Studies On The Preparation, Evaluation And Application Of Curcumin Bonded Silica Gel Stationary Phase For HPLC

It is well known that there is abundant herb plant resource in our country. Traditional Chinese Medicine is very valuable treasure derived from nature. In order to make full and deeply use of them, this thesis focused on the exploration of their new chromatographic separation usage based on the natural chemical structural ligand. The scheme dealt with the preparation of separation materials with both chromatographic and pharmaceutical functions by chemical modification of the medicinal components onto the surface of silica gel for high performance liquid chromatography (HPLC). It is an attractive subject with great significance for the researches of theory and practice in the overlapping area among modern chromatography and related applied sciences to study the chromatographic retention mechanism and application of such novel separation materials.In this thesis, a new curcumin-bonded silica gel stationary phase (CCSP) was prepared so as to study its chromatographic functions and exploit its application for the first time. Its structure was characterized by electrospray mass spectrometry creatively, besides ordinary Fourier transform infrared spectrometry, elemental analysis and thermal analysis. Different structural solutes were used as probes to study the chromatographic property of CCSP systematically, including the basic chromatographic performance and the characteristic chromatographic separation evaluations. The probes covered a wide structural range from common probes, simple polar compound samples to complicate Chinese herbs extracts. Meanwhile, the retention mechanisms were also discussed.The results showed that CCSP exhibited excellent separation abilities for many polar compounds due to its large aromatic rings electronic system and various action sites. It has a promising prospect in the separations of basic compounds and many polar compounds for chromatography. Additionally, materials modifed with natural medical ingredients have potential application in the field of antibacterial medicines, which provides a new way pointing to the modern deep-processing of active components of TCM in the future. The major contents are as follows: 1. The recent advances of the study in the preparation, chromatographic retention mechanisms and their application of novel bonded silica gel stationary phases for HPLC at home and abroad were reviewed systematically. The application prospect of the active components from plants as the bonded ligands for HPLC and related separation technologies in the future was also discussed. Above all will contribute to the research foundation and start point to explore new natural medicinal ligand bonded stationary phases.2. An herb medicinal active component curcumin was chemically immobilized to the surface of silica gel as stationary phase by the solid-liquid phase reaction method under mild conditions by usingγ-glycidoxy-propyltrimethoxy-silane (KH-560) as coupling reagent for high performance liquid chromatography. The structure of new curcumin-bonded stationary phase (CCSP) was characterized by Fourier transform infrared spectrometry, elemental analysis, thermal analysis and mass spectrometry. The preparative scheme and reaction conditions were also partially optimized via investigation under different reaction conditions. The result showed that the curcumin ligand was successfully immobilized on the surface of spherical silica gel with a 0.129 mmolg^-1 of bonded amount, and CCSP had high physical and chemical stability. In addition, the bonded amounts of the packings in different batches were similar to each other, which indicated that the above preparation procedure was practical and reliable.3. Biphenyl was used as probe to determine the column efficiency (8221 plates per meter) in methanol-water (55:45, v/v) mobile phase. The stability of CCSP in strong organic solvent and acidic mobile phase were investigated, respectively. The basic chromatographic properties of CCSP, including hydrophobicity, charge transfer capacity, hydrogen bonding capacity, steric selectivity and ion exchange capacity were systematically evaluated, according to an authorized column validation method proposed by Kimata's group. Subsequently, various structural probes and conventional ODS were employed so as to profound study of new CCSP for a wide apllications in the future. The intensive chromatographic properties lie on the hydrophobicity, charge transfer and recognization abilities for aromatics and their positional isomers. The advantages of CCSP were showed in the separation procecesses by using probes, including many neutral, basic and acidic compounds. For example, nonploar aromatic carbonhydrons, polar and ionized aromatic acids and amines, water-soluble vitamins and nucleosides etc. Some comparative study was also carried out by adopting as reference under the same conditions. According to the chromatographic behavior of probes on CCSP, some retention mechanisms of new packing were also proposed, which will partially be used as the theoretic foundation for its practical application in the future. The results indicated that CCSP performed as a versatile reversed-phase material with a relatively weak hydrophobicity, especially, various action sites located in the curcumin ligand, such as hydrophobic, hydrogen bonding, dipoledipole, charge transfer action sites for different analytes. Hence, CCSP was endowed advantage over only hydrophobic ODS in rapid separations of polar compounds.4. Eleven basic