Buffer Exchange On Hitrap Desalting - GE HEALTHCARE AKTAprime plus Quick Reference Instructions

Buffer exchange on HiTrap Desalting

1
Preparing the buffers
•
Use high purity water and chemicals.
•
Filter all buffers through a 0.45 µm filter before use.
Buffer (port A1):
20 mM sodium phosphate, 0.15 M NaCl, pH 7.0
Prepare at least 500 ml eluent.
* When performing buffer exchange use the appropriate buffer.
2
Preparing the sample
Pass the sample through a 0.45 µm filter.
The maximum recommended sample volume is 1.5 ml.
3
Preparing the system
a) Place the inlet tubing from port A1 (8-port valve)
and port B (2-port valve) in the 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 20) 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 the same sample is applied repeatedly, a
Superloop™ can be used. For information of how to
use it see the instructions for the Superloop.
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 Desalting HiTrap
Templates
Application Template
Desalting
HiTrap Desalting
11-0027-48 AC, 2007-08 • p4 11-0027-48 AC, 2007-09 • p4
*
Controlled By:
Desalting.
Set Sample Inj. Vol
(00.0 ml) 00.0
Run Application Template
Press OK to start
Run data displayed
c) Enter the sample volume and press
template.
Desalting HiTrap Desalting
Theoretical gradient in
Template.
%B
100
Priming
& Equilibration
50
6
Total separation time = 9 min + sample application time
5
Typical result
AU
–– UV 280 nm
280nm
––
Conductivity
0.15
Histidine-tagged
protein
0.10
0.05
Inject
0
0 1
OK to start the
Application
Elution
Sample
3
Sample: Histidine-tagged protein in
20 mM sodium phosphate,
0.5 M sodium chloride,
0.5 M imidazole, pH 7.4
Column: HiTrap Desalting 5 ml
Buffer (A1): 20 mM sodium phosphate,
0.15 M sodium chloride, pH 7.0
min
Min