Separation And Retention Mechanism Of Alkaloids From Corydalis Saxicola Bunting On Rosin-based Polymer LC Stationary Phase

Chinese herbal medicine has been widely used in prevention and treatment of diseases for a long time,and research on its chemical composition and pharmacological effects is attracting more and more attention.Corydalis saxicola bunting is one kind of Zhuang medicine commonly used in Guangxi,and its active ingredients are regarded as a series of alkaloids with various species and similar properties.Due to the similar structure,separation of alkaloids usually are difficult,that leads to problems in quality control of Corydalis saxicola bunting and corresponding products.In the present dissertation,two new chromatogra phic stationary phases were developed for the separation and determination of alkaloids in Corydalis saxicola bunting,and satisfactory results were obtained.On this basis,the separation mechanism of the alkaloids on these stationary phases was discussed.Using EGMRA as a cross-linking agent,Methacrylic acid（MAA）and 2-Hydroxyethyl methacrylate（HEMA）as functional monomers,and alkylated silica gel as the carrier,two reversed-phase weak cationexchange liquid chromatographic stationary phases Ru131 and Ru132 were synthesized and characterized by IR,SEM,TG,EA and BET,respectively.The results showed that both MAA and HEMA were successfully grafted onto the surface of the alkylated silica gel,and the formed microspheres had uniform particle size and good thermal stability,and are suitable as liquid chromatography stationary phases.With alkylbenzenes,polycyclic aromatic hydrocarbons,phenols and flavonoids as probes,the performance of the synthesized Ru131 and Ru132 chromatographic columns were evaluated and compared with C18 column.The experimental results show that both columns have a reversed-phase chromatographic mode with a certain degree of hydrophobic interaction.It is further found that the main retention mechanism of Ru131 chromatographic column for separation of the above probes is due to the interaction of hydrophobic,Coulomb force and hydrogen bonding between Ru131 stationary phase and the probe compounds,while the retention mechanism of the above probes on Ru132 chromatographic column is the hydrophobic and hydrogen bonding forces.On the Ru131 column,the effects of the mixing ratio of the monomers in the stationary phase,the buffer concentration,p H and acetonitrile concentration of the mobile phase and the column temperature on the separation of dehydrocarvedine,palmatine hydrochloride and berberine hydrochloride were investigated.Based on the experimental result,the optimal chromatographic conditions for separation of the three alkaloids on Ru131 column were : the mixing ratio of the monomers as 5:2,the column temperature as 30 ℃,and the mobile phase was acetonitrile/0.005mol/L p H 3.0 phosphate buffered solution（20:80 v/v）with the flow rate of 1.0 m L/min.The three alkaloids were detected at 347 nm.A liquid chromatographic method was developed for the determination of dehydrocarvedine,palmatine hydrochloride and berberine hydrochloride in Corydalis saxicola bunting on the Ru131 column.The measured contents were 0.9894%,0.6655% and 0.1694%,respectively.The total content of the three alkaloids was 1.824%.In addition,separation of the three alkaloids on Ru132 column the effect of mobile phase composition and column temperature were investigated.The optimal chromatographic conditions were found as: column temperature 30 ℃.The mixing ratio of acetonitrile /0.010mol/L phosphate buffered saline solution（p H 3.0）is 20:80（v/v）,The flow rate is 1.0 m L/min,and the detection wavelength λ is 347 nm.The retention mechanism of dehydrocarvedine,palmatine hydrochl oride and berberine hydrochloride on Ru131 and Ru132 columns has been investigated.It has been found that there are hydrophobic interaction and Coulomb force between the three alkaloids and the stationary phase on Ru131,and the retention was mainly attributed to hydrophobic effect.The retention mechanism on the Ru132 column for dehydrocavitine,palmatine hydrochloride and berberine hydrochloride is the result of hydrophobic interaction and hydrogen bonding interaction.