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Troubleshooting Filtration

The first step to troubleshooting (or avoiding having to troubleshoot) any sample preparation or analytical method is to select the correct method and equipment in the first place.  However, this does not guarantee that problems may not arise.  The following article will look at selecting the correct syringe filter, where to find relevant information on selecting a syringe filter, and what common problems may arise during filtration using syringe filters.

Filtration Basics

Filtration is a very simple and cost effective technique which can improve the quality of a sample by removing particulate material that may be incompatible with the analytical technique (Figure 1).  Unfiltered particulates can often clog instrument interfaces and HPLC columns creating high backpressure or blocking devices altogether.  At the very least this problem is likely to reduce operating lifetimes of columns and instruments.

Unfortunately filtration does little to purify the sample from a chemical standpoint by removing undesired contaminant species from solution.  Therefore, it may be pertinent to combine filtration with other more chemically selective forms of sample preparation (i.e. liquid-liquid extraction, solid phase extraction) if interfering contaminants must be removed.

An exception to this is ultrafiltration which uses membrane filters of very controlled low specific porosity.  These membranes are capable of removing large species from the solution such as proteins.  Ultrafiltration is a purification technique in its own right; however, it has limitations such as the facile processing of samples and the ability to automate the process.

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Figure 1: Filtration.


There are several types of filtration products which can be used for different applications (Table 1).  The type of filtration product needs to be chosen based on the desired filtration outcome and the subsequent analytical analysis.  For example, filter paper which is capable of removing particulates which are greater than 40 µm in size would not be suitable for appropriate filtration of a sample which is being injected into an HPLC instrument in which the column has a particle size of 5 µm, this would lead to catastrophic blockages. 

However, if particularly dirty samples of for example, river water were being analyzed an initial filter using filter paper to remove larger particles followed by filtration using a syringe filter (which would be blocked by large particulates) would be appropriate.  If the sample contains matrix components or interferences which need to be removed as well as particulate material then the use of solid phase extraction (SPE) cartridges would be a good choice.

Filtration Media Products Use
Filter paper Cellulose Removal of large particles (> 40 µm)
Membrane filters Nylon, PTFE, polypropylene, polyester, PES etc. Removal of small particulates (< 10 µm)
Functionalized membranes Ion exchange, affinity membranes Removes particulates and matrix interferences
SPE cartridges Silica and polymer based
SPE discs PTFE and fiber glass based

Table 1: Filtration media.


Selecting a Syringe Filter Membrane

When selecting a syringe filter for a specific application there are several features of the filter which need to be considered, including the type of membrane, filter size (diameter), and porosity.

When selecting the membrane material which the syringe filter is made from the sample diluent (solvent, pH etc.) and the nature of the analyte must be considered.  The chemical compatibility of the different membrane materials must be considered in conjunction with the type of diluent (Figure 2).  As can be seen from Figure x all samples can initially be considered as either aqueous or organic.  Membranes such as polyethersulfone (PES) are suitable for all aqueous samples and will also be suitable for the filtration of some samples containing organic solvents. 

Most manufacturers will provide extensive chemically compatibility tables which take into account organic solvents, acids, bases, aqueous components (i.e. hydrogen peroxide), and solution pH (Table 2) so as to avoid damaging the syringe filter through inappropriate use. 

When considering the chemical compatibility of a syringe filter it may also be necessary to take into account the material which the housing that holds the membrane is made of.  Most commonly the housing is made from either polypropylene (PP) or methacrylate butadiene styrene (MBS) which will exhibit different chemical resistances to solvents, acids, bases etc. and could be damaged by some of these components or suffer leaching of material from the filter which would contaminate the filtered sample.


Figure 2: Consideration of sample composition when selecting a syringe filter.

Filter PP PES CA PTFE RC Nylon GF  
Housing MBS PP
Acetone ** - - ** ** ** ** - **
Acetonitrile * - - ** ** N/A ** - **
Chloroform * - - ** ** ** ** - **
MeoH 98% ** * - ** ** ** ** ** *
THF * - - ** ** ** ** - **
Toluene - - * ** ** ** ** - **
HCl, 20% ** ** - ** - - ** * *
H2SO4, 20% ** * - ** * - ** * **
NH4OH, 25% ** * * ** * * * - *
NaOH, 1N ** ** - ** * * * - **
H2O2 ** ** - ** - - ** - **
1-14 ** - - ** - - ** - **
1-13 ** ** - ** - - ** - **
3-14 ** * - ** * ** ** - **
3-12 ** ** - ** ** ** ** * **
4-8 ** ** ** ** ** ** ** ** **

Table 2: Chemical compatibility of syringe filter membranes and housing.  Polypropylene (PP), polyethersulfone (PES), cellulose acetate (CA), polytertafluoroethylene (PTFE), regenerated cellulose (RC), nylon, glass fiber (GF), methacrylate butadiene styrene (MBS).  **  Compatible, *  Limited compatibility, -  Not compatible, N/A  Not analyzed.


The following PDF documents from several syringe filter manufacturers provide more extensive chemical compatibility information and are a great resource to aid in the selection of syringe filters.

Thermoscientific Labware Chemical Resistance Table

Thermoscientific Chemical Resistance Summary

Thermoscientific Physical Properties Table

Agilent Captiva Syringe Filters

Pall Life Sciences Sample Preparation and Mobile Phase Filtration


As well as the sample solvent composition, the type of analytes present in the sample need to be considered.  Depending on the chemical nature of an analyte, the membrane in a syringe filter might nonspecifically bind the analyte, resulting in inaccurate quantitation, especially for low concentration samples.  Glass fiber filters tend to bind strongly to proteins, peptides, and oligonucleotides.  Each membrane type will vary in its binding properties.

