1. Liquid Chromatography - Method Development and Validation

2. Types of Chromatography
Paper Chromatography (Ascending, Descending)
Column Chromatography
Thin Layer Chromatography
Gas Chromatography
Liquid Chromatography

3. Basic Types of Liquid Chromatography
Ion Exchange Chromatography
Size Exclusion Chromatography
Supercritical Fluid Chromatography/ SFE
Liquid-Solid Chromatography (Adsorption)
Liquid-Liquid Chromatography
(Partitioning)

4. Size Exclusion Gel Particle

5. Modes of Liquid-Liquid Chromatography
Reverse Phase
Normal Phase

6. Liquid-Liquid Chromatography (HPLC) (Partitioning) (most common LC)
Polarity
Like dissolves like
Distribution Coefficient /Capacity factor/
Selectivity factor
pH & pKa
Henderson Hasselbalch Equation

7. Schematic Diagram of HPLC

8. Components of HPLC Pump (Isocratic/HP/LP Gradient) Pump heads
Autosampler
Injector
Column
Detector
Data Recorder

9. Schematic Diagram of Injector

10. HPLC/UPLC Column

11. Separation Process on Column

12. Silanol deactivation
Base Deactivation

13. Silanol deactivation Contd.
End Capping

14. Types of Detectors
UV/PDA nm, most common 254 nm Refractive Index
ELSD (Evaporative light scattering)
Fluorescence
Conductivity
ECD

15. HPLC in 1990’s

16. HPLC – Recent Model Waters Alliance

17. Ultra Performance Liquid Chromatography (UPLC)- Waters Aquity

18. Comparison HPLC UPLC Dead volume 0.65 mL 0.25 mL Pressure 6000 psi
Run time
Abt 10 fold
Abt 1 fold
Flow cell volume
8 µL
0.5 µL

19. System Suitability Theorotical Plates (N) Tailing Factor (T)
Resolution (R)
Relative Retention Time (RRT)
Kapacity Factor/Selectivity Factor (K)
Relative Standard Deviation (RSD)

20. Adjustments to chromatographic systems
pH of mobile phase
Conc. of Buffer
Organic Ratio of mobile phase
Column Length
Particle Size
Flow rate
Injection Volume
Column Temperature

21. Method Development Literature survey Solubility
pKa - Henderson Hasselbalch Equation
Selection of wavelength
Selection of column-based on compound
Chemistry (Size exclusion, ion exchange,
MW, chromophore)
Selection of buffer
Selection of org. modifier based on UV cut offs

22. Method Development Contd.
Selection of pH
Optimization of chromatographic
Conditions(flow,inj. vol, pressure, RT, R,
N, T, solution stab, filter compatibility)
Forced Degradation – Stability Indicating
Method
System suitability finalization

23. Method Validation (ICH & USP)
Specificity
Forced Degradation (Stability Indicating)
Filter paper study
Solution stability
Linearity
Range
Accuracy (Bias)
Repeatability (method precision)
Intermediate Precision
Reproducibility (Interlaboratory Precision)
Limit of Detection
Limit of Quantification
Robustness
Statistical analysis

24. Thank You
Questions?

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