Sample Preparation – Manual Solid Phase Extraction

What is solid phase extraction?

Solid phase extraction (SPE) is a technique that is used to clean up and concentrate samples prior to analysis by gas chromatography (GC) or high performance liquid chromatography (HPLC).

This often involves the use of SPE cartridges which are cartridges that contain a chromatographic sorbent bed, the analytes will remain on the sorbent bed and the sample matrix liquid will pass through the column. The sorbent is washed to remove any unwanted interferences, an elution solvent is then used to elute the analytes from the column.

Types of sorbent and their uses

There are four main types of SPE sorbent phases: non-polar, polar, ion exchange and mixed mode.

Non-polar SPE sorbent phases contain non-polar functional groups, e.g. C18, C8, phenyl, cyclohexyl etc. The analyte will interact  with the sorbent surface by van de Waals interactions. A non-polar sorbent is typically chosen for the extraction of non-polar functional groups from polar matrices such as water.

Polar sorbent phases contain polar functional groups such as diol, aminopropyl and unbonded silica, for example. Polar sorbents are used to extract polar analytes from non-polar matrices, the polar functional groups of the analyte can interact with the sorbent bed via dipole-dipole or hydrogen bonding interactions.

Ion exchange sorbent phases are available in both anionic and cation form, they are used to extract analytes with either basic (cationic) or acidic (anionic) functional groups. Anionic-exchange sorbents consist of positively charged functional groups and cationic-exchange sorbents consist of negatively charged functional groups. These surfaces will interact with an oppositely charged analyte by electrostatic interactions.

Mixed mode sorbents have two or more different modes of retention, the most common mixed mode sorbents have hydrophobic and ion-exchange groups on their surface.

The elution stage

In order for an analyte to be eluted from a sorbent surface the interaction between the analyte and the sorbent must be disrupted.

For non-polar surfaces this means using a relatively non-polar solvent such as methanol. The reverse is true for polar analytes being eluted from polar sorbent beds, a relatively polar solvent should be used to elute the analyte, such as ethyl acetate.

Eluting an analyte from an ion-exchange sorbent bed is possible by three different mechanisms – by use of a buffer with high ionic strength, by altering the pH with the addition of either an acid or a base or by the use of buffers that have counterions with a high affinity for the sorbent surface.

To elute and analyte from a mixed mode sorbent bed both of the mechanisms by which the analyte is bound to the surface need to be disrupted. For example, analytes on sorbent beds that have both hydrophobic and ion-exchange functionality are often eluted by using a non-polar solvent with an acid, base or appropriate buffer.

Sorbent selection

When choosing the correct sorbent for SPE it is important to consider the target analyte, the sample matrix and the quantity of sample to be analyzed.

The SPE sorbent that is used will be determined by the functional groups of the analyte as well as the polarity.  Any properties that may be unique to the analyte should also be considered, including but not limited to ionic properties, aromatic rings, and polarity.

The sample matrix is also important, aqueous samples are best separated using reversed phase SPE (i.e. non-polar analyte with a non-polar sorbent). Samples that are in organic solvents are best suited to normal phase SPE (polar analyte with a polar sorbent).

Often some sample preparation is required in order to get the sample into the correct matrix for the sorbent being used. This often happens with biological samples that may need to be transferred into a non-polar solvent before they can be used with a polar sorbent bed.

Sorbent mass

Sorbent mass selection is dependent upon the sample concentration and the analytical technique that is being used. The mass of retained compounds that is held by a given mass of sorbent is called the sorbent capacity.

Step by Step – Solid Phase Extraction

The first step in SPE is to pretreat the sample – this is important as it optimizes the analyte retention as the sample in made more compatible with the SPE method. How the sample is treated will depend on the analyte, the sorbent and the sample matrix.

Next, is the sorbent bed is conditioned – this is done in order to remove any impurities that may have been left behind during the manufacturing process. Conditioning also has the benefit of activating the sorbent which promotes interaction with the analyte.

After conditioning the sorbent is equilibrated just prior to loading the sample. Equilibration is important as it establishes a sorbent chemistry environment similar to the sample. If this is not performed then the sample may change the chemistry of the SPE sorbent, leading to irreproducible results and poor analyte recovery.

Next, the sample is loaded onto the SPE column. When loading the sample onto the sorbent it should be added at a controlled rate so that the sample is able to interact efficiently with the sorbent bed.

After loading the sample is washed which removes contaminants and cleans up the sample. The properties of a wash solvent are that is has a stronger elution strength than the sample solvent, but a weaker elution strength than the elution solvent.  This ensures that the analyte is not eluted but the impurities/ unwanted contaminants are.
Finally, an elution solvent is used to remove the sample from the sorbent bed. An elution solvent is chosen based on its capability to disrupt the interact between the analyte and the sorbent bed

Solid Phase Extraction Applications

SPE is regularly used in a variety of different industries such as pharmaceutical, forensics, environmental and food/agrochemical.

It is a highly versatile technique and negates the need for the use of many different organic solvents – like those that are used for liquid-liquid extraction (LLE). Less solvent consumption also means less exposure to potentially toxic reagents. SPE also has more separation power than that of LLE and has greater accuracy.

Solid phase extraction (SPE) is a technique that is used to clean up and concentrate samples prior to analysis by gas chromatography (GC) or high performance liquid chromatography (HPLC).
SPE is regularly used in a variety of different industries such as pharmaceutical, forensics, environmental and food/agrochemical.

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