Chemical Methods

Extract AOAC Official Method 960.45Vitamin A in Margarine: Spectrophotometric Method First Action 1960 Principle Unsaponifiable portion of margarine is chromatographed on adsorption column consisting of two segments of activated and standardized alumina separated by middle segment of alkaline alumina. (If eluate fraction containing vitamin A is colored, it must be further chromatographed on column of MgO.) Top segment of alumina column prevents caking and initiates separation of vitamin A from carotene and other interfering substances. Middle section of alkaline alumina separates persistent interference that cannot be separated by other adsorbents. Final portion of column is nonfluorescent and provides suitable background for observing vitamin A fluorescence on column, thus facilitating control of the chromatography. Carotene elutes from column first, second fraction of eluate is discarded, and third fraction contains vitamin A. Better control of chromatography than is possible by observing colored and fluorescent bands on column is achieved by observing fluorescence in 1 mL portions from final parts of fluorescent and colored eluates. This technic achieves separation of vitamin A from impurities that cause erroneously high values. Adequacy of separation is determined from ratio of A of chromatographed vitamin A solution at 310 and 325 nm. A of sample solution at 325 nm multiplied by factor 18.3 gives concentration of vitamin A in units/mL.