Paper chromatography is a technique in which separation of the components of a mixture is achieved through the action of a single liquid phase in a process similar to adsorption chromatography in columns and in which a sheet of paper of suitable texture and thickness acts as the adsorbent. Since the natural water content of the paper, or selective inhibition of a hydrophilic component of the liquid phase by the paper may be regarded as a stationary phase, a partitioning of the solute between two phases may contribute to the separation of components.
Alternatively, a two-phase system may be adopted in which the paper is impregnated with one of the phases, which then remains stationary and the chromatogram is developed by slow movement of the other mobile phase over the sheet. Development may be ascending in which case the mobile phase is carried up the paper by capillary forces, or descending in which case the mobile phase is also assisted by gravitational force.
Related: Principle of HPLC
Related: Principle of HPLC
(a) A vapour-tight tank of glass, porcelain or stainless steel provided with inlets for addition of solvent or for releasing internal pressure and so designed that the progress of the chromatographic run can be observed without opening the tank.
(b) A rack of corrosion-resistant material about 5 cm shorter than the inside height of the tank, to serve as support for the solvent trough and for antisiphoning rods which in turn, hold the chromatographic sheets.
(c) Glass troughs, longer than the width of the chromatogram sheets and holding a volume of solvent greater than that needed for one chromatographic run.
(d) Antisiphoning rods of heavy glass to be supported by the rack, and running outside of, parallel to and slightly above the edge of the glass trough.
(e) Chromatographic sheets of special filter paper not less than 25 mm wide and not wider than the length of the troughs, cut into strips to a length approximately equal to the height of the tank; the paper is cut so that the mobile phase runs in the direction of the grain of the paper. A fine pencil line is drawn horizontally across the filter paper at a distance from one end such that, when the sheet is suspended from the antisiphoning rods with the upper end of the paper resting in the trough and the lower portion hanging free into the chamber, the line is located a few cm below the rods. Care should be taken to avoid contaminating the filter paper by excessive handling or by contact with dusty surfaces.
Descending Paper Chromatography
MethodThe substance under examination is dissolved in a suitable solvent. The volumes of the resulting solution are applied by means of micropipette in 6 to 10 mm spots along the pencil line not less than 3 cm apart. If the total volume to be applied would produce spots of a diameter greater than 6 to 10 mm, it is applied in separate portions to the same spot, each portion being allowed to dry before the next is added, or alternatively, the solution may be applied in the form of bands (10 to 20 mm x 2 to 6 mm unless otherwise specified) along the pencil line.
Use sufficient of the saturating solvent prescribed in the monograph to form a 25 mm layer in the bottom of the tank. Close the tank and allow to stand for 24 hours at room temperature.
Suspend the spotted chromatographic sheet in the tank by the use of antisiphoning rods which hold the upper end of the sheet in the solvent trough. It is important to ensure that the portion of the sheet hanging below the rods is freely suspended in the tank without touching the rack or the tank walls or the fluid in the tank.
Close the tank and allow the paper to stand in it for 1.5 hours. Introduce through the inlet into the solvent through a sufficient quantity of the prescribed mobile phase, close the tank and allow development to proceed for the distance or the time prescribed in the monograph, protecting the paper from bright light during development. Remove the paper from the tank and allow it to dry in air at the temperature specified in the monograph. Visualise the spots as described in the monograph. The paper section(s) (see under Apparatus) predetermined to contain the separated components may be cut out and eluted by an appropriate solvent, and the solutions may be made up to a known volume and quantitatively analysed by appropriate chemical or instrumental methods.
Ascending Paper Chromatography
MethodThe test materials are applied to the chromatographic sheets as directed under Descending Paper Chromatography and above the level to which the paper is dipped into the developing solvent. The top of the tank contains a device from which the paper is suspended, and which is capable of being lowered without opening the tank. In the bottom of the tank is a trough to contain the mobile phase into which the paper may be lowered.
Use sufficient of the prescribed mobile phase to form a 25 mm layer in the trough. If a two-phase system is used, both phases are added. If prescribed, pour the saturating solvent between the trough and the walls of the tank. Close the tank and allow to stand for 24 hours at room temperature. Insert the prepared paper into the tank, close the lid and allow to stand for 1.5 hours. Lower the paper into the mobile phase and allow development to proceed for the distance or the time prescribed in the monograph, protecting the paper from bright light during development. Remove the paper from the tank and allow it to dry in air at the temperature specified in the monograph.
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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