Chromatography

Chromatography Systems

Depending on the needs of your application, chromatography can be performed in a simple, gravity-fed, open column, in a centrifuge tube, or in an automated, fully plumbed, and pump-driven system.

A typical automated chromatography system has four parts: a pump, an injection valve, a column, and a detector. The pump delivers the mobile phase such as a solvent or buffer. The injection valve allows a defined amount of sample to be pumped onto the column. The column holds the stationary phase media, also called column packing or resin. The detector is a real-time monitoring device, which allows the researcher to determine the amount of components in the eluate as they are eluted from the column.

A basic column chromatography system using a pump and detectors.

As research needs evolve, many enhancements can be made to this basic setup such as the addition of an autosampler; a fraction collector; multiple detectors to monitor conditions such as temperature, conductivity, and pressure; and a variety of injection valves to automate sample delivery and manage multiple columns.

Chromatography is routinely used for two primary purposes: analytic work (identifying and determining the amounts of various components in a sample, for example, in metabolomics, forensics, and food testing) and preparative work (the purification of single components for further study or use as final products, for example, antibody-based therapeutics).

Preparative chromatography is a widely used laboratory technique for the enrichment or isolation of a specific component from a mixture. Preparative chromatography systems nearly always involve a fraction collector and/or a valve arrangement to allow the collection of discreet fractions of the eluate over time.

Analytic chromatography commonly utilizes a method known as high-performance liquid chromatography (HPLC — generally performed at high pressure and thus alternatively known as high-pressure liquid chromatography). HPLC has become the technique of choice for the identification, quantification, and purity analysis of both small molecules and proteins. When paired with detection systems such as a mass spectrophotometer, sample components can be separated, identified, and quantified in a matter of minutes.

Bio-Rad offers a complete line of laboratory-scale instruments that are flexible, upgradeable, and easy to use. For assistance with selecting an optimal system configuration, please use the chromatography system configuration tool below.

Column Chromatography for Protein Purification

Biomolecules can be divided into four broad categories: amino acids (proteins and peptides), nucleic acids (DNA and RNA), carbohydrates (sugars, cellulose, and similar semipolar organic compounds), and lipids (fats, fatty acids, and sterols — mostly nonpolar hydrocarbon-based organic compounds).

Column chromatography is especially important in protein purification. Proteins are complex and fragile molecules that often must maintain their conformation to retain biological activity. Nucleic acids, lipids, and carbohydrates require no special handling and can often be separated and analyzed using many techniques; in contrast, proteins require gentler handling and may undergo denaturation or unfolding when subjected to the harsher conditions used to purify other classes of molecules. Thus, column chromatography is the principal method for protein purification.

Bio-Rad offers a wide range of media for diverse research needs. Use the table below to select the best media for your application.

What Molecules Can Be Purified by Chromatography?

Chromatography Media Selection Guide

* B, bottle; C, cartridge (1 ml or 5 ml); GC, gravity column; SC, spin column; HPLC, high-pressure column; MPC, medium-pressure column.
** +, low suitability; ++, moderate suitability; +++, suitable; ++++, high suitability.

Prepacked Analytical-Scale Chromatography Media Cartridges by Application

* Binding capacity based on bulk media; check individual instruction manuals for run conditions and specifications.

Common Chromatography Method Elements

Chromatographic methods used in protein purification usually involve direct binding or interaction with the stationary phase via affinity, ion exchange, or hydrophobic interactions. Size exclusion chromatography (SEC), also referred to as gel filtration or gel permeation, is the only method that does not involve direct binding.

In all purification methods, there are common elements to a chromatography run.

• The column is equilibrated in a mobile phase or buffer to ensure uniform column conditioning
• The sample is applied — with a pipet in an open column, with an injection valve and loop in a plumbed system or, in the case of a large sample volume, with a pump
• Proteins of interest are eluted from the column using isocratic or gradient flow of the mobile phase
• The proteins are detected in the eluate using a detector. An optical absorbance detector is often used to measure the light absorbed at specified wavelengths by molecules in the eluate stream. The most common wavelengths used are in the UV range:
  • 190–230 nm, for peptide bonds
  • 280 nm, for the aromatic rings found in some amino acids
• The recorded absorbance values are plotted against time or volume as a chromatogram
• The chromatogram is a record of the run and is also used to analyze protein characteristics such as size, charge, and concentration, providing an indicator of column packing quality or other separation issues
• Finally, the proteins eluted from the column are collected in a fraction collector for subsequent analysis.

A typical absorbance chromatogram.