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Oasis HLB Cartridges and 96-Well Plates
CONTENTS
I. INTRODUCTION
II. SAMPLE PRE-TREATMENT
a. Biological Samples
b. Solid Samples: Soil, Whole Foods, Tissue
c. Aqueous Samples: Water, Beverages
d. Non-Aqueous Liquid
III. SOLID PHASE EXTRACTION FOR ACIDIC,
NEUTRAL, AND BASIC COMPOUNDS USING
OASIS HLB
IV. OASIS HLB PROTOCOL CHARTS
a. Generic Method Oasis HLB SPE
b. Strategy for Optimizing the Generic SPE Method
V. OASIS HLB 20 BOTTLE
OPTIMIZATION APPROACH
VI. TROUBLESHOOTING
a. Adjustment to Optimize Recoveries
I. OASIS HLB
Universal Sorbent for Acidic, Neutral, and Basic Compounds
Oasis® HLB is a Hydrophilic-Lipophilic-Balanced, water-wettable,
reversed-phase sorbent for all your SPE needs. It is made from a
specific ratio of two monomers, the hydrophilic N-vinylpyrrolidone
and the lipophilic divinylbenzene. It provides superior reversed-
phase capacity with a neutral polar ‘hook’ for enhanced retention of
polar analytes.
Waters® has built a family of SPE sorbents which inherit some
key features of this unique substrate: stability at pH extremes
and in a wide range of solvents, extraordinary retention of polar
compounds, and a relative hydrophobic retention capacity 3X
higher than that of traditional silica-based SPE sorbents like C18.
Water-wettable Oasis Sorbents exhibit excellent retention capacity
for a wider polarity spectrum of analytes, even if the sorbent bed
runs dry during conditioning or sample loading. This means that
your SPE methods will be more rugged and robust, obviating the
need for repeat preparation.
The advantage of having higher retention capacity [k] is that
more analytes are retained with less breakthrough, improving the
recovery and overall reproducibility of your SPE method.
Available in five particle sizes [60 μm, 30 μm, 25 μm, 15 μm, and
5 μm], Oasis HLB sorbent, in cartridge, plate, or column format,
allows you to select the appropriate product based on the volume,
viscosity, and turbidity of your sample.
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III. SAMPLE PRE-TREATMENT
a. Biological Samples
This section contains recommendations for preparing your
biological samples (plasma, serum, urine, etc.) prior to solid-
phase extraction.
1. Prepare acidified or basified water diluents.
Note: (To prepare 4% phosphoric acid, Dilute 4.7 mL of 85%
phosphoric acid (the most common available formulation) to
100 mL final volume with water. Or 11.76 mL to 250 mL). To
prepare 5% concentrated ammonia, dilute 5 mL of concentrated
ammonia solution to 100 mL with water. Users will need to
prepare large volumes 100 -250 mL) of pretreatement buffer to
accommodate multiple samples and ensure consistency in the
buffer composition.)
2. Dilute plasma or urine, 1:1 with acidified or basified water. Add
10 to 50 μL of internal standard.
Note: Final concentration should be no more than 10% organic
otherwise protein precipitation can occur and polar compound
retention may be compromised.
3. If necessary, clarify samples by centrifugation at 8,000 x g
for 10-30 minutes.
b. Solid Samples: Soil, Whole Foods, Tissue
1. Homogenize the sample with an appropriate solvent to obtain
an aqueous based or an organic solvent based extract of the
sample. Initial extraction conditions are chosen to maximize
analyte recovery, while minimizing matrix interference. In
many cases, it may be beneficial to add buffers, dispersive
salts, or co-solvents to improve extraction efficiency.
2. Adjust the initial extract to optimize analyte retention onto
the SPE sorbent. This can include pH adjustment, solvent
adjustment, or solvent exchange through evaporation and
reconstitution (refer to Sections IV and V). It may be necessary
to centrifuge or filter the sample prior to loading.
If sufficient dilution has occurred, the sample may be treated in a
manner similar to an aqueous sample.
