Perhaps the most common cause of HPLC column degradation is blockage. This is usually caused by a build-up of particles either in the solvent stream or in the sample. Typical symptoms of column blockage are a steady increase in back pressure and deterioration in peak shape.
Particles in the solvent stream can also cause problems in the pump head, as they can wear piston seals over time and lead to increased maintenance costs. However, it is also important that any filtering process does not adversely affect the sample, either by retaining compounds of interest or by adding peaks to the sample from the filter itself.
Most filters selected for HPLC have a porosity of 0.45 microns. Most high quality syringe filters are “absolute rated”, meaning that no particles larger than 0.45 µm will pass through the filter. This is fine for traditional HPLC columns with particle sizes of 5 µm or larger, because the gaps between the silica beads are large enough to allow 0.45 µm particles to pass easily. However, for smaller particles (e.g. 3 µm) and UHPLC columns, the gap between the silica is smaller and it is recommended to use a 0.2 µm filter, otherwise a build-up of particles can occur and cause a steady blockage over time.
Using a certified filter that has been properly tested for each 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 problems with 'cheap' filters is the lack of consistency.
It is important that the filter membrane is compatible with the sample and the solvent in which it is dissolved. A filter compatibility chart can be helpful to ensure the best choice is made. Please contact us if you have any questions.
Nylon filters 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 wrong membrane can lead to membrane degradation and elution into the column, which is undesirable. Sometimes the wrong membrane is simply difficult to use - aqueous samples with PTFE membranes, for example. PTFE is hydrophobic by nature and resists water. Using an alternative membrane is usually much easier and the results will be as good or better.
For highly particulate samples, a membrane with a pre-filter can greatly increase the filtration capacity. Pre-filters usually consist of one or more layers of glass microfibre, which remove the larger particles and prevent the main membrane from blocking as quickly.
The size of the filter usually has a direct effect on the volume it can filter; the larger the filter, the greater the surface area and the greater the capacity. However, the actual design of the housing can also have a big effect. Well designed 25 mm filters can actually outperform poorly designed 30 mm filters.
While the amount that can be filtered depends on how many particles are in the solution, typically 4 mm filters can be used to filter up to 5 ml and 13/17 mm filters can be used for samples up to about 10 ml. These are recommended for many HPLC samples and can save money over larger filters.
If a larger amount of sample needs to be filtered then a 25 mm or 30 mm filter should be used. Many people use this size and still only use a small amount for HPLC analysis.
For ion chromatography it is essential that the filter does not elute ions into the sample. Special PES (polyethersulfone) filters are available that have been tested specifically for ion chromatography and we recommend that these are used for this application.