5 Building an SPE method
5.1 Typical components of an SPE workflow
To establish a context for the use of the Otto SPEcialist PPM, it is helpful to review the typical
SPE method building process.
Most SPE methods consist of multiple steps in which a liquid (solvent, buffer, or sample) is added
to the SPE column or SPE plate well, and is then pushed through the sorbent bed by a positive
gas pressure exerted on top of the consumable.
The typical process for building an SPE method involves performing the following procedures, in
order:
1. A solvation or “conditioning” of the sorbent bed (optional depending on the exact sorbent
chemistry and extraction mechanism being used).
2. A pre-equilibration (step or steps), just prior to adding the sample. The pre-equilibration
prepares the sorbent bed to retain the desired analytes from the sample during sample
application.
3. A sample application. In this step, analytes from the sample are retained on the sorbent
bed.
4. A wash (step or steps) to selectively remove undesirable interferences from the sorbent
bed while still retaining the analytes of interest.
5. An elution in which the now-purified analytes are recovered from the sorbent bed.
Requirement:
If two successive protocol solvents are immiscible with one another, you must
perform one or more steps involving drying of the sorbent bed.
You obtain maximum analyte recoveries and method reproducibility when critical parameters in
the protocol are well-controlled. Along with consistency in composition of the reagents used in the
protocol, and similar consistency in the SPE consumable itself, control of flow rates through the
SPE columns is also essential. To this end, the Otto SPEcialist is designed to effect a highly
reproducible and controlled flow.
For maximum throughput and productivity, most laboratories want to execute an SPE method in
the shortest time possible. However, processing liquids too quickly through the SPE column can
lead to irreproducibility, especially during the sample application and elution steps.
A common reference for useful flow rates during sample application or elution is "a drop every
two to three seconds" or slower. Assuming an average “drop volume” of approximately 50 μL
correlates to a flow rate of approximately 1.0 to 1.5 ml/min. It is possible to employ much faster
flow rates without compromising results. Unfortunately, due to the variability of flow resistance
among commercial SPE consumables, as well as differences in sample viscosities, there are no
absolute settings for gas pressure that you can use to generate these flow rates. Therefore,
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