To ensure proper operation of your HPLC system, filtration of both the mobile phase and sample is of paramount importance. To minimize dwell volume and sample dispersion, the tubing within an HPLC system typically gets narrower as the mobile phase and sample move through the various components of the instrument. Unfiltered mobile phase and/or samples will inevitably lead to clogs and over-pressurization of the system.
Filtering mobile phases
If the mobile phase will be mixed by the pumping system (in the case of a binary or ternary pumping system), it is usually not necessary to filter the organic phase if a good grade of solvent is used. However, you should filter any buffers to remove undissolved particulates. This filtration step should be done even if you use a high-purity buffer, such as ACS Grade.
If the HPLC method requires a premixed two- or three-component mobile phase, you should filter all components separately and then mix them. Since vacuum filtration is best for filtering, mixing components before filtering could cause the more volatile components of the mobile phase to evaporate and change the effective percentage of each component.
When filtering a mobile phase, it is the norm to use a 0.45 micron filter. For sub-two-micron columns where the frits may be as small as 0.5 micron, consider a 0.2 micron filter. A 0.2 micron filter will prevent any small particles from accumulating on the frit and rendering the column useless. When choosing a filter material, take care that the filter will tolerate the liquid being filtered.
The aqueous part of the mobile phase may be a breeding ground for microbial growth, both in the mobile phase bottle itself and in any degassing unit through which the mobile phase travels. When you see any cloudiness in the bottle, you should immediately change this liquid. It is best practice to write the date the buffer was prepared on the bottle, as well as an expiration date. If you have a degasser channel, periodically flush it with isopropyl alcohol to clean out any microbial buildup.
Filtering samples
Sample filtration is a necessary step for any material that will be injected into an HPLC system. Regardless of how the sample is prepared, there is always a chance that small particles will remain in the matrix. As with mobile phase filtration, take care to use the appropriate filter porosity.
With sample filtration, make sure that the filter is compatible with the sample matrix and analytes. If the sample filter has any affinity for the compounds being analyzed, there may be unwanted sample retention. One material that is gaining in popularity is regenerated cellulose. This material has excellent chemical resistance against standard aqueous and organic solvents and has very low affinity for biological samples, resulting in the highest sample recovery rates.
By taking the proper precautions, you can avoid troublesome flow blockages in your HPLC system. To learn more about reducing flow blockages, listen to an Agilent podcast, and visit the product page for Agilent syringe filters.