What is Paper Chromatography?
Planar chromatography processes are carried out on specialised paper in paper chromatography (PC), a subtype of planar chromatography.
Because it can be used to isolate, identify, and quantify both organic and inorganic substances, PC is thought to be the most straightforward and commonly applied of the chromatographic procedures.
German physicist Christian Friedrich Schonbein was the first to mention it (1865).
Types of Paper Chromatography
Paper Adsorption Chromatography
Silica or alumina impregnated paper functions as an adsorbent (stationary phase) and a solvent as the mobile phase.
Paper Partition Chromatography
Water or moisture found in the pores of the cellulose fibres in filter paper serves as the stationary phase, while a different mobile phase is used as the solvent. Paper partition chromatography is the most common type of paper chromatography in general.
Principle of Paper Chromatography
Instead of adsorption, partition is the major guiding factor in separation. Dispersion occurs between a stationary phase and a mobile phase. In the cellulose layers of filter paper, water serves as the stationary phase. Organic solvents and buffers make up the mobile phase. The growing solution transports the sample as it ascends the stationary phase. Based on how strongly they adsorb onto the stationary phase and how quickly they disintegrate in the mobile phase, the sample components will readily separate.
Instrumentation for Paper Chromatography
- Stationary phase & papers used
- Mobile phase
- Developing Chamber
- Detecting or Visualizing agents
1. STATIONARY PAPERS AND PHASE
- Different grades of Whatman filter papers include No. 1, No. 2, No. 3, No. 4, No. 20, No. 40, No. 42, etc.
- In general, the paper has 98–99% cellulose and 0.3–1% hemicellulose.
Other modified papers
- acid- or base-washed filter paper
- Paper with glass fibres.
- Papers treated with methanol, formamide, glycol, glycerol, etc. are called hydrophilic papers.
- Papers that may be utilised for reverse phase chromatography are hydrophobic due to the acetylation of OH groups.
- You may also make silica, aluminium, or ion exchange resin impregnations.
2. PAPER CHROMATOGRAPHY MOBILE PHASE
- You can use pure solvents, buffer solutions, or a combination of solvents.
Hydrophilic mobile phase
- Isopropanol: ammonia:water 9:1:
- Methanol : water 4:1
- N-butanol : glacial acetic acid : water 4:1:5
Hydrophobic mobile phases
- cyclohexane, isopropanol, and dimethyl ether are the components of kerosene.
- The most often used solvents are polar solvents, but the decision is made based on the characteristics of the material that has to be separated.
- A combination of solvents with the appropriate polarity may be used if pure solvents do not provide acceptable separation.
3. CHROMATOGRAPHIC CHAMBER
- Glass, plastic, and stainless steel are only a few of the materials used to construct the chromatographic chambers. Most people prefer glass tanks.
- They are available in a range of sizes dependent on the type of development and the length of the paper.
- Solvent vapour should be completely saturated in the chamber environment.
Steps in Paper Chromatography
Paper chromatography entails putting the sample mixture on a piece of filter paper, dipping the paper’s edge in a solvent, and permitting the solvent to climb up the paper by capillary action. Basic acts consist of
1. Selection of Solid Support
Exceptional cellulose paper with well-defined porosity, great resolution, little sample diffusion, and favourable solvent movement rates.
2. Selection of the Mobile Phase
- Several combinations of organic as well as inorganic solvents might well be utilised, based on the analyte.
- However, Acetic acid, A good solvent for separating amino acids, is water (12:3:5).
3. Saturation of Tank
- To get a higher resolution, filter paper is wrapped around the inside of the tank before the solvent is added.
4. Sample preparation and loading
- If the solid sample is to be utilised, it is dissolved in a relevant solvent. Using a micropipette, a sample (2–20 ul) is applied to the baseline as a spot and allowed to air dry to prevent diffusion.
5. Development of the Chromatogram
- Filter paper with a sample loaded on it is gently dipped into the solvent up to a height of 1 cm, and the solvent front is allowed to approach the edge of the paper.
- Different development methods are available.
- The solvent flows against gravity like the traditional kind.
- The spots are preserved in a chamber with a mobile phase solvent at the bottom and at the bottom of the paper.
- The solvent holder is located at the top of a dedicated chamber where this is done.
- The solvent runs down the paper while the spot is retained at the top.
- As a result of the solvent moving from top to bottom in this procedure, descending chromatography was coined.
- Ascending-descending chromatography is a combination of the previous two methods.
- The only difference between ascending and descending is the length of separation.
CIRCULAR / RADIAL DEVELOPMENT
- On a circular piece of paper, a Spot is retained in the centre.
- The solvent evenly disperses across the whole structure after flowing through a central wick.
6. Drying of Chromatogram
The solvent front is identified after development and allowed to cure in a dry cabinet or oven.
- Iodine vapour, ninhydrin, and other staining agents were used to identify colourless analytes.
- Radiolabeled or fluorescently tagged analytes were identified by detecting the related radioactivity or fluorescence.
Certain compounds in a mixture go about as far as the solvent, whilst others remain significantly nearer to the beginning point. For a particular molecule, the range of the solvent stays unchanged so long as other variables, such as the type of paper and the specific composition of the solvent, remain unchanged. Rf represents the distance travelled relative to the solvent.
Consequently, “Rf value” is computed for every isolated component in the resulting chromatogram to get a measurement of the quantity of displacement of a component during a paper chromatography experiment. A component’s Rf value is a numerical representation of its distance from the application point.
Applications of Paper Chromatography
- To verify that medications are pure,
- To identify adulterants,
- Find the toxins in the food and drink,
- In the investigation of fermentation and ripening,
- For the purpose of drug and dope detection in both people and animals,
- In cosmetics research,
- biochemical lab analysis of the reaction mixtures.
Advantages of Paper Chromatography
- Paper chromatography needs a very small amount of quantitative data.
- In comparison to other chromatography techniques, paper chromatography is less expensive.
- The paper chromatography method may be used to identify both unidentified inorganic and organic substances.
- In comparison to other analytical techniques or apparatus, paper chromatography takes up very little space.
- Outstanding resolving capacity
Limitations of Paper Chromatography
- Paper chromatography cannot be used for large sample volumes.
- Chromatography is ineffective for quantitative analysis on paper.
- Paper chromatography cannot separate complex mixtures.
- Compared to HPLC or HPTLC, less accurate