Developing the Chromatogram involves three stages:
- Sample preparation
- Sample application
- Developing of the chromatogram
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.