Image Credit: Porvair Sciences Limited
1. Fresh standards are best
When stored for long periods of time, some analytes are susceptible to degradation by light and heat and can precipitate out of solution. This will impact the calibration standards and recovery values of a run, and a procedure should be started with freshly made standards.
2. Optimise loading pH
pH can have a huge effect on any SPE technique and can be utilized to enhance retention of compounds on an SPE product. For instance, the reversed phase technique can be used to adjust pH to neutralize any charge on an analyte.
This will improve interactions with the resin and make the analyte more hydrophobic. For basic compounds, adjust 2 pH ABOVE pKa and for acidic compounds, adjust 2 pH units BELOW pKa.
3. Know your compounds
Properties of the compounds being analyzed can have a huge effect on which type of SPE can be carried out. The pKa and Log P of the analytes of interest are two of the most crucial values. pKa is the point at which the analyte is half ionized and half un-ionized, and Log P is the measure of hydrophobicity of a compound.
4. Loose-packed vs. composited
Loose-packed material can lead to numerous problems for the sorbent bed, including compression, channeling, voiding and inconsistencies in bed weight to bed height ratio differences because of lack of compression of the top frit after loose filling, as seen in Figure 1.
Figure 1. Microlute™ CP, the composited sorbent vs. traditional loose-packed SPE. Image Credit: Porvair Sciences Limited
Recovery and reproducibility of testing can be negatively influenced by all of these factors. Consider composited products such as the Microlute® CP range, which have been designed to eliminate these issues and enhance both reproducibility and recovery of SPE techniques.
5. Sorbent conditioning
Once the pKa of a sorbent is known, the sorbent bed can be activated and ready to bind analytes by conditioning. The silica is bonded with a functionalized moiety which is usually lying flat on the surface of the particles in most silica-based SPE sorbents.
The conditioning step (utilizing a water-miscible organic solvent, e.g., MeOH) gives them the most surface area for analytes of interest to interact/bind to as it causes these bonded functionalities to ‘stand up’ on top of the particles.
This step may not be required if the sorbent is water-wettable, but it can increase cleanliness of extracts by undertaking a solvent wash of the sorbent bed.
6. Refine wash and elution strength
Wash and elution strength can be tweaked to give the cleanest extracts When using a mixed mode or reversed phase SPE product. Log P is the ideal property to help to select these settings.
- Low Log P - Decrease the organic strength of the elution solvent to inhibit hydrophobic interferences whilst still eluting the analyte of choice.
- High Log P - Utilize stronger organic strengths in the wash steps.
7. Know your sorbent
In order to adjust pH of solutions on load and wash steps without deionizing compounds and resins (weak ionic resins only), the pKa of the sorbent being used must be known and utilized with the analytes pKa.
8. Go with the flow (rates)
In order to achieve a successful reversed phase SPE workflow, flow rates are crucial. The retention mechanism is heavily dependent on Van der Waals forces of interaction between the analytes of interest and sorbent.
During an SPE procedure, fast flow rates hinder the analyte of interest’s ability to bind to the sorbent media properly and can lead to a breakthrough on load and wash steps of an SPE technique. A slow and steady flow of samples is best and a user should aim for a flow rate that is below or equal to 1 drop a second (~1 ml /minute).
9. Dry the SPE resin before elution
When dry, silica is extremely susceptible to stationary phase collapse. Before completing the final wash step of the SPE procedure, do not let silica-based resins dry out at any point – this is particularly important during the load step.
Limiting the possible interactions between analytes and bonded phase stops reduced recoveries. The below tips apply to both polymeric and silica resins:
- After undertaking the wash step(s), the resin must be dried using vacuum or positive pressure until the resin is fully dry. Positive pressure/ vacuum should be used for at least three minutes or until no wash solvent is visible.
- If the technique requires a dry down and reconstitution step, the dry downtime is significantly decreased by drying the resin as water is present in most wash solvents, which is much harder to evaporate than most organic solvents.
- If a dry down step is not required, not drying the SPE resin after the wash step can cause the final solution for injection to be less concentrated, leading to reductions in sensitivity.
10. Optimal dry down
Make sure that the chosen dry down technique is vigorous enough to visibly disturb the surface of the elution solvent but not so vigorous to cause the possibility of expelling the solution out of the wells as this will cause severe %RSD and recovery differences. As the level of solvent will move during dry down, this can be quite difficult.
In order to overcome this, evaporators that can be programmed or automatically adjusted for efficient solvent evaporation, such as the Porvair Sciences Ultravap® range of evaporators, should be used. These permit dry down to be optimized throughout every stage of the process without any risk of sample contamination.
About Porvair Sciences Limited
Porvair Sciences, specialists in the manufacture of microplate products, serve Life Sciences, Biotechnology, R&D and Molecular Biology with microplate solutions for all applications, from sample preparation to high throughput screening via our global distributor network.
Our range includes vacuum manifolds, sealers, evaporators and microtiter plates in all popular styles; deep well and shallow well storage plates, assay plates, luciferase reporter gene plates and liquid handling reagent reservoirs. We also provide custom microplate products for life science research. Our vacuum manifolds, essential for 96-well SPE sample preparation, are designed to work with most popular filter plates, including Waters, Millipore, Qiagen, Whatman, GE Healthcare, Varian, Biotage and, of course, Porvair.
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