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Sunday, December 13, 2009

Estimation of α - Tocopherol [Vitamin E]

How to  estimate the alfa-Tocophorol content of given sample

Alfa - Tacopherol occurs widely distributed in plant and animal tissue but only in trace amounts. There are 5 naturally occurring to copherols α-, β-, γ- and δ  θ  amongst which α- tocopherol shows maximum biological activity. It is an antistenlity vitamin. The esters of vitamin. Are more stable and hence vitamin E is incorporated in formulation inform of esters i.e. acetate or palmitate.

Principle: - The method of estimation is based on reduction of ferric ions by tocopherol to ferrous ions which form a red coloured complex with bipyridol i.e. 2- dipyridyl alcohol. The absorbance of complex is measured at 520 nm. This method is trost commonly used because it is simple and gives reproducible results and is applicable for most natural products like fruits tissue, organs etc. However this method is applicable only in estimation of free vitamin E and not esters. Esters should be saponified and interfering substance such as vitamin A arytenoids etc. should be removed from standard prior to estimation.

Procedure: - For sample: Take known amount of sample [1ml] containing [0.1 – 0.7 gm] of α - tocopherol. Rinse quantitatively in long necked flask with 20 ml absolute alcohol. Add 3 ml of freshly prepared koH solution [50%] and pinch of antioxidant to prevent oxidation of formed vitamin during de-etherification. Heat on water bath and reflux for an hour. During etherification this content of flask should be vigorously boiling. The saponified mixture is cooled and rinsed into separating fennel with 10 ml water. It is extracted 3 times with 25 ml portion of solvent ether. All other extracts are combined and washed with 50 ml water. The ether layer is passed tho’ anhydrous sodium sulfate bed and evaporated on stem bath. To assay to control the residue is dissolved in alcohol.


Calculation and observations:-
Conc. of standard = 250 gm / ml
Conc. of sample = unknown ‘X’

Absorbance of standard = 0.467
Absorbance of sample = 0.198

Conc. of sample = conc. of std./Absorbance of std x Absorbance of sample
                           

= 250 /0467x* 0.198


= 105.99 gm / ml of alfa -tocopherol



For standard:-Replace 2 ml of standard solution and repeat.

For colour development: - Dissolve residue in methanol accurately measured and take 0.5 ml of solution for co lour development. To an aliquot add 0.5 ml Fecl3 soln, shake well and add 0.5 ml of bipyridol solution. Make up the volume to 5 ml with methanol and measure the absorbance immediately.
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