Paper Chromatography

  Paper Chromatography

Paper Chromatography (PC) was first introduced by German scientist Christian Friedrich Schonbein (1865)

PC was considered to be the simplest and most widely used of the chromatographic techniques because of its applicability to isolation, identification and quantitative determination of organic and inorganic compounds

 Paper chromatography was later replaced by thin layer chromatography (TLC):

Separations on paper were often very slow (often up to 10 or 20 h), and spots tended to be much more diffuse than, for example, separations on cellulose TLC plates

 Basis of separation in Paper Chromatography – Adsorption Chromatography or Partition Chromatography ???

 It seems to be very obvious that the stationary phase in paper and thus it should be adsorption Chromatography but in Paper chromatography its not the paper that plays a role in separation; however it is the moisture present in paper that plays role in separation and therfore the basis of separation in paper chromatography is Partition hence it is Partition Chromatography.

 Types of Paper Chromatograph

Paper Chromatography is a Partition Chromatography but its mechanism of separation can be changed by modifing the paper

  • Paper Adsorption Chromatography - Paper impregnated with silica or alumina acts as adsorbent (stationary phase) and solvent as mobile phase


  • Paper Partition Chromatography - Moisture / Water present in the pores of cellulose fibers present in filter paper acts as stationary phase & another mobile phase is used as solvent

Paper Chromatography Principle of Separation


The principle of separation is mainly partition rather than adsorption. Cellulose layers in filter paper contains moisture which acts as stationary phase & organic solvents/buffers are used as mobile phase

 Stationary Phase in Paper Chromatography

Whatman filter papers of different grades like No.1, No.2, No.3, No.4, No.20, No.40, No.42 etc are used

In general this paper contains 98-99% of α-cellulose, 0.3 – 1% β -cellulose

Modified Paper Chromatography

  • Modified Papers – acid or base washed filter paper, glass fiber type paper
  • Hydrophilic Papers – Papers modified with methanol, formamide, glycol, glycerol etc
  • Hydrophobic papers – acetylation of OH groups leads to hydrophobic nature, hence can be used for reverse phase chromatography
  • Impregnation of silica, alumna, or ion exchange resins can also be made

Mobile Phase in Paper Chromatography

Pure solvents, buffer solutions or mixture of solvents

Hydrophilic mobile phase -

  • Methanol : water 4 : 1
  • N-butanol : glacial acetic acid : water 4: 1 :5
  • Isopropanol : ammonia : water 9:1:2

Hydrophobic mobile phases dimethyl ether: cyclohexane kerosene : 70% isopropanol

  Types of Paper Chromatography

Ascending Paper Chromatograph - In the ascending mode of development the paper is suspended so that the lower edge is below the level of the solvent, and the solvent moves up via capillary action. An alternative to suspending the paper was to form a self-supporting cylinder from the paper

Descending Paper Chromatography - In descending paper chromatography the upper end of the paper is immersed in a solvent contained in a suspended trough;  solvent sustained by gravity and will continue so long as there is solvent to feed it. This had the useful consequence that a sheet of any (practical) length could be used. In addition, the solvent could be allowed to run off the end of the paper, thus extending the chromatographic run if needed to improve resolution, or enabling compounds to be eluted from the paper and collected for further experiments. 


Horizontal or Circular Paper Chromatography -

a spot of the sample to be analysed was placed at the centre of a circular filter paper. Then a short wick is made by making parallel incisions. This wick is then cut to an  appropriate size and bent so that it dipped into the solvent contained in a Petri dish

Two-dimensional Separations on Paper -

Where separations were not achieved in a single development, it was often possible to achieve the desired result using a second solvent system of different composition and development in a second dimension at 900 to the original direction of chromatography