CHROMATOGRAPHY (PART 2)

6- Gingerol

Has an extra water molecule compared to 6- Shogaol.

6- Shogaol

- The dehydrated form of 6- Gingerol.
- Has a more pungent smell and a spicier taste.

Heating (Dehydration)

6- Gingerol can be converted to 6- Shogaol by?

- Identification of Active Ingredients and Contaminants in Pharmaceutical Raw Materials and Preparations.
- The analysis of herbals used as drugs or food supplements.

USES OF TLC

Column Chromatography

A solid-liquid technique in which the stationary phase is solid and the mobile phase is a liquid.

- Stationary phase is held in a narrow tube through which the mobile phase is forced under pressure or under the effect of gravity.
- While the mobile phase moves, the different components of the mixture also move at different rates.

PRINCIPLE OF COLUMN CHROMATOGRAPHY

- Silica (Polar Compounds)
- Alumina (Less Polar Compounds)
- Calcium Carbonate
- Calcium Phosphate
- Magnesia
- Starch

STATIONARY PHASE OF COLUMN CHROMATOGRAPHY
Adsorbents:

- Petroleum Ether
- Carbon Tetrachloride
- Cyclohexane
- Ether
- Acetone
- Benzene
- Toluene
- Esters
- Water

STATIONARY PHASE OF COLUMN CHROMATOGRAPHY
Solvents:

Wet Method
Dry Method

Packing Techniques for Stationary Phase

Dry Method

Packing Techniques for Stationary Phase (COLUMN CHROMATOOGRAPHY)
- The stationary phase (such as silica, alumina, or cellulose) is packed into the column in dry form.
- Once the stationary phase is packed, the mobile phase is flushed through to make the whole column wet and ensure equilibrium in the stationary phase.

- Air bubbles may be trapped between the mobile and stationary phases, leading to uneven packing.
- Cracks may form in the adsorbent material, which can affect the flow characteristics and separation quality.
- The separation may not result in clear bands due to uneven packing.

COLUMN CHROMATOOGRAPHY
Disadvantages of Dry Method

Wet Method

Packing Techniques for Stationary Phase (COLUMN CHROMATOOGRAPHY)
- The stationary phase is made into a slurry by combining it with the mobile phase.
- The slurry is then packed inside the column.

- The slurry allows the stationary phase to settle uniformly in the column, ensuring even packing.
- No air bubbles are trapped, and there are no cracks in the column adsorbent.
- The bands eluted from the column are uniform and ideal for separation.

COLUMN CHROMATOOGRAPHY
Advantages of Wet Method

Eluate

The liquid passing down the column is referred to as the?

Eluites

The fractions containing the separated compounds are called?

Particle Size of the Solid Stationary Phase
Column Dimensions
Uniformity of Packing
Temperature of the Column
Quality of Solvents
Concentration of solutes

Factors Affecting the Separation using Column Chromatography

Particle Size of the Solid Stationary Phase

Factors Affecting the Separation using Column Chromatography
Decrease of size improves separation

Column Dimensions

Factors Affecting the Separation using Column Chromatography
Efficiency has been improved by increasing the length/width ratio of the column

Uniformity of Packing

Factors Affecting the Separation using Column Chromatography
Non-uniform packing results in irregular movement of solutes through column and less uniform zone formation (e.g. tailing)

Tailing

Where the separated compound forms an uneven shape, making it more difficult to collect fractions.

Temperature of the Column

Factors Affecting the Separation using Column Chromatography
The speed of elution increases at higher temperatures

Quality of Solvents

Factors Affecting the Separation using Column Chromatography
Solvents having low viscosities is giving better results.

Concentration of solutes

Factors Affecting the Separation using Column Chromatography
Substances of high concentration move slowly

Isocratic Elution
Gradient Elution Techniques

Elution Techniques

Isocratic Elution

Elution Techniques
Same solvent composition is used throughout the whole process of separation.

Gradient Elution Techniques

Elution Techniques
Solvents of increasing elution strength are used throughout the separation process.

Chloroform → Ethyl Acetate → Methanol

Example of Gradient Elution Techniques

- Separation of mixture of compounds
- Purification process
- Isolation of active constituents

Applications of Column Chromatography