Introduction to FPLC

The FPLC consists of a pump and a column which will withstand high pressure so separations can be carried out relatively quickly. For a detailed description there is a FPLC system handbook which is particularly useful for trouble shooting. For use of individual columns follow the "instructions" (in the green folder) which accompany each one.

Gel filtration

 

Columns 1 Superdex 75 HR 10/30 and 2 Superdex 200 HR 10/30, one to be used with detergent and one NOT to be used with detergent.

Samples

The bed volume of these columns is about 20-24 mls, therefore for optimum resolution keep sample volumes to 0.5ml or less. The sample loops have to be full to avoid air getting onto the column ie for the 0.5ml sample loop you need at least 0.5ml (can be made up with buffer). Samples must be free of particulate matter by filtration or spinning eg 10-15 min at 13000g in a microfuge.

Buffers

Have buffers at room temperature and degas and filter before use. For gel filtration only one buffer is used and is fed into both pumps so put both inlet pipes into the one buffer bottle..

Setting up a run.

1. Follow column instructions (particularly important for ion exchange columns) to detach any column attached and not wanted and replace with appropriate gel filtration column.

2. Wash pumps. The pumps are usually washed manually into the buffer to be used.

a) Press manual and then step forward until valve. Enter 1.3 and press do store. This changes the valve position to disconnect the column from the pumps and allow a faster flow. Look at the diagram on the valve which explains the flow.

You will hear the valve position change.

b)Press step forward until wash A.B.0.0 Enter 1.1 and press do store. The pumps will begin to move and after about 5 minutes the display will read A.B 0.0. Press end. The valve position will return to its default position, 1.1,.which means the column is connected to the pumps.

3. Wash column. The column can be washed manually or with a program with at least a column volume of buffer. Set the pressure limits for the pumps, 1.5MPa for the gel filtration columns (see "instructions").

Manual. Press manual and step forward until ml/min. Enter 0.5 and press do store. To stop press end.

Method Run wash method, currently method 9 in bank 5.To check press method file and enter 5 in bank. Step forward until list and enter 9. Press do store. The method will be printed. Press end and then enter 9 and press do store. The method can be curtailed by pressing end.

4. Base line. Before beginning a run make sure the base line on the chart recorder is O.K.

Switch on UV lamp and chart recorder. mV should be set at 0.1mV. Baseline is set at 10% deflection. Baseline should not change when range is altered. The flow cell is 0.5cm therefore absorbance is 50% of that detected with the usual 1cm cell in the spectrophotometer. Therefore for a range of 0.5 the maximum absorbance/deflection is equivalent to 1 in the spectrophotometer. Usually sample is diluted 1:5 after separation.

5. Fraction collector. Set fraction size, a convenient volume is 0.5 ml. The fraction collector is controlled by the FPLC so no other settings have to be made.

6. Loading a sample. Wash out loop. Sample is injected with valve in (default) position 1.1. The transparent tube allows you to see the sample filling up the loop and emerging at the other end. Make sure it is full, with no bubbles. The system will tolerate the odd small bubble but it is good practice to operate bubble free, particularly to avoid bubbles in the flowcell which affect the absorbance record. Leave the syringe attached, otherwise the sample will run out. 

7. Running a method. Bank 5 contains the methods supplied with the machine and should not be altered. There is a copy in the pocket inside the front cover of the manual. If you want to alter a method copy it to another bank. The gel filtration method is method 2 in bank 5. Check it by listing it (see above in 3.). Check you are in bank 5 and with run method on the screen enter 2 and press do store. Method 2 is designed to collect fractions for a column volume (48 X.5ml) after the void volume of the column has been through.

8. After a run. Salt is corrosive so must be removed from the column and pumps. Follow column instructions: wash the pumps into degassed and filtered H2O (with .05% azide) if the column and/or pumps is going to be left in it for any length of time. Then wash column as before with at least one column volume. For overnight storage this is sufficient but for longer periods of time repeat the process and store pumps/column in degassed and filtered 20% ethanol in H2O. If removing column make sure you follow instructions and block off the connections on machine and column properly ie fill the connector loop. Wash sample loop out as well. 

9. Finally fill in log book which will remind you to switch off lamp by holding the button down until light goes out and put caps on pens.

 

Ion exchange chromatography

 

Columns HiLoad 16/10 Q Sepharose, Mono Q HR5/5 and Mono S HR 5/5.

 

Samples

For samples > 0.5ml use the Superloops. There is a 10ml and a 50ml Superloop. To attach follow "instructions" Make sure sample is free from particulate matter. The sample does not have to be exactly 10 or 50 ml, that is the maximum that can be delivered.

 

Buffers

To form a gradient both pumps are used, A delivering starting (low salt) buffer and B delivering eluting (high salt) buffer. Put inlets into correct room temperature, degassed and filtered buffers.

 

Setting up a run.

1. Follow column "instructions" to attach ion exchange column taking particular care with the HiLoad 16/10 Q Sepharose which employs a transit syringe to eliminate air from the system.

2 Wash pumps as described for gel filtration except that A and B are washed into different buffers. If the pumps are in ethanol, wash B into low salt buffer first then high salt buffer to avoid precipitation of salt.

3. Wash column according to "instructions".Set the pressure limits for the pumps, 0.3MPa for HiLoad 16/10 Q Sepharose.and 5MPa for Mono Q and S (see "instructions"). To remove ethanol, wash the column with a column volume of buffer A (low salt) at low flow rate eg 1ml/min for HiLoad 16/10 Q Sepharose. The column should have been regenerated after the last use but if you want to repeat elution with high salt buffer, wash the column with buffer B: press manual and enter 100 for conc B %. This will mean just buffer B is delivered onto the column. The column is then equilibrated with 5 column volumes of buffer A at a higher flow rate eg 5ml/min for HiLoad 16/10 Q Sepharose. .

4. Base line as for gel filtration. Make sure both pens are working as red marks the gradient.

5. Fraction collector. Set fraction size, for column size 16/10 and 5/5 a convenient volume is 3 or 1ml respectively.

6. Loading a sample. Inject sample into Superloop or sample loop. Check that method is designed to deliver sufficient volume ie the volume delivered while the valve is set at 1.2 when the pumps are connected to the sample loop.

7. Running a method. See gel filtration. There are methods for Mono Q and Mono S HR5/5 in bank 5, method 0 and 1 respectively. There is a method for purification of Ig on HiLoad 16/10 Q Sepharose in bank 1, method 3. This is designed for Ig preps in 50mM NaCl with buffer A and B containing 50mM NaCl and 1M NaCl respectively. For Ig amounts up to 50mg, use the Mono Q HR5/5 column with bank 5, method 0.

8. After a run. Follow column "instructions" for regeneration i.e. wash with a column volume of high salt buffer B and then regenerate with 5 column volumes buffer A. To store HiLoad 16/10 Q Sepharose wash both pumps with low salt buffer A and then pumps/column with 50mM Tris-HCl pH8 containing 20% ethanol and remove column using transit syringe. Wash pumps with 20% ethanol. For Mono Q/S follow "instructions" i.e for Mono Q wash pumps/column sequentially with 10ml of 100 mM Na2SO4, 5 ml H2O and 5 ml 20% ethanol. NB remember to wash pump B with low salt buffer before washing with ethanol. Detailed instructions are provided for cleaning up columns more thoroughly.

9. Finally as for gel filtration.