Preparation And Application Of Novel Organic Monolithic Capillary Columns For Hydrophilic Interaction Chromatography

Hydrophilic interaction liquid chromatography(HILIC)is a complementary separation mode for normal phase chromatography(NPLC)and reversed phase chromatography(RPLC).It holds the key to the effective separation of polar and ionic compounds.Researchers have developed a series of hydrophilic columns for the separation and analysis of actual samples,but there are still deficiencies in some areas.On the one hand,there are less highly hydrophilic chromatographic materials;on the other hand,the preparation methods of monolithic column normally complex.In this paper,the hydrophilic polymer monolithic column was selected as the subject.Two strong hydrophilic organic polymer monolithic columns were fabricated by selecting more hydrophilic organic monomers and developing a simple preparation method,and thses columns were applied in hydrophilic interaction chromatography for polar molecules.The main contents are as follows:An easy single-step thermal treatment "one-pot" approach for the preparation of poly(pentaerythritol tetraglycidyl ether-co-poly ethylene imine)organic monolithic capillary columns was developed successfully.The column was prepared by the epoxy-amine ring-opening polymerization of pentaerythritol tetraglycidyl ether(PTE)with poly(ethylene imine)(PEI)using acetonitrile(ACN)and polyethylene glycol 600(PEG 600)as the porogenic system at 60 ? for 12 h.The obtained monolith was homogeneous and permeable.It achieved the high-efficiency separation of polar molecules including amides,nucleosides,bases,phenols,and benzoic acids in capillary liquid chromatography(cLC).The highest column efficiency reached ca.101000 N/m(for guanine)on monolith poly(PTE-co-PEI)at 0.64 mm/s,and satisfactory chromatographic performance with column effciencies ranged from 45500 to 97000 N/m was achieved for the four amides.A typical hydrophilic interaction liquid chromatography retention mechanism was observed with high organic solvent contents(>60%ACN).Also,the polymer-based monolithic column was successfully applied to separate the tumor markers.Furthermore,the poly(PTE-co-PEI)monolith could be easily modified with 1,2-epoxydodecane,which reacted with the amino groups presented on the surface of the poly(PTE-co-PEI)monolith.Hydrophobic interactions were observed during the separation of alkylbenzenes and anilines on the post-modified poly(PTE-co-PEI)monolith.Together,these results confirm the feasibility of the epoxy-amine ring-opening polymerization reaction during the fabrication of a monolithic column with high efficiency for cLC applications.A new two-step ring-opening approach was presented for synthesizing a highly hydrophilic sulfoalkybetaine-type monolithic column for using in HILIC.The scaffold column was fabricated by first the amine-epoxy polymerization of PTE with PEI,which was subsequently reacted with 1,4-butane sultone.The internal pore size and the skeleton of the monolithic column were adjusted via optimizing parameters such as monomers,porogen and polymerization temperature.The resulting monolithic column obtained homogeneous structure,good permeability,mechanical stability and good reproducibility.The chromatographic separation performance of the monolithic column was evaluated and applied using five types of polar small molecules,BSA tryptic digest and five tumor markers.It was found that multiple interactions such as hydrogen bonding,electrostatic interaction and so on dominate the retention of analytes.All of these analytes exhibited good separation performance,which was more prominent than that of poly(PTE-co-PEI)monolithic column.Three phenolic isomers and nine benzoic acid compounds,in particurlarly,obtained higher selectivity and separation efficiency.In addition,peak compression occurs in thymidine,guanine,catechol,and gentisic acid,resulting in utral-high column efficiency(?100000 N/m).Further studies on the causes of compression will be carried out in the next chapter.The mechanism of peak compression phenomenon appeared on the sulfoalkybetaine-type monolithic column was studied in depth.Two analytes with utral-high column efficiency(thymidine and gentisic acid)were selected as the main test subjects.The effects of solvents,analyte concentrations,mobile phase polarity and mobile phase additives on column efficiency and peak shape were explored.The results showed that the aprotic sample solvent(ACN)can promote the peak compression;the influence of the polarity change of the mobile phase on the peak compression is much greater than that of the sample concentration and the injection volume;different mobile phase additives can cause peak compression of different analysis.For instance,catechol and gentisic acid are only sensitive to ammonium acetate and trifluoroacetic acid(TFA),respectively.Peak compression may occur,when the above three conditions are match simultaneously.