Paper Chromatography : Sample Preparation and Application


Spotting of Paper Chromatographic Sheet (Image Courtesy :

Developing the Chromatogram involves three stages:

  • Sample preparation
  • Sample application
  • Developing of the chromatogram

Sample Preparation

Samples can come either as liquids or solids. Liquids can be spotted without any pretreatment. However, solid samples need to be dissolved in a suitable solvent as only liquids can be applied to the chromatography paper.

Spotting and Developing

Spotting of the sample is done with the help of a capillary tube or automated applicator.. The sample is applied as a neat spot on a horizontal line drawn with a pencil close to one edge. Allow the spot to dry and then immerse the paper in the developing chamber as per the selected technique with the marked spot above the solvent level.

The solvent begins to move and draws the sample components differentially along with it. At the end of the development take out the paper and mark the solvent front with another line .Allow the paper to dry in drying cabinets with the provision of electrical heating before visualisation

Commonly Encountered Problems

  • Overlapping and oversized spots- Such spots can be differentiated by using other techniques such as two-dimensional chromatography
  • Uneven advance of solvent front – It is a common problem which can lead to inaccuracies in calculation of Rf values due to uneven advance of spots. The reasons for this could be unevenness in cutting of paper sheet or not sufficient solvent in chamber to travel up the plate
  • Streaking – high concentrations often lead to streaking instead of single well resolved spots. The problem can be overcome by dilution of the sample
  • Washing away of applied spot leads to errors. Sample spot gets submerged in the developing solvent if care is not exercised at time of immersion of paper into the developing chamber.
  • Spotting above or below the marked pencil line can lead to errors in calculation of Rf values

Spot visualization is a pre-requirement for interpretation of the chromatogram. Visualisation techniques will be taken up in a subsequent article.

About Dr. Deepak Bhanot

Dr Deepak Bhanot is a seasoned professional having nearly 30 years expertise beginning from sales and product support of analytical instruments. After completing his graduation and post graduation from Delhi University and IIT Delhi he went on to Loughborough University of Technology, UK for doctorate research in analytical chemistry. His mission is to develop training programs on analytical techniques and share his experiences with broad spectrum of users ranging from professionals engaged in analytical development and research as well as young enthusiasts fresh from academics who wish to embark upon a career in analytical industry.


  1. Ezhil gurusamy says:

    How a small 1-2 millimetres spot sample applied on paper chromatography is becoming a big coin size shape spot

    • The sample is applied as a concentrated spot but when it moves on the paper with the help of developing solution the solvent disperses it in two dimensions alongwith the upward movement. This results in spreading and area increase.

  2. how the spot occurs?

    • Spot results when sample component or standard is moved along the plate by the developing solvent. It gets noticed under normal light or after derivatization and with help of a UV viewing chamber.

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