Care and use manual, Short elution gradient 2.1 x 50 mm, Iv. typical chromatographic conditions – Waters Oasis HLB Direct Connect HP 2.1 x 30 mm Column for Automated Sample Preparation with Waters UPLC with Online SPE Technology User Manual
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[ Care and Use ManUal ]
Oasis HLB Direct Connect HP 2.1 x 30 mm Column
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IV. TyPICaL CHROmaTOgRaPHIC CONDITIONS
The analytical process begins with loading a sample on to the extraction
column via the SPE injector port (valve A). The loading pump (QSM)
using 100% aqueous mobile phase (with or without additives) at
a flow rate of 2 mL/min moves the sample from the injection loop
on to extraction column A. During the loading step, analytes of
interest will be trapped on the extraction column, while un-retained
interferences will be discarded to waste. After completing the loading
step, the extraction column is washed to remove mildly retained inter-
ferences. After the wash step, the trapped analytes on extraction column
A are eluted with a gradient from the ACQUITY BSM on to a UPLC ana-
lytical column for peak re-focusing. During the elution step of extraction
column A, extraction column B is regenerated then re-equilibrated
(using the QSM) to original conditions for the next analysis. The events
on each of the parallel extraction columns are shown in Figure 2.
Figure 2: Timeline showing the events occurring on each of the
extraction columns during an online SPE UPLC analysis
Typical mobile phase conditions to use for the load, wash and elu-
tion of the Oasis HLB Direct Connect HP 2.1 x 30 mm column will
depend on the analytes of interest. The pH of the mobile phase will
affect the retention behavior of analytes.
For a typical application consisting primarily of basic compounds,
such as the analysis of pharmaceuticals in water, a loading condi-
tion using a high pH aqueous mobile phase would usually provide
good retention of the analytes of interest. An example of this type
of loading mobile phase is 2% ammonium hydroxide in water,
pH 10. To wash mildly retained interferences from the extraction
column without eluting the compounds of interest the percentage
of organic in the mobile phase can be increased without changing
the pH. For the example of basic pharmaceuticals in water, the wash
could be set at 20% methanol with 2% ammonium hydroxide.
Figure 2: Timeline showing the events occurring on each of the extraction columns during an online SPE UPLC analysis
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Time
Time
Time
5
4
3
2
1
0
-2
-4
-6
Acquisition
Start
Loading
SPE
wash
Flush
Flush
Re-equilibration
Recon.
5%
95%
SPE A
SPE B
Elution
Re-equilibration
6
5
4
4.04
4.36
3
2
1
6
5
4
3
2
1
0
Short Elution Gradient
2.1 x 50 mm