[Readers Insight] How to Choose the Right SPE Cartridge?

In chromatographic sample preparation, Solid-phase extraction (SPE) is an essential skill for every analyst. Yet choosing a right SPE cartridge can be remarkably tough, especially when facing a cabinet of cartridges full of abbreviations: C18, HLB, MCX, MAX, WCX, WAX... Almost every chromatographer has once faced a desperate situation where they picked an SPE cartridge, only to find either the target analyte not retained at all or the impurities co-eluted with the target, bringing severe matrix effects.

In fact, an SPE cartridge is not just a filter, but a simplified and fast offline chromatographic column. Choosing the right SPE cartridge from a dozen of candidates determines a large proportion of success. Only by a right cartridge can the target analyte be retained effectively, cleaned up selectively, and eluted efficiently.

Before examining the target compound, examine the sample solvent first. 

If the sample has an aqueous matrix (e.g. plasma, urine, surface water, fermentation broth), reversed-phase SPE is usually the first choice, which extracts the target analyte by its hydrophobicity.

Traditional C18 cartridges are appropriate for moderately to highly hydrophobic compounds, but they have a significant limitation of being potentially sensitive to drying out. If the sorbent bed dries completely after conditioning, the alkyl chains may collapse, significantly reducing recovery.

Polymer HLB cartridges (hydrophilic-lipophilic balance), on the other hand, are an all-purpose option in reversed-phase SPE which are less susceptible to performance loss caused by the sorbent bed drying out. They incorporate hydrophilic groups (pyrrolidone) to balance hydrophilic and hydrophobic interactions, thus applicable for a broad range of polar and nonpolar compounds.

If the sample is pure oil, or has a nonpolar organic matrix, while the target analyte has polar functional groups, normal-phase SPE is more appropriate.

Silica, alumina, or Florisil sorbents, which rely on polar interactions to selectively retain polar compounds, are commonly used in normal-phase mode. Depending on the application, they can be used to retain either polar impurities or polar target analytes.

Normal-phase SPE is commonly used for the cleanup of food and environmental samples, particularly in applications involving pesticide residues and dioxins.

If the sample matrix is too complex (e.g. biological plasma, wastewater sludge extracts), conventional SPE cartridges may fail to provide sufficient cleanup, and the resulting extract may still contain substantial amounts of interfering substances. In this case, mixed-mode ion exchange SPE cartridges can be particularly useful.

These cartridges combine reversed-phase hydrophobic interactions and electrostatic attraction. They remove neutral interferences using water or pure methanol effectively, then elute the target analytes selectively by adjusting the pH.

If the target compound is basic (positively charged), cation exchange cartridges are selected.

For most weakly basic pharmaceutical compounds containing secondary or tertiary amine groups, MCX cartridges (mixed-mod strong cation exchange) are a suitable choice, followed by concentrated ammonia-methanol solution for elution.

For strongly basic compounds (e.g. quaternary ammonium salts) which may bind too strongly to MCX sorbents and become difficult to elute, WCX cartridges (weak cation exchange) may be a good alternative. By acidifying the mobile phase during elution, the carboxyl groups on WCX sorbents become protonated and electrically neutral. The electrostatic attraction between the analytes and the sorbent is therefore eliminated, allowing the analytes to be released.

If the target compound is acidic (negatively charged), anion exchange cartridges are selected. MAX cartridges (mixed-mode strong anion exchange) may be used for weak acids (e.g. carboxylic acids), and WAX cartridges (weak anion exchange) may be used for strong acids (e.g. sulfonic acids, including per- and polyfluoroalkyl substances (PFAS) that contain sulfonic acids).

Once the SPE sorbent has been determined, the next step (where beginners can easily make mistakes) is choosing suitable sorbent mass and cartridge size. In general, SPE involves two critical processes: retaining the target analytes during sample loading and releasing them during elution. The sorbent mass must be sufficient to handle the total amount of compounds retained without compromising recovery.

Under relatively clean conditions, the typical static adsorption capacities of silica-based sorbents (e.g. C18) are approximately 1% to 5% of the sorbent mass, while those of polymeric sorbents (e.g. HLB, MCX) are approximately 10% to 15%. However, these values are empirical for small molecules under ideal conditions; in practice, the molecular structure of the target analyte, sample loading solvent, and competing impurities in the matrix cause the dynamic working capacities to be considerably lower.

Besides referring to the cartridge manufacturer's technical specifications for capacity information, it is recommended to keep the total amount of target analytes and interfering compounds retained on the sorbent below approximately one-third (1/3) of its static adsorption capacity as a preliminary estimate. This provides a safety margin and helps prevent sample breakthrough during loading.

If the cartridge is too large, for example a 500 mg cartridge on a blood sample containing nanogram-level target analyte, it would take too much solvent (e.g. 10 mL) to elute, too much time (e.g. 2 hours) to evaporate, and the strong retention would cause elution to be more difficult and recovery to be lower.

On the contrary, if the cartridge is too small, for example a 30 mg cartridge on dozens of milliliters of loaded sample, the matrix interferences would saturate the sorbent's retention sites, causing the target compounds to pass directly through the cartridge without being retained.