15. Paper Chromatography

Operation TOP > Operation 1 > Paper Chromatography

General Remarks

As an example of how to operate paper chromatography, the movie explains the analysis of Methyl Red and Methyl Orange (developing solvent: isobutyl alcohol (1), concentrated ammonia (1), distilled water (50)) which is carried out in "Organic Chemistry Experiment".

In paper chromatography, the filter paper serves as the stationary phase and support. The sample moves along with the developing solvent when the edge of filter paper is soaked into the solvent. Separation of the compounds takes place depending on their motilities.
Under fixed conditions including the stationary phase, the developing solvent, and temperature, the Rf value generally remains constant. Accordingly, the analytes are identified based on their Rf values as well as the shapes and colors of the spots.
Do not spot too much sample at once on a filter paper because a large spot gives improper results.
Keep the filter paper clean by handling its end and putting on a clean paper.

Lightly draw a circle with approximately 10 mm radius at the center of a filter paper with a pencil.
Draw cross marks on the circle as the starting points of chromatography.
Cut in the paper to make a solvent-drawing segment as depicted above.
Take the sample solution with a capillary, spot the solution lightly on one of the starting points, and dry the spot with an electric heater. Repeat this operation a few times to concentrate the sample in the small spot, which should be less than 3 mm in diameter.
Use a new capillary when a different sample is spotted.

Pour the developing solvent into a Petri dish until the bottom is completely covered. Be careful not to wet the rim of the Petri dish.
Fold the solvent-drawing segment to touch the developing solvent in the Petri dish.
Put another Petri dish with the same size on the filter paper as a lid and wait for the development.
Stop the development just before the front of the developing solvent reaches the edge of the Petri dish.

Take out the filter paper and immediately mark the front of the developing solvent with a pencil.
Dry the filter paper with an electric heater.
Record the shape and color of the spots.
Calculate Rf values.
Rf ＝ a/b
a = The distance from the starting point to the gravity center of the sample spot.
b = The distance from the starting point to the front of the developing solvent.