Note: If necessary, filter samples for suspended solids
(Eg; Environmental/Waste water/Water analysis/Food, etc.).
IV. SOLID PHASE EXTRACTION FOR ACIDIC,
NEUTRAL AND BASIC COMPOUNDS USING
OASIS HLB
1. Place Oasis HLB cartridge or plate on the vacuum manifold
and set the vacuum to 5” Hg.
2. Condition with Methanol.
3. Equilibrate with Water
a. In each case (conditioning and equilibration) add the
solvent before applying vacuum.
4. Switch off the vacuum pump or Stop vacuum by closing the
valve ( before switching off the vacuum pump, please reduce
the vacuum to the lowest possible setting).
5. Load your diluted sample.
6. Switch on or open valve at lowest possible vacuum and
gradually increase as needed in order to load the entire
sample onto the sorbent bed.
7. Switch off the vacuum pump or stop vacuum by closing the valve.
8. Apply 5% methanol in water wash solvent.
9. Switch on vacuum to 5” Hg (adjust/increase as needed).
10. Pull vacuum for another 30 sec to a minute to eliminate
residual wash solvent.
11. Switch off the vacuum pump or stop vacuum by closing the
valve (before switching off the vacuum pump, please reduce
the vacuum to the lowest possible).
12. Release vacuum and discard waste fluids, insert collection
device and replace the cover.
13. Apply 100% organic elution solvent and let it flow through
by gravity before switching on the vacuum pump.
c. Aqueous Samples: Water, Beverages
Adjust pH to maximize analyte retention on the SPE sorbent.
Buffer salts and dispersive agents may be used to increase
partitioning onto the SPE sorbent. Pretreatment to remove
suspended matter prior to SPE treatment may include filtration or
centrifugation.
d. Non-Aqueous Liquid
When appropriate the sample may be diluted with aqueous buffers
and organic co-solvents for reversed-phased or mix-mode SPE.
14. Switch on or open valve at lowest possible vacuum and
gradually increase as needed.
15. Pull vacuum for another 30 sec to a minute (to collect all
elution solvent).
16. Remove collection device.
17. Evaporate / Reconstitute or Dilute as needed.
18. Transfer to vial or plate for analysis.
19. If using plates, cover prior to analysis.
Oasis HLB Cartridges and 96-Well Plates
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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 PROTOCOL CHARTS
a. Generic Method Oasis HLB SPE
Prepare sample
(typically acidfy)
4a: Condition
Methanol
4b: Equilibrate
Water
4c: Load
Sample solution
4d: Wash
5% methanol in water
4e: Elute
Methanol
Evaporate and reconstitute
(optional)
Avoid using methods developed for C18 or other silica-based cartidges
(see note below)
b. Strategy for Optimizing the Generic SPE Method
Prepare sample solution
either in acid or base
Condition
Methanol
Equilibrate
Water
Load
Sample solution
Wash 1
5% methanol in water
Acidic Compounds Basic Compounds
Wash 2
2% formic acid in methanol/water
Elute
5% NH4OH in methanol/water
5% NH4OH in methanol/water
2% formic acid in methanol/water
The strategic procedure to obtain cleaner extracts start with the
generic method through the first wash.
Wash 2
Elute
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 C18 or other silica-based
sorbents may not be appropriate for the polymeric Oasis HLB sorbent.
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 pKa of
the compound) to increase retention.
The pH is raised for basic compounds (above the pKa 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.
Oasis HLB Cartridges and 96-Well Plates
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2% NH4OH in MeOH/Water
2% HCOOH in MeOH/Water
Retention Factor (k)
Acids Retained Better Bases Retained Better
Acids (HA)
Neutrals
Bases (BH
Figure 1. Retention Factor Versus pH For Acids, Bases, and Neutrals.
+ )
Low Retention for Bases
Recommended pH range of Silica
Theory
(pKa = 4.8)
pH
Recommended pH range of Oasis
(pKa = 9.0)
Low Retention for Acids
®
B
A-
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 BOTTLE
OPTIMIZATION APPROACH
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.
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.
