Bio-surfactant Applied In Microemulsion Capillary Electrokinetic Chromatography

Microemulsion electrokinetic chromatography (MEEKC) is a separation technique on the basis of micellar electrokinetic chromatography. MEEKC can separate charged and un-charged, hydrophilic and lipophilic substances simultaneously. Rhamnolipid is a widely used bio-surfactant. It can form stable microemulsion without co-surfactant owing to its high surface activity. The thesis firstly used a small content of rhamnolipid in microemulsion in MEEKC to solve the problems such as high Joule heat, long analytical time, and severe baseline noise existed in typical microemusion system. The thesis mainly included the following three parts.1. Crude rhamnolipids was purified with liquid-liquid extraction. With HPLC, there were only three peaks in chromatogram. HPLC-MS was applied to identify three constituents. It was testified that they belong to the group of rhamnolipids. The content of these three kinds of rhamnolipids were determined with HPLC, and the average molecular weight of rhamnolipid was calculated out to be 523.22 according to the area normalization method. Furthermore, influencing factors including the concentration of rhamnolipid, pH, concentration of co-surfactant and content of organic solvent on microemulsion stability were investigated, which was very helpful for utilization of rhamnolipid as surfactant in microemulsion to improve MEEKC performance.2. Without co-surfactant, a rhamnolipids microemulsion was firstly prepared and was used in microemulsion electrokinetic chromatography to determine three corticosteroids, namely prednisone, hydrocortisone and prednisolone in cosmetics. The effects of pH of the running buffer, concentration of rhamnolipid, ionic strength, type and concentration of oil, capillary temperature, separation voltage and injection voltage and time were investigated, the optimum microemulsion consisted of 0.1%(w/w) rhamnolipids, 0.8%(w/w) n-heptane and 99.1% borax buffer (80 mmol/L, pH9.2). With separation voltage of 20 kV, electrokinetic injection of 3 s/10 kV and capillary temperature at 20℃, prednisone, hydrocortisone and prednisolone were baseline separated within 9.4 min. RSDs of migration time for three analytes were all less than 0.2% and RSDs of peak area were less than 5.0% (n=7). Excellent linearity was obtained ranged from 5 to 100 mg/L, the detection limits based on ratio of signal to noise of 3 were 1.0, 1.1 and 1.3 mg/L for three analytes respectively. The method has been applied to determination of the three corticosteroids in actual cosmetic samples by simply extraction pretreatment, the recoveries of three analytes were between 81.6% and 108% and RSDs were less than 4.8%. Compared with typical SDS microemulsion system, rhamnolipids microemulsion used in MEEKC is beneficial to shortening the analysis time dramatically.3. Rhamnolipid-SDS microemulsion system was applied in MEEKC to analyze water- and fat-soluble vitamins simultaneously. According to the investigation of separation efficiency, we found rhamnolipid microemulsion system used in MEEKC could not baseline separated fat-soluble vitamins. On the other hand, with typical SDS microemulsion, it took so much time to analyze water- and fat-soluble vitamins simultaneously, and poor resolution was gotten. The mixed surfactant using rhamnolipid and SDS provided more stable microemulsion and better performance in MEEKC. Furthermore, it was testified that n-butanol is the key factor for separation fat-soluble vitamins, and acetonitrile as organic solvent modifier is very important to improve resolution at some extent. According to the comparison of microstructure of different kinds of microemulsion, it was found that SDS microemulsion has the larger charge-to-mass ratio than rhamnolipid microemulsion. With the increase of n-butanol, the charge-to-mass of microemulsion system increased, correspondingly. By investigating influence of composition of microemulsion on separation efficiency, the optimized microemulsion consisted of 0.1% Rhamnolipid(w/w)- 0.4% SDS(w/w)- 0.8% n-heptane(w/w)- 6% n-butanol(v/v)- 15% acetonitrile(v/v). Then, MEEKC conditions were optimized as follows: capillary temperature at 20℃, separation voltage at 20 kV and electrokinetic injection with 3 s/10 kV. Under the optimum conditions, 4 water- and 3 fat-soluble vitamins were baseline separated within 24 min. Analysis time shorten with excellent separation efficiency owing to low concentration of SDS and addition of butanol to loosen structure of microdroplet. Good linearity were over two magnitudes for all vitamins with R greater than 0.993. The detection limits of vitamin B₁, B₆, B₅, B₁₁, A, D₃ and E were 9.0, 4.0, 6.9, 15, 73, 20 and 21μg/mL respectively. By reparative injection sample 5 times under the optimum conditions, RSDs of migration time and peak area were all less than 1.7 % and 4.9 % respectively. The method was applied to determine of water- and fat-soluble vitamins in commercial multivitamin pharmaceutical formulation with acceptable quantitative results corresponding to label claim. Good accuracy and precision were obtained with recoveries between 88.0% and 105%. The developed method is suitable for the analysis of water- and fat-soluble vitamins simultaneously.