Waters Certified Sep-Pak - Manual (Page 3)

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Certified Sep-Pak Cartridges 3
c. SPE Procedure Steps
The following section describes the steps involved in a complete
solid-phase extraction procedure:
1. Pretreatment
Solid samples (soil, tissue, etc.)
• Shake, sonicate, or use soxhlet extraction.
- Extract sample with polar organic solvent (methanol,
acetonitrile) for polar analytes.
- Extract sample with organic solvent and drying agent
(dichloromethane, acetone) for non-polar analytes and
multi-residue extraction.
Non-Aqueous liquid
• If the sample is soluble in water, dilute it with water for
reversed-phase SPE.
• If the sample is soluble in hexane, dilute it with (or exchange to)
hexane for SPE.
Wastewater
• Filter or centrifuge as necessary.
2. Condition
For reversed-phase sorbents, preconditioning of the sorbent with
an organic solvent, such as methanol, acetonitrile, isopropanol, or
tetrahydrofuran, is usually necessary to obtain reproducible results.
Without this step, a highly aqueous solvent cannot penetrate the
hydrophobic surface and wet the sorbent. Thus, only a small fraction
of the sorbent surface area would be available for interaction with the
analyte. For the same reason, it is important not to let silica-based
SPE cartridges dry out between the solvation step and the addition of
the sample. A complete preconditioning of a reversed-phase cartridge
includes the solvation step and an equilibration with a low-strength
solvent such as water or buffer.
3. Load
When the analytes of interest are not retained by the sorbent,
this is called analyte breakthrough. For some methods, such as
pass-through cleanup, analyte breakthrough is desirable and is
maximized for those specific methods. However, in all other cases,
analyte breakthrough is unwanted and contributes to poor recovery
and method reproducibility. Breakthrough occurs when:
• There is too high an organic concentration in the load solution for very
polar analytes. Dilute sample at least 1:1 with water or buffer prior to
loading.
• The analytes are bound to proteins, they may pass through
the sorbent. Ensure that analytes are not bound to proteins by
acidifying or basifying the sample.
• Sorbent is overloaded by the matrix component. Therefore, it is
important to choose the correct sorbent mass (see table 1).
• The flow rate of the load step is too fast. There is not enough
contact time between the analytes and the sorbent. Look at the drops
and adjust the vacuum so that you see discrete droplets, not a stream
of liquid.
Table 1. Choice of Cartridges Based on Sample Size
Sample Size Cartridge
10-100 mL 3 cc/200 mg or 6 cc/500 mg
100-500 mL 3 cc/200 mg or 6 cc/500 mg
500-1000 mL 6 cc/500 mg (LP) or 6 cc/1 g
4. Wash
The wash steps are designed to remove unwanted matrix components
that remain from the loading step. The ideal wash solvent removes
only the matrix while keeping the analytes bound to the sorbent. For
complex samples this is impossible, so the wash steps are optimized
using pH, solvent strength, and solvent polarity to remove as much
matrix as possible while maintaining acceptable analyte recovery.