.5 ml petroleum ether, stopper tubes with minimum cilicone grease and shake mechanically for 10 minutes.
8. A layer separates clearly and completely, if not centrifuge lightly.
9. Transfer 2 ml of heptane or petroleum ether to a clear glass tube with stopper.
10. Add 4 ml thiourea borax reagent stopper and shake again for 10 minutes.
11. A blank with 2 ml heptane and 4 ml thiourea borax reagent should also be prepared.
12. Suck off the heptane layer and transfer the reagent layer to colorimeter tube and read unknown against blank in a spectrophotometer at 430 nm.
13. Plot a graph of standard citric acid solution i.e. od versus concentration of citric acid in µg and determine unknown.