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CHROMATOGRAPHY INTRODUCTION 621 ( chromatography-introduction-621 )

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First Supplement to USP 40–NF 35 Physical Tests / á621ñ Chromatography 1 á621ñ CHROMATOGRAPHY INTRODUCTION Chromatographic separation techniques are multistage separation methods in which the components of a sample are dis- tributed between two phases, of which one is stationary and the other is mobile. The stationary phase may be a solid or a liquid supported on a solid or a gel. The stationary phase may be packed in a column, spread as a layer, distributed as a film, or applied by other techniques. The mobile phase may be in a gaseous or liquid form, or a supercritical fluid. The separation may be based on adsorption, mass distribution (partition), or ion exchange; or it may be based on differences among the physicochemical properties of the molecules, such as size, mass, and volume. This chapter contains general procedures, defini- tions, and calculations of common parameters and describes general requirements for system suitability. The types of chroma- tography useful in qualitative and quantitative analyses employed in USP procedures are column, gas (GC), paper, thin-layer (TLC) [including high-performance thin-layer chromatography (HPTLC)], and pressurized liquid chromatography [commonly called high-pressure or high-performance liquid chromatography (HPLC)]. GENERAL PROCEDURES This section describes the basic procedures used when a chromatographic method is described in a monograph. These pro- cedures are followed unless otherwise indicated in the individual monograph. Paper Chromatography STATIONARY PHASE The stationary phase is a sheet of paper of suitable texture and thickness. Development may be ascending, in which the solvent is carried up the paper by capillary forces, or descending, in which the solvent flow is also assisted by gravitational force. The orientation of paper grain, with respect to solvent flow, is to be kept constant in a series of chromatograms. The machine direction is usually designated by the manufacturer. APPARATUS The essential equipment for paper chromatography consists of a vapor-tight chamber with inlets for the addition of solvent and a rack of corrosion-resistant material about 5 cm shorter than the inside height of the chamber. The rack serves as a sup- port for solvent troughs and antisiphon rods that, in turn, hold up the chromatographic sheets. The bottom of the chamber is covered with the prescribed solvent system or mobile phase. Saturation of the chamber with solvent vapor is facilitated by lining the inside walls with paper wetted with the prescribed solvent system. SPOTTING The substance or substances analyzed are dissolved in a suitable solvent. Convenient volumes delivered from suitable micro- pipettes of the resulting solution, normally containing 1–20 mg of the compound, are placed in 6- to 10-mm spots NLT 3 cm apart. DESCENDING PAPER CHROMATOGRAPHY PROCEDURE 1. A spotted chromatographic sheet is suspended in the apparatus, using the antisiphon rod to hold the upper end of the sheet in the solvent trough. [NOTE—Ensure that the portion of the sheet hanging below the rods is freely suspended in the chamber without touching the rack, the chamber walls, or the fluid in the chamber.] 2. 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. 3. After equilibration of the chamber, the prepared mobile phase is introduced into the trough through the inlet. 4. The inlet is closed, and the mobile solvent phase is allowed to travel the desired distance down the paper. 5. The sheet is removed from the chamber. 6. The location of the solvent front is quickly marked, and the sheet is dried. 7. The chromatogram is observed and measured directly or after suitable development to reveal the location of the spots of the isolated drug or drugs.



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