Basic Knowledge of HPLC

 Liquid Chromatography 

The term liquid chromatography, as used in the compendia, is synonymous with high-pressure liquid chromatography and high-performance liquid chromatography. LC is a separation technique based on a solid stationary phase and a liquid mobile phase.

Stationary Phase: Separations are achieved by partition, adsorption, or ion-exchange processes, depending on the type of stationary phase used. The most commonly used stationary phases are modified silica or polymeric beads. The beads are modified by the addition of long-chain hydrocarbons. The specific type of packing needed to complete an analysis is indicated by the “L” designation in the individual monograph (see also the section Chromatographic Columns, below). The size of the beads is often described in the monograph as well. Changes in the packing type and size are covered in the System Suitability section of this chapter.

Chromatographic Column: The term columnincludes stainless steel, lined stainless steel, and polymeric columns, packed with a stationary phase. The length and inner diameter of the column affects the separation, and therefore typical column dimensions are included in the individual monograph. Changes to column dimensions are discussed in the System Suitability section of this chapter.  Compendial monographs do not include the name of appropriate columns; this omission avoids the appearance of endorsement of a vendor’s product and natural changes in the marketplace. See the section Chromatographic Columns for more information.

Mobile Phase: The mobile phase is a solvent or a mixture of solvents, as defined in the individual monograph.

Apparatus: A liquid chromatograph consists of a reservoir containing the mobile phase, a pump to force the mobile phase through the system at high pressure, an injector to introduce the sample into the mobile phase, a chromatographic column, a detector, and a data collection device.

Gradient Elution: The technique of continuously changing the solvent composition during the chromatographic run is called gradient elution or solvent programming. The gradient elution profile is presented in the individual monograph as a gradient table, which lists the time and proportional composition of the mobile phase at the stated time.

Procedure

1. Equilibrate the column and detector with mobile phase at the specified flow rate until a constant signal is received.
2. Inject a sample through the injector, or use an auto-sampler.
3. Begin the gradient program.
4. Record the chromatogram.
5. Analyze as directed in the monograph

• SYSTEM SUITABILITY
  System suitability tests are an integral part of gas and liquid chromatographic methods. These tests are used to verify that the chromatographic system is adequate for the intended analysis.
  The tests are based on the concept that the equipment, electronics, analytical operations, and samples analyzed constitute an integral system that can be evaluated as such.
  Factors that may affect chromatographic behavior include the following:
  • Composition, ionic strength, temperature, and apparent pH of the mobile phase
  • Flow rate, column dimensions, column temperature, and pressure
  • Stationary phase characteristics, including type of chromatographic support (particle-based or monolithic), particle or macropore size, porosity, and specific surface area
  • Reverse-phase and other surface modification of the stationary phases, the extent of chemical modification (as expressed by end-capping, carbon loading, etc.)