Clinical Chemistry, Vol 20, 802-811, Copyright © 1974 by the American Association for Clinical Chemistry

Spectral Interpretation and Kinetic Studies of the Fe³⁺-H₂SO₄ (Zak) Procedure for Determination of Cholesterol

¹ Analytical Chemistry Division, National Bureau of Standards, Washington, D. C. 20234.

Spectral changes and kinetic data presented for the Zak reaction show the pseudo-first-order character of the consecutive oxidative reactions undergone by cholesterol and selected steroid dienes. These data give excellent support to the proposed formation of the homologous series of steroidal enylic cations which are the absorbing species in the spectrophotometric cholesterol determination. Typical thermodynamic quantities of G, H, S and E_a are also reported for several different reactions. Low acetic-sulfuric acid ratios evidently result in the stabilization of the lower members of the homologous enylic series. Although excellent reproducibilities are obtained for premixed and non-premixed Zak reagents, it is necessary to use the premixed reagent if good temperature control is required, as would be the case for kinetic methods of analysis run under the conditions we used. The isosbestic point observed at 503 nm may also be used as an analytical wavelength because it is less sensitive to variables such as temperature, time, and steroid concentration.

Submitted on March 26, 1974
Accepted on May 7, 1974