Paper Chromatography Basic Principles and Facts

How Paper Chromatography is Done and Its Usefulness in Science

Oct 4, 2009

Paper chromatography, even today, has useful application for scientific investigations, and it reveals how chemists can separate and identify specific chemical compounds.

Scientists always seem to be thinking, seeking, experimenting, and finding important new scientific facts – that is the very nature of scientific inquiry. In a sense, scientists are restless minds and souls, who are never satisfied with the status quo. A brief look at paper chromatography shows how science tools and techniques provide information for multiple inquiries and experiments.

Paper Chromatography Basic Principles, Uses and Value

Paper chromatography is one of the earliest known molecular separation techniques. Paper chromatography procedures were first used by the Romans who used gall nut extracts impregnated into paper to estimate ferrous sulphate and copper acetate. The Romans also used snail pigments to stain togas, according to Morgan and Wilson, in "An Early Description of Paper Chromatography?" , Chromatographia (2004) 60: 135-136. These authors also describe F. F. Runge's use of colored inorganic salts to spread and react with other chemicals on paper to visualize new compounds and color reactions.

M. S. Tswett, a Russian botanist, was one of the first chemists in the early 1903 to actually develop and promote chromatography as a useful technique to separate chlorophyll within a powder-packed, glass cylinder (classical column chromatography) and to use filter paper for capturing chlorophyll. Since these chlorophyll studies related directly to plant and animal pigments, both botanists and zoologists began to experiment with the technique.

Paper chromatography is inexpensive, rapid and provides graphic, clear results. Solutions containing known, or unknown, chemical compounds are spotted and dried in sufficient quantity onto filter paper. Next, the paper is appropriately suspended in a suitable solvent. The solvent interacts with the paper and is drawn into the paper fibers by capillary action. Some molecules are more soluble in the solvent than others, and these differences mainly account for varying rates of compound movement. The chromatograph runs until the solvent front reaches the top of the paper. The paper is dried and compounds that are visible are recorded and identified by comparing to known standards. Amino acids, sugars, lipids and nucleic acids can be identified by spraying with appropriate reagents to detect these specific compounds. For example, ninhydrin may be sprayed over the paper, and as many as 20 different kinds of naturally-occurring, amino acids may be detected.

Paper Chromatography Results and Interpretations

Paper chromatography is a rapid and reproducible procedure – as long as all conditions are controlled and maintained. Variables in chromatography include:

All the above variables are controlled and monitored. The solvents used for extraction, and the solvent used for development are important. When the chromatographic run is complete the Rf value may be calculated (for long linear runs) as follows: the solvent distance traveled less the total distance of solvent travelled from the spotted origin to the end of the paper in mm. This solvent front is the larger denominator, and the distance run by each detected compound is listed as the numerator. Migration examples: pigment = 45 mm, solvent = 50 mm; Rf (Retardation factor) value = 45/50 = 0.9 is the Rf value.

Paper chromatography is economical, simple, reproducible and accurate for the separation and identification of many important organic and inorganic compounds. Chromatography's value as a teaching tool of scientific inquiry will continue into the future, even though more sophisticated, chemical separations tools are, and will be, available. Thin-layer chromatography is another unique technique that is valuable for the detection of drugs and other chemical compounds.

Wandersee, J.H., et al. eds. 1996. Bioinstrumentation, Tools for Understanding Life. National Assoc. Biol. Teachers, Reston, Va. 329 pp

Paper Chromatography. 1976. 15th ed., Micropaedia of Encyclopedia Brittanica. Chicago, Illinois

The copyright of the article Paper Chromatography Basic Principles and Facts in Scientific Research Methods is owned by Donald Reinhardt. Permission to republish Paper Chromatography Basic Principles and Facts in print or online must be granted by the author in writing.