Studies On The Preparation, Characterization And Application Of Baicalin-bonded Stationary Phase

The medicinal components of traditional Chinese herbs can chemically beanchored on the surface of silica gel and used as excellent selectors forchromatographic separations and enrichments in pharmaceutical analysis and chiralseparation because their unique natural structures with separation and medicinaldouble fuctions can provide various interaction sites for analytes, especially naturalproducts of herbs. It has significant meaning for the modernization of TraditionalChinese Medicine to develop novel separating materials with active ingredients andexploit new scheme to make full use of the abundant herb resources in our country,which is an attractive subject with great promising prospect for the researches oftheory and practice in the overlapping area among modern chromatography andrelated applied sciences. In this thesis, a new baicalin-bonded spherical silica gelstationary phase (BBSP) was prepared for the first time. Its structure andchromatographic performance were characterized by spectroscopic methods and highperformance chromatography with solute probes. Some chromatographic mechanismof the new bonded stationary phase for different kind of analytes was systematicallystudied. Meanwhile, the baicalin-bonded amorphous silica gel was used to enrichflavonoids of Scutellariae Radix. The major contents are as follows:1. The advances of high performance liquid chromatography bonded silica gelstationary phase and technologies used for separating and purifying activecomponents of traditional Chinese herbs were reviewed systematically. Theemphases were laid on two parts: the former was preparation, chromatographicproperty and application of bonded silica gel stationary phases for HPLC, thelatter dealt with definitions, characteristics and applications of traditional &modem separation and purification technologies. The developing prospect ofBBSP was expected. All these studies were used as the start point for furtherstudies in this thesis.2. The solid-liquid state continuous reaction method was employed to prepare a new baicalin-bonded stationary phase (BBSP) containing natural antibacterialcompound for HPLC, usingγ—aminopropyltriethoxy silane (TM550) as couplingreagent. The amiopropyl-bonded silica gel (ABSP) as precursor was prepared inthe first step. Then BBSP was obtained via the reaction between the amino groupsof ABSP and the acyl chloride of baicalin. The hydrophobic balicalein layercovered the silica gel can provide the protection for BBSP from acidic medium.Its chemical structure was characterized by elemental analysis, diffuse reflectanceinfared Fourier transform spectroscopy, thermal analysis, electrospray massspectrometry and chemical analysis. The preparative scheme and reactionconditions were partially optimized. The results showed that baicalin ligand wassuccessfully immobilized on the surface of silica gel with a low bonded amountabout 0.11 retool/g, which implied the hindrance effect of baiealin bulk group.Additionally, the bonded amounts of the packings in different batch were similarto each other, which indicated the above preparation procedure was practical andreliable.3. The baicalin-bonded stationary phase was packed into stainless steel column byslurry method. Its column efficiency was determined to be 8970 plate number permeter. The stability of the new column was first investigated by using acidicmobile phases. The basic chromatographic properties of BBSP, includinghydrophobicity, steric selectivity, hydrogen bonding capacity and ion exchangecapacity, were evaluated by the authorized method proposed by Kimata group.Furthermore, some probes, such as PAHs, nitrogen-containing compounds,positional isomers, phenolic compounds, nucleosides and bases, flavonoids wereemployed to characterize the chromatographic performance of BBSP, and clarifythe separating mechanism as compared with ODS. These probes have differentkind of chemical structures, dissociation and polarity, and their chromatographicbehavior should define the chromatographic property of BBSP for practicalapplication in the future. The results showed that BBSP not only possessesexcellent hydrophobicity of ODS, but also exhibites strong hydrogen bondingcapacity andπ-πinteraction, relatively strong ion exchange capacity, working aselectron acceptor in charge transfer. BBSP was more suitable for basic compounds because it can shield the side effect of silanol residues by internal embeddedamide group and glucoside. In addition, BBSP has a good promising prospect inthe separation and enrichment of flavonoids from many medicinal plants becauseof the strong infinity to flavonoids.4. Baicalin-bonded amorphous silica gel (BBS) for extracting active componentsfrom traditional Chinese herbs was synthesized and characterized with similarmethods of BBSP, such as IR, MS and elemental analysis. Based on theenrichment result of baicalin on BBSP, the similar mutual interaction between theligand of the bonded phase and the solutes in solution can be observed, whichillustrates a new path for the fast capture of interesting compounds from thecomplicate herb system. The results showed that BBS exhibits a good capacity toenrich baicalin in solution even with its low bonded amount. The absorptivecapacity (Qe) was about 214.26mg/g.5. BBS was applied to enrichment and purification of flavonoids of ScutellariaeRadix as a new type of separating material. Several main flavonids in theScutellariae Radix solution, such as bacalin, balicalein, wogonoside and wogonin,were identified by HPLC-ESI-MS. The enriching and purifying effects of BBSwere preliminarily investigated and evaluated based on the total amount of theenriched flavonoid in both static mode in solution and dynamic mode byhome-made SPE column. The usual UV spectroscopy and HPLC were employedto monitor the flavavonoids. It was found that BBS has selective enrichment forflavonoids of Scutellariae Radix. The static enrichment capacity for flavonoidswas about 81.9551mg/g. The interactions for baicalin and baicalein are relativelystronger than wogonoside and wogonin. It was still required further to study thepreferable enrichment-eluting condition for BBS. Besides chromatographicseparation function, BBS should be new materials for antibacterial and medicinalpurpose.