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Fundamentals of SPE Mechanisms

Sample preparation accounts for up to 60% of the time taken for the analytical process. Therefore, it is important to select the correct sample preparation technique and understand the underlying separation mechanism(s) in order to fully maximize the process to give optimum results.  Solid-phase extraction (SPE) is a highly selective mode of sample preparation, akin to the familiar principles of column chromatography.  The range of currently available SPE sorbent chemistries and new technologies makes this technique applicable to a wide variety of analytes in a myriad of application areas. 

However, when compared to other sample preparation techniques such as protein precipitation, liquid-liquid extraction (LLE) or QuEChERS, SPE can be considerably more time consuming and often requires a much greater effort toward method development.  However, the rewards are certainly worth it, with highly selective extractions typically producing very pure samples.

In this article we will cover some of the basic scientific principles behind SPE in order to allow the correct mode of extraction to be selected through an understanding of how analytes interact with and are separated by the sorbent. 


SPE Mechanisms

SPE sorbent phases fall into four primary categories, non-polar, polar, ion exchange (cation and anion), and mixed mode.


Non-Polar SPE

Non-polar SPE phases contain non-polar functional groups such as C18, C8, C6, C4, C2, phenyl, cyclohexyl, and cyanopropyl.  Interaction between the analyte and sorbent surface is via Van der Waals (sometimes called dispersive) forces.  These sorbent types are used for the extraction of molecules which contain non-polar functional groups from predominantly polar matrices (i.e., water). 

The interaction between the analyte and the sorbent surface group is facilitated by polar solvents which repel the analyte from the solution phase and more strongly onto the sorbent surface.  In order to elute analytes from any sorbent surface the interactions between the analyte and SPE functional groups must be disrupted.  In the case of non-polar surfaces, this can be achieved through the use of solvents with some non-polar character (i.e., less polar than water, such as MeOH, MeCN, IPA, THF etc.). 

As mentioned previously, this extraction mode is highly amenable to the extraction of non-polar analytes from polar matrices and is applicable in areas such as the analysis of pharmaceuticals, environmental samples, and clinical samples.1,2


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Figure 1: Non-polar SPE.

Polar SPE

Polar SPE phases are used for the extraction of polar analytes from non-polar matrices.  The sorbent surface has polar functional groups such as diol, aminopropyl, cyanopropyl, unbonded silica, alumina, and FlorisilTM.  Analytes containing polar functional groups can interact and, therefore, be retained at the sorbent surface via dipole-dipole or hydrogen bonding interactions. 

In order to maximize analyte-sorbent interaction, non-polar solvents should be used, and, to disrupt these interactions to elute the analyte, a solvent with some polar character should be used (e.g., THF, ethyl acetate, IPA, MeCN, MeOH, H2O, and combinations of water-miscible organics with water).  If the polar analyte is already in a polar media such as a biological sample (which would not allow good retention on the sorbent surface) a simple solvent exchange (via liquid-liquid extraction) into a non-polar solvent can be performed prior to the SPE extraction.


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Figure 2: Polar SPE.


Ion Exchange

Ion exchange media comes in both anionic and cationic forms for the extraction of analytes with basic (cationic) or acidic (anionic) functional groups.  Cation exchange sorbents will contain surface groups which are negatively charged and the reverse is true for anion exchange materials.  Both surface types interact with the oppositely charged analyte via electrostatic (ionic) interactions. 

There are three mechanisms for disrupting analyte/sorbent interactions:

  1. Using a high ionic strength buffer.  The large number of ions within the buffer will disrupt the analyte/sorbent interactions and elute the analyte. 
  2. Altering the pH through addition of an acid or base.  This can work in two ways.  The pH can be adjusted so that the analyte is neutralized and eluted from the SPE sorbent or the functional groups on the surface can be neutralized to similar effect.  As a general rule in deciding whether to raise or lower the pH, to neutralize an acidic functional group lower the pH 2 units below the pKa (of the analyte or surface functional group).  Conversely, for basic functional groups, increasing the pH two units above the pKa (of the analyte or surface functional group) will deprotonate the functional group, rendering it neutral.
  3. Utilizing buffers which contain counter ions which have a high affinity for the sorbent surface.  If the analyte is strongly retained by the surface and the interaction cannot be disrupted by using a high ionic strength buffer.  These high-affinity ions can readily displace the analyte which may have a lower affinity for the sorbent surface.  This effect is dramatically more pronounced in anion exchange than in cation exchange.  The relative selectivity of different counter ions is shown in Figure 3.

