A Study On The Separation And Purification Of Astaxantion From Haematococcus Pluvialis And The Analysis Of Its Isomers

Because of the particular function of Chinese herbal medicine in curing the difficult and baffling diseases, a rapid and efficient separation technique should immediately be required for the separation of the active ingredients from natural products or druggery. However, owing to the similarity of druggery molecular isomer on physical and chemical properties, it is very difficult to study the separation and analysis of the isomers from the active ingredients. It will be very significant that the separation and analysis of the astaxanthin isomers studied by high performance liquid chromatography (HPLC).The work in this dissertation is as follows:1. The activity components of Haematococcus pluvialis were separated by means of saponification (saponification liquid: 5% KOH-CH3OH) after the dichloromethane and methanol extraction, then purificated by column chromatography and recrystallization. Astaxanthin was obtained by the separation and purification from Haematococcus pluvialis.2. The different disruption technologies on the fresh cell of haematococcus pluvialis were discussed by means of ultrasonic, freezing and grinding methods respectively. The results show that the ultrasonic method adapts to the cell disruption of haematococcus pluvialis. The optimized conditions are ultrasonic power 315 W, disruption time 20 min. The content of astaxanthin (0.98%) from haematococcus pluvialis using acetone as the blank sample were determined by spectrophotometer.3. HPLC was used to analyze the isomers of pure astaxanthin. The separation conditions of astaxanthin isomer were optimized by the selection of the chromatography column, eluted methods, mobile phase, mobile phase ratio and the column temperature. The conditions of HPLC after optimization are as follows: C30 column (5μm,4.6×250 mm), the dichloromethane / acetonetrile (1:1) as mobile phase, isocratic elution, the wavelength determined at 487 nm, column temperature 35℃, flow rate 1 mL/min.4. The astaxanthin isomers were identified by using LC-MS. The chromatography conditions are as follows: Kromasil C18 chromatography column (4.6μm,4.6×50 mm), the solution of acetonitril / water (9:1) with 5% HCOOH as mobile phase, isocratic elution, the wavelength determined at 487 nm, column temperature 35℃, flow rate 1 mL/min. Three peaks of astaxanthin isomers were detected by using MS (ESI) with the ESI ion source using the positive ion or negative ion scan mode. HPLC-MS results showed that three isomers were preliminaly identified from the pure astaxanthin.