Simultaneous Fluorometric Estimation of Chlorophylls and Pheophytins in Refined Edible Fats and Oils

Abstract

It has been believed that chlorophylls are not remained in refined edible fats and oils, because their contents are below the limit of detection by spectrophotometric analysis. In this experiments, photo fluorometric quantification was investigated to evaluate simultaneously the content of chlorophylls and pheophytins in refined edible oils.Chlorophylls a and b, and pheophytins a and b were prepared respectively from acetone extracts of spinach and/or followed by the treatment of oxalic acid. Sixteen mililiters of actone solution of each component (concentration ca. 0.15mgl) was added into 3.64g of blank oil, which was prepared through the removal of photosensitizer in edible plant oils by using a activated carbon-Celite column. By the measurement of fluorescence intensity in four measurement systems (Ex 425-Em 663, Ex 454-Em 646, Ex 404-Em 670 and Ex 434-Em 655nm) by spectrophotofluorometer, the following calculation formulas were introduced;Chl a=(10.8F663425+0.307F646454-3.30F670404-4.91F655434)×10-3mg/kg oilChl b=(0.566F663425+5.09F646454-0.199F670404-0.950F655434)×10-3Phy a=(-8.09F663425-0.214F646454+11.7F670404+2.74F655434)×10-3Phy b=(-4.81F663425-1.71F646454+1.23F670404+7.92F655434)×10-3In the case of practical evaluation of refined edible oils, the fluorescence intensity of blank oil was subtracted from that of sample solution, dissolving 3.64g oil in 16ml of spectrum grade acetone. Thus, the indentity and concentration of four components may be accurately and rapidly determined within the concentration range from 0.05 to 1mg/kg oil as the total chlorophylls.As the results of fluorometric quantification on several refined edible oils, it was confirmed that “chlorophylls” was presented at 0.06 to 0.3ppm as the sum of four components, in which pheophytin a showed the highest content above 60%.