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In paper chromatography the adsorbent 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. Since the natural water content of the paper, or selective imbibition of a hydrophilic component of the liquid phase by the paper fibers, may be regarded as a stationary phase, a partitioning mechanism may contribute significantly to the separation.
Alternatively, a two-phase system may be used. The paper is impregnated with one of the phases, which then remains stationary (usually the more polar phase in the case of unmodified paper). The chromatogram is developed by slow passage of the other, mobile phase over the sheet. Development may be ascending, in which case the solvent is carried up the paper by capillary forces, or descending, in which case the solvent flow is also assisted by gravitational force.
Differences in the value of RF have been reported where chromatograms developed in the direction of the paper grain (machine direction) are compared with others developed at right angles to the grain; therefore, the orientation of paper grain with respect to solvent flow should be maintained constant in a series of chromatograms. (The machine direction is usually designated by the manufacturer on packages of chromatography paper.)
Descending Chromatography
In descending chromatography, the mobile phase flows downward on the chromatographic sheet.
Apparatus— The essential equipment for descending chromatography consists of the following:
A vapor-tight chamber provided with inlets for addition of solvent or for releasing internal pressure. The chamber is constructed preferably 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. Tall glass cylinders are convenient if they are made vapor-tight with suitable covers and a sealing compound.
A rack of corrosion-resistant material about 5 cm shorter than the inside height of the chamber. The rack serves as a support for solvent troughs and for antisiphon rods which, in turn, hold up the chromatographic sheets.
One or more glass troughs capable of holding a volume of solvent greater than that needed for one chromatographic run. The troughs must also be longer than the width of the chromatographic sheets.
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 2.5 cm 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 across the filter paper at a distance from one end such that, when the sheet is suspended from the antisiphon 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 centimeters below the rods. Care is necessary to avoid contaminating the filter paper by excessive handling or by contact with dirty surfaces.
Procedure— The substance or substances to be analyzed are dissolved in a suitable solvent. Convenient volumes, delivered from suitable micropipets, of the resulting solution, normally containing 1 to 20 µg of the compound, are placed in 6- to 10-mm spots not less than 3 cm apart along the pencil line. 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.
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. The bottom of the chamber is covered with the prescribed solvent system. Saturation of the chamber with solvent vapor is facilitated by lining the inside walls with paper that is wetted with the prescribed solvent system. It is important to ensure that the portion of the sheet hanging below the rods is freely suspended in the chamber without touching the rack or the chamber walls or the fluid in the chamber. The chamber is sealed to allow equilibration (saturation) of the chamber and the paper with the solvent vapor. Any excess pressure is released as necessary. For large chambers, equilibration overnight may be necessary.
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 equilibration of the chamber, the prepared mobile solvent is introduced into the trough through the inlet. The inlet is closed and the mobile solvent phase is allowed to travel the desired distance down the paper. Precautions must be taken against allowing the solvent to run down the sheet when opening the chamber and removing the chromatogram. The location of the solvent front is quickly marked, and the sheets are dried.
The chromatogram is observed and measured directly or after suitable development to reveal the location of the spots of the isolated drug or drugs. The paper section(s) predetermined to contain the isolated drug(s) may be cut out and eluted by an appropriate solvent, and the solutions may be made up to a known volume and quantitatively analyzed by appropriate chemical or instrumental techniques. Similar procedures should be conducted with various amounts of similarly spotted reference standard on the same paper in the concentration range appropriate to prepare a valid calibration curve.
Ascending Chromatography
In ascending chromatography, the lower edge of the sheet (or strip) is dipped into the mobile phase to permit the mobile phase to rise on the chromatographic sheet by capillary action.
Apparatus— The essential equipment for ascending chromatography is substantially the same as that described under Descending Chromatography.
Procedure— The test materials are applied to the chromatographic sheets as directed under Descending Chromatography, and above the level to which the paper is dipped into the developing solvent. The bottom of the developing chamber is covered with the developing solvent system. If a two-phase system is used, both phases are added. It is also desirable to line the walls of the chamber with paper and to saturate this lining with the solvent system. Empty solvent troughs are placed on the bottom of the chamber, and the chromatographic sheets are suspended so that the end on which the spots have been added hangs free inside the empty trough.
The chamber is sealed, and equilibration is allowed to proceed as described under Descending Chromatography. Then the developing solvent (mobile phase) is added through the inlet to the trough in excess of the solvent required for complete moistening of the chromatographic sheet. The chamber is resealed. When the solvent front has reached the desired height, the chamber is opened and the sheet is removed and dried.
Quantitative analyses of the spots may be conducted as described under Descending Chromatography.