For Cation Exchangers:

Ba2+ > Pb2+ > Ag+ > Cu+ > Ca2+ > Fe2+ > Mg2+ > K+ > Mn2+ > RNH32+ > NH4+ > Na+ > H+ > Li+

For Anion Exchangers:

Benzene sulfonate- > Citrate- > HSO4- > NO3- > HSO3- > NO2- > Br- > Cl- > HCO3- > HPO4- > formate > acetate > propionate > F- > OH-

To change to a more highly selective ion, pass 2-5 bed volumes of 1N solution of the new counter ion through the sorbent.

To change to an ion with lower selectivity, pass 5-65 bed volumes (depending on how much less selective the new counter ion is in comparison to the current counter ion) of 1N solution of the new counter ion through the sorbent.

Figure 3: Relative selectivity of counter ions for ion-exchange SPE.

Ion-exchange sorbents can be further classified as either weak or strong exchangers depending on the type of ionic group bonded to the surface. 

Strong cation exchangers contain an acid functional group, such as a sulfonic acid, which is ionized over the entire pH range. 

Weak cation exchange sorbents have surface functional groups such as carboxylic acids which are negatively charged at high pH, but neutral at low pH. 


Strong anion exchangers are comprised of quaternary ammonium groups that are ionized (positively charged) over the full pH range. 

Weak anion exchangers have primary, secondary, or tertiary amine moieties which will be ionized at low pH, but neutral at high pH. 


The flow diagram shown in Figure 4 can be used to select an appropriate ion exchange sorbent.

Figure 4: Ion exchange selection chart (including analyte and sorbent properties).


For both anion and cation exchange, a protic environment is required for ion exchange to occur (water is highly protic).  Some biological samples like urine or certain cell growth buffers exhibit very high salt content (thus, high ionic strength) and may require dilution to reduce the ionic strength prior to application to the SPE sorbent in order to facilitate retention.


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Figure 5: Cation exchange SPE.   Figure 6: Anion exchange SPE.


Mixed-mode sorbents exhibit two or more primary retention mechanisms with most common mixed-mode sorbents having hydrophobic and ion-exchange functional groups attached to the surface.  Hydrophobic groups can vary from short chain, less retentive (but more selective) C2 moieties to highly retentive C18.  The ion-exchange functionalities can be anion or cation exchangers, or a combination of both. 

There are two methods of manufacturing mixed mode sorbents, either by bonding the sorbent concurrently with different functional group chemistries or by blending discrete sorbents in appropriate ratios.  The blending approach is preferable due to the ease of bonding a single functional group to the silica surface reproducibly.  Furthermore, if different retentive properties are required, different ratios of single functional group sorbents can be blended. 

The development of protocols using mixed-mode sorbents can be more involved than with sorbents which have a single retention mechanism; however, very clean extracts from highly complex matrices can be achieved. 

In order to elute analytes, which will be retained via two retention mechanisms, from the surface, both retentive interactions must be disrupted.  With sorbents which have both hydrophobic and ion exchange groups, this will often involve the use of mixtures of non-polar solvents with either appropriate buffers, acids, or bases.   


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Figure 7: Mixed-mode SPE.

  1. Gilart, N.; Borrull, F.; Fontands, N.; Marce, R. M. Trends Environ. Anal. Chem. 2014, 1, e8-e18.
  2. Spietelun, A.; Marcinkowski, T.; de la Guardia, M.; Namiesnik, J. J. Chromatogr. A 2013, 1321, 1-13.
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