Observations on the Zak Cholesterol Reaction: Glacial Acetic Acid Purity, a More Sensitive Absorbance Peak, and Bromide Enhancement

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Abstract
An examination of the absorbance spectrum of the Zak cholesterol reaction, made with two brands of commercially available reagent-grade glacial acetic acid, showed not only the expected peak at 560 mµ, but an additional, higher peak at about 490 mµ. A specially purified glacial acetic acid did not show this 490 mµ peak. Conversely, increasing the ratio of color reagent to glacial acetic acid from 0.8 to 1.2 (v/v) greatly increased the height of the absorbance peak at 480 mµ and eliminated the 560 mµ peak. Under optimum conditions of time and color-reagent concentrations, the 480 mµ peak affords approximately double the sensitivity for cholesterol as compared to the 560 mµ peak. Finally, it was observed that the reported interference of bromide and iodide ions in the Zak reaction results almost entirely from an enhancement effect on the color by the cholesterol itself, and that the addition of about 100 µg. NaBr per milliliter of glacial acetic acid prior to reaction produced the full enhancement of 50 per cent greater color, and virtually eliminated interference at 560 mµ from additional amounts of bromide or iodide salts.

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