Chromatography

- GC and HPLC
 Contents
1. Chromatography – A Introduction:

2. Use of Chromatography

3. How Chromatography works

4. Types of Chromatography

5. Principles of GC

6. Instrumentation of GC

7. Application of GC

8. Advantages of GC

9. Principles of HPLC

10. Instrumentation of HPLC

11. Application of HPLC

12. Advantages of HPLC

13. References
 Chromatography – An Introduction
Chromatography is derived from thee Greek word `Chroma’
meaning colour and `Graphy’ means writing.

Chromatography is a technique for separating mixtures into

their components in order to analyze, identity, purity and
quantify the mixture of components.

It gives the ability to separate molecules using partitioning

characteristics of molecule.
Use of Chromatography:

1. Pharmaceutical company - Determine the amount of each

chemical found in new product
2. Hospital - detecting blood or alcohol levels in a patient’s blood
stream.

3. Law Enforcement – to compare a sample found at a crime

scene to samples from suspects
4. Environmental agency- determine the level of pollutants in
the water supply.
5. Manufacturing plan – to purify a chemical needed to make a
product.
TYPES OF CHROMATOGRAPHY:
Liquid chromatography: Separates liquid samples with a liquid
solvent (mobile phase ) and a column composed of solid beads
(stationary phase).

Gas chromatography: separates vaporized samples with a carrier

gas(mobile phase) and a column composed of liquid or of a solid
beads(stationary phase).

Paper chromatography: separates dried liquid samples with a

liquid solvent(mobile phase) and a paper strip(stationary phase)

Thin- layer chromatography: separates dried liquid samples

with a liquid sample (mobile phase) and a glass plate covered with
a thin layer of alumina or silica gel(stationary phase).
GC - Principle :
* Partition of molecules between gas (mobile phase) and liquid
(stationary phase).

Most Common Stationary Phases:

1. Separation of mixture of polar compounds

Carbowax 20M (polyethylene glycol)

2. Separation of mixtures of non-polar compounds

OV101 or SE-30 (polymer of methylsilicone)

3. Methylester of fatty acids

DEGS (diethylene glycol succinate)
Schematic Diagram for GC Instrumentation
Practical Requirements of GC:

 Carrier gas
 Flow regulators & Flow meters
 Injection device
 Columns
 Temperature control device
 Detectors
 Records & Integrators
Types of GC Detectors :

 Flame Ionization Detector :

 Thermal Conductivity Detector :

 Electron Capture Detector :

GC – Applications:

 Qualitative analysis
 Checking the purity of a compound
 Presence of impurities
 Quantitative analysis
GC ADVANTAGES :

 Very good separation

 Time (analysis is short)
 Small sample is needed - ml
 Good detection system
 Quantitatively analyzed
 Cost of instrument is low
 More accuracy
 Highly sensitive
HPLC Principle :
High performance liquid chromatography is
basically a highly improved form of column
chromatography.

Instead of a solvent being allowed to drip through a column under

gravity, it is forced through under high pressures of up to 400
atmospheres. That makes it much faster.
How does HPLC works
Practical Requirements of HPLC:

 A Solvent reservoir & mixing

system
 A high pressure pump
 Sample inlet pump
 A Column
 Detector
 Recording Unit
HPLC Detectors

 UV Detectors

 Refractive index Detectors

 Conductivity Detectors
HPLC Applications

 Qualitative Analysis

 Checking the purity of the compound

 Presence of impurities

 Quantitative analysis