Studies On The Preparation, Evaluation And Application Of A New Magnolol-bonded Silica Gel Stationary Phase For HPLC

The active ingredients of Chinese herbal medicine have extensively been studied in pharmacological functions for the treatment of diseases, by contrast, their separation functions still were not explored. Herbal raw materials is readily available and contains various active sites in the their natural chemical structures, which facilitate to the exploration of chromatographic function, avoiding the complicate synthesis and purification. In addition, due to the antibacterial functions of herbal active ingredients these stationary phases will be more suitable for long-term separation of urine and other biological samples with longer column life as comparison with conventional modified silica. In this theses, a new magnolol-bonded silica stationary phase (MSP) was first prepared. Its chemical structure of magnolol on the stationary phase was characterized by various instrumental methods. The basic chromatographic property was systematically evaluated by using different structural probes, such as common organic alkaline, acid and neutral polar compounds. The related separation mechanism of MSP was also studied in reversed-phase liquid chromatography. The results showed that the new magnolol column exhibited versatile separation ability for series of polar aromatic compounds because of its various sites. We expect these new materials can be used for industrial chromatographic separations of green products in the future. The main research contents are as follows:1. The recent advances on the preparation, chromatographic retention mechanisms and their applications of novel natural products based silica gel stationary phases for HPLC at home and abroad were briefly reviewed. The application prospect of the natural active components as the bonded ligands for high-performance liquid chromatography in the future was also discussed. Above all will contribute to the theoretical basis for the exploration of new magnolol-bonded stationary phase in this study.2. By the solid-liquid continuous reaction method, the y-mercaptopropyl bonded silica (MPSP) was prepared as the precursor for the next synthesis. Following that a phenylcarbamoted magnolol was obtained with phenyl isocyanate, which was chemically linked to MPSP by a thiol-enyl addition reaction through a free radical Initiator AIBN. Finally a new magnolol-bonded silica stationary phase (MSP) was produced after a series of post-processing. The chemical structure of MSP was characterized by Fourier transform infrared spectroscopy, elemental analysis and thermogravimetric analysis. The results showed that natural component magnolol have been successfully bonded onto the surface of silica gel. The ligand bonding concentration of MSP was calculated to be about72μmol/g from the carbon content results of elemental analysis. It was also found that the stationary phase has good thermal stability because most weight loss occurred above300℃. The prepared magnolol-bonded phase was packed into a stainless steel column (4.6i.d.×150mm) under constant pressure and used as analytical column for HPLC.3. The retention time of biphenyl as solute probe on the new MSP column was19.44min by using methanol-water (60:40, v/v) as mobile phase. The number of theoretical plates was measured to be16423.55plates/m for biphenyl. According to evaluating method proposed by Kimata et al, some basic chromatographic property of magnolol-bonded silica stationary phase for liquid chromatography were respectively evaluated, including hydrophobicity, hydrophilicity, ion exchange and stereoselectivity of the chromatographic performance. The results showed that the new magnolol bonded silica stationary phase exhibited an excellent reversed-phase chromatographic property for a broad-spectrum compounds, which is similar to conventional C18phase (ODS). However, some good chromatographic performance of MSP compared with ODS may be due to various interactions between the stationary phase and solutes, such as hydrophobic interaction, hydrogen-bonding, dipole-dipole,π-π stacking and steric effect. Obviously, the magnolol ligand with various sites for analytes could contribute to the enhancement of selectivity and separation efficiency.4. In order to further study the separation ability and retention mechanism of the new MSP, some common ionized organic alkaline, acidic and polar compounds were selected as test solutes including eleven anilines, five pyridines, eight benzoic acids, four phenylalanine derivatives and twelve phenols in reversed-phase mode. The compared study was also carried out under the same conditions with commercial ODS column as a reference. The experimental results indicated that MSP has not only excellent reversed-phase chromatographic characteristics, but also is a kind of versatile stationary phase with multi-sites, which enable to the comprehensive separation mechanism mainly comprised of hydrophobic, hydrogen-bonding, dipole-dipole,π-π stacking and charge transfer interactions. For example, the better separations of benzoic acids and phenols can be achieved on the new MSP in a relatively short analysis time, instead of ODS. Therefore, MSP has an application advantage in the rapid routine analysis of the benzoic acids and phenols.5. To understand its application in drug analysis of purines, pyrimidines, pterins and flavonoids complex mixtures, the separations of several biological drugs including four purines, eight pyrimidines, four pterins and five flavonoids as polar representative samples were investigated on the new MSP column, respectively. To clarify the separation mechanism, a commercial ODS column was also tested under the same chromatographic conditions. We observed that the high selectivities and fast baseline separations of the above drugs were achieved by using simple mobile phases. Although there is no sealing treatment, the peak shapes of basic drugs containing nitrogen such as purines, pyrimidines and pterins were symmetrical on MSP, which indicated the the magnolol as ligand with multi-sites could shield the side effect of residual silanol groups on the surface of silica gel. Although somewhat difference of separation resolution, it was found that the elution orders of some drugs were generally similar on both MSP and ODS. We can make some conclusion that the hydrophobic interaction should played a significant role in the separations of the above basic drugs, which was attributed to their reversed-phase property in the nature. However, MSP could provide the additional sites for many polar solutes, which was a rational explanation for high selectivity of MSP. For example, in the separations of purines, pyrimidines and pterins to these compounds on MSP, hydrogen-bonding interaction played leading roles besides hydrophobic interaction. Some solute molecules (such as mercaptopurine, vitexicarpin) and MSP can form the strong π-π stacking in the separation process. All improved the separation selectivity of MSP for the above drugs.6. To explore its application in drug analysis, the magnolol-bonded phase (MSP) was also employed to separate and preliminarily identify two commercially available drugs, dexameth acetate adhesive tablets and cortex magnoliae officinaalis by reversed-phase liquid chromatography-mass spectrometry (LC-MS) using simple methanol and water as mobile phase. The results showed that MSP has fast and efficient separation ability to complicate components of prescription drugs. The chemical structures of components in drug samples were preliminarily identified by electrospray ionization mass spectrometry(ESI MS). The magnolol-bonded silica stationary phase with natural ligand will have a broader prospect in the preparative chromatography to purify natural products and develop their potential, especially active components extraction and purification from local medicinal herbal resources.7. A rapid analytical method of simulate determinating potassium guaiaclosulfonate hemihydrate,4-acetamidophenol and caffeine in the compound paracetamol and methylephedrine oral solution was carried out on the mode of reversed-phase high-performance liquid chromatography. These compounds were baseline separate in the simple mobile phase which consist of methanol and second water. Good results were both acheived in the testing of the real sample and the spiked samples. The established method is able to match the rapid routine analytical requirements of the actual sample because of its convenient, fast and accurate.