Cation Exchange - GE HEALTHCARE AKTAprime plus Quick Reference Instructions

Cation exchange

1
Preparing the buffers
•
Use high purity water and chemicals.
•
Filter all buffers thtough a 0.45 µm filter before use.
Start buffer (port A1):
50 mM sodium acetate, pH 5.5
Elution buffer (port B):
50 mM sodium acetate, 1.0 M NaCl, pH 5.5
Prepare at least 500 ml of each eluent.
Alternative buffers:
Start buffer (port A1): 20 mM sodium phosphate, pH 7.0
Elution buffer (port B): 20 mM sodium phosphate, 1.0 M NaCl,
pH 7.0
Start buffer (port A1): 20 mM sodium citrate, pH 3.0
Elution buffer (port B): 20 mM sodium citrate, 1.0 M NaCl, pH 3.0
2
Preparing the sample
a) Adjust the sample to composition of binding buffer by:
•
diluting the sample in binding buffer or
•
by buffer exchange using HiTrap Desalting or HiPrep
26/10 Desalting.
b) Pass the sample through a 0.45 µm filter.
3
Preparing the system
a) Place the inlet tubing from port A1 (8-port valve)
in the binding buffer and the tubing from port B
(2-port valve) in the elution buffer.
b) Place the three brown waste tubings in waste.
c) Connect the column between port 1 on the injection
valve (7-port valve) and the UV flow cell (see Ordering
information on next page for suitable columns).
d) Fill the fraction collector rack with 18 mm tubes
(minimum 40) and position the white plate on the
fractionation arm against the first tube.
e) Connect a sample loop large enough for your sample
between port 2 and 6 on the injection valve. Use a
syringe to manually fill the loop.
Note: If a Superloop is needed, additional information
is supplied in the instructions for Superloop.
11-0027-48 AC, 2007-09 • p34
4
Selecting Application Template and
starting the method
a) Check the communication to PrimeView. At the lower
right corner of the screen the text
prime should be displayed.
b) Use the arrow and OK buttons to move in the menu
tree until you find Cation Exchange HiTrap
Templates
Application Template
Cation Exchange
HiTrap SP
c) Enter the sample volume and press
template.
Note: If a 5 ml column is preferred, see cue card on
p.36.
Theoretical gradient in Cation Exchange HiTrap SP
Template.
%B
100
Priming
& Equilibration
50
Sample
Wash 1
11 10
Total separation time = 63 min + sample application time
Controlled By:
Set Sample Inj. Vol
(00.0 ml)
Run Application Template
Press OK to start
Run data displayed
OK to start the
Application
Wash 2
Elution
Re-equilibration
20 17 5
SP.
00.0
Min