á591ñZINC DETERMINATION
The need for a quantitative determination of zinc in the Pharmacopeial insulin preparations reflects the fact that the element is an essential component of zinc-insulin crystals.In common with lead,zinc may be determined either by the dithizone method or by atomic absorption.

Dithizone Method
Select all reagents for this test to have as low a content of heavy metals as practicable.If necessary,distill water and other solvents into hard or borosilicate glass apparatus.Rinse thoroughly all glassware with warm dilute nitric acid (1in 2)followed by water.Avoid using on the separator any lubricants that dissolve in chloroform.
Special Solutions and Solvents—
ALKALINE AMMONIUM CITRATE SOLUTION— Dissolve 50g of dibasic ammonium citrate in water to make 100mL.Add 100mLof ammonium hydroxide.Remove any heavy metals that may be present by extracting the solution with 20-mLportions of Dithizone Extraction Solution(see Lead á251ñ)until the dithizone solution retains a clear green color,then extract any dithizone remaining in the citrate solution by shaking with chloroform.
CHLOROFORM— Distill chloroform in hard or borosilicate glass apparatus,receiving the distillate in sufficient dehydrated alcohol to make the final concentration 1mLof alcohol for each 100mLof distillate.
DITHIZONE SOLUTION— Use Standard Dithizone Solution(see Lead á251ñ),prepared with the distilled Chloroform.
STANDARD ZINC SOLUTION— Dissolve 625mg of zinc oxide,accurately weighed,and previously gently ignited to constant weight,in 10mLof nitric acid,and add water to make 500.0mL.This solution contains 1.0mg of zinc per mL.
DILUTED STANDARD ZINC SOLUTION— Dilute 1mLof Standard Zinc Solution,accurately measured,with 2drops of nitric acid and sufficient water to make 100.0mL.This solution contains 10µg of zinc per mL.Use this solution within 2weeks.
TRICHLOROACETIC ACID SOLUTION— Dissolve 100g of trichloroacetic acid in water to make 1000mL.
Procedure— Transfer 1to 5mLof the preparation to be tested,accurately measured,to a centrifuge tube graduated at 40mL.If necessary,add 0.25Nhydrochloric acid,dropwise,to obtain a clear solution.Add 5mLof Trichloroacetic Acid Solutionand sufficient water to make 40.0mL.Mix,and centrifuge.
Transfer to a hard-glass separator an accurately measured volume of the supernatant believed to contain from 5to 20µg of zinc,and add water to make about 20mL.Add 1.5mLof Alkaline Ammonium Citrate Solutionand 35mLof Dithizone Solution.Shake vigorously 100times.Allow the chloroform phase to separate.Insert a cotton plug in the stem of the separator to remove any water emulsified with the chloroform.Collect the chloroform extract (discarding the first portion that comes through)in a test tube,and determine the absorbance at 530nm,with a suitable spectrophotometer.
Calculate the amount of zinc present by reference to a standard absorbance-concentration curve obtained by using 0.5mL,1.0mL,1.5mL,and,if the zinc content of the sample extracted exceeds 15µg,2.0mLof the Diluted Standard Zinc Solution,corrected as indicated by a blank determination run concomitantly,using all of the reagents but no added zinc.