Fast Protein Liquid Chromatography - FPLC System Components

FPLC System Components

A typical laboratory FPLC consist of one or two high-precision pumps, a control unit, a column, a detection system and a fraction collector. Although it is possible to operate the system manually, the components are normally linked to a personal computer or, in older units, a microcontroller.

1. Pumps: GE/Pharmacia systems utilize two two-cylinder piston pumps, one for each buffer, combining the output of both in a mixing chamber. Waters systems use a single peristaltic pump which draws both buffers from separate reservoirs through a proportioning valve and mixing chamber. In either case the system allows the fraction of each buffer entering the column to be continuously varied. The flow rate can go from a few milliliters per minute in bench-top systems to liters per minute for industrial scale purifications. The wide flow range makes it suitable both for analytical and preparative chromatography.

2. Injection loop: A segment of tubing of known volume which is filled with the sample solution before it is injected into the column. Loop volume can range from a few microliters to 50ml or more.

3. Injection valve: A motorized valve which links the mixer and sample loop to the column. Typically the valve has three positions for loading the sample loop, for injecting the sample from the loop into the column, and for connecting the pumps directly to the waste line to wash them or change buffer solutions. The injection valve has a sample loading port through which the sample can be loaded into the injection loop, usually from a hypodermic syringe using a Luer-lock connection.

4. Column: The column is a glass or plastic cylinder packed with beads of resin and filled with buffer solution. It is normally mounted vertically with the buffer flowing downward from top to bottom. A glass frit at the bottom of the column retains the resin beads in the column while allowing the buffer and dissolved proteins to exit.

5. Flow Cells: The effluent from the column passes through one or more flow cells to measure the concentration of protein in the effluent (by UV light absorption at 280nm). The conductivity cell measures the buffer conductivity, usually in millisiemens/cm, which indicates the concentration of salt in the buffer. A flow cell which measures pH of the buffer is also commonly included. Usually each flow cell is connected to a separate electronics module which provides power and amplifies the signal.

6. Monitor/Recorder: The flow cells are connected to a display and/or recorder. On older systems this was a simple chart recorder, on modern systems a computer with hardware interface and display is used. This permits the experimenter to identify when peaks in protein concentration occur, indicating that specific components of the mixture are being eluted.

7. Fraction collector: The fraction collector is typically a rotating rack that can be filled with test tubes or similar containers. It allows samples to be collected in fixed volumes, or can be controlled to direct specific fractions detected as peaks of protein concentration, into separate containers.

Many systems include various optional components. A filter may be added between the mixer and column to minimize clogging. In large FPLC columns the sample may be loaded into the column directly using a small peristaltic pump rather than an injection loop. When the buffer contains dissolved gas, bubbles may form as pressure drops where the buffer exits the column; these bubbles create artifacts if they pass through the flow cells. This may be prevented by degassing the buffers, or by adding a flow restrictor downstream of the flow cells to maintain a pressure of 1-5 bar in the effluent line.