Polyethersulfone (PES) membranes are particularly suitable for the filtration of samples conating proteins as they exhibit very low protein binding.  PES is also more heat resistant than most other membranes and can be used up to 100 °C.  These types of membranes are also often used for ion chromatography.

Cellulose acetate membranes exhibit very low protein binding (< 24 µg/cm2) and are ideal for aqueous and biological samples.

Polypropylene membranes are amenable to use with a wide variety of organic and aqueous based samples.  They can be used to filter samples containing proteins as they are low protein binders and can be used with strong acids and bases without the need for prewetting.

Nylon membranes also show very high protein binding (~225 µg/cm2), however, they are extremely low in extractables and mechanically very strong.  They also possess good thermal stability up to 50 °C and due to their hydrophilic nature show good compatibility over a broad range of aqueous and organic samples and can be thought of as a good general filter for HPLC samples.

Polyvinylidene fluoride (PVDF) membranes show very low protein binding (~15 µg/cm2) amking it a good choice for general biological sample filtartion.

Polytetrafluoroethylene (PTFE) membranes are chemically resistant to nearly all solvents, acids, and bases.  It also shows good thermal stability and has low extractables.  If this membrane is to be used with aqueous samples it will require prewetting prior to use due to its hydrophobic nature.

Regenerated cellulose is a hydrophilic solvent resistant membrane that exhibits low protein binding making it amenable to applications such as tissue culture media filtration and general biological sample filtration.

If the sample is particularly particulate laden or viscous then single membrane filters may get clogged by larger particulates before the sample actually reaches and passes through the syringe filter.  When working with these types of samples it can be advantageous to use a syringe filter which contains a prefilter.  The prefilter will trap larger particulates which protects the final membrane from fouling and allowing higher volumes of sample to be filtered before the filter clogs.   

Multi-layered filters can filter 4-6 times more volume than a membrane-only filter.  However, prior to selecting this type of filter it is important to note that prefilters are usually made of glass fibre, which can lead to higher levels of extractables leaching from the syringe filter and contaminating the filtered sample or a greater amount of analyte binding especially with proteins, peptides, and oligonucleotides.  In such cases it might be better to filter samples through a filter which contains an inert/low protein binding filter and a prefilter. 

If analyte binding is expected, then processing pure standards of the analyte(s) through the filtration device and then analysing the filtrate using the appropriate analytical technique can help to determine the degree to which the analyte(s) are being lost.

Selecting Syringe Filter Size

The ideal filter diameter (2-50 mm) balances filtration performance with the risk of extractables and analyte binding, and depends on the sample volume (Table 3).  Larger filters enable fast filtration using low pressure which minimizes the chance of bursting; however, they may have higher hold-up volumes which could trap samples resulting in sample loss and poor quantitation.  The use of larger filters also risks analyte loss due to nonspecific binding and may introduce higher levels of extractable impurities.

Sample Volume (mL) Filter Diameter (mm) Hold-up volume (µL) Filtration Area (cm2)
< 1 4 10 0.1
1-10 13 25 0.65
10-150 25 100 3.6
10-150 33 80 4.5

Table 3: Syringe filter diameters.  Note diameters may vary slightly between manufacturers.


Selecting Syringe Filter Porosity

The porosity of the syringe filter should be considered in conjunction with the porosity of the column inlet frit and/or packing material diameter so that any particulates which would be large enough to block the column inlet frit, or column itself, are removed prior to reaching the HPLC system.  For example if a column is packed with particles which are less than 2 µm a 0.2 µm UHPLC filter should be used.  Syringe filters with a 0.2 or 0.45 µm porosity are suitable for the filtration of samples which will be analysed using columns packed with particles > 2 µm.


Syringe Filters for Specific Applications

Many manufacturers will list syringe filters which are suitable for specific types of application (Table 4) this takes a lot of the guess work out of selecting a syringe filter.  Although, it is still good practice to check the chemical compatibility with respect to a particular sample.

Type of Sample Recommended Syringe Filter Alternative Syringe Filter
HPLC, UHPLC, LC-MS, GC Regenerated cellulose (RC) PTFE or nylon
ICP-MS PTFE Glass fiber/PTFE (high particulate samples)
CE Regenerated cellulose (RC) Nylon
Undiluted organic solvents PTFE Nylon
Protein analysis, samples with biomolecules (buffers) Polyethersulfone (PES) Regenerated cellulose (RC) or cellulose acetate (CA)
Tissue culture media Polyethersulfone (PES) Regenerated cellulose (RC) or cellulose acetate (CA)
High particle load samples (organic solvent) Glass fiber/PTFE  
High particle load samples (aqueous solvent) Glass fiber/nylon  

Table 4: Syringe filters for particular applications.


Minimizing Extractable Contamination

Extractables are undesired artefacts contributed to the sample fluid from the filter device i.e. membrane or housing formulation component, or a component introduced during the manufacturing or packaging process.  These components can be extracted from the filter by the sample solvent.  Signals from extractable impurities can pose problems as they will produce erroneous signals within the chromatogram or mass spectrum which could result effect quantitative results.  However, these signals can be reduced significantly simply by rinsing the filter with 1 mL of the sample solvent prior to carrying out filtration of the sample (Figure 3).


Figure 3: Effect of rinsing on extractables.


Determining Filtration Efficiency

It is essential that the efficiency of the sample filtration method is assessed in order to confirm both accuracy and precision (Equation 1). 


The sample chosen should be the specific sample under investigation, however, if this is not available or a number of samples are under investigation then a representative sample should be chosen.


You may also like…

The CHROMacademy Essential Guide Webcasts:

Troubleshooting Sample Preparation

Choosing the Correct Sample Preparation Technique

Understanding and Improving SPE

CHROMacademy eLearning Module:

Primary Sample Preparation Techniques

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