구성 HPLC Method Development Part 1. General Chromatographic Theory Part 2. Overview of HPLC Media Part 3. The Role of the Mobile Phase in Selectivity Part 1 첫 번째 시간으로 General Chromatographic Theory 관련 내용을 소개 드리고자 합니다. 세부내용 A. The Liquid Chromatographic Process 1. The beginning of Liquid Chromatography 2. Basic of Chromatographic Separation 3. The Liquid Chromatographic Process 4. Mechanisms of Interaction in RP Chromatography 4.1 Hydrophobic Interactions 4.2 Polar Interactions 4.3 Ion-exchange Interactions B. Chromatographic Measurements 1. Chromatographic Measurements 2. The Void Volume 3. The Capacity Factor 4. Peak Asymmetry 5. Peak tailing (due to Secondary Interactions) 6. Peak tailing (due to Sample Overloading) 7. Peak Fronting (due to Sample Overloading) 8. Peak Fronting (due to Sample Solvent Effects) 9. Selectivity 10. Column Efficiency 11. The van Deemter Equation 12. Column Efficiency is a Function of Particle Size 13. Effect of Particle Size on Efficiency 14. Effect of Column Length on Efficiency 15. Balancing Column Length and Particle Size 16. Effect of Flow Rate on Efficiency 17. Quick Review: C. Resolution: The Goal of Chromatography 1. Resolution: The Goal of Liquid Chromatography 2. Resolution: The Relative Effectiveness of k’, a and N 3. The Impact of Efficiency on Resolution 4. Optimizing Efficiency for Maximum Resolution 5. The Impact of Capacity Factor on Resolution 6. Optimizing Capacity Factor for Maximum Resolution 7. The Impact of Selectivity on Resolution D. Method Development Exercise 1: Optimizing to Reduce Analysis Time and Increase Productivity 1. Mupirocin Impurity Profile 2. Mupirocin: Original Method 3. Step 1. Adjust k' for better Resolution 4. Step 2. Optimizing Efficiency and Length 5. Step 3. Optimize the Flow Rate 6. Mupirocin: Intermediate Method 7. Step 4. Switch to Core-Shell Media 8. Mupirocin: Final Optimized Method Part 2 두번째 시간으로 HPLC Media & Selectivity 관련 내용을 소개 드리고자 합니다. 세부내용 A. Chromatography Media 1. Fully porous silica 2. Core-shell particles 3. Monolithic rods 4. Organosilica hybrid 1. Fully Porous Silica Particles 1) Advantages: - Default HPLC particle from prep to UHPLC - High mechanical strength - Excellent efficiency - Highly amenable to modulation of material characteristics 2) Disadvantages: - Dissolution of silica at pH > ~7.5 2. Core-shell particles 1) Core-Shell Particle Size Distribution 2) Chromatographic Efficiency 3) Fully Porous Efficiency 4) Chromatographic Efficiency 5) The Core-Shell Advantage 6) Core-Shell Performance 7) Advantages: - Improved efficiency compared to fully porous - Improved resolution and sensitivity - Shorter analysis times usually 8) Disadvantages: 1) Silica dissolution at alkaline pH 2) May have reduced loading capacity 3) Low retention can be a drawback 3. Monolithic rods 1) Monolithic Silica Rod 2) Advantages: - Extremely low pressure and resistance - Analysis of dirty, viscous samples - No bed shifting/voiding due to compression 3) Disadvantages: - Silica dissolution at alkaline pH - Limited stationary phase options - Efficiency limited 4. Organosilica hybrid 1) Using Alkaline Mobile Phase 2) Kinetex EVO Surface Chemistry 3) Improved Peak Shape for Bases 4) Organosilica Hybrid Material 5. Advantages: 1) Expanded pH stability range from ~pH 1-12 2) Allows for improved retention of polar bases 3) Often provides improved peak shapes 6. Disadvantages: 1) Limited sources of media 2) Reduced stationary phase options 3) Increased cost 7. Review HPLC Media 1) Fully porous Silica Particle: - General workhorse - Many particle sizes and stationary phases - Low sensitivity to system dead volume 2) Core-Shell Particle: - Improved efficiency  leading to increased Resolution & Sensitivity - Decreased analysis times due lower surface 3) Organosilica Hybrid Particle: - Expanded pH stability range - Same performance characteristics as fully porous - Optimal for high pH applications 4) Monolithic Rod: - Low backpressure; reduced clogging - Ideal for analysis of “dirty” samples (e.g. plasma) - Efficiency ~3um fully porous spherical silica 8. Physicochemical Parameters 1) Particle Shape 2) Particle Size 3) Pore Size 4) Surface Area 5) Carbon Load B. Reversed-Phase Bonded Phases 1. Bonded Phases 1) Stationary Phase Bonding 2) Not All C18 Phases Are Equal 2. Alkyl-Bonded Phases 1) Alkyl Phase Selectivity 3. Phenyl-Bonded Phases 1) Phenyl Selectivity 2) Tip: Methanol to Enhance Phenyl Selectivity 4. Polar Embedded/End capped Phases 1) Polar C18 Phases 2) Enhanced Polar Selectivity 5. Stationary Phases Summary 1) HPLC Method Development 2) Column Selection Model C. Method Development Exercise 2: Media Selection and Phase Screening 1. Testosterone Epimer Analysis 1) Particle Selection 2) Alkyl Phase Screening 3) Alternative Column Screening 4) Evaluation of Phenyl Phase 5) Evaluation of Embedded and Hybrid 6) Final Method Part 3 세번째 시간으로 The Role of the Mobile Phase in Selectivity 관련 내용을 소개 드리고자 합니다. 세부내용 A. Reverse-Phase Solvents 1. Reverse-Phase Solvents 2. Solvent considerations 3. Solvent Strength: Acetonitrile versus Methanol 4. Solvent Selectivity 5. Solvent Selection Screening (For Isocratic Methods) 6. Solvent Selectivity for Phenyl Phases B. Buffers and the Role of Mobile Phase pH 1. Buffer Selection for RP-HPLC 2. Effect of pH on Stationary Phase 3. Effect of pH on Analyte Behavior 4. Effect og pH on Analyte Retention Behavior 5. Optimizing Mobile Phase Selectivity C. Method Development Exercise 3: Optimizing Mobile Phase and Stationary phase 1. Optimizing Mobile Phase and Stationary Phase 2. Nicotine and Metabolites: Initial Low pH Screen 3. Nicotine and Metabolites: High pH Screen to Increase Retention 4. Nicotine and Metabolites: Final Method using Hybrid Particle D. Gradient Analysis 1. Gradient analysis 2. Gradient Slope 3. Effect of Gradient Slope on Resolution 4. Effect of Starting % Organic on Resolution F. The Use of Temperature in Method Development G. Method Development Exercise 3: Gradient Optimization and Phase Screening 1. Clinical Testing for Barbiturates 2. Optimizing Mobile Phase and Stationary Phase 3. Initial Scouting Gradient on Core-Shell C18 4. Gradient Slope Optimization 5. Other Potential Method Development