Waters Oasis HLB Cartridges and 96-Well Plates - Manual (Page 4)

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[ CARE AND USE MANUAL ]
Oasis HLB Cartridges and 96-Well Plates
Figure 1. Retention Factor Versus pH For Acids, Bases, and Neutrals.
Summary
With the generic SPE method, one method yields good results for
a wide range of compounds. With an optimized method, cleaner
extracts can be obtained.
V. OASIS HLB 20 BOT T LE
OPTIMIZATION AP P ROACH
Chemical and chromatographic principles may be applied to
optimize methods on Oasis HLB. Selectivity is dramatically
enhanced by tuning pH, as well as the ratio of organic solvent to
water, in the mobile phase to manipulate retention.
If analytes or interferences are ionizable, then, as highly polar
entities in their charged states, they may be eluted in weak mobile
phases. If, by changing pH, they are converted to neutral form,
they are retained primarily by the strength of their hydrophobic
interaction with the sorbent surface. Stronger mobile phases, with
higher organic solvent concentrations, will then be required for
successful elution.
Published work by Waters chemists clearly demonstrates the
benefits of such a 2-D [two-dimensional] process on Oasis HLB.
The theory of retention, wash-elute studies for alprenolol, and
successive selectivity improvements made by refining the Oasis
HLB method are summarized in the figures below.
Wash/Elution Steps: 20 Bottle Optimization for Oasis HLB
The 20 Bottle Optimization method for Oasis HLB is set up first
by spiking the analyte into saline and loaded it into the wells of
the 96-well plate or 20 cartridges of Oasis HLB. We prepare the
20 bottle of solvents as described bellow. First, we start with 5%
MeOH with base wash step to remove proteins, to prevent the wells
from clogging, as well as putting bases in a neutral state for more
retention by reverse phase.
The 5% MeOH wash step (removes salts and proteins) in the
generic Oasis HLB method is weak enough that the analyte should
not wash off. Use the washes with increasing percentages of
organic solvent on identical replicate samples.
Plot the response against the organic solvent ratio, and determine
what percent organic to use for the wash and elution step After
running the 2-D optimization (“20 Bottle Optimization”), it is
important to select a wash step that is not too strong, or you may
lose your analyte, and is the most effective in the removal of
unwanted components.
It is also recommended to select the elution organic solvent ratio
that is just strong enough to elute the analyte and retain the most
hydrophobic interferences on the sorbent.
2% NH4OH in MeOH/Water 2% HCOOH in MeOH/Water
20 Bottle Optimization Oasis HLB: Example
■ ■
Wash 1: Base with 5% MeOH (remove proteins to prevent
clogging of wells; bases are in neutral state for more retention).
■ ■
Wash 2: Base with 40% MeOH (removes hydrophilic bases
and neutrals and all acids).
■ ■
Wash 3: 100% Water (removes residual ammonium hydroxide).
■ ■
Elute: Acid with 70% MeOH (101% recovery from rat plasma).
Note: Depending on matrix and sensitivity requirements, additional
washes may be required.
pH
Retention Factor (k)
Neutrals
Bases (BH + )
Theory
A -
B
Acids (HA)
Recommended pH range of Silica
Recommended pH range of Oasis ®
(pKa = 4.8)
Acids Retained Better Bases Retained Better
Low Retention for Acids
Low Retention for Bases
(pKa = 9.0)