Learn Paper Chromatography in detail, including its principle, apparatus, ascending and descending procedures. A complete guide for pharmaceutical and laboratory professionals.
Paper Chromatography (Apparatus and Procedure)
Introduction to Paper Chromatography
Paper Chromatography is a widely used analytical technique for separating the components of a mixture. It works through the movement of a liquid phase over a sheet of specially prepared filter paper, which acts as the stationary phase.
In this method, separation occurs due to differences in how individual components interact with the mobile phase (solvent) and the stationary phase (paper). Depending on the system used, separation may involve:
- Adsorption mechanisms, similar to column chromatography
- Partitioning between two liquid phases, where natural water in the paper or an impregnated phase acts as the stationary phase
This technique is simple, cost-effective, and commonly used in pharmaceutical analysis, quality control laboratories, and research settings.
Principle of Paper Chromatography
In Paper Chromatography, the components of a mixture distribute themselves between:
- Stationary phase – Water molecules present in the paper fibers or a phase impregnated into the paper
- Mobile phase – A solvent that moves through the paper by capillary action (and gravity in some methods)
The separation occurs because different compounds travel at different speeds based on:
- Solubility in the mobile phase
- Affinity for the stationary phase
- Molecular size and polarity
Development can occur in two main ways:
- Ascending Paper Chromatography – Solvent moves upward by capillary action.
- Descending Paper Chromatography – Solvent moves downward, assisted by gravity.
Apparatus Used in Paper Chromatography
The following apparatus is required for performing Paper Chromatography:
(a) Chromatography Tank
A vapor-tight tank made of glass, porcelain, or stainless steel. It should:
- Allow solvent addition or pressure release
- Permit observation without opening the chamber
(b) Support Rack
A corrosion-resistant rack approximately 5 cm shorter than the tank height. It supports:
- Solvent trough
- Anti-siphoning rods
- Chromatographic sheets
(c) Glass Trough
- Longer than the width of chromatogram sheets
- Holds sufficient solvent for one complete run
(d) Anti-Siphoning Rods
Heavy glass rods placed parallel to the solvent trough. They:
- Support chromatographic sheets
- Prevent solvent backflow
(e) Chromatographic Sheets
Special filter paper:
- Minimum width: 25 mm
- Cut according to tank height
- Grain direction aligned with solvent flow
A fine pencil line is drawn horizontally across the paper to mark the sample application line. Care must be taken to avoid contamination from handling or dust.
Descending Paper Chromatography
Method
In Descending Paper Chromatography, gravity assists solvent movement, making development faster and often more efficient.
Procedure:
- Dissolve the test substance in a suitable solvent.
- Apply 6–10 mm spots along the pencil line, at least 3 cm apart.
- Allow each application to dry before adding more solution if needed.
- Add saturating solvent to form a 25 mm layer in the tank.
- Close the tank and allow saturation for 24 hours at room temperature.
- Suspend the chromatographic sheet using anti-siphoning rods.
- Ensure the paper hangs freely without touching tank walls.
- Allow to stand for 1.5 hours.
- Introduce the prescribed mobile phase.
- Allow development for the specified time or distance.
- Protect from bright light during development.
- Remove and air dry the paper.
- Visualize spots as directed in the monograph.
If required, separated spots can be cut, eluted, and quantitatively analyzed using chemical or instrumental methods.
Ascending Paper Chromatography
Method
In Ascending Paper Chromatography, the solvent travels upward by capillary action.
Procedure:
- Apply the test sample as described in descending method.
- Ensure the application point is above the solvent level.
- Add prescribed mobile phase to form a 25 mm layer in the trough.
- If using a two-phase system, add both phases.
- Add saturating solvent if required.
- Close the tank and allow saturation for 24 hours.
- Insert prepared paper and allow to stand for 1.5 hours.
- Lower paper into mobile phase without opening the tank.
- Allow development for prescribed time or distance.
- Protect from bright light.
- Remove and air dry as specified.
Ascending Paper Chromatography is simple and commonly used for routine laboratory analysis.
Advantages of Paper Chromatography
- Simple and economical technique
- Requires minimal equipment
- Suitable for small sample quantities
- Useful in pharmaceutical quality control
- Effective for separating amino acids, dyes, alkaloids, and drugs
Frequently Asked Questions (FAQ)
1. What is Paper Chromatography used for?
Paper Chromatography is used to separate and identify components of mixtures, especially in pharmaceutical analysis, food testing, and biochemical studies.
2. What is the difference between ascending and descending Paper Chromatography?
In ascending Paper Chromatography, the solvent moves upward by capillary action. In descending Paper Chromatography, gravity assists solvent movement, making it faster and sometimes more efficient.
3. What acts as the stationary phase in Paper Chromatography?
The stationary phase is either the water naturally present in the paper fibers or a liquid phase impregnated into the paper.
4. Why is the chromatography tank kept closed for 24 hours?
The tank is kept closed to allow vapor saturation, ensuring consistent solvent development and better separation results.
5. How are spots visualized after development?
Spots may be visualized under UV light or by using specific detecting reagents as prescribed in the monograph.
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