Preparation Of Polar Embedded Mixed-Mode Liquid Chromatography Stationary Phase

Objective:High performance liquid chromatography（HPLC）is considering as an effective analysis and separation technique and has been used to separate and purify various compounds.However,with the development of society,the single-mode chromatography stationary phase is often limited in the separation and analysis of complex samples,and the mixed-mode high performance liquid chromatography（MHPLC）with multiple retention mechanism shows the potential of separating complex samples.Therefore,it is significant to develop and design new mixed-mode HPLC columns with high selectivity and flexibility.Based on the functional groups modified on the silica,two multi-mode stationary phases,the ionic liquid embedded C18 and cellulose co-functionalized stationary phase（1）and polar-embedded phenyl dendritic stationary phase（2）,were synthesized.Moreover,the separation mechanism and chromatographic properties of the two types of stationary phases were also studied.Methods:（1）A novel multi-mode stationary phase with enantiomeric separation ability named as Sil-VMB-NCAD was successfully synthesized by combining ionic liquid embedded octadecyl chains and derivative cellulose on the silicon sphere through thiol-ene click chemistry reaction.The following methods were used to characterize the synthesized stationary phase and its intermediate products,including fourier transform infrared spectroscopy（FT-IR）,elemental analysis（EA）,thermogravimetric analysis（TGA）and nuclear magnetic resonance spectroscopy（NMR）.Hydrophobic analytes（alkylbenzenes,polycyclic aromatic hydrocarbons and positional isomers）,ionic compounds（phenols）and anilines were used to investigate the chromatographic behavior of the Sil-VMB-NCAD column.Meanwhile,to further study the multiple retention mechanism of the prepared column,retention time varied with the composition of the mobile phase and the p H values of mobile phase was studied.In addition,the chiral separation ability of the prepared column was investigated using warfarin,ketoprofen,1-phenylpropanol,and styrene oxide.（2）1,4-butanediol diglycidyl ether（BDDE）and aniline were grafted on the silicon sphere surface for preparing the polar-embedded phenyl dendritic multi-mode stationary phase（Sil-BD-AN）.Results of FT-IR,scanning electron microscope（SEM）,EA and TGA confirmed the successful construction of the multi-mode stationary phase.The multiple separation abilities of Sil-BD-AN column were evaluated using different substances including polycyclic aromatic hydrocarbons（PAHs）,anilines,alkylbenzenes,nucleic bases,nucleoside and positional isomers.Then,the retention abilities of the prepared column were examined by researching impacts of chromatographic conditions（injected volume,column temperature and acetonitrile content）on retention of analytes.In addition,the content of p-phenylenediamine（PPD）in the real sample of hair dye was also determined using the Sil-BD-AN stationary phaseResults:（1）The chemical shifts in the NMR spectra of C18 ionic liquids were consistented with the data of the characteristic groups（methylene and imidazole rings）from the literature.Derived cellulose exhibited different absorption peaks compared to the IR absorption spectrum of cellulose.The stretching vibration of N-H appeared at 3293 cm^-1,and peaks at 1637 cm^-1 and1562 cm^-1 were the stretching vibration of C=O and C=C,respectively.The above results confirmed that acryloyl chloride and 3,5-dimethylphenyl isocyanate have been successfully bounded to the cellulose.In addition,chemical shifts of the target groups（methyl,methylene,carbonyl,and phenyl rings）were presented in the FT-IR spectra of the final synthesized stationary phase material,Sil-VMB-NCAD.The data of TGA and EA proved that the corresponding functional groups were successfully modified to the surface of silica in each synthesis step of stationary phase material.Above results confirmed that Sil-VMB-NCAD has been successfully synthesized.PAHs,alkylbenzenes and positional isomers were achieved baseline separation in the prepared mixed-mode stationary phase under RPLC mode.Three phenols were separated on the prepared Sil-VMB-NCAD column in IEC mode.The separation of anilines was realized on the prepared column under multiple retention mode.In addition,several chiral enantiomers（warfarin,ketoprofen,1-phenylpropanol,and styrene oxide）were successfully isolated in the RPLC mode.（2）These characteristic peaks of target groups were appeared in the FT-IR spectra of the prepared material.Peaks at 2954 cm^-1 and 2873 cm^-1were ascribed to the stretching vibration of–CH₃ and-CH₂.These two peaks appeared in the spectrogram of Sil-BD,indicating the successful grafting of BDDE on the Sil-BD materials.In addition,the peak intensity of Sil-BD-AN at 2954 cm^-1 and 2873cm^-1 were enhanced compared with Sil-BD,and peaks at 1654 and 1466 cm^-1were ascribed to characteristics of phenyl on Sil-BD-AN.The data of TGA and EA proved that the corresponding functional groups were successfully modified to the surface of silicon sphere in each synthesis step of stationary phase material.Above results confirmed that stationary phases has been successfully synthesized.Different analytes,such as PAHs,alkyl benzenes,positional isomers,nucleic bases and nucleosides,were separated on the Sil-BD-AN column in the corresponding retention mode.In addition,three isoforms of phenylenediamine were successfully isolated on the prepared column,and the p-phenylenediamine（PPD）cotent in the commercial hair dye was was calculated as 26.8μg mg^-1based on the fitted linear equation（y=29.269x+29.739,R²=0.9993）.Conclusion:（1）In this study,a polar-embedded multi-mode chromatographic column with enantioseparation capability was successfully prepared.The chromatographic behaviors of the three types of hydrophobic substances（positional isomers,PAHs and alkylbenzenes）proved that the Sil-VMB-NCAD column can realize the separation of non-polar substances under RPLC mode.Then,the retention properties of the anionic compounds on the multi-mode stationary phase testified that the prepared column can possess IEC mode.In addition,the separation behavior of anilines proved the multi-mode retention mechanisms of the synthesized Sil-VMB-NCAD stationary phase.Moreover,the prepared column also successfully separated four chiral substances and showed good chiral separation ability in multiple mode.In conclusion,the novel MHPLC stationary phase material can execute chiral and achiral separation and show the potential to separate complex compounds.（2）In this work,a polar-embedded phenyl dendritic multi-mode stationary phase was synthesized.Different analytes including PAHs,alkyl benzenes,nucleic bases,nucleoside,and positional isomers were separated on the Sil-BD-AN column,which demonstrated the RPLC and HILIC retention modes of the preparation column.The good RSD values of retention time and the peak area affirmed the excellent stability of the prepared column.Furthermore,the prepared column realized the accurate determination of PPD content in the commercial hair dye.To sum up,the prepared multi-mode column can be used as a tool for separating complicated specimens and has broad application potential in the analytical field.