Chemical compatibility in HPLC samples

Efficient high-performance liquid chromatography (HPLC) analysis requires removal of particulates that could interfere with accurate detection of the analyte of interest. Filtration prior to HPLC serves to reduce impurities but risks introducing new contaminants in the form of extractables.
Awareness and selection of syringe filter components based on their chemical compatibility to solvents reduces the risk of new contaminants interfering with HPLC analyses.

What are common methods of sample preparation?

Accurate HPLC analyses require suitable sample preparation to maximize purity and ensure the analyte concentration is within the range of detection. Common methods for sample preparation include:

  • Dilution
  • Centrifugation
  • Filtration

Dilution or centrifugation of individual samples might be sufficient to minimize insoluble particles for HPLC. Sample filtration using a filtration device adds an extra layer of security, because the membrane physically segregates insoluble particles to help prevent irregular chromatogram peaks. Syringe and syringeless filters are a convenient option to process multiple samples for autosampling.

However, filtration devices themselves can be a source of extractables with the potential to influence data quality, dependent on their compatibility with the solvents used in sample preparation.

What are extractables?

Extractables are compounds released from a component, such as a filter membrane or housing, that is in contact with the sample solvent. The quantity of extractables varies depending on the solvent resistance of the component, as well as temperatures and pressures involved.

How can extractables affect HPLC?

These chemicals can interfere with HPLC analyses if detectable by the downstream analytical method. Extractables can introduce data noise, which reduces data quality and results in otherwise unnecessary HPLC troubleshooting. This is particularly a concern if the extractable has the same retention time as the analyte of interest.

If the extractable contribution is inconsistent across filters and so it elutes at an inconsistent rate, this situation is much more challenging to account for in analysis.

What strategies can help reduce extractables?

It is important to choose a filtration membrane that has high chemical compatibility with the mobile phase solvents used in the lab. Polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), and regenerated cellulose (RC) membranes are popular choices, because they are highly compatible with common mobile phase solvents such as purified water, acetonitrile, and methanol.

Users are encouraged to obtain data from manufacturers about the level of extractables found in their filtration membrane. If such data are unavailable, users can test their preferred membrane with blank value samples (i.e., samples consisting of only the mobile phase solvent).

When practical, a good lab practice is to discard the first 1 or 2 mL of sample after syringe filtration. Most extractables will be eluted in these first drops.

What other considerations might influence membrane choice?

Reducing variation in materials across the lab can benefit overall consistency. One way to reduce variation is to standardize on one or two membrane types, such as PTFE or RC, and use them in a variety of different formats. For example, the same membrane materials would then be used in membrane circles for mobile phase filtration, syringe filters for manual preparation, and syringeless filters for automated filtration.

Another benefit of standardization is that fewer consumables will need to be stocked. Indeed, it is not always possible or economical to have every combination of housing and membrane material on the shelf.

Regenerated cellulose offers some distinct advantages over other membrane types. Read about the benefits of RC here.

Which membrane material should I use for my samples?

Try the Whatman filter selector to find out if you are using the most appropriate filtration solution for your samples. To discuss any challenges you are facing, please contact Cytiva's Scientific Support.


Laboratory Filtration – Principles and chemical compatibility chart