SPE Troubleshooting: Why Won't My Sample Flow Through the SPE Cartridge?
- Photo: Phenomenex: SPE Troubleshooting: Why Won't My Sample Flow Through the SPE Cartridge?
- Video: Phenomenex: Why Your Sample Won’t Flow Through an SPE Cartridge | SPE Tip
In this troubleshooting tip, Genevie Hodson, Senior Manager of Technical Support and Applications at Phenomenex, addresses a common solid phase extraction (SPE) question:
"My sample won't flow through my SPE cartridge. Why is this happening?"
Several factors can contribute to flow problems during SPE. A systematic troubleshooting approach can help identify the root cause and restore proper cartridge performance.
Identifying When the Flow Problem Occurs
The first step in troubleshooting is determining when the flow issue appears.
Ask the following question:
- Does the problem occur during the sample loading step?
- Or does it occur before sample loading, during cartridge conditioning or when running blanks?
The timing of the problem can help identify its underlying cause.
Cause 1: Viscous or Cloudy Samples
Description
Samples that are thick, highly viscous, or contain large amounts of particulate matter can restrict flow through the SPE sorbent.
Recommended Actions
Before SPE, the sample should be:
- Diluted
- Centrifuged
- Filtered
Another option is selecting a sorbent with a larger particle size.
Example
- Strata-X – 33 µm particle size
- Strata XL – 100 µm particle size
The larger particle size of Strata XL is better suited for viscous samples and can improve flow performance.
When This Problem Appears
If sample viscosity or particulate content is the cause, the flow issue will typically occur during the sample loading step.
Cause 2: Insufficient Manifold Pressure
Description
- Flow problems may originate from the SPE manifold or instrumentation.
- The applied pressure must be sufficient to generate proper flow through the cartridge.
Recommended Actions
Check:
- The SPE manifold
- Associated instrumentation
- Individual manifold positions
In some cases, a specific position within the manifold may be responsible for the problem.
When This Problem Appears
If manifold pressure is the issue, poor flow will typically be observed during:
- Conditioning steps
- Blank runs
Cause 3: Immiscible Solvents
Description
Using solvents that are not mutually miscible can interfere with flow through the SPE cartridge.
Recommended Actions
Always use solvents that are miscible with each other.
Example
- Hexane
- Water
These solvents are immiscible and may cause flow problems if used together.
When This Problem Appears
Flow issues caused by solvent incompatibility are generally observed during:
- Conditioning steps
- Blank runs
Cause 4: Precipitation and Protein Build-Up
Description
- Sample constituents or proteins may precipitate and clog the sorbent bed.
- This blockage can significantly restrict or stop flow through the cartridge.
Recommended Actions
Possible corrective actions include:
- Diluting the sample
- Reducing sample volume
- Avoiding denaturing solvents
- Washing the sorbent with a low ionic strength buffer before the regular wash step
When This Problem Appears
If protein precipitation or sample-derived precipitates are responsible, the flow problem will typically occur after the sample has been loaded.
Systematic Troubleshooting Approach
When investigating SPE flow issues, use a systematic elimination strategy.
Key Principle
- Evaluate one variable at a time and isolate potential causes individually.
- This approach helps identify the true root cause rather than changing multiple parameters simultaneously.
Summary of Common SPE Flow Problems
Cause / Typical Observation Point / Suggested Solution
- Viscous or cloudy sample: During sample loading - Dilute, centrifuge, filter, or use larger particle size sorbent
- Insufficient manifold pressure: Conditioning or blanks - Check manifold and instrumentation
- Immiscible solvents: Conditioning or blanks - Use only miscible solvents
- Precipitation or proteins: After sample loading - Dilute sample, reduce volume, avoid denaturing solvents, use low ionic strength buffer wash
Conclusion
Flow problems in SPE can arise from several common sources, including viscous samples, insufficient manifold pressure, incompatible solvents, and precipitation of sample components or proteins. Determining exactly when the problem occurs during the SPE workflow is a critical first step in troubleshooting. A systematic, one-variable-at-a-time approach allows the root cause to be identified efficiently and corrective actions to be implemented.
