Unit 4: Separation Techniques-II (Chromatography)
Course Code: CHM-DSC-352
Paper Name: Analytical Chemistry
Table of Contents
1. Basic Principles of Chromatography
Chromatography is a physical method of separation in which the components to be separated are distributed between two phases: a Stationary Phase and a Mobile Phase.
Key Mechanism
Separation occurs because different components of a mixture travel at different speeds through the stationary phase based on their affinity for each phase. Components with a higher affinity for the mobile phase move faster, while those with higher affinity for the stationary phase move slower.
2. Classification of Chromatographic Methods
Chromatography is classified based on the physical state of the phases and the mechanism of separation.
| Type | Stationary Phase | Mobile Phase | Mechanism |
|---|---|---|---|
| Paper | Liquid (water in cellulose) | Liquid | Partition |
| TLC | Solid (Silica/Alumina) | Liquid | Adsorption |
| Column | Solid | Liquid | Adsorption |
| HPLC | Solid (Fine particles) | Liquid (High Pressure) | Adsorption/Partition |
3. Paper and Thin Layer Chromatography (TLC)
Paper Chromatography
Uses a specialized paper as the support. It is primarily a Partition chromatography technique where separation occurs between the water held in the paper fibers and the moving solvent.
Thin Layer Chromatography (TLC)
A thin layer of adsorbent (like silica gel) is coated on a glass or plastic plate. It is faster and provides better resolution than paper chromatography.
Rf Value (Retention Factor): The ratio of the distance traveled by the substance to the distance traveled by the solvent front.
Rf = Distance moved by solute / Distance moved by solvent front
4. Column Chromatography
Used for large-scale preparative separations. The stationary phase is packed into a glass column, and the mixture is loaded at the top.
[Image of the setup for column chromatography]- Elution: The process of washing a compound through the column using a mobile phase (eluent).
- Adsorbents: Common materials include Alumina (Al2O3) and Silica Gel (SiO2).
5. High Performance Liquid Chromatography (HPLC)
HPLC is a highly improved form of column chromatography. Instead of letting a solvent drip through a column under gravity, it is forced through at high pressures (up to 400 atmospheres).
- Speed: Separation is much faster due to high pressure.
- Efficiency: Uses very small particle sizes for the stationary phase, providing a much higher surface area for interaction.
- Sensitivity: Highly sensitive detectors allow for the analysis of trace amounts of substances.
6. Exam Focus: Tips and FAQs
- Rf Value: Remember that Rf values are always less than 1. If your calculation gives >1, you have inverted the ratio.
- Selection of Solvents: The "eluting power" of a solvent depends on its polarity. For adsorption chromatography, polar solvents move solutes faster.
- Comparison: Be prepared to explain why HPLC is superior to classical column chromatography (speed, resolution, and automation).
Frequently Asked Questions
Q: What is the stationary phase in paper chromatography?
A: It is technically the water molecules trapped in the cellulose fibers of the paper, not the paper itself.
Q: How do you visualize colorless spots on a TLC plate?
A: Using UV light, iodine vapors, or specific spraying reagents (like Ninhydrin for amino acids) that react to form colored products.
Q: What is "Reverse Phase" HPLC?
A: It is a common mode where the stationary phase is non-polar and the mobile phase is polar. This is the opposite of "Normal Phase" chromatography.