This document provides an overview of high performance liquid chromatography (HPLC). It discusses the basic principles of chromatography and how HPLC works to separate compounds. HPLC uses a stationary phase and mobile phase to separate samples based on properties like polarity. Different separation modes are used like normal phase, reversed phase, ion exchange, size exclusion, and affinity chromatography. Instrumentation includes the column, detector, pump, injection port, and auto-injector. Various detectors can be used like UV/Vis detectors and photo diode array detectors. HPLC provides high resolution, sensitivity, repeatability and is useful for analyzing small samples and purifying compounds.

1. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
DR. AKHTAR ALI
FIRST YEAR
RESIDENT(PG)
DEPARTMENT OF
PHARMACOLOGY
DR. S.N. MEDICAL
COLLEGE
JODHPUR ( RAJ.)

2. Basic principal of chromatography

3. Concept of chromatography
Chromatography is an analytical method in which compound are physically
separated prior to measurement.
The main purpose of chromatography is to separate and quantify the target
sample in the matrix.
chromatography
Liquid
chromatography
Gas
chromatography
Supercritical-fluid
chromatography

4. History of chromatography

5. Why use HPLC?
High resolution
High sensitivity
Good repeatability
Small sample size
Easy to fractionate the sample and purify
Non destructive

6. Scope of HPLC

7. Flow Diagram of HPLC

8. Chromatogram

9. Separation mechanism
Compound are separated because the molecules move at
different rates in the column.
Due to different interaction between stationary phase and
different sample , the molecules move at different rate, therefore
separation can be done.

10. Separation modes
Normal phase chromatography
Reversed phase chromatography
Ion chromatography
Size exclusion chromatography

11. Normal Phase chromatography
First developer used
- CaCO3 as separation column
- Petroleum ether as solvent
Stationary phase: Polar property
Mobil phase: Non-polar property
This combination is defined as Normal phase
chromatography

12. Normal Phase chromatography
Mobil phase: Non-polar
Organic solvent:
Hexane,
Benzene,
Methylene chloride,
Chloroform,
Diethyl ether etc.
Stationary phase:

13. Reversed Phase chromatography
Stationary phase: Non-polar property
Mobil phase: Polar property
This combination is defined as reversed
phase chromatography

14. Staionary phase
C18 (ODS) type
C8 (octyl) type
C4 (butyl) type
Cyno type
TMS type
Mobile phase
Water/buffer + Organic solvent
Organic solvent:
Methanol
Acetonitrile etc.
Buffer:
Phosphate buffer
Acetate buffer etc.
Reversed phase chromatography

15. What is the interaction

16. Hydrophobicity

17. Retention Time and Hydrophobicity

18. Increase of solvent polarity

19. Effect of stationary phase

20. Peaks:
1. Methyl benzoate
2. Ethyl benzoate
3. n- Propyl benzoate
4. n- Butyl benzoate
Effect of Stationary phase

21. Reversed phase Ion-Pair Chromatogr

22. Ion-Pair Reagent
Anion compounds
-Tetra-n-butylammonium hydroxide (TBA)
Cation compounds
-Butanesulfonic acid sodium salt (C4)
-Pentaesulfonic acid sodium salt (C5)
-Hexanesulfonic acid sodium salt (C6)
-Heptanesulfonic acid sodium salt (C7)
-Octanesulfonic acid sodium salt (C8)
-Decanesulfonic acid sodium salt (C10)

23. Reversed Phase Ion Pair

24. Reversed Phase Ion Pair
Advantage:
Share several features with reversed phase HPLC- column and
mobile phase.
By selecting ion pair reagent, concentration or pH, selectivity can
be improved.
Ion and natural compound can usually be done in the same run.
Disadvantage:
Equilibration time is long.
Dedicated columns are often recommended.
Beware of ion pair reagent precipitation in organic solvent eg.
Methanol, acetonitrile, etc.

25. Polarity of common organic
functional groups and
solvents
Organic solvents:
•Hexane
•Carbon tetrachloride
•Ether
•Benzene
•Methylene chloride
•THF
•Iso-propanol
•Chloroform
•Ethyl acetate
•Acetonitrile
•Methanol
•Water
Functional groups:
•Alkane
•Alkyl halide
•Alkene
•Dienes
•Ethers
•Ketones
•Aldehydes
•Amines
•Alcohol
•Phenols
•Carboxylic acid
•Sulfonic acid
Non-polar
Polar

26. Ion Exchange chromatography
Anion exchange Cation exchange

27. Example of anionic and cationic
ion chromatography
Cationic
Anionic

28. Size Exclusion Chromatography
Purpose of SEC
GPC (Gel Permeation chromatography)
- Molecular weight measurement of polymer
GFC (Gel filtration chromatography)
- Separation of protein

29. Affinity chromatography

30. Choice of liquid chromatography

31. Instrumentation of HPLC
Column
Detector
Mobil phase
Pump
Injection
Port
Auto-injector

32. Isocratic System

33. High-pressure gradient system

34. Low-pressure gradient system

35. Selection of Detector

36. PDA Detector

47. Thank you