Alkaloid content in Ashwagandha Root

Method for the estimation of  Alkaloids content

Reagents & solvents  Required:

Diethyl ether, Methanol, Dilute ammonia solution, Chloroform, 1N Sulphuric Acid, 0.5N Sulphuric Acid

Glass-wares and Apparatus Required:   Stoppered flask, Measuring cylinder, Funnel, Separating funnel, Beaker, Filter paper and pipette. 

Procedure:

Weigh accurately about 3.0gm sample (Fine powder) in a 250mL stoppered conical flask. Add 75mL of a mixture of diethyl ether and methanol (4:1). Shake well and set aside for 15 minutes and add 5mL of dilute ammonia solution and shake frequently during one hour. Transfer the liquid to a separating funnel through cotton plug. Now wash the cotton with 25mL mixture of diethyl ether and methanol (4:1) and then with diethyl ether. Add 30mL of 1N H₂SO₄ and shake well, allow separating and running off the lower layer into another separator. Continue the extraction 1^st with 25mL and then with 20mL of mixture of 5 volume of 0.5N H₂SO₄ and 1 volume of methanol. Collect all the acid solution in another separating funnel. Wash the combined acid solution first with 10mL and then with two successive portions each of 5mL of chloroform, washing each chloroform layer with the same 20mL of 0.5N sulphuric acid contained in another separator. Reject the chloroform layer. Combined all the acid solution. Make acid solution neutral with dilute ammonia solution (Use litmus paper to check pH) and add 5mL in excess. Shake with successive quantities, 5x10mL chloroform. Wash each chloroform extract separately with the same 5mL of water and filter the chloroform layer through whatman no.1 filter paper over a bed of sodium sulphate into a pre- weighed evaporating dish. Evaporate on water bath, until about 2mL remains. Add 2mL of ethyl alcohol and continue the evaporation on water bath. Repeat the process using two further quantities, each of 2mL of ethyl alcohol and dry the residue for 1 hour at 80^oc and weigh the dish and calculate total alkaloids.       

Calculate the percentage of Total alkaloids as:

             (A – B ) X 100

          ------------------------

                      W

A = Weight of Beaker + Residue

B = Weight of Empty Beaker

W = Weight of material taken

For raw material : 10gm sample (20 #) is to be taken in the case of raw material and the quantity of  (diethyl ether : methanol) ratio shall be increased accordingly. Rest of the method is same as above.