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Selecting Filters for HPLC and UHPLC

Why filter your sample?

Perhaps the most common cause of degradation of HPLC columns is blockage. This is normally caused by a build up of particulates that are either present in the solvent stream or in the sample. Typical symptoms of a column blocking are a steady increase in back pressure and poorer peak shape.

Particles in the solvent stream can also cause problems in the pump head, as they can wear the piston seals over time and result in increased maintenance bills. However it is also important is that any filtering process does not adversely affect the sample, either by retaining compounds of interest or adding peaks to the sample from the filter itself.

Selecting the correct pore size

Most often filters chosen for HPLC have a 0.45µm porosity. Most quality syringe filters are “absolute rated” which means that no particles larger than 0.45um will pass through the filter. This is OK for traditional HPLC columns that have 5µm particle sizes or larger because the gaps between the silica spheres are large enough to allow particle of 0.45µm to pass through easily. However for smaller particles (e.g. 3µm) and UHPLC columns the gap between the silica is smaller and it is recommended to use a 0.2µm filter otherwise a build-up of particles can occur and cause a steady blockage over time.

Using a certified filter that is properly tested for every batch will ensure that there is no variation in the filter membranes and that the pore size really is an absolute size, as well as confirming that the membrane is not contributing to peaks in your chromatography. One of the biggest headaches with 'cheap' filters is lack of consistency.

The filter membrane

It is important that the filter membrane is suitable for use with the sample and solvent it is dissolved in. A list of filter compatibilities can be helpful to ensure the best selection is made. If you have any questions please feel free to contact us.

Nylon filters are have polar surfaces and should not be used with proteins and peptides as they may bind the sample. Low binding membranes such as Regenerated Cellulose (RC) or PVDF should be used instead.

Using the incorrect membrane can result in membrane degradation and elution into the column which is not desirable. Sometimes the incorrect membrane is just hard to use - aqueous samples with PTFE membrane for example. PTFE is naturally hydrophobic and resists water. Using an alternative membrane is usually a lot easier and the results will be just as good or better.

Where high particulate samples are used, a filter membrane with a pre-filter can greatly increase filtration capacity. Pre-filters are generally one or more layers of glass microfibre which will remove the large particles and prevent the main membrane from blocking as quickly.

Filter size

The size of the filter will normally have a direct affect on the volume it can filter – the larger the filter, the bigger the surface area and the larger capacity. However the actual design of the housing can have a large effect also. Well designed 25mm filters can actually out perform poorly designed 30mm filters.

Whilst the amount that can be filtered will depend on how many particulates are in the solution, typically 4mm filters can be used to filter up to 5ml and 13/17mm filters can be used for samples up to about 10ml. These are recommended for many HPLC samples and can save money over larger filters.

If a larger amount of sample is to be filtered then a 25mm or 30mm filter should be used. Many people use this size and still use only a small amount for HPLC analysis.

Ion chromatography

For ion chromatography it is essential that the filter does not elute ions into the sample. Special PES (polyethersulfone) filters that have been tested specifically for Ion chromatography are available and we recommend using these for this application.