Introduction to Medium-Pressure Chromatography

Medium pressure chromatography has been used for more than a century to separate and purify molecules. It is typically performed as part of the larger laboratory workflow, either to separate a mixture of molecules — preparative chromatography — or to quantitate highly purified molecules or compounds — analytical chromatography. Medium pressure chromatography has many applications that can be classified either by the type of chromatographic method used (for example, ion exchange vs. affinity chromatography) or by the types of molecules being isolated and their characteristics (such as antibodies or organic acids).

Flexibility
Medium-pressure chromatography systems can perform all types of chromatography. They are routinely used for high-resolution ion exchange and high-resolution size exclusion separations. Both multi-dimensional purification schemes and simple tagged recombinant protein purification applications can be easily performed.

High resolution
Medium-pressure chromatography systems produce enough pressure to accommodate higher resolution chromatography media (5–15 µm beads).

Automation
Most medium-pressure chromatography systems use software or control systems for unattended operation. Although some low pressure systems offer basic automation, medium pressure systems are designed to operate without user intervention via intuitive software.

Backpressure
Medium-pressure chromatography systems cannot move the buffer along a flow path when the pressure capabilities of the system exceed the resistance or backpressure after the pump. The following factors will increase backpressure and should be considered in the setup and use of the system:

• Tubing inner diameter and/or connections
• Lower temperatures like those in coldrooms or refrigerators. Low temperature increases the viscosity of the solvent or buffer and thereby increases the backpressure. This will be more pronounced with buffers containing a high concentration of dissolved salts (for example, 1 M NaCl)
• Certain buffer gradients
• Smaller beads or lower porosity in the chromatography media

Sample application
Buffer pumps are used to pump samples onto the column because the column backpressure is relatively high. The plumbing can vary depending on the volume or clarity of the sample. Some examples are:

• With relatively small sample volumes (<5 ml) a fixed volume sample loop plumbed to an injection valve is generally used. The sample is manually injected or automatically pumped into the loop while it is not in line with the flow path. When the loop is full, the injection valve is switched and the sample loop is placed in line with the flow path. The sample flows out of the loop and onto the column. Usually after 3–5 loop volumes the loop is switched out of the flow path and the separation continues
• With sample volumes >5 ml, a special, variable volume sample loop plumbed to an injection valve is used. The sample is manually injected or automatically pumped into the loop while it is not in line with the flow path. The variable volume loop has two chambers with a movable seal between them. The design accommodates a range of volumes without diluting the sample with buffer. When appropriate, the injection valve is switched to place the sample loop in line with the flow path. The sample flows out of the loop and onto the column. Usually after 3–5 loop volumes, the loop is switched back out of the flow path and the separation continues
• For sample volumes >20 ml, there are two options:
  • A dedicated sample pump can be used to apply the sample directly onto the column. Because the pump is separate from the gradient pumps, it can be cleaned thoroughly with minimal risk of cross-contamination
  • The system or gradient pumps can be used to apply the sample. Although this is often used with larger volume samples, the pumps must be thoroughly cleaned and sanitized between runs to prevent cross contamination and fouling of the piston, seals, and check valves. Additionally, a fine filter mesh (0.22 µm) is recommended to remove particulates from the sample

Medium-pressure chromatography systems follow the basic flow path: buffer reservoir to pump, to injection valve, to column, to UV detector, to conductivity monitor, to pH monitor, to a fraction collector or outlet valve. Medium-pressure systems are often used for high resolution separations so it is important to minimize all tubing lengths and dead volumes. Extra volume in tubing or components will cause "band broadening," the diffusion of distinct peaks into one another.

Fluidics setup and flow path design can be easily achieved using software and instrumentation to indicate the order in which parts should be connected, for example, using the Point-to-Plumb™ and guided fluidics selection tools on the NGC systems. Application-specific flow paths can be easily plumbed using a series of available template designs.

Fig. 1. Medium-pressure chromatography system setup and flow path.

Medium-pressure chromatography is used for the full range of chromatographic separations including all protein purification techniques. Complex gradients, or changes in the proportion of two buffers, are easily possible with medium-pressure chromatography systems. These gradients can be generated by a switch/proportioning valve before the pump inlet or by varying the speed of two separate pumps. Both strategies can produce accurate, reproducible gradients. Medium-pressure systems can be designed to meet the requirements of the application, from a simple inject and elute strategy to a fully automated system using additional modules and accessories such as an autosampler, application-specific valves, gradient mixers, fraction collectors, and UV, conductivity, pH, refractive index, and other detectors.

Bio-Rad offers two families of medium-pressure chromatography systems, the NGC platform and the BioLogic DuoFlow™ platform. Both systems are modular, allowing you to easily scale up and add on new capabilities. The NGC system offers new system capabilities and tools to enable a simpler workflow. The Point-to-Plumb feature provides a guided light path to allow quick visualization of where to plumb your system and the Tier Rotate™ feature reduces path length to streamline your separations. Explore the NGC system tour to learn more and simply design your own personalized NGC system using our configurator tool.