Waters Oasis HLB Cartridges and 96-Well Plates - Manual (Page 3)

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[ CARE AND USE MANUAL ]
Oasis HLB Cartridges and 96-Well Plates
Note: For the Load and Elute steps, it is recommended that flow rates should not exceed 1.0 ml/min, In all other steps, flow rates up to 5 ml/
min are acceptable.
Note: You may need to momentarily increase the vacuum to start the flow of aqueous solutions.
IV. OASIS HLB P ROTOCOL CHARTS
a. Generic Method Oasis HLB SPE
Avoid using methods developed for C
18
or other silica-based cartidges
(see note below)
■ ■
The fast Oasis HLB generic method is ideal for
LC/MS/MS analysis.
■ ■
The generic method is an excellent starting point since
it works for a wide range of compounds, yielding high
recoveries (> 85%) and consistent results (< 5% RSD).
■ ■
When cleaner backgrounds are required for higher
sensitivity or selectivity, the generic protocol can be
optimized using a straighforward method development
strategy.
The strategy uses the full pH range (pH 1 to pH 14) and varies the
organic solvent level.
Note: Wash and Elute steps developed for C
18
or other silica-based
sorbents may not be appropriate for the polymeric Oasis HLB sorbent.
b. Strategy for Optimizing the Generic SPE Method
The strategic procedure to obtain cleaner extracts start with the
generic method through the first wash.
By adjusting the pH of the additional washes to increase analyte
retention, higher concentrations of organic solvent may be applied
to remove interferences.
■ ■
The pH is decreased for acidic analytes (below the pK
a
of
the compound) to increase retention.
■ ■
The pH is raised for basic compounds (above the pK
a
of the
compound) to increase retention.
■ ■
The pH is then changed to elute the analyte. The % solvent
in the Wash 2 and Elute steps is determined by varying the
% methanol in 10% increments at each pH.
■ ■
Analyze the Wash 2 and Elute samples to determine
optimum % methanol.
■ ■
Select the highest % methanol in Wash 2 that does not
remove any analytes.
■ ■
Select the lowest % methanol in the Elute step that elutes
the analytes.
Prepare sample
(typically acidfy)
Prepare sample solution
either in acid or base
4a: Condition
Methanol
Condition
Methanol
4b: Equilibrate
Water
Equilibrate
Water
4c: Load
Sample solution
Load
Sample solution
4d: Wash
5% methanol in water
Wash 1
5% methanol in water
4e: Elute
Methanol
Evaporate and reconstitute
(optional)
Wash 2
2% formic acid in methanol/water
Wash 2
5% NH
4
OH in methanol/water
Elute
5% NH
4
OH in methanol/water
Elute
2% formic acid in methanol/water
Acidic Compounds Basic Compounds