CHROMATOGRAPHY

Color

Chroma means?

To write

Graphein means?

Chromatography

Is a collective term for a set of laboratory techniques for the separation, identification, and determination of the chemical components in a complex mixture.

Mikael Zweth

Who first used chromatography to separate plant pigments using a column of calcium carbonate or chalk?

Calcium Carbonate or Chalk

In Mikael Zweth's initial experiments with chromatography in 1906, what material did he employ as the stationary phase to separate plant pigments?

Chlorophyll
Carotenes
Xanthophylls

Mikael Zweth did a test on the separation of plant pigments such as:

Green

Mikael Zweth did a test on the separation of plant pigments such as:
Chlorophyll

Orange

Mikael Zweth did a test on the separation of plant pigments such as:
Carotenes

Yellow

Mikael Zweth did a test on the separation of plant pigments such as:
Xanthophylls

Stationary Phase
Mobile Phase/ Eluent

Main Components of Chromatography

Stationary Phase

Main Components of Chromatography
- Remains fixed in place.
- A layer or coating where the analyte is mainly placed for analysis.

Mobile Phase/ Eluent

Main Components of Chromatography
- A solvent that flows through the stationary phase.
- Responsible for carrying the components of the mixture through the stationary phase/medium being used.

- Works by allowing the molecules present in a mixture to distribute themselves between a stationary and a mobile phase/medium. 
- Depends on the AFFINITY of the compounds into the mobile and stationary phase 
- Molecules that spend most of their time in the mobile phase are carried along faster, while molecules adhering to the stationary phase moves slower.

Principles of Chromatography

Affinity

Is the strength of adhesion of molecules of interest.
In chromatography:
- It refers to the strength of adhesion to the stationary phase and mobile phase.
- Differences in this “property” may cause the differential separation of mixture components.

Adsorption
Solubility

Properties that Command Affinity

Adsorption

Properties that Command Affinity
- Property of how well a component of the mixture sticks to the stationary phase.

Solubility

Properties that Command Affinity
- Property of how well a component dissolves in the mobile phase.

- Used for separation of different compounds such as amino acids, proteins, and carbohydrates.
- Identification and control of technical products such as drugs, hormones, and vitamins.
- Helpful for qualitative and quantitative separation and analysis of complex mixtures.
- Useful for the determination of molecular weights of proteins.
- Concentration of substances from dilute solutions.

Applications of Chromatography

Paper Chromatography
TLC and HPLTC
Column Chromatography

TYPES OF CHROMATOGRAPHY
On the basis of chromatographic bed shape

Adsorption Chromatography
Partition Chromatography
Ion Exchange Chromatography
Molecular Exclusion Chromatography

TYPES OF CHROMATOGRAPHY
On the basis of the interaction of solute to the stationary phase

High Performance Liquid Chromatography
Gas Chromatography

TYPES OF CHROMATOGRAPHY
On the basis of the physical state of the mobile phase

Preparative Chromatography
Analytical Chromatography

TYPES OF CHROMATOGRAPHY

Preparative Chromatography

TYPES OF CHROMATOGRAPHY
- It seeks to separate the components of a mixture for further use [and thus is a form of purification]
NOTE:
- Considered as a form of purification.
- Sometimes also a form of concentration.

Analytical Chromatography

TYPES OF CHROMATOGRAPHY
- It normally operates with smaller amounts of materials and seeks to measure the relative proportions of analytes in mixtures
NOTE:
- Not just to identify the specific constituents or components present.
- But also to quantify how much of each component is present, based on estimation through chromatography.

Archer John Porter Martin
Richard Laurence Millington Synge

Paper Chromatography was a method invented by the British Biochemists?

Paper Chromatography

A technique that involves placing a spot or dot of sample solution or mixture onto a strip of filter paper, which acts as the stationary phase.
NOTE:
A chromatographic technique that uses paper sheets or strips as the adsorbent, acting as the stationary phase, through which a solution is made to pass.

Thin Layer Chromatography

- Method for identifying substances and testing the purity of compounds.
- Components of the samples will separate on the stationary phase according to how much they adsorb on the stationary phase versus how much they dissolve in the mobile phase.

Is a thin, uniform layer (0.25 mm thick) of adsorbent (usually silica gel, alumina or cellulose) coated on a plate.

