Dionex UltiMate 3000, Установка системы и приложений

Страница: 37 / 73

UltiMate 3000 - System Installation and Application
33
3.5 Comprehensive 2D LC
This application supports online comprehensive two-dimensional separation of peptides:
While the autosampler's injection valve is in
Load position, sample is drawn by the syringe
through the needle and placed in the sample loop. The needle pair withdraws liquid from the
well or vial and then descends into the wash port. The injection valve is switched to the
Inject
position, allowing the capillary pump to direct the sample to the ion exchange column (SCX).
The peptides that are not retained are collected on trap column 1 (
→
Fig. 18, no. 12) that is in-
line with the ion exchange column. Valve 2 is in position 1-2 . In parallel, valve 1 is in position 1-
2 to flush the trap column 2 (
→
Fig. 18, no. 15) via the loading pump. (For information about
how to install the trap columns, refer to section 3.7.)
At the end of the loading step, valve 2 is switched to position 10-1 , setting trap column 2 in line
with the ion exchange column. Trap column 1 is now in line with the loading pump for a
desalting step. When desalting is finished, valve 1 is switched to position
10-1 . This connects trap
column 1 and the reverse phase column, thus allowing the separation of the peptides collected on
trap column 1. During that time, a salt gradient is started. The peptides eluting from the ion
exchange column are trapped on trap column 2, collecting a new peptides fraction.
At the end of the separation on the reverse phase column, valve 1 is switched to position 1-2 to
condition trap column 1 via the loading pump (acetonitrile is removed). When trap column 1 is
ready again, valve 2 is switched to position
1-2 . Trap column 1 is flushed with the salt buffer
while trap column 2 is equilibrated with the loading solvent.
At this stage, a new salt gradient starting from the highest salt percentage reached in the previous
one can be started to load peptides on trap column 1. Simultaneously, the peptides previously
collected on the trap column 2 are being separated, with valve 1 in position
10-1 .
The process can be iterated, running an increasing salt concentration on the ion exchange column.
In this way, peptides are collected on one trap column while a reverse phase separation is
performed for the peptides collected on the other trap column during the previous iteration.
Please note: The right choice of the time allowed for desalting and reconditioning the
trap columns is crucial for this application.
Please note: The application comprises 2 pumps (LPG-3400 and DGP-3600) and two
FLM-3300 flow managers (
→
Fig. 17, page 34). Therefore, adapt the
names under which the devices are identified in the installation
environment and in the Chromeleon client in the Chromeleon Server
Configuration program. Keep in mind that you may have to adapt the links
to the associated control panels as well.