Paper Chromatography is a simple, economical, and widely used separation technique that belongs to the family of planar chromatography. In this method, a strip of specially selected filter paper acts as the stationary phase, while a suitable solvent or solvent mixture acts as the mobile phase. As the solvent rises through the paper by capillary action, different components of a mixture move at different rates, resulting in separation.
This technique is especially useful for separating amino acids, sugars, dyes, plant pigments, and other polar substances. Because it requires minimal equipment and offers quick results, paper chromatography is used extensively in teaching laboratories, research settings, and pharmaceutical quality control.
Principle
Paper Chromatography works on the principle of partition between two phases:
- The stationary phase – a thin layer of water molecules held within the pores of filter paper
- The mobile phase – an organic solvent or mixture that moves up the paper
Solutes distribute between these phases according to their solubility and polarity. More polar compounds tend to remain closer to the stationary phase, whereas less polar compounds move faster with the mobile phase. The difference in migration results in separation.
Theory of Paper Chromatography
The process of separation depends on two major forces:
(a) Propelling Force
The propelling force is the upward movement of the mobile phase caused by capillary action. This force pulls the solvent and solutes through the paper fibers. The stronger the capillary attraction, the faster the solvent front travels.
(b) Retarding Force
The retarding force opposes the movement of the solutes. This force arises due to adsorption, partitioning, or interaction of solute molecules with water molecules trapped in the paper. A solute with higher affinity for the stationary phase travels slower.
Advantages of Paper Chromatography
- Simple, inexpensive, and does not require sophisticated equipment
- Suitable for separating polar molecules such as amino acids and sugars
- Low sample quantity required
- Several samples can be run simultaneously
- Good visualization and easy documentation
Disadvantages of Paper Chromatography
- Resolution is lower compared to TLC and HPLC
- Not suitable for non-polar or volatile samples
- Environmental variables (humidity, temperature) affect reproducibility
- Longer development time than TLC
Types of Paper Chromatography
- Ascending Paper Chromatography
- Descending Paper Chromatography
- Ascending–Descending Chromatography
- Radial (Circular) Paper Chromatography
- Two-Dimensional Paper Chromatography
Steps Involved in Paper Chromatography
The uploaded PDF outlines the major steps, which are expanded below in detailed form.
1. Choice of the Filter Paper
Selecting the right grade of filter paper is essential. Requirements include:
- Uniform texture and thickness
- High purity and minimal contamination
- Good capillary rise characteristics
- Suitable pore size for effective separation
Whatman No. 1 is most commonly used, although specialized papers may be selected based on the nature of the analysis.
2. Preparation of the Paper
The paper should be handled carefully to avoid contamination. It may be marked lightly with pencil to indicate the baseline and solvent front. The paper can also be pre-treated or impregnated with reagents depending on the type of separation required.
3. Preparation of the Solution
The sample is dissolved in a suitable solvent, typically a polar solvent such as water, methanol, or ethanol. The concentration should be appropriate to avoid overloading the paper.
4. Application of the Sample
The sample is applied on the baseline using a capillary tube or micropipette. Important considerations include:
- Small and uniform spot size
- Adequate spacing between multiple samples
- Avoiding spreading or blotting
5. Choice of the Solvent
The solvent system should provide optimal separation. Common solvents include:
- Butanol–acetic acid–water
- Phenol–water
- Alcohol–water mixtures
Solvent selection depends on solute polarity and the desired separation efficiency.
6. Stationary Phase
Water adsorbed in the cellulose fibers of the filter paper acts as the stationary phase. This water layer helps partition solutes during their migration.
7. Development of the Chromatogram
The prepared paper is placed in a chromatography chamber containing the chosen mobile phase. The solvent moves through the paper by capillary action. Different development methods include:
- Ascending method: Solvent moves upward
- Descending method: Solvent moves downward due to gravity
- Radial method: Solvent moves outward in circular patterns
- Two-dimensional method: Paper is developed in one direction, dried, rotated, and redeveloped
During development, the chamber should be saturated with solvent vapor, and the paper must not touch the walls of the container.
8. Drying the Chromatogram
After the solvent reaches an appropriate height, the paper is removed and dried in air or a hot air oven. Drying ensures fixation of separated components for visualization.
9. Location of the Compound
Many compounds are colorless and require visualization using:
- UV light
- Iodine chamber
- Ninhydrin spray (for amino acids)
- Silver nitrate or ferric chloride reagents
Spots become visible through color formation or fluorescence.
10. Quantitative and Qualitative Analysis
Paper Chromatography is primarily qualitative, but can also provide semi-quantitative information.
- Qualitative analysis: Identification based on Rf values
- Quantitative analysis: Measuring spot intensity or using densitometry
Rf (Retention factor) is calculated as:
Rf = Distance traveled by solute / Distance traveled by solvent front
Precautions in Paper Chromatography
- Use clean glassware and dry chromatography chambers
- Avoid touching the paper surface with bare hands
- Spot size must be small and well-defined
- Maintain constant temperature and humidity
- Use freshly prepared solvent systems
Applications
- Identification of amino acids, sugars, and plant pigments
- Purity testing of pharmaceuticals
- Analysis of inks and dyes
- Separation of small polar compounds
- Monitoring biochemical reactions
- Detection of adulterants in herbal drugs
- Forensic analysis
Detailed Notes:
For PDF style full-color notes, open the complete study material below: