Basic Steps for SPE Columns Operation

The solid phase extraction column is a sample pretreatment device for extracting, separating, and concentrating which is developed from a chromatography column. The separation selectivity of the solid phase depends on the retention strength of the components that can be retained, so the selection of the solid phase will depend on the shape of the analyte and the sample solvent.

Operating solid-phase extraction (SPE) columns involves several key steps to effectively extract, purify, and concentrate target analytes from a sample matrix. Here are the basic steps for operating SPE columns:

Composition of SPE

1. The column tube: is made of serum-grade polypropylene in the shape of a syringe.
2. Sintered mat: fixation and filtration.
3. Fixed phase: commonly bonded silica gel materials.

Materials Needed:

  • SPE columns with the appropriate sorbent material.
  • Sample to be processed.
  • Solvents for conditioning, sample loading, washing, and elution.
  • Vacuum manifold or positive pressure system (optional).
  • Collection tubes or vials for eluate.


  1. Select the Appropriate SPE Column:
    • Choose an SPE column with a sorbent phase that is suitable for your analytes and sample matrix. Consider factors like polarity, pH, and compatibility.
  2. Condition the SPE Column:
    • Prior to use, condition the SPE column by passing a solvent through it. Use a solvent that is compatible with your sorbent material. The purpose of this step is to remove impurities and activate the sorbent. Typically, the solvent used for conditioning is the same as the one used for elution.
  3. Equilibrate the Column:
    • After conditioning, equilibrate the SPE column with a small volume of a solvent that matches your sample’s composition. This ensures that the sorbent is in the proper state for sample loading.
  4. Load the Sample:
    • Load your sample onto the conditioned and equilibrated SPE column. This can be done manually by pipetting the sample onto the column or using an automated system. Allow the sample to pass through the sorbent bed.
  5. Retention and Interference Removal:
    • During sample loading, the target analytes are retained on the sorbent phase based on their specific interactions, while interfering substances pass through the column.
  6. Wash the Column:
    • Wash the SPE column with a suitable solvent or solvent mixture to remove impurities, matrix components, or unretained substances. This step ensures that only the analytes of interest are retained in the column.
  7. Dry the Column (Optional):
    • In some cases, particularly for certain analytes and applications, you may want to dry the sorbent bed by applying vacuum or air pressure for a short duration to remove residual moisture.
  8. Elution:
    • Elute the retained analytes from the sorbent by passing an appropriate elution solvent through the column. The elution solvent changes the chemical environment of the analytes, breaking their interaction with the sorbent and allowing them to be collected in the eluate.
  9. Collect the Eluate:
    • Collect the eluate, which contains the concentrated analytes, in suitable collection tubes or vials.
  10. Concentration (Optional):
    • If necessary, concentrate the eluate to reduce its volume and increase analyte concentration using techniques such as evaporation or nitrogen blowdown.
  11. Analytical Analysis:
    • Analyze the concentrated eluate using your chosen analytical technique, such as HPLC, GC, mass spectrometry, or others, for quantification and identification of the target analytes.
  12. Quality Control:
    • Perform quality control checks, including the use of standards, blanks, and replicates, to ensure the accuracy and precision of your SPE process and subsequent analysis.

Proper optimization of each step, including solvent selection, flow rates, and sorbent compatibility, is essential for successful SPE column operation. Additionally, following best practices for handling and disposal of solvents and waste materials is crucial for laboratory safety and environmental compliance.

In general, SPE improves the technique of sample preparation.

SPE can be used for sample pretreatment of gas chromatography, liquid chromatography, infrared spectroscopy, mass spectrometry, NMR, UV, and AAS.
The solvent strength of the positive solid phase increased with increasing polarity. The solvent strength of the inverse-phase solid phase increased with the increase of non-polarity. Common solvents are water, methanol, isopropyl alcohol, acetonitrile, and sometimes acetone and dichloromethane.