Retention Behaviors Of Strongly Polar Compounds From Traditional Chinese Medicine In Hydrophilic Interaction Chromatography

Reversed phase liquid chromatography (RPLC) is currently the most popular method in the field of high performance liquid chromatography (HPLC) as it is effective for varities of organic compounds. However, many polar compounds are difficult to be separated by RPLC. Contrary to RPLC, hydrophilic interaction chromatography (HILIC) use polar stationary phase and aqueous/organic mobile phase and can provide an approach to effectively separate polar compounds, thus it has been used in the separation and analysis of carbohydrates, amino acids, peptides, proteins, nucleotides, pharmaceutical drugs, etc. In this thesis, the retention behaviors of six polar compounds from traditional Chinese medicine were investigated systematically with HILIC on three of the most widely used stationary phase by changing organic solvent types and concentration, additives (salt and organic acid) in mobile phase and column temperature. The obtained results are as follows:1. The influences of organic solvents type and concentration on retention behaviors of solutes were investigated by changing concentration of acetonitrile, methanol and tetrahydrofuran in mobile phase. Generally, the retention curves of six solutes exhibited "U" shape with the change in the concentration of water from 0 to 100% (v/v) in mobile phase, indicating a mixed-mode of hydrophilic interaction and reversed phase chromatography. The retention curves of "U" shape were related with properties of organic solvents and stationary phase and molecular structures of solutes. On the same stationary phase, the retentions of solutes were strongest when acetonitrile was used in mobile phase.2. The influences of different concentration of HCOONH4, CH3COONH4, HCOONa and CH3COONa on retention behaviors of solutes were studied. The retention times of all solutes increased with increase of salt concentration on diol silica column, while the retentions of acidic solutes were found to decrease with increase of salt concentration on aminopropyl and 8-glucolactone bonding silica column. The reason of this phenomenon was elucidated. Also, it was found that influences of salt type on retentions of solutes were not consistent with Hofmeister effects.3. The influences of CH3COOH, HCOOH and TFA on retention behaviors of solutes was studied. The results indicated that the retentions of neutral solutes had less variation, but retentions of acidic solutes had more variation upon adding organic acid in mobile phase. In analysis of traditional Chinese medicine, the amount of HCOOH and CH3COOH added should be 0-0.5%, and TFA should be 0-0.1% in the mobile phase.4. The influences of column temperature on retention of solutes were investigated in the range 10-50℃. As temperature raised, retentions of all solutes decreased gradually on diol silica column, while had a different variation trend in aminopropyl andδ-glucolactone bonding silica column. By Van't Hoff method, the driving force for the transfer of solutes from mobile phase to stationary phase was deduced.