Study On The Retention Mechanism In Hydrophilic Interaction Chromatography Using Stoichiometric Displacement Theory

Hydrophilic interaction liquid chromatography (HILIC) provides an alternative approach to effectively separate small polar compounds on polar stationary phases by overcoming the shortage of reversed phase chromatography(RPLC) in not capable of retaining highly polar compounds and normal phase chromatography(NPLC) being difficult to provide sufficient solubility for polar compound. HILIC can provide good resolution and selectivity for small polar molecules, peptides and proteins samples. However, arguments still exist concerning the retention mechanisms of HILIC, and there are advantages as well as shortcomings in retention models which have been proposed, including the classic mechanisms of absorption and partitioning. Research of protein in HILIC is still too limited. Stoichiometric displacement theory for retention (SDT-R) is a theoretical model which gives a thorough description of the retention behavior of solutes in RPLC. SDT-R can be regarded as a universal theory for solute separation and a general retention mechanism of biopolymers in HPLC, validity of which has been confirmed in several modes except size exclusion chromatography(SEC). The advantages and characteristics indicate the potential application of SDT in HILIC mode. In this thesis SDT was applied to investigate the retention mechanisms of polar solutes in HILIC, including small molecule compound and protein. Theoretical model and retention equation for solutes under typical HILIC mobile phase as well as the wide concentration range are derived and tested by fitting with retention value of solutes obtained from experiment. Description on the mobile phase-solute-stationary phase relationship was given theoretically. Adsorption and desorption energy changes were also analyzed according to the concept of thermodynamic fraction. All the facts are evidence that SDT is suitable and accurate in the study of retention mechanism under HILIC mode, which is of great significance for the development of HILIC. This thesis was divided into five parts as following:1. Review:This chapter gives a detailed review on the characteristics and application of HILIC, especially the development of research on retention mechanism in HILIC as well as the principle and application of SDT. Significance of the application of SDT in the study of retention mechanism of HILIC was also discussed.2. Study on retention mechanism of small polar compounds in HILIC:Theoretical retention equation of Igk' vs lg[H2O] according to SDT was derived and well-fitted with the experimental lgk' of 15 small polar compounds on 5 HILIC columns with different ligands, based on which the parameters lgI and Z were obtained. Advantages and superior of SDT was observed through the comparison with adsorption and partitioning mechanism, including the explaination of physical meaning of the parameters in the equation as well as the relationship among mobile phase, solutes and stationary phase. The parameter j and lg? was obtained from the slope and intercept of the linearity oflgI vs Z. It is obsearved that j is very close to the theoretical value of water, which is strong proof that water is displacer in HILIC, and acetonitrile is the diluting agent of water. Results of simulation in molecular orientation indicate the dispersion force still can't be ignored in HILIC. Discussion of the retention process in HILIC and explaination of the linearity of lgI vs Z was also obtained based on SDT.3. Study on retention mechanism of protein in HILIC:Similar to the method in chapter 2, theoretical retention equation of lgk' vs lg[H2O] according to SDT also works well with the experimental lgK' of standard protein on 4 HILIC columns with different ligands. The values of lgI and Z obtained were found to be much larger than that of small molecules. Good linearity was also achieved from the plots of lgI vs Z, indicating that retention behavior of protein in HILIC is in accod with SDT, and small j was observed than the theoretical value of water. From the effect of formic acid as additive in mobile phase to protein and three small molecule compounds which can't be eluted in pure acetonitrile/water system, conclusion was drawn that formic acid that was added to help eluting protein was the main reason to the decrese ofj in protein rentention.4. Application of SDT in dual rentention mechanism:Principle of dual HILIC/RPLC mechanism of solutes with different polarity was investigated in detail using the synthesized HILIC column with ?-CD as ligand. "U-shape" plots were observed in the relationship between lgk' of three kinds of solutes with different polarity and the volume fration of water in a wide concentration range, the left and right side of which represent HILIC and RPLC mode, respectively. The effect of partition coefficient lgP to HILIC and RP mode indicated that retention in RPLC increases with the lgP of solutes, while it is not the same way in HILIC. A four parameters equation was derived based on SDT which was used to fit the "U-shaped" curves observed. Results indicated that the experiment values were consistent with the theoretical values. The parts of HILIC and RPLC on the "U-shaped" curves can both fit the derived equation well with all the linear correlation coefficients greater than 0.99, which indicated SDT provide a universal way to explain the the dual HILIC/RP retention mechanism. SDT can act as theoretical guidance for the development of HILIC, including the exploration of new dual-function stationary phases in order to improve separation efficiency.5. Thermodynamic study on retention of solutes in HILIC:Adsorption and desorption energy changes were analyzed according to the concept of thermodynamic fraction. Based on the linearity of lgI vs 1/T and Z vs 1/T, Gibbs free energy changes(?G), enthalpy changes(?H) and entropy changes(AS) can be written as the addition of adsorption and desorption energy which are independent of each other and can be calculated separately. It is also obsearved that ?G, ?H and ?S calculated from the addition of their adsorption and desorption fraction all fit well with the results obtained by van't Hoff plot, and explaination from the point of adsorption and desorption process was clearly given about the thermodynamic information, which indicated that it is reliable to use the method of adsorption and desorption to understand retention of in HILIC.