How to Estimate of Vitamin A by Carr-price Method

It is based on the measurement of unstable blue co lour produced by vitamin A soln in an anhydrous alcohol free chloroform and a sat a soln of antimony dichloride in anhydrous alcohol free CHcl3 maximum absorpn is observed around 620 nm. The blue co lour is very unstable and is necessary to determine within 8-10 sec. after addition of reagent. The reagent should be handled carefully as it is corrosive apparatus must be clean and dry as reagent is V sensitive to moisture.
Thoroughly purified vitamin A is a viscous yellow oil freely sol in methanol, ether, acetone and CHcl3 it is highly susceptible to atmospheric O2 . It is destroyed by UV light etherification of along. of vitamin A with palm tic or acetic acid produces some what stable comp. Hence vitamin A is prepared as oily solns. of acetate or palm tic.
1 iu = 0.344 mg of vitamin A acetate
= 0.3 ug of vitamin A.

Reagents: – 1) NIL alcohols KoH 2) Sbcl2 reagent. (Carr-price reagent). Take 25 gm of sbcl2 in 100 ml of CHcl3. Decant the clear soln and store in amber colored bottle.

Procedure: – Weigh accurately 1 gm of sample in r.b.f Add 15 ml freshly prepared ale. KoH and reflux for 30.45 mins, cool and wash the reflux condenser c about 10 ml water. Add 500-100 ml water and transfer to a separating tunnel. Rinse the flask with 25 ml water and about 20 ml ether; add the content to the separating tunnel. Add 50 ml and extract by shaking Dastard the ag. Layer wash ether layer with 25 ml water. Continue the washing if necessary (till free from alkali). Allow the ether extract to stand for 10 min and draw off any separated water. Filter the ether extract into a e.f.. thru several gms of anhydrous Na2So4 placed on a filter paper in a tunnel fines the separating tunnel once with 20 ml ether and add to flask evaporate the whole extract to 5 ml on a steam bath. Heat extract at r.t. for sometime. Take up the residue in CHCI3 and 3 ml of Carr price reagent read absorbance in photoelectric colorimeter.

Procedure: – Std. Curve.

Prepare a series of t.t containing 0.15 iu of vitamin A soln make up the volume to 3 ml take the soln to barrette. Add to it 3 ml of sbcl3 reagent and read absorbance with in 8 – 10 sec. Draw calibrate curve.

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