Regenerated cellulose in HPLC filters

High-performance liquid chromatography (HPLC) is a versatile technique for chemical analysis that employs a range of different solvents depending on the application.
HPLC benefits from filtration during sample preparation to remove particulates. Please see our other article on HPLC sample preparation for more detail around filtering.

Why is a broadly compatible filter useful?

Many different filter materials are available to suit, for example, acids, bases, and organic solvents.

The use of a filter that is broadly compatible with many different solvents, such as those using regenerated cellulose (RC), introduces consistency into lab operations. By using the same filter type across multiple samples, a source of analytical variation is removed. This makes investigating the root cause of spurious results easier and reduces the time spent deciding on what filter to use. This idea of standardization can be extended by use of the same standard membrane in multiple device formats. This ensures that whether you are filtering mobile phase or sample, or whether you are using automated or manual filtration you are still exposing your system to a single membrane type.

What is regenerated cellulose?

Regenerated cellulose consists of pure cellulose without the addition of any wetting agent. After solubilization and casting, it results in a uniform, reproducible membrane for filtration.

This manufacturing process produces a hydrophilic membrane with spontaneous wetting properties and compatibility with both organic and aqueous solutions. The RC membrane is also mechanically stable and has a good wet strength.

How does RC make HPLC sample preparation easier?

Regenerated cellulose filters are broadly compatible with many different solvents. These solvents include acetic acid, acetone, acetonitrile, chloroform, ethanol, ethers, methanol, THF, and water. This broad solvent compatibility simplifies workflows. Compatibility tables provide further information.

RC membranes also have low levels of extractables. These substances are released from the filter and can potentially influence a measurement by absorbing light at the same wavelength as compounds in the sample (Table 1). Reducing the level of extractables helps to minimize interference in HPLC data.

Table 1. Levels of UV-absorbing materials from RC filter devices exposed to common HPLC solvents

 

Solvent
Product
Water Methanol Acetonitrile
214 nm 254 nm 214 nm 254 nm 214 nm 254 nm
SPARTAN™  30 mm, 0.45μm - mAU
9.5
1.4 5.7 3.7 4.5 1.9
SPARTAN 30 mm, 0.2 μm - mAU
2.8
2.8 4.2 3.1 5.4 1.6
SPARTAN 13 mm, 0.2 μm - mAU
1.0
0.3 3.0 1.3 0.8 0.8

 

Another major aspect of filter selection is the protein binding characteristics of the filter material. High levels of nonspecific protein binding can reduce protein recovery substantially. This loss can be detrimental to the results of an experiment by reducing the quantity of proteins available for analysis and decreasing the signal-to-noise ratio.

RC filter devices demonstrate low protein binding (Table 2), making them appropriate for determining protein levels with minimal effect on quantitation. For filters designed to prepare difficult-to-filter samples, such as sugar syrup, this is less of a consideration because reduced sample recovery is expected.

Table 2. Low protein-binding filters show high levels of protein recovery when filtering a model solution of bovine serum albumin (BSA).

 

Filter device
Recovery of 1 mg/mL BSA (%) SD Sample loss (μg) Recovery of 0.5 mg/mL BSA (%) SD Sample loss (μg)
SPARTAN 13 mm, 0.2 μm
98
0.6 40 97 0.3 26
SPARTAN 13 mm, 0.45 μm
99
0.7 21 99 0.9 9
Whatman GD/X  25 mm RC, 0.2 μm
86
1.9 284 78 2.2 222
Mini-UniPrep PP, RC, 0.2 μm
95
0.9 20 94 2.5 11
 

Which filtration devices use RC membranes?

A wide range of Whatman filtration devices are available with regenerated cellulose membranes. These devices include syringe filters with and without prefilters, as well as syringeless filters for use with autosamplers. One family of devices, SPARTAN syringe filters, is HPLC-certified.

Recent testing results suggest that standardizing on a membrane, such as RC, for typical HPLC sample preparation reduces the variation associated with sample preparation and increases lab process consistency.

Try our Whatman Filter Selector App to find out if you are using the most appropriate filtration solution for your samples. To discuss any challenges you are facing, please contact GE's Life Sciences Scientific Support.