Bio-Rad BioLogic Signal Import Module (SIM) - Manual (Page 186)

MULTIPLE COLUMNS ADVANCED SYSTEM APPLICATIONS
9-5
9.3 MULTI-DIMENSIONAL CHROMATOGRAPHY
Multi-dimensional chromatography is used to perform multi-step chromatographic purifications in a single
automated run. This technique is particularly useful for purifications done on a routine basis. Multi-dimensional
experiments usually involve some type of affinity purification step followed by a high-resolution step such as
ion exchange, hydroxyapatite or size exclusion chromatography. Table 9-1 shows a few common
multi-dimensional chromatography experiments types.
Table 9-1. Common Multidimensional Chromatography Experiments
Experiment Type Dimension 1 Dimension 2 Dimension 3
2D Affinity Desalt
2D Affinity Size Exclusion
3D Affinity Desalt Ion Exchange
Most of the common protein purification techniques can be incorporated into multidimensional
chromatography experiments. Generally, purification techniques that are sample independent provide the
greatest benefit when included as part of a multidimensional experiment. Examples include: affinity
chromatography of proteins tagged with His tags, glutathione-s-transferase and maltose binding protein; size
exclusion chromatography and ion exchange chromatography.
Both two- and three-dimensional chromatography experiments can be performed using the BioLogic
DuoFlow system. The type and complexity of experiments that you can run will depend on the hardware
installed on your system. Maximizer and Pathfinder systems, with their increased valve capacity, can run the
most complex experiments.
System Setup
When setting up a multi-dimensional chromatography protocol it is important to consider a few essential
factors. First of all, sequential purification protocols must be compatible. In other words, the material
collected in one dimension must be suitable for injection in the next dimension in terms of composition, pH,
concentration etc. Another factor to consider is the number of fractions to be collected and the capacity of
your fraction collector. Although fractions can be collected during the entire experiment it is usually best to
only collect fractions during the final dimension in order to avoid running out of tubes. This is particularly
important if you are purifying multiple samples.
The setup of a multidimensional experiment can be complex and requires that care be taken in plumbing the
valves and equilibrating the system. The placement of valves, pumps and detectors should be planned so as
to minimize tube lengths. Prior to connecting columns to the system make sure that all the tubing has been
filled with buffer.
A few plumbing diagrams have been supplied in the BioLogic software online help that show how to plumb
both two and three-dimensional chromatography experiments. Method templates designed for these
plumbing diagrams are available in a file named "Multi-D Templates.zib" located in the BioLogic directory.