1

12/09/2023

2

12/09/2023

1. Introduction
2. Classification of chromatographic methods
3. Principle of chromatography
4. High performance liquid chromatography (HPLC)
5. Gas chromatography (GC)
6. Thin layer chromatography (TLC)

Invention of Chromatography

Mikhail Tswett

Russian Botanist

(1872-1919)

Mikhail Tswett invented chromatography in 1901 during his research on plant pigments.

He used the technique to separate various plant pigments such as chlorophylls, xanthophylls and carotenoids.

Original Chromatography Experiment

Later

Start: A glass

column is filled

with powdered

limestone

(CaCO₃).

End: A series of colored bands is

seen to form,

corresponding to

the different pigments in the original plant extract. These bands were later determined to be chlorophylls, xanthophylls and carotenoids.

An EtOH extract

of leaf pigments

is applied to the

top of the column.

​

EtOH is used to

flush the pigments

down the column.

Chromatography: (Greek = chroma “color” and graphein “writing” ) Tswett named this new technique chromatography based on the fact that it separated the components of a solution by color.

Common Types of Chromatography

Tswett’s technique is based on Liquid Chromatography. There are now several common chromatographic methods. These include:

Paper Chromatography

Thin Layer Chromatography (TLC)

Liquid Chromatography (LC)

High Pressure Liquid Chromatography (HPLC)

Ion Chromatography

Gas Chromatography (GC)

6

12/09/2023

Definition:

Chromatography is defined as a procedure by which solutes are separated by dynamic differential migration process in a system consisting of two or more phases, one of which moves continuously in a given direction and in which the individual substances exhibit different mobilities by reason of differences in adsorption, partition, solubility, vapor pressure, molecular size, or ionic charge density.

7

12/09/2023

Mobile Phase:

The Phase that travels through the column (gas or liquid) – transport sample through the column.

Stationary Phase:

Immiscible solid or liquid phase that fixed in place in the column or on a solid support – retain analytes within the column.

Band or Zone:

• Area across which analyte is distributed on column
• Zones of different analytes gradually separate as bands progress down column

8

12/09/2023

• Sample
• Mobile phase (eluant)
• Stationary Phase
• Detection method
• Chromatogram

Thin Layer chromatography

Column Chromatography

9

12/09/2023

• Method to separate components in a mixture based on different Distribution coefficients between the two phases.
• Chromatography categorized on the basis of interaction between solute and stationary phase

• Mobile phase either gas or liquid
• Stationary phase either liquid or solid
• Liq/Liq (Partition)
• Liq/Sol (Adsorption)
• Gas/Liq (Partition)
• Gas/Sol (Adsorption)

​

10

12/09/2023

11

12/09/2023

Sample

Mobile

time

Response

A

B

Figure:

Schematic diagram showing the separation of compounds A and B. and the output of the detector response at various stages of elution

The process of:

• Addition of sample
• Mobile elution process
• Separation mechanism
• Retention time ?
• Detection by, UV lamp, UV detector, other detectors.
• Eluted bands / collection
• Chromatogram? (function of retention time versus detector response)
• Partition coefficient K’
• k’ = C_s/C_M

12

12/09/2023

13

12/09/2023

Principles of (TLC)

14

12/09/2023

Basic Steps of TLC Technique

15

12/09/2023

• Slurry of the active material is uniformly spread over the plate by means of a commercially available spreader.

​

• Air-drying overnight, or oven-drying at

80-90 °C for about 30 minutes.

​

• Ready to use thin layers (pre-coated plates)

are commercially available.

Preparation of the Plate

16

12/09/2023

Sample Application

∙

17

12/09/2023

Locating of the Spots

R_f = b/a

For Colored Compounds:

18

12/09/2023

R_f = b/a

For Colorless Compounds:

19

12/09/2023

Applications of TLC Technique

Identification of Unknown Compounds

20

12/09/2023

Analysis of Reaction Mixture

∙

Start. mat.

Rxn. mixt.

∙

Product

∙

21

12/09/2023

Chromatogram Development

• Avoid direct contact between the sample and the solvent system.
• The tank or chamber is preferably lined with filter paper.
• As the developing solvent travels up the plate, it dissolves the sample and carries it up; the sample distributing itself between the moving solvent and the stationary phase.

22

12/09/2023

Determination of the Purity of a Product Compound

23

12/09/2023

Quantitative Determination of

an Unknown Concentration

24

12/09/2023

Instrumentation of HPLC

25

12/09/2023

HPLC Detector

Characteristics of Typical HPLC Detectors :

26

12/09/2023

HPLC Recorder

27

12/09/2023

What is the Applications of HPLC ?

Qualitative Analysis

Quantitative Analysis

Purification of Compounds

Identification of Compounds

Separation of Mixture Components

Peaks correspond to

individual components

28

12/09/2023

Quantitative Analysis

External Standard Method

29

12/09/2023

GC

30

12/09/2023

31

12/09/2023

GC Column

• Packed column
• ~ 3-6mm inner diameter tubing, 1-5 m long
• used for preparative separations or to separate gases that are poorly retained
• lower resolution
• small, uniform particle size decreases Eddy diffusion (requiring higher pressures)

• open tubular (more common):
• 0.1-0.5 mm inner dia., 10-100 m long
• 0.1-5 μm thick sp coated on inner walls
• higher resolution, shorter analysis times, greater sensitivity compared to packed columns

Detectors

• Flame Ionization Detector (FID):

​

​

​

​

​

​

32

12/09/2023

Detectors

• Flame Ionization Detector (FID):
• organic solutes are burned in flame producing CH radicals and eventually CHO⁺
• CH ^. + O ^. → CHO⁺ + e^-
• CHO⁺ ions are collected by cathode, produces current as the response

33

12/09/2023

34

12/09/2023

Applications of GC ?

Qualitative Analysis

Quantitative Analysis

Identification of Compounds:

Peaks correspond to

individual components

Separation of Mixture Components:

Retention time comparsion

​

​

Pyrolysis gas chromatography

It is used for the identification of non-volatile materials (plastics, natural and synthetic polymers, and some microbiological materials.

​

It is based on the fingerprint chromatogram for the sample, which results from its thermal dissociation and fragmentation.

35

12/09/2023

Quantitative Analysis

External Standard Method

36

12/09/2023

Food Analysis

Analysis of foods is concerned with confirm the presence

and determination the quantities of the analytes (lipids,

proteins, carbohydrates, preservatives, flavours, colorants,

and also vitamins, steroids, and pesticide residues).

​

Drug Analysis

​

GC is widely applied to identification of the active components, possible impurities as well as the metabolites.

Aspects of GC Applications:

37

12/09/2023

Forensic Analysis

​

In forensic cases, very little sample is available, and the

concentration of the sample components may be very low.

​

GC is a useful due to its high sensitivity and separation efficiency.

Environmental Analysis

​

The environmental contaminants; e.g. dichlorodiphenyltrichloro-

ethane (DDT) and the polychlorinated biphenyls (PCBs) are present

in the environment at very low concentrations and are found among

many of other compounds.

​

GC, with its high sensitivity and high separating power, is mostly

used in the analysis of environmental samples.

38

12/09/2023