Introduction to Column Chromatography Methods and Instrumentation

Column chromatography is a method commonly used to separate molecules in complex mixtures. Unlike planar chromatography, in column chromatography, the stationary phase or resin is packed into a column. The mobile phase is then passed through the packed stationary phase to achieve separation. The purpose of this separation can be analytical or preparative.

Both gas chromatography and liquid chromatography separations are performed on columns. Our discussion here focuses on liquid column chromatography systems.

Related Topics: Chromatography, Liquid Chromatography Principles

Column Chromatography Systems
Preparative Chromatography

Fig. 1. A basic chromatography system using a pump and detectors.

Column chromatography is performed on a packed, three-dimensional stationary phase inside a glass, plastic, or metal column and can be used for both preparative and analytical purposes. Column chromatography techniques encompass a broad range of applications based on diverse solid and liquid phase chemistries and column configurations, enabling precise analyses as well as resolution of complex mixtures that would be difficult or impossible to separate by other means.

There are many systems available for liquid chromatography, and the choice of system is dictated by the complexity of the desired separation. The mobile phase can be passed over the stationary phase by gravity flow, using centrifugal force, or using an automated pump-based system. Pump-based systems can range from simpler low-pressure chromatography systems consisting of a peristaltic pump and a fraction collector to complex medium-pressure chromatography systems and high-pressure liquid chromatography (HPLC) systems that include multi-wavelength detectors and buffer blending capabilities.

Column chromatography is therefore classified according to the type of fluid flow system used:

  • Gravity chromatography
  • Low-pressure chromatography
  • Medium-pressure chromatography (including fast protein liquid chromatography)
  • High-pressure/high-performance liquid chromatography (HPLC)

Gravity chromatography is commonly used for small-scale, preparative chromatography. Low- and medium-pressure chromatography systems are commonly used for medium-scale preparative applications. Medium-pressure systems are more versatile and can be used for both analytical and preparative purposes. High-pressure systems permit high-resolution separation of either proteins or small molecules and are most commonly used for analytical purposes.

Gravity Chromatography
Gravity Chromatography
Fig. 2. Disposable plastic chromatography columns. Bio-Rad offers a wide selection of empty and prepacked gravity and spin chromatography columns.

As its name implies, gravity chromatography uses gravity to pass sample and buffers across the column resin. Although prepacked gravity columns are commercially available, it is not uncommon for users to pack their own columns. Small-volume columns (~1.5 ml) designed for quick flowthrough by spinning in a microcentrifuge provide a convenient method for rapidly purifying many small samples. Elution from the column is often followed by quantification of  protein or nucleic acid concentration in collected fractions using colorimetric methods such as Bradford or Lowry assays (perhaps followed by SDS-PAGE) or by fluorometry or spectrophotometry, typically in the UV-visible range. Gravity chromatography is a low-cost, simple method of preparative chromatography, however it does not afford high resolution.

Applications: small-scale, rapid preparative chromatography of nucleic acids, peptides, and proteins.

Visit our Gravity Chromatography page to learn more.

Low-Pressure Chromatography
Low-Pressure Chromatography
Fig. 3. A typical low-pressure chromatography system. Bio-Rad carries a range of low-pressure chromatography columns for use with the BioLogic™ low-pressure chromatography system and other systems.

Low-pressure chromatography systems are operated at less than 50 psi (0.35 MPa). These systems require a sample pump and are often equipped with fraction collectors, gradient capabilities, and detectors to monitor column elution. Low-pressure systems are compatible with prepacked columns and offer more flexibility and higher resolution than gravity chromatography. Nonetheless, low-pressure chromatography is not a high-resolution chromatography method suitable for complex protein purifications.

Applications: medium-scale preparative chromatography of native or recombinant proteins.

Visit our Low-Pressure Chromatography page to learn more.

Medium-Pressure Chromatography
Medium Pressure Chromatography
Fig. 4. A modern medium-pressure chromatography system. Empty and prepacked medium-pressure chromatography columns are available for use with the NGC™ medium-pressure chromatography system and other systems.

Medium-pressure chromatography, also referred to as fast protein liquid chromatography, is more suitable for complex purifications and can also be used for analytical purposes. Medium-pressure chromatography is conducted at operating pressures that are actually rather high, up to 3,500 psi (24 MPa). Medium-pressure chromatography systems often include additional capabilities such as column switching valves, advanced gradient capabilities, and multi-wavelength detectors. These systems are compatible with a wide range of prepacked columns and resins and can therefore be used for simple recombinant protein purifications as well as for complex analytical purposes.

Applications: preparative and analytical chromatography of a wide variety of molecules, ranging from nonvolatile organics to nucleic acids, peptides, and proteins.

Learn more on our Medium-Pressure Chromatography page.

High-Pressure Liquid Chromatography (HPLC)
High pressure liquid chromatography (HPLC)
Fig. 5. Bio-Rad’s Aminex® prepacked HPLC columns are popular for small-molecule analysis.

High-pressure liquid chromatography (HPLC), alternatively known as high-performance liquid chromatography, is conducted at very high pressures — up to 5,000 psi (34 MPa). HPLC is a powerful analytical tool providing high resolution and sensitivity, with the ability to detect concentrations down to parts per trillion while having very small sample requirements (in the microliter range).

Because HPLC systems can be operated at higher pressure, the resin particle size can be decreased, thereby increasing the resin’s resolution.

Since a much wider variety of compounds are analyzed using HPLC, HPLC systems usually have several types of detectors. A common combination is a UV-absorbance detector in combination with an evaporative light scattering detector (ELSD) and/or a mass spectrometer (MS). The combination of HPLC and mass spectrometry, a technique for determining the mass-to-charge ratios of molecular ions created by fragmenting the analytes after elution using electrospray, electron-impact, or other ionization methods, yields HPLC-MS, often abbreviated LC-MS,  a very powerful analytic tool used for applications such as environmental sampling, drug discovery, and proteomic studies.

HPLC, like lower-pressure forms of chromatography, can be used to separate molecules based on size (size exclusion chromatography, or SEC), hydrophobicity (hydrophobic interaction chromatography, or HIC), or charge (ion exchange chromatography, or IEX). Unlike other forms of liquid chromatography, HPLC is not suitable for affinity chromatography.

Applications: preparative and analytical chromatography of a wide variety of molecules, ranging from nonvolatile organics to nucleic acids, peptides, and proteins.

Bio-Rad does not manufacture HPLC systems, but we do carry a full selection of HPLC columns.

Column Chromatography Resources


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