Paper chromatography is an analytical technique used for separation of mixtures into their components for identification (Figure 1), purification, and quantification. It is a useful technique as it is quick, has high resolution and ease of detection when separating small samples. The mobile phase is usually an alcohol solvent mixture, while the stationary phase is a strip of chromatography paper.

Figure 1. Paper Chromatography is used to separate components of a plant
Figure 1. Paper Chromatography is used to separate components of a plant

PC involves same basic principles as thin layer chromatography. The stationary phase is cellulose of chromatography paper, which acts as the supporting matrix. Mobile phase (a liquid or a gas) is a developing solution that travels up the stationary phase and carries samples along with it. Two-dimensional paper chromatography also known as two-way chromatography, involves using two solvents and rotating paper at 90° in between them.

The factors affecting separation of components in PC are thickness of layer, degree of saturation (a fully equilibrated paper contain up to 20% of aqueous stationary phase), low Rf, concentration of spotting solution, temperature, and purity of solutes.

Stationary Phase

Most commonly used papers as stationary phase are Whatman no. 1, Whatman no. 3 and Schull 2043. Hydrophobic as well as hydrophilic solvents can also be used as stationary phase depending upon type of analysis.

Mobile Phase

Solvent is used as a mobile phase and type of solvent used depends upon type of sample to be analyzed. For example, in pigment identification from a leaf of the spinach plant, the stationary phase is typically a piece of chromatography paper with a dried spot of the plant extract near one end. The mobile phase used is an acetone-ligroin mixture, a nonpolar (hydrophobic) solvent mixture.

Sample application and Component Detection

A small spot of the solution containing the mixture sample is applied to the chromatography paper (Figure 2) about 2 cm away from the base of the paper. The sample gets absorbed.  Later the paper is dipped into a suitable solvent in a sealed chamber (Figure 1).

Figure 2. Sample application on chromatography paper
Figure 2. Sample application on chromatography paper

The solvent moves up the paper by capillary action. Rf value is calculated. Rf value is defined as the ratio of the distance traveled by the solute to the distance traveled by the solvent, which is calculated using the formula shown below.

For example, if  the compound travels 2.8 cm and a solvent front travels 3.0 cm, the Rf is 0.933 as calculated below (see figure 1).

Figure 1: Calculation of Rf
Figure 1: Calculation of Rf

Applications of Paper Chromatography

Among all the chromatography methods, paper chromatography is the oldest; being inexpensive, and rapid, it still remains popular.

  • Paper chromatography is used for sequencing DNA and RNA, detecting blood or alcohol levels in bloodstream, and analysis of metabolism of herbicides by bacteria.
  • The classic use for this technique is separation and identification of dyes and pigments in ink and paint samples.