Adsorbent
in paper chromatography is a sheet of paper of suitable texture and
thickness. Chromatographic separation may proceed through the action
of a single liquid phase in a process analogous to adsorption
chromatography in columns. The partitioning mechanism may contribute
significantly to the separation since the natural water content of
the paper, or selective imbibition of a hydrophilic component of the
liquid phase by the paper fiber, may be regarded as a stationary
phase.
Two-phase
system may be alternatively used where chromatogram is developed by
slow passage of the other, mobile phase over the sheet. Either
ascending or descending, which the solvent is carried up the paper
capillary forces or solvent flow is also assisted by gravitational
force, respectively.
Rf
values may vary in the chromatograms developed in the direction of
the paper grain with others developed at right angles to the grain,
thus, orientation of paper grain with respect to solvent flow should
maintain constant in series of chromatograms.
Descending
Chromatography/Ascending Chromatography
In
descending chromatography, the mobile phase is downward on the
chromatographic sheet which assisted by gravitational force. In
ascending chromatography, the lower edge of the sheet is dipped into
the mobile phase to permit the mobile phase to rise on the
chromatographic sheet by capillary action.
Apparatus
- Vapor-tight chamber – the chamber is constructed of glass, stainless steel, or porcelain and is so designed as to permit observation of the progress of the chromatographic run without opening of the chamber. It should be provided with inlets for addition of solvent or releasing internal pressure.
- Rack corrosion-resistant material – about 5cm shorter than the inside height of the chamber. Serves as support for solvent troughs and antisiphon rods which, in turn, hold up the chromatographic sheets.
- One or more glass trough – capable of holding a volume o solvent greater than that needed for one chromatographic run.
- Heavy glass antisiphon rods – to be supported by the rack and running outside of, parallel to, and slightly above the edge of the glass trough.
- Chromatographic sheets – of special filter paper at least 2.5cm wide and not wider than the length of the troughs are cut to a length approximately equal to the height of the chamber. A fine pencil line is drawn horizontally acrss the filter paper at a distance from one end such that.
- Substances to be analyzed are dissolved in a suitable solvent. The spot is delivered with micropipette containing 1 – 20 mcg of compound and placed 6 – 10mm not less than 3cm apart the pencil line.
- The spotted chromatographic sheet is suspended in the chamber by use of the antisiphon rod, which holds the upper end of the sheet in the solvent trough. Solvent prescribed must be covered the bottom and walls of the chamber. Solvent vapor should saturate the chamber.
- A volume of the mobile phase in excess of the volume required for complete development of the chromatogram is saturated with the immobile phase by shaking. After saturation, introduce the mobile solvent through the inlet. Closed the inlet and allow the mobile solvent to travel on the paper. Avoid run down the mobile solvent while removing of chromatogram. Mark the location of solvent front and dried.
- Observe and measure directly or after suitable development to reveal location of the spots of the isolated development.
