Study On The Purification Method Of Phosphatidylcholine With High Content

Natural phospholipids, main constituents of biological membranes, have a lot of important biological functions to all living organisms. High-content of phosphatidyl-choline (PC) is the fraction products of phospholipids extracted mainly from soybean or egg yolk. It not only can prevent the occurrence of hepatitis, fatty liver and diseases in heart and blood vessel, but also has functions of lowering cholesterol amount and anticancer. Therefore, World Health Organization (WHO) lists it as one of the nine longevity food. With the improvement of people's life, the demand of high-content PC is bigger and bigger. However, our country lags in manufacture technology and spends much on importing expensive high-content PC from foreign countries each year. How to refine PC from soybean or egg yolk becomes an international attracting project in recent years. And it is significant, necessary and imperative in our country.The project focuses on the three parts as following.First, the purification techniques of high-content ePC and sPC were investigated systematically and roundly. The raw materials are egg yolk powder or soybean concentrated lecithin. The quality indexes of ePC and sPC products fit for the national medicine standard. This is a new point of this paper.The process of ePC includes two parts such as solvent extraction and adsorption separation. We studied the influence of extraction time, temperature, ethanol concentration, solvent volume and times on the purity and recovery of PC with single factor design and orthogonal design. Neutral lipids (NL) and glycolipids (GL) in powder was removed by acetone. The purity of product is 68.0%, which reaches the national requirement of powder lecithin. The total recovery is 95.5%.In the adsorption process, 100~200 mesh coarse silica gel-â…¡ was selected as the adsorbent by comparing the separation results of three different adsorbents (200~300 mesh coarse silica gel, 80~120 mesh coarse silica gel-â…¡ and 100~200 mesh coarse silica gel-â…¡). Based on the adsorption separation commonness, TLC was utilized toscreen out mobile phase system. Hexane-isopropanol-water was determined as mobile phase. The optimal proportion of mobile phase (46:46:8) was obtained by considering retention time and resolution of PE and PC. After optimizing temperature, flow rate, injection concentration and sample weight, we obtained ePC with the content as high as 94%, which exceeds the requirement of medicine on PC. The recovery of PC in this process is 60.9%.The adsorbent, mobile phase and optimal loadability had been paid more attention in the investigation of sPC preparation from soybean concentrated phospholipids. We put forward a separation method that soybean concentrated phospholipids were directly used as raw material in column chromatography (CC). High-content PC reaching the medicine standard was obtained. Compared with the traditional method that PC was refined with CC after solvent extraction or superficial critical CO2 to remove impurities, the method is simple, low-consumption and valuable in industrial application. 100-200 mesh coarse silica gel-II was selected as adsorbent by comparing purity, recovery and solvent consumption. Hexane-isopropanol-water (46:46:7.4) was chosen as mobile phase. The optimal loadability of CC is 0.086 gPC/ (g silica gel). The purity of refine product is as high as 85.5% at a recovery of near 100 % on the optimal condition hereinbefore.Second, Adsorption equilibrium amount of sPC and ePC on 100-200 mesh coarse silica gel- II were obtained by using static adsorption method in different temperatures and concentrations. The isotherm was fitted by Freundlich and Langmuir equations respectively. And Langmuir equation matches the experiment results well. The general mass transfer model, including axial dispersion, external-film mass transfer and pore diffusion, was further applied to describe the adsorption process of for ePC and sPC on fixed bed with silica gel- II. This is a new point of the work. The pore diffusion coefficient and mass transfer coefficent were obtained by optimization the adsorption breakthrough of ePC and sPC. And these coefficients' values were used to calculate the elution curves of ePC at different flow rates and temperatures. The results show the model can predict the adsorption process of sPC and ePC in the fixed bed of silica gel. The effects of flow rate, initial inset concentration and columntemperature on adsorption breakthrough curves were studied dynamically. All these results are helpful to industrial design and scale-up.Third, a new method of analyzing phospholipids contents is brought forward. Comparison on phospholipids analysis was made between three detectors, i.e., ELSD, UV, and RI. The results show that ELSD is the optimum detector in phospholipids analysis. The gradient ternary solvent procedure of hexane-isopropanol-water was determined by carefully adjusting the ratio of three solvents and flow rate, and switch time of intermediate gradients. It is the first time to baseline resolve ten phospholipids and regress the calibration curves of PE, PI, PC, SM and LPC. Six kinds of soybean lecithin and egg yolk lecithin with these phospholipids were quantified. The coefficient of variation (C.V.) of retention time for five phospholipids are all less than 0.5% and the C.V. of peak areas are less than 6%. Study results prove the method has advantages such as fast, good resolution, high sensitivity in detection, good repeatability and accuracy.