Paper Chromatography

Return to Main Menu

Paper chromatography is one method for testing the purity of compounds and identifying substances. Paper chromatography is a useful technique because it is relatively quick and requires small quantities of material.

Separations in paper chromatography involve the same principles as those in thin layer chromatography. In paper chromatography, like thin layer chromatography, substances are distributed between a stationary phase and a mobile phase. The stationary phase is usually a piece of high quality filter paper. The mobile phase is a developing solution that travels up the stationary phase, carrying the samples with it. Components of the sample will separate on the stationary phase according to how strongly they adsorb to the stationary phase versus how much they dissolve in the mobile phase.

Video: Chromatography process (same as TLC Process video) ( 5.83 M )

Choose a developing chamber that can be sealed well. The chamber should be large enough to hold the paper that is to be developed.

The chamber should be clean and dry before use.

Add the mobile phase to the chamber so that it is about 2 cm deep. Seal the chamber tightly and let the chamber stand overnight if possible. Why? The larger the chamber, the longer it should stand.

What is wrong with this student's paper chromatography chamber setup? Answer

Cut a square piece of high-quality filter paper to fit into your development chamber. With a pencil, draw a straight line about 3 cm from the bottom edge of the paper.

Video: Prepare the paper ( 4.61 M )

First, each sample should be dissolved in an appropriate solvent to make about a one percent solution (0.01 g sample/1 g solvent). Less than one milliliter of solution will be needed for the experiment. Then the dissolved samples may be spotted to the paper.

Video: Spotting the samples ( 3.13 M ) Text description

If a larger quantity of sample is needed for the experiment than is provided by one application, the solution may be re-spotted.

Video: Re-spotting ( 1.12 M ) Text description

All spots on the chromatogram should be 2 to 2.5 cm away from the edges of the paper and from each other.

After preparing the chamber and spotting the samples, the paper is ready for development. Be careful to handle the paper only by its edges, and try to leave the development chamber uncovered for as little time as possible.

Initially, the chromatogram should be suspended in the chamber without touching the solvent. To suspend the chromatogram, to the top of the paper and thread a piece of string throught the paper clip. Then tape the string to the outside of the chamber to hold the chromatogram in place. The paper should hang in the development chamber overnight, if possible.

After the chromatogram has hung in the chamber, immerse the paper's bottom edge into the developing solvent.

Allow the chromatogram to dry in a well-ventilated area.

A student has developed a chromatogram as shown in the picture above. Will this chromatogram yield good results? Answer

A student removed this chromatogram from the development chamber and allowed the solvent to dry. What did he forget to do? Answer

If the spots can be seen, outline them with a pencil.

If the spots are not obvious, the most common visualization technique is to hold the paper under an ultraviolet lamp. (Caution: Do not look directly into the lamp!) Many organic compounds can be seen using this technique. Outline the spots with a pencil.

The R_f value for each spot should be calculated. R_f stands for "ratio of fronts" and is characteristic for any given compound. Hence, known R_f values can be compared to those of unknown substances to aid in their identifications.

(Note: R_f values often depend on the temperature, solvent, and type of paper used in the experiment; the most effective way to identify a compound is to spot known substances next to unknown substances on the same chromatogram.)

In addition, the purity of a sample may be estimated from the chromatogram. An impure sample will often develop as two or more spots, while a pure sample will show only one spot.

Related Modules: Thin Layer Chromatography, Gas Chromatography