Solid phase extraction (SPE) is a sample preparation technique often used by chromatographers prior to analysis. SPE is most often used to remove interfering compounds from a sample, although it can also be used to enrich/concentrate analytes of interest in the sample. SPE makes use of a solid phase material (there are many to choose from) that functions to retain the interfering substances, while solvents elute the sample, which is collected and analyzed.
SPE can therefore improve analytical results in HPLC, GC, IC, and MS analyses by reducing the complexity of a sample, reducing baseline interferences, and/or increasing detection sensitivity. Employing solid phase extraction sample preparation can also reduce harmful compounds introduced into the chromatography system thereby extending the longevity of the analytical column and instrument. It is, therefore, easy to see why SPE is widely used by chromatographers in pharmaceutical, environmental, forensics, and food safety applications.
SPE uses significantly smaller volumes of solvent compared to liquid/liquid extraction (LLE) and supported liquid extraction (SLE) methods.
Solid phase extraction employs the same basic principles as chromatography. SPE makes use of the same HPLC column solid phase packing materials in a single-use container such as plastic tubular cartridges (shown in Figure 1), 96-well plates, or pipette tips. Like HPLC columns, there are many stationary phase options to choose from, such as reversed phase, ion exchange, normal phase, and mixed mode phases (see Table 1 and the SPE Solid Phase Selection page). Unlike HPLC, SPE is typically performed as a low resolution and very low-pressure method designed to prepare samples by removing interfering substances or concentrating a sample.
The most common steps to performing SPE are shown in Figure 1. SPE methods are based on these steps.
Purpose: To optimize the sample for effective analyte retention. Consider the following when pre-treating a sample prior to application to the SPE product:
Table 2 provides general SPE pre-treatment examples for various sample matrices.
|Table 2 Basic sample pre-treatment approaches for common sample
|Dilute with an equal volume of water or suitable buffer prior to applying the sample to the SPE column. Buffer choice and pH considerations are dependent upon the compound of interest in the sample.
|Blood is similar to serum and plasma, apart from the presence of whole red blood cells. Dilute with an equal volume of water or buffer to ensure that compounds of interest are free in solution.
|Dilute with an equal volume of water or suitable buffer prior to applying the sample to the SPE column.
|Dilute samples with non-polar organic solvents such as hexane due to the non-polar nature of the matrix.
|Homogenize sample with a non-polar solvent.
|Ointments are typically either water-based or oil-based. For and Creams water-based products, dissolve in a polar solvent such as methanol. For oil-based products, dissolve in a non-polar solvent such as hexane.
|Pre-treatment is dependent upon the particulate content of the sample. Some samples can be applied directly to the SPE product. For samples heavily laden with particulates, filtration/centrifugation may be necessary.
|Soil and Sludge
|Analytes can be difficult to adsorb onto the sorbent material. Samples are typically extracted using a non-polar solvent such as hexane, then using a polar sorbent material for the SPE process.
|Fruits and Vegetables
|Homogenize sample with a polar solvent such as methanol and subsequently dilute with water if required.
|Crude Oil Products
|Dilute sample with a non-polar solvent such as hexane.
|Typically diluted/homogenized with water or suitable buffer.
|Meats and Soft Drinks
|Dilute sample with water.
Purpose: Column conditioning prepares the sorbent for effective interaction(s) with the compounds of interest.
Purpose: To re-equilibriate the column
Purpose: To apply the sample at an appropriate flow rate to maximize retention of the analyte to the stationary phase.
Purpose: To wash the stationary phase using an intermediary solvent to remove impurities bound less strongly to the sorbent than the compounds of interest.
Purpose: To elute and collect the analyte. This step is designed to selectively recover the analyte(s) by disrupting the analyte-sorbent interaction.
96 well plate formats are ideal if one is processing a large number of small samples simultaneously while in cartridge format, you can process a couple dozen samples at a time. SPE manifolds are often used for helping to process samples.
SPE cartridges, also referred to as SPE columns, are single-use plastic tubes. Thermo Scientific SPE cartridges are available in a variety of sizes with a capacity of from 1mL to 150mL. Cartridge format is used to process a limited number of samples at a time. 3mL cartridges are a popular cartridge size and a good starting point for many applications.
Here are some considerations when determining the best cartridge size to use:
Table 3 provides typical sample, sorbent, and elution solvent loading volumes for reference to help in determining a cartridge size to use.
|Table 3 sample, sorbent, and elution solvent used in methods for different sizes of SPE cartridges
|Minimum Elution Volume
Well plate formats are ideal if one is processing a large number of small volume samples simultaneously. Bed weights are typically lower 2mg – 30mg begin common and are often used for bioanalytical sample analysis where samples are often complex, lower in concentration and volume. Processing is often high throughput in nature.
Table 4 illustrates the broad range of solid phase extraction phases and formats offered to solve sample preparation need of a broad spectrum of samples, sample volume, and application requirements, as well as throughput constraints.
|Table 4. Thermo Scientific SPE phases in cartridge and well plate format
|Normal Phase / Reversed Phase
|Mixed Mode Ion Exchange
|MM Cation Exchange
|MM Anion Exchange