Estimation of phytosterol can be done taking cholesterol as standard and using Liberman-Burchard reagent. Take 0.5, 1, 1.5, 2, 2.5 ml of the Standard solution of cholesterol (prepared by dissolving 10 mg of standard cholesterol in 10 ml chloroform) in separate test tubes. Then, add 2 ml of the L.B. reagent to all tubes and make the final volume equal in each tube by adding chloroform. Cover all the tubes with black carbon paper and keep in dark for 15 minutes. The same volume containing chloroform & LB reagent will save as blank/control. Then, set zero of UV/VIS spectrophotometer with blank at 640 nm. Determine the absorbance of all standards (six tubes) were on Spectrophotometer and plot standard graph. Finally, determine absorbance of 3 ml of sample solutions after adding the Liberman-Burchard reagent and chloroform. Now with the help of calibration graph plotted with std. you can estimate the total phytosterol.
@Yogesh Tripathi Sir. Can you please provide the reference for the procedure described.
Thanks and best regards
S.M. Sabir, I. Hayat and Gardezi, S.D.A. 2003. Estimation of Sterols in Edible Fats and Oils. Pakistan Journal of Nutrition, 2 (3): 178-181.
Preparation of L B Reagent: Cool acetic anhydride and concentrated sulfuric acid in ice water or in the freezer compartment. To a 500-ml amber glass bottle fitted with a polyseal cap, add 220 ml of cold acetic anhydride and 200 ml of glacial acetic acid (room temperature). Mix by inversion and add 30 ml of coid concentrated sulfuric acid. The reagent is ready for immediate use and is stable for more than 6 months when stored at 4 0C in the dark. When stored at room temperature, the reagent shows signs (darkening) of deterioration after 1-2 weeks.
Lieberman–Burchard is a reagent used in a colourimetric test to detect phytosterol / cholesterol, which gives a deep green colour. This colour begins as a purplish, pink colour and progresses through to a light green then very dark green colour. The colour is due to the hydroxyl group (-OH) of cholesterol reacting with the reagents and increasing the conjugation of the un-saturation in the adjacent fused ring.
Dear Olga Lock! Thanks for yours suggestion but that be too expensive and out of our budget.
Do you think Instead of chloroform if we use DMSO or Acetone as a solvent for ours samples, will it interfere with ours results (LB reagent)?
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