Influences Of Different Factors On Glycocholic Acid Extraction From Bovine Bile And The Methodology Research Of Determining The Glycocholic Acid By HPLC

This study aimed to investigate the process of using ethanol precipitation and column chromatography to extract Glycocholic acid (GCA) from bovine bile, the influence of the flow rate, the adding amount of NaCl during salting out, the pH value and other factors on the extraction amount and purity of Glycocholic acid(GCA).So that build a more optimized extraction technology route from bovine of GCA from bovine bile. And a high performance liquid chromatography (HPLC) methodology were preliminarily developed and verified to determine GCA in the research.According to the orthogonal table L9(34), four factors and three levels orthogonal experiment design were used to research and analysis of the extract of GCA from bovine bile during the trial, flow velocity, add the amount of adding NaCl by salting out, extraction of pH value and proportion of mobile phase on the purity of GCA and the influence of amount of extracting determined the quality and quantity of GCA. The orthogonal experiment results showed that the most optimal combination of GCA extraction is that the volume by a 200 mL of column chromatography for separation and purification,55 g per column chromatography silica gel as a stationary phase, using mixture of chloroform and n-butanol elution solvent as mobile phase, each column sample loading quantity is 100 mL bovine bile, the velocity was level 1, the pH value was 2, the ratio of mobile phase was 3, and the amount of NaCl salting out was for level 1. By this way,0.3104 g GCA was extracted and its purity is up to 96.25%, and the recovery rate over 65% from 100 mL bovine bile. The test condition of HPLC method was: wavelength 210 nm, column temperature was 30℃, mobile phase acetonitrile:water= 35:65, flow rate 0.5 mL/min. The preliminary study methodology research is stable, and this feasible method can be used for the detection of GCA.The results showed, the extraction amount of GCA was larger than the other ways using orthogonal experiment designed optimal combination process,and it’s purity was higher, and the method was easy to do, and the cost was lower and production cycle was short. The HPLC method is stable and reliable.