Solid Phase Extraction, SPE Cartridges



Silica Gel

Solid Phase Extraction is a fast and efficient sample preparation technique for cleanup or concentration for analysis, HPLC, GC, TLC, UV, IR and an excellent tool for method development that is used by most chromatographers today.

SPE is a form of digital (step-wise) chromatography designed to extract, partition, and / or adsorb one or more components from a liquid phase (sample) onto a stationary phase (adsorbent or resin). An adsorbed substance can be removed from the adsorbent by step-wise increase of elution strength of the eluent (step gradient technique). SPE extends a chromatographic system's lifetime, improves qualitative and quantitative analysis, and the demand placed on an analytical instrument is considerably lessened.

In general, SPE is used for three important purposes in state-of-the-art analyses:

  • Concentration of the analyte (up to factor 10.000 - increase of chromatographic sensibility / improved limits of detection)
  • Removal of interfering compounds (protection of subsequent analyses like HPLC, GC, TLC, UV or IR spectroscopy,…)
  • Changing an analyte's environment to a simpler matrix more suitable for subsequent analyses.

Advantages of SPE compared to classical liquid-liquid extraction:

  • Lower consumption of solvents
  • Faster - enormous time savings
  • Lower costs per sample
  • Potential for automation
  • High consistency in individual sample handling
  • More specific selectivity because of the broad range of adsorbents and different retention mechanisms
  • Optimisation of extraction by variation or adjusting of the solid phase and chromatographic conditions

Main steps of the SPE procedure

  1. Conditioning Of The Adsorbent
    Conditioning of the adsorbent is necessary in order to ensure reproducible interaction with the analyte. Conditioning, also called solvation, results in a wetting of the adsorbent and thus produces an environment which is suitable for adsorption of the analyte. Nonpolar adsorbents are usually conditioned with 2-3 column volumes of a solvent, which is miscible with water (methanol, THF, -propanol etc.), followed by the solvent in which the analyte is dissolved (pure matrix, e. g. water, buffer). Polar adsorbents are conditioned with nonpolar solvents. After the conditioning step, the adsorbent bed must not run dry to maintain solvation (deconditioning).


  2. Sample Application (adsorption)
    Sample application can be performed with positive or negative pressure with a flow rate of ~3 ml/min. Sample volumes vary from a few milliters up to liters.





  3. Since analytes can be either adsorbed on the SPE packing material or directly flow through while the interfering substances are retained, there are two possible procedures that can be followed. The first procedure is accomplished by following Step 2: Sample Application, then Step 3: Washing and then Step 4: Elution. However, in cases where washing is unnecessary, step three can be omitted such that Step 2: Sample Application is then followed by Step 4: Elution.


  4. Washing Of The Adsorbent (optional)
    Washing of the adsorbent is usually achieved with a special wash solution; however, in some cases it may not be necessary. If the polarity difference between the wash solution and the eluent is very large, or if both are not miscible, drying of the adsorbent bed after washing is recommended to improve elution and recovery.





  5. Elution
    Elution with a suitable eluent should not be too fast. The elution speed depends on the column or cartridge dimensions and the quantity of adsorbent (about 1 ml/min).

    Without Step 3With Step 3
    Analyte molecules show no interaction with the adsorbent.Analyte molecules are enriched on the adsorbent.
    Interfering components and solvent molecules (matrix) are retained.Interfering components and solvent molecules (matrix) are not retained.
    Analyte molecules are "washed" from the adsorbent.Remaining interfering components are washed from the adsorbent.
    The solid phase is simply used to "filter" the sample.The analyte is removed from the adsorbent by elution with a suitable solvent

4 Step MethodReverse PhaseNormal PhaseIon Exchange
Step 1: Conditioning1: Rinse packing Bed with 3-5 mL methanol. 2: Rinse packing bed with 3-5 mL water or buffer. Don't let packing bed dry before adding sample.Rinse packing bed with 3-5 mL of non-polar solvent. Don't let packing bed dry before adding sample.Rinse packing bed with 5 mL of de-ionized water or low ionic strenght buffer (e.g. 0.0001M-0.01M).
Step 2: Sample IntroductionApply sample solutions to the top of the packing bed. Push or draw the sample through the bed at a flow rate of 1-5 mL/min. Collect sample for analysis if desired compound has passed through the packing bed with out being retained.Apply sample solutions to the top of the packing bed. Push or draw the sample through the bed at a flow rate of 1-5 mL/min. Collect sample for analysis if desired compound has passed through the packing bed with out being retained.Apply sample solutions to the top of the packing bed. Push or draw the sample through the bed at a flow rate of 1-2 mL/min. Collect sample for analysis if desired compound has passed through the packing bed with out being retained.
Step 3: WashIf the desired compound was retained, wash off any weakly retained interfering compound(s) with a polar solvent.If the desired compound was retained, wash off any weakly retained interfering compound(s) with a non-polar solvent.If the desired compound was retained, wash off any weakly retained interfering compound(s) with a de-ionized water or low strength buffer.
Step 4:ElutionElute desired compound with 1-2 mL of a non-polar solvent and collect for analysis.Elute desired compound with 1-2 mL of a polar solvent and collect for analysis.Elute desired compound with 1-5 mL of a high salt concentration solution (e.g. 0.1-0.5M) or change elution buffer pH such that the sample compound is no longer ionized and collected for analysis.