GE Healthcare (formerly Amersham Biosciences) provides an instrument for fast protein liquid chromatography (FPLC) wherein proteins of various sizes can be readily purified using different kinds of columns. The instrument I use is a dual-pump system which is largely sufficient for most of our chromatography applications. FPLCs with four pumps are also available which provide a facility for running four different solutions simultaneously. The flow rate of the pump can get as low as 0.01ml per minute and can reach up to 10ml per minute. The pumps require a constant flow of 20% ethanol in water which serves as a lubricant. The machine is provided with an online UV detector which can be set for a maximum of three wavelengths covering a range from the far UV region (214nm) to the visible range (700nm).
I occasionally purify proteins within the molecular weight range 40 – 60kDa using this system. I usually use a buffer system which reaches a maximum of 500mM salt toward the end of the run. The salt and pH conditions, of course, depend on the type of column used. Online pH and conductivity detection units are also available from GE Healthcare and need to be purchased separately. I use the conductivity meter sparingly, only when I am monitoring protein desalting, in cases where manual gel filtration cannot be carried out.
Injection loops of different volumes are available. Loops of 10 – 150ml, termed superloops, are available from GE Healthcare; 100 – 150ml loops can also be purchased. A fraction collector that can gather up to 100 fractions either dropwise or at specified time intervals is provided with the AKTA (BASIC) FPLC System. Although the fraction collector can be automated, I prefer manual collection when the peaks elute.
One major drawback is that the instrument should not be kept at 4°C, as it tends to spoil the PCBs in the instrument. I generally place the column, buffers and samples in a cold chamber, with the instrument outside the chamber, for purifying temperature sensitive proteins. This operation might become cumbersome in cases of highly temperature sensitive proteins.
I find the software provided with the instrument extremely easy to handle. I run it in a multi-user environment and I have found no problems with it. Default methods for almost every column supplied by GE Healthcare are available with the software. The instrument can either be controlled manually or using programmed runs. I have saved a series of default programs for different kinds of proteins and conditions. This helps when re-purification of several batches has to be carried out. I also feed in the pressure limits of different columns (based on the specifications of the manufacturer). This option is extremely useful as the software automatically shuts down the pumps and pauses the run when the pressure exceeds the maximum cut-off. I have saved a couple of expensive columns thanks to this feature!
In the current versions of the software, the UV lamp can be switched on when required during the run. In the version I use, the lamp is automatically switched on when the instrument is started. This sometimes leads to wastage of lamp lifetime, especially when column cleaning is carried out. I suggest new buyers look for this feature when they are investing in this instrument.
R. Mahalakshmi
Scientist
Bharathiar University