Empore™ 96-Well Solid Phase Extraction UR Plate
03 Mar 2009

Empore™ Solid Phase Extraction Plates are designed for the solid phase extraction of analytes from 96 samples. The Universal Resin sorbent is a terpolymer based on styrenedivinylbenzene and designed to provide good retention of a wide range of analytes. A single reversed phase method can often be used to isolate and concentrate a variety of acidic, basic and neutral compounds which may reduce method development time.

The Empore 96-Well Universal Resin plate offers both polar (hydrophilic) and nonpolar (hydrophobic) functionalities. The polar functionality provides good wetting (hydrophilic) characteristics which allow for sorbent drying with minimized loss of recovery/reproducibility. This functionality imparts selectivity for ionized species. The nonpolar (hydrophobic) functionality allows for reverse phase selectivity similar to a silica based C8 without the secondary silanol interactions.

Product Information
The Empore™ UR 96-Well Solid Phase Extraction Plates are available in a standard well, 1.2 mL volume size (Cat. 6045SD) and a deep well, 2.5 mL volume size (Cat. 6345SD). If sample or reagent volumes exceed the volume of the well, multiple aliquots of solution may be used.
UR (Universal Resin)

Instructions For Use – Generic SPE Method for the UR Plate
1. Condition: Insert a waste tray in the vacuum manifold. Place the manifold collar and Empore™ plate onto the manifold. Add 100 μL of methanol to each well and wait 30 seconds, then apply enough vacuum to remove the methanol before proceeding to Step 2.

2. Rinse: Add 200 μL of water or buffer to each well. Apply vacuum until all wells have drained.

3. Load: Add a minimum of 100 μL of prepared sample to each well. Apply vacuum until all wells have drained.*

4. Wash: Add at least 500 μL of water to thoroughly rinse the extraction disk, prefilter and well. Apply vacuum until all wells have drained. Repeat with a second aliquot of water or buffer.

5. Elute: Replace the waste tray with a collection plate. Align collar and nozzle with collection plate wells. Add 150 μL of acetonitrile or methanol to each well. Wait 30 seconds. Apply vacuum until all wells have drained.

* Suggested flow rate for the load and elution steps is 2 mL/minute. Adjust vacuum settings as needed to obtain adequate flow rates. Viscous samples may require greater than 8 inHg (0.3 bar) to obtain a 2 mL/minute flow rate during the loading step.

Suggestions for Optimizing Recoveries
• Add acid to samples if analytes are highly bound to serum or plasma proteins.

• Adjust the sample pH two units above the pKa of the analyte for basic analytes or two units below the pKa of the analyte for acidic analytes to suppress ionization and enhance the recovery of acidic and basic analytes.

• If sample flow problems are encountered when adding samples directly to extraction disk plate: – Dilute sample up to 1:4 with water or buffer.
– Centrifuge samples and add the supernatant to the
extraction disk plate.

Conditioning
• Recoveries of most analytes are not negatively impacted if the sorbent dries out. To minimize variability in results, it is suggested that vacuum not be applied for longer than 1 minute after the wells have drained.

• A vacuum setting of 8 inHg (0.3 bar) or greater is recommended for the rinse step.

Sample Loading
• Flow rates of 2 mL/min generally provide a good starting point.

• Evaluate sample loading at both low (5-7 inHg/0.17-0.24 bar) and high vacuum (15-20 inHg/0.5-0.68 bar) and examine the effect on analyte recovery. If an analyte has low affinity for the sorbent, it may need to pass more slowly through the sorbent bed for sufficient attraction to occur.

• For samples less than 100 μL in volume, dilute with water or buffer to 100 μL or more.

Wash
• Water is suggested as a first wash to remove proteins that may precipitate and occlude the membrane.

• Use water or buffer to increase the recovery of highly polar analytes.

• For cleaner eluates/improved chromatography, evaluate the following:
– Keep wash composition constant and evaluate varying wash volume (at least twice the load volume).
– Compare multiple consecutive washes to a single aliquot.
– Keeping the wash volume constant, increase the organic concentration in increments of 5% to determine the amount of organic that results in the cleanest chromatography without loss of analyte.

• To increase selectivity
– Use an acidic organic solvent wash of 0.1 to 1.0% acid (v/v) for acidic analytes.
– Use a basic organic solvent wash of 0.1 to 1.0% ammonium hydroxide (v/v) for basic analytes.
Elution
• Elution solvent composition should be optimized for individual analytes and LC mobile phase compatibility.

• Wait 30 seconds for the elution solvent to soak into the extraction disk and begin desorbing analyte before applying the vacuum.

• A flow rate of 2 mL/min generally provides a good starting point.

• Evaluate eluting at both low (5-7 inHg/0.17-0.24 bar) and high vacuum (15-20 inHg/0.5-0.68 bar) and examine the effect on analyte recovery. If an analyte has a strong affinity for the sorbent, elution may need to occur more slowly to allow adequate desorption.

• Determine the minimum effective elution volume.
– Increase elution volume in 25 μL aliquots. For example, compare single 25, 50, 75, 100 μL aliquots.
– Compare a single larger volume aliquot of eluting solvent to two smaller volume aliquots.

• To increase sensitivity for dissociable analytes:
– Compare 100% organic to 70-90% organic with 1% acetic acid (v/v) as the elution solvent
for basic analytes.
– Compare 100% organic to 70-90% organic with 1% ammonium hydroxide (v/v) as the elution solvent for acidic analytes.

• To reduce evaporation and reconstitution of the eluate (for the direct injection of the eluate onto the LC/MS system):
– If additional dilution is necessary, add water or buffer directly to the collection plate.

Note: Empore Solid Phase Extraction Products are intended for solid phase extraction during scientific research only. These products are not intended for use in medical devices or in assessment and treatment of clinical patients.

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