Estimation and Micro Estimation of Citric Acid | Biotechnology

In this article we will discuss about the estimation and micro estimation of citric acid.

Estimation of Citric Acid:

Titrable Acidity Method:

Requirements:

1. Fermented broth.

2. 0.1 (N) NaOH.

3. 1.0(N) Succinic acid.

4. Phenolphthalin.

5. Titration set.

Procedure:

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:

Titrable acidity:

Normality of NaOH using 1 (N) succinic acid = 0.07(N)

Volume of NaOH required = X ml

.

Micro Estimation of Citric Acid:

Requirements:

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).

Procedure:

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.