Fabrication Of Double-Functionalized Polymer Monolithic Column Based On TAIC-MMA And Its Chromatographic Characteristics

Since the birth of high performance liquid chromatography?HPLC?,it has been widely used in various fields due to its high efficiency,rapidity and automation.However,with the continuous improvement of separation and analysis work,the stationary phase as the core of HPLC has become a research hotspot.In recent years,organic polymer monolithic columns have been applied to the separation and analysis of small molecular substances and proteins successfully.However,due to the low efficiency of the monolithic column in the separation of small molecular substances,the selectivity in the separation of proteins is poor,thus the further applications of the HPLC wan limited.In this paper,we have synthesized the framework material of poly?TAIC-MMA-EDMA?monolithic column and use these two monomers as comonomers to improve the column efficiency and protein selectivity.The introduction of vinyl ferrocene,graphene oxide and?-cyclodextrin functionalized modification of the framework material,realizing the small molecule of benzene series,traditional Chinese medicine Notoginseng and Schisandra,complex biological samples and chiral drug ibuprofen separation and purification.In addition,the effect of crosslinker trimethylolpropane triacrylate?TMPTA?on the efficiency of monolithic column was also investigated.1.Triallyl isocyanurate?TAIC?and methyl methacrylate?MMA?were used as double monomers,ethylene glycol dimethacrylate?EDMA?as a cross-linking agent,1,4-butanediol,1-dodecanol,and N,N-dimethylformamide as tri-porogens,and azobisisobutyronitrile?AIBN?as initiator.The optimized monolithic columns were investigated by scanning electron microscopy,a nitrogen absorption-desorption apparatus,and mercury intrusion porosimetry.The effect of mobile phase composition and temperature on chromatographic behavior was studied in detail as well.The results showed that homemade monolithic columns not only have relative uniform porosity structure,low back pressure,and high thermal stability,but also show high column efficiency,the theoretical plates number of the new polymer monolithic column exceeded 31,000 plates m^-1.Good resolution was achieved for HPLC separation of small molecules,including polycyclic aromatic hydrocarbons,isomers of phenols,and the Chinese herbal medicine notoginseng.Methodological validation of six drugs in notoginseng show that correlation coefficients of the linear regressions were in the range of 0.9989⁰.9997;the value of relative standard deviation for precision were in the range of 1.37%⁴.21%for intra-day and 0.34%⁵.31%for inter-day,respectively;the values of accuracy expressed by recovery were in the range of 100.57%¹02.51%,95.18%¹02.68%,101.61%¹02.82%,100.94%¹02.40%,100.99%¹03.15%and 99.49%¹03.91%for the six drugs in order,respectively.Taking aromatic hydrocarbons as samples,the relative standard deviation values of the run-to-run?n=5?and column-to-column?n=5?based on peak area were<2.01%and<2.39%,and two RSDs based on retention time were<2.56%and<2.23%,respectively.The result showed that the polymer monolithic column has good separation ability and high permeability for small molecular substances.2.On the basis of the above two monomers,a novel ferrocene-based monolith with a network structure was fabricated via in situ free radical polymerization using vinyl ferrocene as the co-monomer within a stainless-steel column?50×4.6 mm i.d.?for the separation of proteins from complex bio-samples,taking merit of the specific absorption of ferrocene to protein,including human plasma,egg white,and standard proteins.The morphology and pore size distribution indicate that the optimized monolith has a relatively uniform structure with the network.The results showed that 26“fractions”were separated from human plasma,and the column efficiency of the aromatic small molecule?naphthalene?was up to 30,560 plates m^-1.The homemade monolith showed excellent selectivity for intact proteins,mainly depending onthe hydrophobic chromatography mechanism of ferrocene.In addition,the electrostatic interaction and hydrogen-bond interaction were theadditional interactions in the chromatographic separation owing to the sandwich structure of ferrocene present in the monolithic column.Methodological validation of two compounds show that correlation coefficients of the linear regressions were in the range of 0.999⁰.9998;the value of relative standard deviation for precision were in the range of 0.49%³.14%for intra-day and1.43%³.94%for inter-day,respectively;the values of accuracy expressed by recovery were in the range of 99.80%¹02.52%and 101.48%¹05.82%for the six drugs in order,respectively.Taking aromatic hydrocarbons as samples,the relative standard deviation values of the run-to-run?n=5?and column-to-column?n=5?based on peak area were1.25%².41%?1.17%².83%and two RSDs based on retention time were 1.09%².72%?1.43%².81%,respectively.Methodological verification shows that the small molecule analysis method established by the monolithic column has good linearity,precision,accuracy,repeatability and reproducibility.3.In order to diversify the functional groups of the monolithic column,graphene oxide?GO?was introduced into the monolithic column,and a poly?GO-TAIC-MMA?monolithic column was prepared by the principle of redox polymerization.The internal morphology and pore size distribution of the polymer were observed by scanning electron microscopy,nitrogen adsorption-desorption method and mercury intrusion method.After optimization,a monolithic column with good mechanical properties and separation performance was obtained,and 8 kinds of aromatic small molecular compounds were effectively separated in 5 minutes.At the same time,5 kinds of main compounds were separated from the traditional Chinese medicine Schisandra.ingredient.The results show that the introduction of graphene material enhances the interaction between the compound and the monolithic column,increases the binding site,and improves the uniformity of the internal pore structure of the monolithic column to a certain extent,and improves its small molecular substance.Methodological verification shows that the small molecule analysis method established by the monolithic column has good linearity,precision,accuracy,repeatability and reproducibility.4.On the basis of the Chapter 2,the allyl?-cyclodextrin??-CD?was introduced as a comonomer,and the??-CD-TAIC-MMA?monolithic column was synthesized by the principle of free radical polymerization.The internal morphology and pore size distribution of the polymer were observed by scanning electron microscopy,nitrogen adsorption-desorption method and mercury intrusion method.The optimized monolithic column achieves chiral resolution of the racemic body ibuprofen,and five main components were separated from the Chinese medicine rhubarb.Methodological verification shows that the small molecule analysis method established by the monolithic column has good linearity,precision,accuracy,repeatability and reproducibility.5.On the basis of the Chapter 2,this chapter replaces ethylene glycol dimethacrylate?EDMA?with trimethylolpropane triacrylate?TMPTA?as a cross-linking agent.Under the condition of other materials unchanged,poly?TAIC-MMA-TMPTA?monolithic column is prepared by in-situ radical polymerization.The optimized polymer monolithic column was characterized by scanning electron microscopy and nitrogen adsorption-desorption method,and its chromatographic behavior was determined.The results show that the pore size distribution of the monolithic column is not uniform,and the benzene small molecule derivative has a certain separation ability,but the separation time is long.In summary,based on the TAIC-MMA dual-function monolithic column,five new polymer monolithic columns were prepared.The prepared monolithic column and the established chromatographic analysis method were successfully applied to the separation and analysis of small molecular substances,biological samples,chiral drugs and active ingredients of traditional Chinese medicines.Methodological verification shows that the five new prepared monolithic columns have good linearity,precision,accuracy,repeatability and reproducibility.