Common Problems in Solid-Phase Extraction: A Practical Troubleshooting Guide
Introduction
Solid-phase extraction (SPE) is a fast, easy and high-performance sample preparation technique that evolved from liquid–solid extraction and liquid chromatography. Compared with liquid–liquid extraction, SPE often gives higher recoveries, better separation of analytes from interferences, simpler workflows and greater reproducibility. SPE is now widely used in pharmaceuticals, food, environmental analysis, customs inspection and chemical industries.
However, routine SPE runs can encounter recurring problems, and due to the differences in experiment settings, their causes vary widely. In this article, we summarize the typical causes and their respective practical fixes — arranged by problem type — so that readers can troubleshoot efficiently.
Problem 1. Low Recovery
Low recovery is the most common problem analysts encounter in SPE. Typical manifestations include unexpectedly low analyte signals from final extract, analyte in load fraction or incomplete elution.
Below are some likely causes and their fixes:
1. Sorbent choice / polarity mismatch.
Cause: The sorbent’s retention mechanism doesn’t match the analyte’s chemistry (e.g., analyte too polar for reversed-phase cartridge, too nonpolar for polar sorbent).
Fix: Choose a sorbent with the appropriate retention mechanism (reversed-phase for nonpolar neutral molecules, polar sorbents for polar analytes, ion-exchange for charged species). If analyte is retained too strongly and resists elution, consider a less hydrophobic or less selective sorbent.
2. Eluent strength or pH insufficient.
Cause: Elution solvent isn’t strong enough (or pH not set to convert analyte to its non-retained form).
Fix: Increase organic percentage or use a stronger eluent; for ionizable analytes adjust pH so the analyte is in the neutral form (or use counter-ion exchange chemistry). Consider adding small amounts of modifiers (acid/base) to improve desorption.
3. Too little elution volume.
Cause: Not enough solvent passed through to fully desorb analyte.
Fix: Increase elution volume in increments and monitor recovery; use multiple fractions if necessary.
Problem 2. Flow Rate
Even in a same batch of SPE cartridges, flow rate may vary. Too fast flow reduces retention and affects the interaction between solvent and sorbent. Too slow flow increases run time.
Flow rate variations may be resulted from these causes:
1. Packing/bed differences (amount, packing density).
Fix: Use a controlled manifold (see the end of this article for an operation instruction of Welch’s SPE Manifold!) or a peristaltic/positive-displacement pump to set reproducible flows. As a rule of thumb many SPE procedures are stable with flows below 5 mL/min; for steps that require tighter control (e.g., controlled elution or equilibration) aim for slower flow.
If flow is too slow but not clogged: apply gentle positive pressure (or vacuum) within manufacturer limits to restore a practical flow rate.
2. Particulate matter / clogging
Fix: Filter or centrifuge samples before loading. Use a glass fiber/prefilter if samples have many particulates.
3. High sample viscosity.
Fix: Dilute the sample with an appropriate solvent (matrix-compatible) to lower viscosity.
Problem 3. Estimate of Sorbent Adsorption Capacity
Sorbent overload causes breakthrough and analyte loss. Therefore, sorbent adsorption capacity must be pre-estimated. Furthermore, if SPE specifications are not given, the capacity can also be used to choose cartridge size.
-
Silica-based: capacity often ≤ 5% of sorbent mass.
Example: a 100 mg/mL C18 cartridge → 100 mg × 5% = 100 × 0.05 = 5 mg of analyte maximum (approximate). -
Polymeric sorbents: roughly 3× the silica capacity, often ≤ 15% of sorbent mass.
Example: a 100 mg/mL HLB cartridge → 100 mg × 15% = 100 × 0.15 = 15 mg. - Ion-exchange resins: described by exchange capacity, typically 0.25–1.0 mmol/g, where 1 mmol/g means 1 mmol of monovalent charged substances can be adsorbed per 1 g of sorbent.
Problem 4. Poor Reproducibility
If high variability between replicates were found between each run, consider the following causes & fixes:
-
Cartridge bed dried out before sample loading.
Fix: Re-activate and re-equilibrate the cartridge (conditioning followed by equilibration in the loading solvent) so the packing is fully wetted. -
Too-high flow during sample application.
Fix: Lower the loading flow; allow sufficient contact time to establish equilibrium. -
Wash solvent too strong (partial elution during wash).
Fix: Allow the wash solvent to soak in briefly, then apply vacuum/pressure and control the flow at ~1–2 mL/min to avoid abrupt stripping of analyte. -
Overloaded cartridge.
Fix: Reduce the sample amount or switch to a higher capacity cartridge.
Problem 5. Unsatisfactory Cleanup
Unproper choice of cartridges, solvents, or strategies may cause SPE cleanup to be unsatisfactory from expectation. These causes and their fixes include:
1. Wrong purification strategy
Cause: Using a mode that retains impurities rather than the analyte (or vice versa) without considering which gives better selectivity.
Fix: In many targeted analyses, retaining the analyte and removing matrix by selective washing gives better cleanup than trying to retain everything except the analyte. If multiple sorbents could work, choose the more selective adsorbent. As a general ranking of selectivity for separating analytes from matrix: ion-exchange > normal-phase > reversed-phase (when appropriate for the chemistry).
2. Wash/elution solvents poorly chosen
Fix: Reoptimize wash and elution conditions (composition, pH, ionic strength) — small changes in organic percentage or pH often have big effects on selectivity.
3. Cartridge contains contaminants / not properly conditioned
Fix: Ensure cartridges are conditioned according to manufacturer recommendations (e.g., wetting solvent → equilibration solvent). If contamination suspected, discard and use fresh cartridges.
Operating Instruction for Welch SPE Manifold
The video below demonstrates the operating instruction for Welch SPE Manifold. Users should follow the instruction to ensure correct operation, so as to get consistent and reliable SPE performance.