compounds containing nitrogen-atom were employed as solute probes so as to further study the separation ability of new curcumin bonded stationary phase for basic compounds, which is important to the bonded silica gel stationary without end-capped process. The effects of mobile phase variables, such as methanol content and pH in mobile phases on the chromatographic behaviors of the basic compounds, as well as the silanophilic activity of CCSP were investigated in detail via the comparative study with ODS. The behavior change was used to clarify the related retention mechanism of the analytes on CCSP. The results indicated that baseline separation of three series of basic compounds on CCSP (no end-capped) could be achieved only with methanol-water as binary mobile phases without addition of buffer solution, respectively. CCSP exhibited better separation selectivity for the above basic compounds than that of ODS. It could also be noticed that the elution order of N,N-dimethylaniline andβ-naphthylamine, N-methylaniline and m-nitrobenzaldehyde were very different under the same reversed-phase chromatographic conditions. Obviously, CCSP had different retention mechanism to the basic compounds as compared with conventional ODS. According to the chromatographic data, some conclusion could be made that the versatile separation ability of CCSP derived from the synergetic interactions between the curcumin and analytes, including hydrophobic, hydrogen-bonding, dipole-dipole, n-Ï€andÏ€-Ï€charge transfer interactions. CCSP had two advantages in the separation for basic compounds. On one hand, the rapid separation of basic compounds can easily be realized because of weak hydrophobicity and multi-sites of CCSP. On the other hand, the side effect of residual silanols on the surface of silica gel was despressed by the polar linkage spacer. Thus, symmetrical peak shapes can be observed by using only methanolwater as mobile phase. Moreover, the unfavorable "U" shape of retention factor (k') of the basic solutes never appeared with increasing of methanol content.5. Subsequently, thirteen aromatic carboxylic acids were used as acidic probes to characterize new CCSP. In the same way, the comparative study with ODS was also done under the same reversed-phase chromatographic conditions. After some investigation of influence factors, it could be found that baseline separation of three series of aromatic carboxylic acids on CCSP were easily achieved, instead of ODS. CCSP behaved as a reversed-phase packing and its separation selectivity for aromatic carboxylic acids was better than that of ODS. The different elution orders of some acidic compounds were also observed on both CCSP and ODS. Among them, five of solutes exhibited stronger affinities to CCSP, which was inconsistent with the hydrophobicities and bonded amount of both packings. It was obvious that CCSP exhibited better separations whether acidic or basic compounds, which was mainly independent of the ionization property of solutes and dependent on the synergetic interactions providing by CCSP. For example, hydrogen bonding, dipole-dipole interactions existed between the phenolic hydroxyl or carbonyl groups of the curcumin and polar groups of acidic compounds. The CCSP containing curcumin ligand had two advantages over ODS. On one hand, the polar and ionized analytes, such as salicylic acid, p-nitrobenzoic acid, 3,4,5-trihydroxybenzoic acid, phthalic acid and o-bromobenzoic acid showed stronger affinities to CCSP because of various synergistic mechanisms. On the other hand, the retention of benzoic acid and o-chlorobenzoic acid can be successfully separated on CCSP within a short time because of its weak hydrophobicity.6. Five water-soluble vitamins and five nucleosides were chromatographied for the further study. Their chromatographic behaviors were studied on both CCSP and ODS, respectively. Some influence of mobile phase variables such as methanol content, pH and ionic strength on the retention behavior of analytes was investigated so as to clarify the retention mechanism. The results showed that a better separation of five water-soluble vitamins on CCSP was achieved by using methanol-0.01molL^-1 NaH₂PO₄ (40:60, v/v, pH3.5) as mobile phase, and the separation of nucleosides was carried out with only water as mobile phases. CCSP had better separation ability for vitamins and provided the different retention order for the analytes as compared With ODS. CCSP could also achieved effective separation of nucleosides and with different selectivity for thymidine and xanthosine from ODS within a relatively short analytical time. The various interactions and weak hydrophobicity of CCSP contributed to its advantages over only hydrophobic ODS in the separations of the above polar compounds.7. CCSP was employed for the preliminary separations of four complicate samples, including salicylic acid liniment, Sichuan garlic Allium Sativum L powder extracts, Sichsandra sphenanthera Rehd. et wils. seed extract and Atractylodes macrocephala Koidz. extract. The separation ability for complicate samples and the chromatographic retention mechanisms on CCSP were studied as compared with ODS. It was found that besides hydrophobic interaction other interactions such as hydrogen bonding interaction, dipole-dipole interaction, n-Ï€andÏ€-Ï€charge transfer interaction could also tailor the chromatographic retention behaviors of analytes and facilitate to the separations. Rapid separations of the above samples can be achieved on CCSP under simple chromatographic conditions with the help of the synergistic interactions.