HPLC

1. HPLC
Presented by
ANU BALA

2. OUTLINE
• INTRODUCTION
• TYPES OF CHROMATOGRAPHY
• RP LIQUID CHROMATOGRAPHY
• INSTRUMENTATION
• CHROMATOGRAM
• METHOD DEVELPOMENT LAYOUT
• ADVANTAGES OF HPLC

3. INTRODUCTION
• High performance liquid
chromatography
• A form of liquid chromatography used to
separate compounds that are dissolved in
solution.
• HPLC instruments consist of a reservoir
of mobile phase, a pump, an injector, a
column, and a detector
• Compounds are separated by injecting a
sample mixture onto the column
• Separation is based on Partition of
compounds towards stationary and
mobile phase

4. TYPES OF CHROMATOGRAPHY
A. Based on modes of chromatography
1. Normal phase mode
2.Reverse phase mode
B. Based on principle of separation
1. Adsorption chromatography
2. Ion exchange chromatography
3. Partition chromatography
4. Size exclusion
C. Based on elution technique
1. Isocratic separation
2. Gradient separation

5. D. Based on the scale of operation
1. Analytical HPLC
2. Preparative HPLC
E. Based on the type of analysis
1. Qualitative analysis
2. Quantitative analysis

7. INSTRUMENTATION

8. HPLC Pump

9. Column

10. Bonded Phases
• C-2 Ethyl Silyl -Si-CH2-CH3
• C-8 Octyl Silyl -Si-(CH2)7-CH3
• C-18 Octadecyl Silyl -Si-(CH2)17-CH3
• CN Cyanopropyl Silyl -Si-(CH2)3-CN

11. Silica
Stationery
Phase
support
The longer the alkyl chains, the longer the
retention time in a reversed phase column.

12. Injector

13. Detector
Detector is a device used in liquid and gas
chromatography to visualize components of the
mixture being eluted off the chromatography column
There are two general types of detectors
1 Destructive detector
2 Non destructive detector

14. UV Detector

15. Photodiode Array Detectors

16. CHROMATOGRAM

17. BAD CHROMATOGRAM

18. METHOD DEVELOPMENT
PHYSICOCHEMICAL PROPERTIES
(i.e. Structure, Solubility, Nature, components)
SAMPLE PREPARATION
(i.e. Concentration, Dilutions)
SELECTION OF HPLC PARAMETERS
1. Mobile phase : Selection of solvents and combinations
Selection of Buffers
Isocratic / Gradient
Flow rate
2. Column: NP/RP
Temperature
3. Detection: Selection of wavelength
4. Chromatography: Parameters of Chromatogram

19. METHOD DEVELOPMENT
PRE METHOD VALIDATION
1. Selection of Upper and Lower Limit of Quantitation
2. Impurity profiling
3. Accuracy and Precision
4. Linearity
5. Selectivity (LOD, LOQ)
6. Stability
Validation

20. Advantages of HPLC
High sensitivity
High performance
Rapid process and hence time saving
Can be used for qualitative as well as quantitative
estimation
Can be used for both analytical and preparative
purpose