Preparation Of Mixed Mode Stationary Phase For Liquid Chromatography

As a multi-component separation and analysis technology,high performance liquid chromatography(HPLC)has become an indispensable technology in analytical science.Chromatographic stationary phase is the core element to determine the separation effect.Traditional chromatographic stationary phases mainly adapt to a single separation mode,such as reversed phase liquid chromatography(RPLC),normal phase liquid chromatography(NPLC),ion exchange liquid chromatography(IEC),hydrophilic liquid chromatography(HILIC).When encountering compound with complex structure and properties,traditional chromatographic stationary phase is often unable to complete the separation and analysis work accurately and quickly,so the development of new mixed-mode chromatographic stationary phase has important practical significance for the wide application and development of HPLC.In this paper,a new method for selective derivatization of binary amino silane was proposed.Three kinds of bonded silica gel stationary phases containing complex ligands were prepared.The chromatographic properties of the prepared stationary phases were evaluated by a series of characterization methods.The specific content includes the following aspects:(1)C22 amide-silane was synthesized by a new method of selective derivation of binary amino groups,and the synthesized product was covalently bonded to silica gel surface to form liquid chromatography stationary phase C22-EDA.The stationary phase was characterized by solid-state nuclear magnetic resonance(NMR),scanning electron microscopy(SEM)and elemental analysis,and it was proved that the synthesized functional modifier was successfully grafted onto the surface of silica gel.The separation of hydrophobic and hydrophilic compounds showed that the new amide modified stationary phase has amphiphilic properties.Compared with conventional C18 stationary phase,polar embedded C22-EDA stationary phase can effectively identify four estrogens,demonstrating good separation potential of C22-EDA for polar estrogen compounds.(2)C22-EDA(U)was prepared by using N,N-dimethylcarbamyl chloride as secondary acylation agent to treat C22 amide silane.The retention mechanism between the fixed phase and analyte was investigated by changing the ratio of acetonitrile in the mobile phase.The results showed that the fixed phase modified by monoamide and the fixed phase modified by amide/urea-based double polar group exhibited RPLC and HILIC properties in the mobile phase with low and high content of acetonitrile,respectively.The isolation of tricyclic antidepressants and sulfonamides showed that the insertion of urea enhanced the carbonyl π-benzene π interaction between the stationary phase and aromatic solute,thereby enhancing the retention of aromatic compounds.Acid and base stability tests showed that the stationary phase can be used in mobile phase environment with pH=2～10 for a long time.(3)On the basis of amide/urea group embedding,1-naphthalyl isocyanate was selected to modify the silane again to obtain the complex ligand modified C22 silane containing three groups of amide/urea/naphthalene group,and bonded to the surface of silica gel to obtain C22-EDA(Nap).The results showed that the modified stationary phase has good separation ability of isomeric aromatic compounds and basic compounds.The electron donor provided the stationary phase special charge transfer ability,so that the stationary phase has good nitro selectivity.In addition,polar nucleoside compounds were successfully separated from the three modified stationary phases in HILIC mode,proving that the three modified stationary phases have the RPLC/HILIC hybrid mode separation ability.