If you've ever worked with FPLC systems in the laboratory or engaged in any other forms of protein purification, you've likely come across the need to introduce cooling when working with labile proteins. This article explores the issues with finding a suitable cooling option for FPLC systems, and offers the modified Foster Chromatography Cabinet supplied by GPE Scientific as the ideal laboratory solution.

FPLC Overview

FPLC, often referred to as fast protein liquid chromatography, fast performance liquid chromatography, or medium pressure liquid chromatography (MPLC) is a separation / protein purification process that focuses on providing the most yield of the target protein while maintaining the integrity and stability of the protein. Unlike HPLC, which is primarily an analytical technique, FPLC focuses on maximising yield from the purification of proteins using medium-pressure chromatography, at a comparatively lower pressure than HPLC (<5 bar) and higher flow rates (1-5 ml/min).

FPLC methods involve separating molecules, primarily based on their size, charge, or hydrophobic interactions. Chromatographic techniques such as size exclusion chromatography, ion-exchange chromatography and affinity chromatography are some of the many medium-pressure liquid chromatography branches you'd likely come across along the way.

General Principles

Standard FPLC strategy involves choosing column materials, typically a solid purification resin (almost always agarose or a polymer material). The protein of interest will bind to the beads of resin while in an aqueous buffer solution (binding buffer), but become dissociated and return to solution in an elution buffer. The mechanism of binding will depend on the type of chromatography resin selected, for example the interaction will be based on charge or ion exchange.  The sample dissolved in 100% binding buffer is injected into a sample loop, and pumped from the sample loop into the chromatography column. The proteins of interest bind to the stationary phase while other components are carried out in the mobile phase. After washing the columns are eluted according to the requirements of the technique selected.  The elution buffer for ion exchange will contain 0.5-1M salt, typically sodium chloride.  During elution the total flow rate of the buffer is kept constant, whilst the proportion of elution buffer to binding buffer is gradually increased from 0% to 100% concentration, over a set period of time, typically 20 column volumes.  This is called a linear gradient elution.

At some point during this process, the bound proteins individually dissociate, causing them to leave the stationary phase and appear in the eluant, which then passes through detectors measuring salt concentration, protein concentration and pH. As each protein is eluted, it appears as a peak on a UV detector at 280nm UV and is collected in the fraction collector for further use.

Problems with Uncooled FPLC

As with any chromatographic techniques, or processes that involve pressure and pumping, heat often comes into play, and is especially a pest when it comes to proteins. Structural analytical techniques such as NMR (Psst... as a side note GPE are premium suppliers of NMR tubes and Benchtop NMR Spectrometers!) require the analyte to keep its structural integrity. Molecular biology researchers looking at the biological activity of proteins cannot afford them to become denatured and lose that activity.

In applications where the temperatures can get very high if not managed, the chromatography columns are at risk of being permanently damaged. Cooling in this instance is essential to protect the often expensive equipment.

On a more precise note, depending on the level of purity required, cooling may also be necessary to achieve consistent binding & elution kinetics of the proteins within the column and maintain a steady flow rate.

The Solution?

Chromatography systems do not inherently come with cooling, and options are limited to find a solution to this. Working in a laboratory with a cold-room? You're in luck! Otherwise, some alternative options include cooled fraction collectors or chromatography columns, neither of which offer a water-tight solution, and often come at higher cost and lead to increased noise levels in the laboratory.

What about simply just using a laboratory refrigerator? They can achieve target temperatures of +4°C, the typical temperature chosen to preserve the protein integrity whilst maintaining flow rate capabilities. The problem comes down to finding a setup that is feasible for the entire FPLC system. How do you deliver power? Where should the fraction collector sit given that the shelves may not be at the correct height intervals? How do you fit longer columns? Can you extract waste? Standard laboratory refrigerators just simply aren't designed to handle FPLC systems.

The Solution.

Having come across the same issues in the past, Jencons Scientific adopted the use and sale of Foster Cabinets modified for FPLC use. A wide range of customisations were introduced to allow the ergonomic placement of ÄKTA FPLC and other medium pressure chromatography systems.

Cabinets come in two sizes - a single and double door version to fit one or two systems, both with the capacity for the fraction collector and longer chromatography columns.

Custom modifications include customer-chosen access ports for comms and waste, internal electric sockets to power the FPLC systems. Two-tier racks for the system and fraction collector, scaffold rods for columns that can't be attached to the FPLC system. Over-temperature cut-outs to prevent the columns overheating, and sliding shelves and steel platforms for additional storage.

Designed and built specifically for FPLC, Foster chromatography cabinets provide the ultimate solution for your ÄKTA and NGC systems.

Adjusting FPLC for cooling

There is little difference in terms of process and protocol when operating at low temperatures for FPLC. The main thing to consider is the buffer conditions. If the buffers were prepared at room temperature, they should ideally be refrigerated along with the FPLC system at least 12h before using them, to ensure their temperature stability. pH should also be checked after the temperature has stabilised, especially with Tris buffers.

Lower liquid temperatures will also lead to higher viscosity of the buffers, and so operating pressures will increase compared to room temperature if all else is equal. Consider adjusting flow rates to avoid exceeding the pressure limit of the column.

If using any of the equipment or buffers at room temperature for other experiments, it is a good idea to let the buffers and all equipment reach room temperature conditions before disassembly to avoid air bubbles.

There are more specifics to be followed depending on your chosen system, which are all slightly different. The user documentation should contain further instructions for operating at cold temperatures, and should be followed ahead of any of the general tips here.

Written By: Robert Williams, Penny Hamlyn