Anion Exchange - GE HEALTHCARE AKTAprime plus Quick Reference Instructions

Anion exchange

1
Preparing the buffers
•
Use high purity water and chemicals.
•
Filter all buffers through a 0.45 µm filter before use.
Start buffer (port A1):
20 mM Tris-HCl, pH 8.0
Elution buffer (port B):
20 mM Tris-HCl, 1.0 M NaCl, pH 8.0
Prepare at least 500 ml of each buffer.
Alternative buffers:
Start buffer (port A1): 20 mM Glycin-NaOH, pH 9.5
Elution buffer (port B): 20 mM Glycin-NaOH, 1.0 M NaCl, pH 9.5
Start buffer (port A1): 20 mM bis-Tris, pH 6.5
Elution buffer (port B): 20 mM bis-Tris, 1.0 M NaCl, pH 6.5
2
Preparing the sample
a) Adjust the sample to composition of start 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 • p32
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 Anion Exchange HiTrap
Templates
Application Template
Anion Exchange
HiTrap Q
c) Enter the sample volume and press
template.
Note: If a 5 ml column is preferred see cue card on
p.36.
Anion Exchange HiTrap Q
Theoretical gradient in
Template.
%B
100
Priming
Elution
& Equilibration
50
Sample
Wash 1
11 10
Total separation time = 63 min + sample application time
Controlled By:
Q.
Set Sample Inj. Vol
(00.0 ml)
Run Application Template
Press OK to start
Run data displayed
OK to start the
Application
Wash 2
Re-equilibration
20 17 5
00.0
Min