Solid Phase Extraction (SPE) is a widely used sample preparation technique in analytical chemistry laboratories to isolate, purify, and concentrate specific target molecules from complex liquid mixtures. This process is a form of low-pressure liquid chromatography, utilizing the differential affinity of compounds between a liquid sample (the mobile phase) and a solid adsorbent material (the stationary phase). The objective of SPE is twofold: to remove unwanted matrix interferences, such as salts or proteins, and to increase the concentration of the target substance, the analyte, before final analysis by instruments like High-Performance Liquid Chromatography (HPLC) or Gas Chromatography (GC). SPE improves the sensitivity and accuracy of subsequent analytical measurements.
The Underlying Chemical Principles
Solid Phase Extraction operates by exploiting selective retention, where the target analyte is temporarily captured by the solid phase while unwanted components pass through. Separation relies on various intermolecular forces, including non-polar interactions (such as hydrophobic interactions) and polar interactions (like hydrogen bonding and dipole-dipole forces).
The sorbent material is typically a finely divided, porous material packed into a cartridge. Analysts select a sorbent whose surface chemistry complements the analyte’s chemical properties, promoting strong but reversible binding. For example, a non-polar analyte is retained on a non-polar sorbent through hydrophobic interactions, partitioning it out of a polar liquid sample.
By manipulating the chemistry of the liquid phase, analysts control the strength of these molecular interactions to achieve separation. Changes in solvent polarity, pH, and ionic strength are tools used to modulate the balance between the analyte’s affinity for the solid sorbent versus its affinity for the liquid solvent.
The Four Stages of Solid Phase Extraction
Solid Phase Extraction is performed through a sequence of four distinct steps.
Conditioning
The first stage is Conditioning, where a solvent is passed through the dry sorbent bed to prepare it for interaction with the sample. This step wets the sorbent material and solvates the functional groups on its surface, creating the proper chemical environment for analyte retention. For example, in reversed-phase extraction, the sorbent is typically conditioned with an organic solvent like methanol, followed by a rinse with water.
Loading
The second stage is Loading, which involves introducing the liquid sample onto the conditioned sorbent. During this step, the target analyte interacts with and binds selectively to the solid phase, while the majority of the liquid matrix and non-retained impurities pass through the column. The flow rate is often controlled to ensure adequate contact time for the analyte to be efficiently captured by the stationary phase.
Washing
Next is the Washing stage, where an intermediate solvent is passed over the sorbent to remove any weakly retained matrix components. The wash solvent is chosen to be strong enough to disrupt the binding of impurities but weak enough to leave the desired analyte firmly attached to the sorbent.
Elution
The final step is Elution, which recovers the purified and concentrated analyte from the sorbent bed using a strong solvent. The elution solvent is selected because it has a high affinity for the analyte, effectively breaking the retentive interactions between the analyte and the solid phase.
Primary Modes of Separation
SPE utilizes several different chemical modes of separation.
Reversed-Phase SPE
Reversed-Phase SPE is the most common mode, relying on non-polar, hydrophobic interactions for retention. It employs a non-polar sorbent, such as silica bonded with C18 hydrocarbon chains, to extract non-polar analytes from polar solvents like water. The analyte is retained by hydrophobic attraction and is subsequently eluted with a more non-polar solvent, such as acetonitrile or methanol.
Normal-Phase SPE
Normal-Phase SPE is based on polar interactions, utilizing a polar sorbent like bare silica or alumina. This technique extracts polar analytes from non-polar liquid matrices, with retention occurring via hydrogen bonding and dipole-dipole forces. Elution of the analyte is achieved by increasing the polarity of the elution solvent.
Ion Exchange SPE
Ion Exchange SPE separates compounds based on electrostatic attraction between charged functional groups. This mode uses sorbents with either positively charged groups (cation exchange) or negatively charged groups (anion exchange) to selectively retain oppositely charged analytes. Retention is disrupted, and the analyte is eluted, by changing the pH to neutralize the analyte’s charge or by using a high-ionic-strength buffer that competes for the binding sites.
Essential Uses in Analytical Science
SPE is used across numerous scientific disciplines where complex samples must be cleaned and concentrated before analysis.
In forensic toxicology, SPE is routinely used to extract drugs of abuse, poisons, and their metabolites from biological fluids like urine, blood, or plasma. This cleanup removes proteins and other biological matter that could damage or contaminate the analytical equipment.
Environmental analysis relies on SPE to detect and quantify trace levels of pollutants in large-volume water samples. Pesticides, herbicides, and pharmaceutical residues are first concentrated from liters of water onto a small SPE cartridge, allowing for accurate measurement.
In the pharmaceutical industry and clinical diagnostics, SPE is used for the isolation and purification of drug compounds and their metabolic byproducts from complex biological matrices. This preparation is essential for quality control, drug discovery, and for accurately measuring drug levels in patients during clinical trials.