Introduction to Gravity Chromatography

Typical gravity column.

As pictured below, a typical gravity column involves a buffer reservoir at the top of the column with flow controlled using a stopcock or a tubing pincher at the column outlet. There are a number of refinements available to assist with the general design:

• Graduated reservoirs for column packing and buffer delivery
• Flow adaptors to minimize head space above the gel bed and minimize disruption of the gel bed during sample loading
• Column bed supports/frits to retain media
• Integral water jacket for temperature control
• Stopcocks to control flow rate
• Luer-Lok end cap fittings for tubing and serial column connections
• Fraction collectors

• Simplicity — a gravity column is easy to design, set up, and operate
• Minimal start up costs — costs are limited to the researchers' needs. No injectors, pumps, detectors, or fraction collectors are required
• Disposable — many disposable prepacked and empty polypropylene columns are available

Gravity chromatography is:

• A manual process requiring constant monitoring
• Not a high resolution method. It is ideal for single step purifications like desalting and affinity
• Time consuming. No automated injectors, pumps, or detectors are used to control sample delivery, flow rate, or fraction analysis

Reproducibility is an issue if care is not taken to normalize methods. Manual sample loading can be a challenge due to the disruption of the column bed both before and after sample loading. Use a flow adaptor to minimize these effects.

Adjusting flow rate

• With a gravity flow system, the buffer reservoir must be placed above the column to ensure flow
• The flow rate can be controlled manually using a stopcock or a simple tubing pincher at the column outlet
• It is highly recommended to measure the volume and flow rate by counting drops/unit time:
  1 drop = 50 µl. Normalized methods will lead to more reproducible separation methods
• Fraction collectors are available that can count drops and dispense fractions defined by drop number or volume

Sample loading

• Manual sample loading is not recommended in size exclusion/gel filtration chromatography. It is critical that the column bed remains undisturbed or the resolution will be compromised
• Bind and elute techniques work well, however, additional column washes become necessary due to the inherent sample dilution impact with this technique

Elution and fraction collection

• It is highly recommended that the user employ a timer during elution and fraction collection to avoid the possibility of the column running dry

Bio-Rad offers a complete line of pre-packed and empty columns for gravity chromatography.

Empty Column Selection Guide

Prepacked and empty columns.

Glass Econo-Column Columns Selection Guide
Choose the correct Econo-Column chromatography column by length and inner diameter The corresponding flow adapter is shown with the columns. A flow adapter is not needed for ID's of 0.5 or 0.7 cm. A dash ( – ) indicates that the column is not available in that configuration (L, ID, or package type).

Jacketed Econo-Column Chromatography Columns Selection Guide
Choose the correct jacketed glass Econo-Column chromatography column by length and inner diameter. The corresponding flow adapter is shown with the columns. A dash ( – ) indicates that the column is not available in that configuration (L, ID, or package type).