20 Bottle Optimization Oasis HLB: Example
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.
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.
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VI. TROUBLESHOOTING
a. Adjustment to Optimize Recoveries
Spike an appropriate volume of reagent water with all analytes and internal/surrogate standards. Follow steps 4a-4e in Section II, but use a
rack to collect the eluates in the Load (4c), Wash (4d), and Elute (4e) steps in separate collection vessels. In addition, repeat step 4e with a
second portion of methanol and collect the eluate. Analyze all four collected fractions. Use the provided table to determine adjustments, if
necessary, to optimize sample recovery.
If the fraction from this step
contains the analyte:
Load (4c)
Wash (4d)
First Elution (4e)
Second Elution (4e repeated)
The Oasis HLB sorbent has been found to retain ionized analytes more strongly than silica-based
reversed-phase sorbents. However, recoveries may be enhanced when analyte ionization is suppressed.
For acidic analytes, adjust the sample pH to at least two pH units below the pKa of the acid. For basic
analytes, adjust the pH to at least two pH units above the pKa of the conjugate acid.
Recoveries of very polar analytes can be increased by using only water (not 5% methanol in water) as
the wash solution.
If an acceptable recovery of analyte(s) is obtained in this fraction (usually > 90%), no adjustments
are necessary.
For very non-polar analytes, methanol may not have adequate elution strength. Stronger solvents
such as acetonitrile or ethyl acetate may be substituted, or used in sequence. In addition, for ionizable
analytes, methanol may need to be modified with the addition of 2% acid or 2% base, as appropriate.
If solvents stronger than methanol or acetonitrile are used for the elution, then a preliminary
conditioning step (see step 4a) should be performed prior to the methanol conditioning step. For
example, if ethyl acetate is to be used as an eluent, condition the cartridge with ethyl acetate, followed
by methanol (4a) and then water (4b).
Make this adjustment for optimum analyte recovery:
Recommended volume for generic methods (assuming 1:1 dilution)
Cartridges 96 well Plate
Cartridges size (cc)/
Sorbent mass (mg)
Condition/
equilibration (ml)
Maximum load
of matrix and
dilution (ml)
Wash (ml) 1 2 4 5 10 40 0.2 0.5 0.5 -1.0 1 - 2 0.2
Elute (ml) 1 2 4 5 10 60 0.05 - 0.2 0.15 - 0.3 0.4 -1.0 0.8 - 2.0 0.025-0.10
1 3 6 12 20 35 5 10 30 60 2
1 2 3 5 10 50 0.2 0.5 0.5 -1.0 0.5 -2.0 0.2
1 2 5 15 30 100 0.5 1 1 - 2 1 -2 0.025 - 0.75
µelution
plate
Note: The above listed sample volumes are for biological samples and for certain types of samples (i.e drinking water) up to 20 times of
sample solution is possible.
Note: Solutions should be made fresh daily.
Load volumes for large volume water analysis
Cartridges size/
sorbent mass
Load (ml water)
total of matrix
and dilution
6 cc
1 cc 3 cc
50 ml 200 ml 500 ml 1000 ml 1000 ml 2000 ml 5000 ml
(200 mg,
30 µm)
6 cc
(200 mg,
60 µm)
12 cc 20 cc 35 cc
Oasis HLB Cartridges and 96-Well Plates
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Oasis 96 Well Plates
All Oasis 96 Well Plates, including µelution plate are designed to work individually as well as on any
automated robotics system.
The specific dimensions are:
96 well plate:
µelution plates:
Length = 5.030 inches
Width = 3.365 inches
Height = 2.015 inches
Length = 5.030 inches
Width = 3.365 inches
Height = 1.501 inches
All Waters Oasis 96 Well Plates and μelution plates meet the ANSI (American National Standards
Institute) guidelines for dimensions of microplates.
Waters, The Science of W hat’s Possible, and Oasis are registered trademarks of Waters Corporation. All other trademarks are the property
of their respective owners.
©2014 Waters Corporation. Produced in the U.S.A. April 2014 715000109EN KP-P DF
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
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