Care and use manual – Waters Oasis HLB Cartridges and 96-Well Plates User Manual
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[ CARE AND USE MANUAL ]
Oasis HLB Cartridges and 96-Well Plates
VI. T ROUBLESHOOTING
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:
Make this adjustment for optimum analyte recovery:
Load (4c)
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.
Wash (4d)
Recoveries of very polar analytes can be increased by using only water (not 5% methanol in water) as
the wash solution.
First Elution (4e)
If an acceptable recovery of analyte(s) is obtained in this fraction (usually > 90%), no adjustments
are necessary.
Second Elution (4e repeated)
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).
Recommended volume for generic methods (assuming 1:1 dilution)
Cartridges
96 well Plate
µelution
plate
Cartridges size (cc)/
Sorbent mass (mg)
1
3
6
12
20
35
5
10
30
60
2
Condition/
equilibration (ml)
1
2
3
5
10
50
0.2
0.5
0.5 -1.0
0.5 -2.0
0.2
Maximum load
of matrix and
dilution (ml)
1
2
5
15
30
100
0.5
1
1 - 2
1 -2
0.025 - 0.75
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
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
1 cc
3 cc
6 cc
(200 mg,
30 µm)
6 cc
(200 mg,
60 µm)
12 cc
20 cc
35 cc
Load (ml water)
total of matrix
and dilution
50 ml
200 ml
500 ml
1000 ml
1000 ml
2000 ml
5000 ml