Preparation And Characterization Of Allitridin Diallyl Trisulfide-bonded Silica Gel Stationary Phase For HPLC

The design, preparation and application of new-style .chromatographicseparation materials with natural ligands, such as active antibacterial compoundsderived from medicinal herbs, have very important meanings to deeply exploit andutilize rich Traditional Chinese Medicine (TCM). The versatile materials can exhibitboth chromatographic separation and antibacterial functions because itspre-organization molecular structure and drug nature, avoiding biologicalcontainment from various complicate samples in the separation process so thatprolong column lifespan. This is an attractive subject in the overlapping area ofmodem chromatography with application sciences, showing important academicvalue. In this thesis, a new diallyl trisulfide-bonded silica gel stationary phase (DTSP)with the ligand from garlic plant for HPLC was first prepared. Its structure andchromatographic performance were characterized and evaluated. Related separationmechanism was systematically studied. Additionally, the antibacterial activity ofdiallyl trisulfide-bonded silica gel was tested and the antibacterial mechanism wasproposed. The major contents are as follows:1. Recent progress of the study of bonded silica gel stationary phases for HPLCwas reviewed from two aspects, including the improvement of traditionalbonded-silica gel stationary phases and the design of new bonded phases. Themain subject focused on the preparation, chromatographic property, retentionmechanism and application of bonded silica gel stationary phases for HPLC. Theapplication prospect of the bonded for HPLC and related technique in the futurewas also discussed. Above all provide the basis and start point for furtherresearch of the subject.2. The solid-liquid reaction method was used for the preparation of diallyltrisulfide-bonded stationary phase (DTSP) via the addition reaction betweencoupling reagentγ-mercaptopropyl bonded stationary phase (MPSP) and diallyltrisulfide in the presence of dicumyl peroxide (DCP) for the first time. The key scheme lies in how to survive the pre-organization natural structure of selectorfor chromatography and its antibacterial activity for medicinal purpose.. It wasreally realized via a moderate addition reaction andγ-mereaptopropyl trimethoxysilane coupling reagent to produce multi-sulfide on the surface of silica gel.DTSP possesses alkyl chain-like ligand, which is similar to octadecdyl-bondedsilica gel phase (ODS) besides its bioactivity. The reaction conditions were alsopartially optimized during the investigation. Their structures were characterizedby elemental analysis, infrared Fourier transfer spectrum, thermal analysis andmass spectrometry. The bonded amounts of MPSP and DTSP were determined tobe 1.47 mmolg^-1 and 0.23 mmolg^-1, respectively. They also have good thermalstability.3. The basic chromatographic performances of DTSP were evaluated according tothe comprehensive evaluating method proposed by authorized Kimata'sgroup.Various structural solutes as probes and ODS as reference were employed so asto deeply study the separation mechanism and chromatographic character ofDTSP. The solutes included PAHs, positional isomers, neutral aromatics, phenolssubstituted benzoic acids, aromatic amines etc. The results showed that besidesits antibacterial. function DTSP can be provide hydrophobic, hydrogen-bonding,Ï€-Ï€,n-Ï€, charge transfer and ion-exchange interactions for different analytes.Synergetie interactions endowed DTSP a versatile packing for the fastseparations of neutral, acidic and basic compounds even positional isomers.Moreover, it is a good commencement for the separations of complicate samples,such as Rheum palmatum L, Tylophora ouata and Garlic powder. It can also befound that diallyl trisulfide-bonded stationary phase exhibited selectively stronginfinity to the sulfides based on the similarity interaction model.4. In order to further clarify chromatographic performance in the separation of polarcompounds; the behavior of some nucleosides, pyrimidines, purines andwater-soluble vitamins on DTSP were studied by HPLC. Meanwhile, thecomparative study in the retention behavior of the above solutes on both DTSPand ODS were also done under the same conditions. The influence of mobilephase variables, such as methanol content, pH value and ionic strength on the retention behavior of the polar compounds were investigated. The betterseparations of nucleosides, pyrimidines, purines and water-soluble vitamins onDTSP can be achieved, respectively. Based on the chromatographic data, it can beincluded that hydrophobic interaction, hydrogen-bonding interaction, n-Ï€,Ï€-Ï€interaction and dipole-dipole interaction are mainly responsible for the retentionbehavior of the above analytes. In addition, to some extent, ion exchangeinteraction on DTSP can.also change the retention behavior. Obviously, DTSPcan provide various sites and synergistie action for polar analytes, whichimproved the separation as compared with ODS. DTSP exhibits the promisingapplication in the separation of polar compounds.5. Diallyl trisulfide is one of high antibacterial active components from garlic plantAllium Sativum L. The chemical linkage of natural active ligands on the surface ofsilica gel has theoretical research and promising applications..The diallyltrisulfide-bonded silica gel (DTS) and theγ-mercaptopropyl-bonded silica gel(MPS) were prepared by using general amorphous silica gel as matrix. The surfacestructure of DTS was characterized by elemental analysis, FT-IR, thermal analysisand eleetrospray mass .spectrometry. The bonded amounts of MPS and DTS weredetermined to be 1.73 mmolg^-1 and 0.27 mmolg^-1, respectively. The antibacterialproperty of the prepared material DTS was investigated by usingantibactedum-circle. The antibacterium-circles of DTS to Staphylococcus aureus,Escherichia coli, Bacillus subtilis, Aerobacter aerogenes and Proteus vulgariswere 9.0mm, 8.1mm, 10.1mm, 9.2mm and 8.9mm, respectively. The influence ofthe content of DTS in beef extract peptone medium on the inhibition effect toStaphylococcus aureus and Escherichia coli by mixing-smearing plate cultivatemethod was also investigated.The results showed that DTS exhibited a goodinhibition effects to the above five bacteria. After chemically bonding of diallyltdsulfide on silica gel, it can be found that not only elimination of the strongodour of Allium Sativum L.,but also preservation the antibacterial activity werefound on the surface of DTS. Meanwhile, the antibacterial mechanism of DTSwas proposed based on the experimental result analysis. A mixing-smearing plateculture method can be provided for the antibacterial test of the hydrophobic particle samples.6. Diallyl sulfides have no response because of their weak polarities and difficultionization in LC-ESI-MS. In this chapter, diallyl sulfides in garlic powder werefirst identified via the introduction of Ag(â… ) induced-ionization. Meanwhile, abovecompounds were quantified by using diallyl trisulfide(DATS) as reference. Agradient program was employed and methanol-water was used as mobile phase.The chromatographic analysis, was carried out on a Waters Symmetry C₁₈column(3.9mm×150mm, 5μm). The flow rate was at 0.5mLmin^-1. Under aboveconditions, all diallyl sulfides were separated within 30rain. The derivativereagent lmmolL^-1 AgNO₃ was injected to post-columu at a 5μLmin^-1 flow rate.The ESI⁺ was used to monitor ions with 3Kv capillary voltage. The results showedthat the 'Rule of sulfur groups' (logk'= 0.4276+ 0.0364n, r=0.9910) was observedaccording to their chromatographic behavior. Based on the principle of similarmolar absorbance coefficients for diallyl sulfides, these compounds in garlicpowder were determined by comparing with DATS, which has obvious advantageover barium sulfate gravimetrie and argentometrie methods.7. The above study focused on the preparation and characterization of new stationaryphase DTSP. However, its chromatographic application for biochemical sampleswill still be requiring to be developed in the next step.