THIN LAYER CHROMATTOGRAPHY
Stationary Phase:

Standard Thin Layer Chromatography

TLC is also called?

Silica Gel

What is the most common use adsorbent of TLC?

Acetone
Hexane
Ethyl Acetate
Toluene

Common Mobile Phase for TLC
Mobile phases for TLC are volatile liquids (+ organic cpds) and are removed after chromatography.

Normal TLC Plates
High Performance TLC Plates

Two types of TLC plates are available, differing in the particle size of the stationary phase:

Normal TLC Plates

TLC PLATES
Has a layer of stationary phase 0.25mm thick and with particle sizes of 5-12 mm.
NOTE:
These plates are the standard TLC plates used for most analyses.

High Performance TLC Plates

TLC PLATES
Having smaller particle sizes of 2-10 mm are used and the layer thickness is only 0.1-0.2 mm.
NOTE:
- Better separation efficiency compared to normal TLC plates.
- Faster chromatographic analysis (takes less time).
- Requires smaller volumes of mobile phase.

Nature of Analytes
Solvent Strength

Mobile Phase for TLC
What primarily determines the retention of compounds in the stationary phase during chromatography?

- Have the highest affinity for the polar silica gel.
- Are retarded the most in the stationary phase.

Mobile Phase for TLC
Analytes with very polar groups or a greater number of polar groups:

Solvent Strength

Mobile Phase for TLC
Refers to the ability of the solvent to elute or move the compounds more quickly.

Low Solvent Strength

Solvent Strength of Mobile Phase:
- Results in strong retention of compounds on the TLC plate.

High Solvent Strength

Solvent Strength of Mobile Phase:
- Leads to low retention of compounds on the TLC plate.

Chloroform
Methanol
Water

Mobile Phase for TLC
What components of the general mobile phase mixture often recommended by the USP when no other is specified?

180:15:1

Mobile Phase for TLC
What is the specific volumetric ratio (v/v) for the general mobile phase mixture of chloroform/methanol/water recommended by the USP?

- Cutting and Activation of TLC Plate 
- Sample Spotting
- Chromatographic Development of TLC Plate
- Removal and Drying of TLC Plate
- Detection or Visualization

TLC Procedure

Ruler
Cutter

TLC PROCEDURE (STEP 1)
Enumerate the tools mentioned for cutting the TLC plate.

- Pre-elution
- Heating of TLC plate above 100∘C

TLC PROCEDURE (STEP 1)
Identify the two methods mentioned for the activation of a TLC plate.

Pre- elution

TLC PROCEDURE (STEP 1)
- Use methanol (or other solvents) to run through the plate to remove water and volatile compounds.

Heating

TLC PROCEDURE (STEP 1)
- Heat the TLC plate above 100°C, typically at 105°C for 15 minutes to remove absorbed water.

Sample Spotting

TLC PROCEDURE
The samples, prepared in a volatile solvent, are placed as spots or bands on the plate parallel to one of its edges.

Spots
Bands

Forms in which samples can be placed onto the TLC plate.

Ethanol
Methanol

Common Solvent for Sample Plotting in TLC

Micropipettes
Capillary Tube

Tools mentioned for depositing small volumes (e.g., 10 microliters) of sample onto the TLC plate usually thru spots.

Linomat Spotter

- Alternative equipment mentioned for applying samples in a band form instead of a spot.
- A syringe is used to deliver specific, predetermined volumes of the sample.
- The TLC plate moves back and forth in the machine during the process.

Chromatographic Development of TLC Plate

TLC PROCEDURE
The plate is inserted vertically in a development tank with a mobile phase at the bottom of the tank. The tank is closed and the mobile phase migrates up the plate by capillary forces.

Removal and Drying of TLC Plate

TLC PROCEDURE
- When the mobile phase has moved a certain distance towards the top of the plate, the TLC plate is removed from the tank and the mobile phase is evaporated from the plate in a fume hood. 
- To completely evaporate the solvent, it is heated to 60°C.

UV Light of 254 nm and 365 nm

TLC PROCEDURE
To facilitate visualization of the noncolored spots or bands on the TLC plate after chromatography, they are viewed under?

UV 254 nm

UV Light Visualization:
Green Light

UV 365 nm

UV Light Visualization:
Blue Light