USP〈621〉 CHROMATOGRAPHY

INTRODUCTION

Chromatographic separation techniques are multistage separation procedures in which the components of a sample are distributed between two phases, one of which is stationary while 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, or distributed as a film, etc. The mobile phase may be gaseous or liquid or supercritical fluid. The separation may be based on adsorption, mass distribution (partition), ion exchange, etc., or may be based on differences in the physicochemical properties of the molecules such as size, mass, volume, etc.

Portions of the present general chapter text that are national USP–NF text, and therefore not part of the harmonized text, are marked with symbols (♦♦) to specify this fact.

♦This chapter describes general procedures, definitions, and calculations of common parameters and generally applicable requirements for system suitability.

The types of chromatography useful in qualitative and quantitative analysis employed in USP procedures are column, gas, paper, thin-layer (including high-performance thin-layer chromatography), and pressurized liquid chromatography (commonly called high-pressure or high-performance liquid chromatography).

GENERAL PROCEDURES

This section describes the basic procedures used when a chromatographic method is described in a monograph. The following procedures 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 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 support for solvent troughs and for 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 micropipets of the resulting solution, normally containing 1–20 µg of the compound, are placed in 6- to 10-mm spots not less than 3 cm apart.

descending paper chromatography procedure

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.]

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.

After equilibration of the chamber, the prepared mobile phase 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.

The sheet is removed from the chamber.

The location of the solvent front is quickly marked, and the sheet is 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.

Source from USP and Please refer to USP for details:https://online.uspnf.com/uspnf/document/1_GUID-6C3DF8B8-D12E-4253-A0E7-6855670CDB7B_8_en-US

plastic pharmaceutical packaging

Polymer Vial 2ml 5ml 10ml for cell and gene therapies