The paper chromatography (PC) can be described as a kind of planar chromatography, where the chromatography processes are performed on a special paper. PC is thought to be the most simple and the most extensively employed of chromatographic techniques due to its ability for isolation, identification, and quantitative analysis for organic as well as inorganic substances. PC was first developed in 1865 by German scientists Christian Friedrich Schonbein (1865).
Types of Paper chromatography
1. Paper Adsorption Chromatography
Silica-impregnated paper Alumina is used as an absorbent (stationary stage) and solvent acts as mobile phase.
2. Paper Partition Chromatography
The water or moisture that’s contained within the pores cellulose fibers in filter paper functions as stationary phase and another phase that is mobile is used to act as a solvent. Paper chromatography is mostly a reference to chromatography using partitions of paper.
Principle of Paper chromatography
The concept of separation is mostly partition instead of the concept of adsorption. Substances are divided between a stationary and mobile phase. Cellulose layers on filter papers contain moisture that serves as a stationary phase. Organic solvents/buffers can be used as mobile phases. The solution is developed and travels along the stationary phase, carrying the sample along. The components of the sample are separated quickly based on how much they adsorb to the stationary phase, and how easily they disintegrate into the fluid phase.
Instrumentation of Paper chromatography
- Stationary phase & papers used
- Mobile phase
- Developing Chamber
- Detecting or Visualizing agents
1. STATIONARY PHASE AND PAPERS
Whatman filter papers in various grades such as No.1, No.2, No.3, No.4, No.20, No.40, No.42 etc
In general , the paper has 99-99% a-cellulose, 0.3 – 1% of b-cellulose.
Other papers that have been modified
- Acid or base washed filter paper
- Glass fiber type paper.
- Hydrophilic Papers – Papers modified by methanol, formamide glycol, glycerol and so on.
- Hydrophobic papers – Acetylation of OH groups results in hydrophobic characteristics, which is suitable for reverse-phase chromatography.
- Impregnation of silica, alumna, or ion exchange resins can also be made.
2. PAPER CHROMATOGRAPHY MOBILE PHASE
Pure solvents buffer solutions, pure solvents, or even mixtures of solvents are acceptable.
Examples-
Hydrophilic mobile phase
- Isopropanol: ammonia:water 9:1:2
- Methanol : water 4:1
- N-butanol : glacial acetic acid : water 4:1:5
Hydrophobic mobile phases
- dimethyl ether: cyclohexane kerosene : 70% isopropanol
- The most commonly used solvents are the polar ones However, the selection of the solvent is contingent on the characteristics of the substance that is to be separated.
- If the pure solvents don’t offer a sufficient separation A mixture of solvents that have polarity suitable to be used.
3. CHROMATOGRAPHIC CHAMBER
- The chambers used for chromatography are constructed from a variety of materials, including plastic, glass and stainless steel. Glass tanks are most commonly used.
- They are offered in various dimensions based on size of the paper and the type of development.
- The chamber’s atmosphere must be filled with solvent the vapor.
Steps in Paper Chromatography
Paper chromatography is the process where a sample mixture is placed on the surface of a filter paper. The edges of the filter paper gets submerged in an solvent, and the solvent rises upwards on the paper through capillary action. The most fundamental steps include:
1. Selection of Solid Support
Fine-quality cellulose paper with well-defined porosity. High resolution and low diffusion of sample and favoring a high speed of moving of solvent.
2. Selection of Mobile Phase
Diverse combinations of solvents, both organic and inorganic could be employed depending upon the specific analyte.
Example. Butanol: Acetic acid water (12:3:5) is an ideal solvent to separate amino acids.
3. Saturation of Tank
The wall inside the tank is covered with the filter paper prior to when the solvent is added to the tank to increase resolution.
4. Sample Preparation and Loading
If the sample is solid to dissolve, it must be dissolving in a solvent that is suitable. A sample (2-20ul) is added to the base line in an area using the micropipette, and then dried to avoid diffusion.
5. Development of the Chromatogram
The sample loaded filter paper is placed in the solvent with care with a minimum height of one centimeter and then waited until the front of the solvent reaches just below the edge of paper.
Different kinds of techniques for development can be employed:
A. ASCENDING DEVELOPMENT
As with conventional types that uses solvent, this one flows against gravity. The spots are placed on the bottom of the paper, and are kept inside a chamber, with mobile phase solvent on the bottom.
B. DESCENDING TYPE
The process is performed in a specific chamber, in which the solvent holder is on highest point. The spot is held at the top, and the solvent runs across the paper. The solvent flows from top to bottom , and it’s known as the descending process chromatography.
C. ASCENDING – DESCENDING DEVELOPMENT
A combination of two methods is known as ascending-descending chromatography. The separation length is only increased the first time an ascending occurs then descending.
D. CIRCULAR / RADIAL DEVELOPMENT
Spot is located in the center of a circular paper. The solvent flows through a wick located at the center and spreads out across all directions evenly.
6. Drying of Chromatogram
Following the developmentprocess, the front of the solvent is marked. The left to dry in an oven or in a cabinet that is dry.
7. Detection
Analytes that are colorless can be can be detected through staining with agents like iodine vapour and ninhydrin, among others.
Analytes that are fluorescently labeled or radiolabeled can be determined by measuring the radioactivity and florescence.
Rf values
Certain components in a mixture go the same distance as the solvent can go, while some remain much closer to the bottom line. The distance traveled relative to the solvent is constant for each compound , as long as other parameters like the kind of paper used and the precise chemical composition remain constant. The distance traveled in relation to the solvent’s composition is known as Rf. Rf value.
In order to measure the amount of movement of a particular component in a paper chromatography study, “Rf value” is calculated for every component in the created chromatogram. A Rf value refers to a numerical number that refers to the distance that the component has traveled from the point of application.
Applications of Paper Chromatography
- For ensuring the quality of pharmaceuticals,
- For the detection of adulterants
- Check for contaminants in food and beverages
- Study of fermentation and ripening,
- To identify dopes and other drugs in both animals and humans.
- In the analysis of cosmetics
- Analysis of reactions in biochemical labs.
Advantages of Paper Chromatography
- Simple
- Rapid
- Paper Chromatography requires very less quantitative material.
- Paper Chromatography is less expensive than other chromatography techniques.
- The inorganic and unknown inorganic as organic compound can be identified using the paper chromatography .
- Paper chromatography doesn’t take up any space when compared with other methods or equipments for analysis.
- Great resolution power
Limitations of Paper Chromatography
- The large volume of sample cannot be used on paper the chromatography.
- Quantitative analysis using the chromatography method is ineffective.
- Complex mixtures cannot be separated using paper chromatography.
- A little less accurate than HPLC or HPTLC
