Title Page
ABSTRACT
Contents
1. Introduction 10
1.1. Extraction and Purification of Liquiritin, Glycyrrhizic Acid and Glabridin from Licorice Root 10
1.2. Protein Binding and High-Performance Frontal Analysis 13
2. Theoretical Background 16
2.1. Types of Chromatography 17
2.2. Retention in Reversed Phase Liquid Chromatography 20
2.3. Determination of Retention Mechanisms in Reversed Phase Liquid Chromatography 23
2.3.1. Exothermodynamic Relationships 23
2.3.2. Analysis of Selectivity 24
3. Experimental 28
3.1. Licorice 28
3.1.1. Chemicals 28
3.1.2. HPLC Analysis 28
3.1.3. Samples and Columns Preparation 29
3.1.4. Extraction and Separation 29
3.2. HPFA 31
3.2.1. Chemicals 31
3.2.2. HPLC Analysis 32
3.2.3. Preparation of Solutions 32
4. Results and discussions 32
4.1. Results and Discussions of Licorice 32
4.1.1. Effect of Different Extraction Solvents 33
4.1.2. Effect of Different Extraction Methods 34
4.1.3. Effect of Different Volumes of Methanol 36
4.1.4. Method Validation 37
4.1.5. Preparative Separation on Different Particles Sized Columns 42
4.2. Results and Discussions of HPFA 46
4.2.1. Determining the Injection Volume 46
4.2.2. Determination of Unbound drugs 51
4.2.3. Determination of Binding Parameters 52
5. Conclusion 54
한글요약 55
Summary in Chinese 56
References 57
Appendix 60
Appendix 1. Study of retention in micellar liquid chromatography on a C18 column on the bases of liner salvation energy relationships 60
Appendix 2. Selective solid-phase extraction of glabridin from licorice root using molucelarly imprinted polymer 61
Appendix 3. Effects of solvents, temperatures and pressures on particle size in aerosol solvent extraction system 62
Appendix 4. Study of retention with ionic liquid on a C18 column on the basic of linear solvation energy relationships 63
List of Publications 64
감사의 글 68
Table 1. Extracted amounts of LQ, GA and glabridin with different solvents 34
Table 2. Extracted amount of LQ, GA and glabridin by 3 steps extraction 39
Table 3. RSDs, recovery rates and LODs of the three compounds from licorice 41
Table 4. Peak width with different injection volumes and particle sizes 46
Table 5. Determination of unbound drugs by HPFA 51
Figure 1. Molecular structures of liquiritin (A), glycyrrhizic acid (B) and glabridin (C) 13
Figure 2. Chemical structures of drug compounds, (A) (+)-Catechin hydrate, (B) Genistein 16
Figure 3. Classification of chromatography techniques 18
Figure 4. Extraction procedure of LQ, GA and glabridin from licorice 31
Figure 5. Effect of different dipping times on the extracted amounts of LQ, GA and glabridin 35
Figure 6. Effect of different ultrasonic times on the extracted amounts of LQ, GA and glabridin 36
Figure 7. Effect of volume of methanol on the extracted amounts of LQ, GA and glabridin 37
Figure 8. Chromatogram of the extracts of licorice by different injection volumes (Column: C18, 5 μm, 4.6*150mm, 10 and 75 μL, FR: 0.5ml/min, 252nm) 43
Figure 9. Chromatogram of the extracts of licorice by different injection volume (Column: C18, 40/63 μm, 4.6*250 mm, 10, 100 and 200 μL, FR: 0.5 mL/min, 252 nm) 44
Figure 10. Chromatogram of the extracts of licorice by different injection volumes (Column: C18, 12 μm, 4.6*250 mm, 10, 100 and 200 μL, FR: 0.5 mL/min, 252 nm) 45
Figure 11. The effect of injection volume on elution profile of drug and 40μM HSA mixed solution. (a) (+)-catechin hydrate, (b) genistein (UV wavelength 280 nm (a), 260 nm (b)) 48
Figure 12. Chromatograms of drugs with different concentrations in 40μM HSA by HPFA. (a) (+)-catechin hydrate, (b) genistein (UV wavelength 280 nm (a), 260 nm (b)) 50
Figure 13. The Scatchard plot for drug-HSA binding. (a) (+)-Catechin hydrate, (b) Genistein (b) Genistein 53