8 Estimation of tryptophan
 

 

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The indole ring of tryptophan gives an orange-red colour with ferric chloride under strongly acidic condition. The colour intensity is measured at 545nm.

 

Materials and Reagents

 

Papain solution

Ferric chloride

Glacial acetic acid

Acetic anhydride

Distilled water

30N H2SO4

Standard Tryptophan

 

Vortex mixer

Spectrophotometer

 

Composition of reaction mixtures

 

A.  Papain solution

Dissolve 400mg technical grade papain in 100ml 0.1 N sodium acetate buffer pH 7.0. Prepare the solution fresh every day.

 

B. Reagent A

Dissolve 135mg FeCl3.6H2O in 0.25ml water and dilute to 500 ml with glacial acetic acid containing 2% acetic anhydride.

 

C. Reagent B

30 N H2SO4

 

D. Reagent C

Mix equal volumes of reagent A and B about one hour before use.

 

E. Standard Tryptophan

Dissove 5mg tryptophan in 100ml water (50|ag/ml).

 

Procedure

 

1.  Weigh 100mg of air-dried, powdered and defatted grain sample into a small test tube

2.  Add 5ml papain solution, shake well and close the tube

3.  Incubate at 65C overnight

4.  Cool the digest to room temperature, centrifuge and collect the clear supernatant

5.  To 1ml supernatant add 4ml reagent C.

6.  Mix in a vortex mixer and incubate at 65C for 15min

7.  Cool the digest to room temperature and read the orange-red colour at 545nm

8.  Set a blank with 5ml papain alone and repeat steps 3-7

9.  Pipette out 0,0.2,0.4,0.6,0.8 and 1ml standard tryptophan and make up to 1ml with water. Develop colour following the steps 5 to 7  

 

Calculation

 

Draw a standard curve. "Substract the absorbance value of the blank from that of the sample and calculate tryptophan content from the graph.

                                         Tryptophan value from graph in μg x 0.096

Tryptophan in grain sample =        -------------------------------------------------------  g/16gN

                                            Percent N in the sample