Solid phase extraction guide

What is SPE?

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.

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SPE sample cleanup and enrichment

Remove interfering compounds by:

  • Selective adsorption of analytes + interferences
  • Selective elution of interferences
  • Selective elution and collection of analytes

Enrich/concentrate analytes by:

  • Large sample load volume
  • Small elution volume
  • Evaporation/reconstitution

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.

How SPE works

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.

SPE stationary phase selection

Performing SPE

Performing SPE

The most common steps to performing SPE are shown in Figure 1. SPE methods are based on these steps.

Overview of SPE Steps

1. Sample pre-treatment

Purpose: To optimize the sample for effective analyte retention. Consider the following when pre-treating a sample prior to application to the SPE product:

  • Adjust sample/matrix composition for proper dilution/ionic strength
  • Ensure that sample is at proper pH for optimum retention
  • Confirm that analytes are free in solution
  • Remove any unwanted particulates via filtration or centrifugation

Table 2 provides general SPE pre-treatment examples for various sample matrices.

Table 2 Basic sample pre-treatment approaches for common sample
Sample Matrix Sample Pre-Treatment
Serum, Plasma 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.
Whole Blood 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.
Urine Dilute with an equal volume of water or suitable buffer prior to applying the sample to the SPE column.
Fats, Oils Dilute samples with non-polar organic solvents such as hexane due to the non-polar nature of the matrix.
Cereals Homogenize sample with a non-polar solvent.
Ointments 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.
Water 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.
Dairy Produce Typically diluted/homogenized with water or suitable buffer.
Meats and Soft Drinks Dilute sample with water.  

2. Column conditioning

Purpose: Column conditioning prepares the sorbent for effective interaction(s) with the compounds of interest.

  • An appropriate solvent conditions the column and activates the ligands on the chromatographic surface
  • The solvent should have similar characteristics (solvent strength, pH, etc.) to the sample to ensure maximum retention of analyte.
  • Prevent the sorbent from drying during the conditioning step (dry sorbent can affect the ability of the analytes to interact); allow about 1mm of last conditioning solvent to remain above the top tube frit

3. Column re-equilibration after column conditioning

Purpose: To re-equilibriate the column

  • Use the same solvent that is used for the sample pre-treatment step (do not let the sorbent dry during the conditioning step)
  • Allow about 1mm of last conditioning solvent to remain above the top tube frit

4. Sample application

Purpose: To apply the sample at an appropriate flow rate to maximize retention of the analyte to the stationary phase.

  • A typical flow rate is 1mL/minute.
  • A high flow rate can lead to inconsistent extractions.

5. Solvent wash to remove interferences

Purpose: To wash the stationary phase using an intermediary solvent to remove impurities bound less strongly to the sorbent than the compounds of interest.

  • Select a wash solvent that is strong enough to remove the interferences, but weak enough to leave the compounds of interest behind
  • Selectively rinse away the less strongly bonded interferences
  • Wash solvent selected according to phase mechanism and analyte properties (a typical wash solution may contain less organic or inorganic salt than the final eluent)

6. Elute 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.

  • Selectively elute the analytes of interest using different solvents
  • A smaller elution volume leads to a more concentrated extract
  • Select an elution solvent that leaves the strongly retained impurities behind
  • Select elution solvent according to phase mechanism and analyte properties
  • For best results, elute compounds of interest using two small aliquots (rather than one large aliquot)
  • Use as small a volume of solvent as possible when you need to dry down the eluent and reconstitute it for purposes of concentrating the sample.

When to use a SPE cartridge versus 96-well format

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 cartridge format

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:

  • Cartridge volume should be selected to closely match the volume of wash solvent use
  • The volume of sample and solvent added can help in determine which size cartridge you need.
  • Typically, one can load a sample that is approximately 5-10% of the sorbent weight in a given SPE cartridge.
  • The amount of sorbent increases along with size of the cartridge.
  • Smaller cartridge sizes contain smaller amounts of sorbent and require less solvent for elution. This can make separation more efficient separation and is advantageous when evaporation is required.
  • Larger cartridges can be used when larger volumes of solvent are required. A larger sorbent amount can also be helpful when analytes are difficult to retain on a column.
  • One should take into consideration of extraction speed and sample capacity. For example, a faster extraction speed or a higher sample capacity requirement will need a larger cartridge.

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
Cartridge Volume Sample Size Sorbent Mass Minimum Elution Volume
1mL 2.5-10mg 50-100mg 100-200mL
3mL 25-100mg 500mg 1-3mL
6mL 25-100mg 500-1000mg 2-6mL
12mL 100-200mg 2000mg 10-12mL

96-well plate format

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.

Thermo Scientific SPE cartridges and well plates

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.

  • Thermo Scientific HyperSep cartridges and well plats are the gold standard in SPE products.
  • Thermo Scientific SOLA and SOLAμ SPE well plates and cartridges are ideal for biosamples, including low-volume samples for bioanalysis.
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