Procedure—
Pipet into a suitable vessel 25 mL of the final washed ether solution of the unsaponifiable fraction obtained as directed for
When Tocopherol Is Present under
Procedure in the
Vitamin A Assay 
571
, and evaporate to about 5 mL.
Without applying heat, remove the remaining ether in a stream of inert gas or by vacuum. Dissolve the residue in sufficient alcohol to give an expected concentration of about 0.15 mg of alpha tocopherol per mL. Pipet 15 mL into a 50-mL centrifuge tube, add about 200 mg of palladium catalyst, stir with a glass rod, and hydrogenate for 10 minutes in the
Hydrogenator, using hydrogen that has been passed through alcohol in a blank tube. Add about 300 mg of chromatographic siliceous earth, stir with a glass rod, and immediately centrifuge until the solution is clear.
Test a 1-mL aliquot of the solution by removing the solvent by evaporation, dissolving the residue in 1 mL of chloroform, and adding 10 mL of
antimony trichloride TS: no detectable blue color appears.
[NOTE—If a blue color appears, repeat the hydrogenation for a longer time period, or with a new lot of catalyst.
]
Pipet 2 mL of the supernatant into a glass-stoppered, opaque flask, add 1.0 mL of a 1 in 500 solution of ferric chloride in dehydrated alcohol,
* and begin timing the reaction, preferably with a stop watch. Add immediately 1.0 mL of a 1 in 200 solution of 2,2
¢-bipyridine in dehydrated alcohol, mix with swirling, add 21.0 mL of dehydrated alcohol, close the tube, and shake vigorously to ensure complete mixing. When about 9½ minutes have elapsed from the beginning of the reaction, transfer part of the mixture to one of a pair of matched 1-cm spectrophotometer cells. After 10 minutes, accurately timed, following the addition of the ferric chloride-dehydrated alcohol solution, determine the absorbance at 520 nm, with a suitable spectrophotometer, using dehydrated alcohol as the blank. Perform a blank determination with the same quantities of the same reagents and in the same manner, but using 2 mL of dehydrated alcohol in place of the 2 mL of the hydrogenated solution. Subtract the absorbance determined for the blank from that determined for the assay specimen, and designate the difference as
AD.
Calculate the alpha tocopherol content, in mg, in the assay specimen taken by the formula:
30.2 AD / (LCD),
in which AD is the corrected absorbance; L is the length, in cm, of the absorption cell; and CD is the content of the assay specimen in the alcohol solution employed for the measurement of absorbance, expressed as g, capsules, or tablets per 100 mL.
