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Please check out our Solid Phase Extraction Equipment sections for more information or to find manufacturers that sell these products
Solid phase extraction (SPE) is a liquid chromatography method of separating a particular compound, or analyte, from a liquid mixture in which it is dissolved or suspended. This is commonly performed when separating an analyte of interest from other impurities in a mixture, for example, in order to use the resulting sample (now more concentrated, and with greater purity) for further analytical or quantitative studies such as mass spectrometry. For example, a simple immunoprecipitation can remove interfering proteins but not phospholipids, which can interfere in downstream mass spectrometry. SPE can remove both, resulting in a mass spec sample that is relatively free from interfering impurities. Common applications of SPE are listed in Table 1.
Table 1 – Applications of SPE
Typically the starting liquid mixture, known as the mobile phase, is passed through a column packed with a substance known as the sorbent to form the stationary phase. The type of column packing chosen for the extraction is key to the separation process, because ideally the analyte being extracted from the mobile phase will interact with the stationary phase in a manner different from the liquid. In other words, the analyte may have a strong affinity for the packing material in the column and thus be retained within the column, while the rest of the mobile phase flows through. Subsequent elution of the analyte then yields a sample of greater purity. Alternatively, the stationary phase may retain impurities in the mobile phase, allowing the compound of interest to flow through and be collected in the eluent.
SPE is a method that could entail endless optimization. To do it well and get on with your experiments, it helps to know the basic components of SPE, so that you can set up conditions for the extraction more easily and work more efficiently. Here is a look at some of the key features of SPE systems, including automated systems for even more hands-off help.
Choosing mobile and stationary phases for SPE
The type of sorbent you choose for the solid phase, and solvents for the mobile phase, depend largely on the nature of your sample or analyte of interest. The primary characteristics of polarity and charge—whether the analyte is polar, nonpolar, or a cation or anion—will help you determine the appropriate stationary phase. Combining this with a solvent of the appropriate hydrophobicity, depending on whether you want the analyte to pass through or be retained by the column, will lead you to the optimal set of SPE reagents.
In general, solvent strength can help to determine how quickly an analyte flows through the packing material of the stationary phase. Travel through the stationary phase will be slower with a weaker solvent, because the analyte will remain associated with the packing material longer. Alternatively, travel is usually faster with stronger solvents, which disrupt the interactions between the analyte and the stationary phase.
How do you decide whether to achieve your SPE objectives by selectively retaining or eluting your analyte?
Ultimately, this will depend on the particular conditions, but broadly speaking, it is helpful to extract your analyte by retaining it in the stationary phase if the analyte is low in concentration, or if there are multiple forms of the analyte present. In the latter case, a subsequent rinse and selective elution with gradually increasing solvent strength can effectively separate the similar analytes. The other option—letting your analyte flow through so the column retains unwanted impurities—is more appropriate when the starting sample has a high concentration of analyte.
Selecting SPE method type
Normal- and reversed-phase SPE
SPE comes in several flavors, foremost among them normal-phase and reversed-phase. Normal-phase SPE uses a polar stationary phase, and a nonpolar mobile phase, with extraction mediated by hydrophilic interactions. Normal-phase SPE is often used to separate uncharged analytes that may be anywhere from hydrophobic to polar. Most stationary phases are comprised of silica that has been bonded to specific functional groups to endow the packing material with the desired characteristics, such as degrees of polarity or hydrophobicity.
Not surprisingly, reversed-phase SPE is the opposite: The analyte is run through a nonpolar stationary phase using a polar mobile phase, and extraction is mediated by hydrophobic interactions. Reversed-phase SPE is usually used for analytes that are slightly polar to nonpolar. Materials commonly used for the stationary phase of reversed-phase SPE include silica with bound alkyl or aryl groups, polymer-coated silica, polymer-based material, and carbon-based material.
Ion exchange SPE
In ion exchange SPE, extraction is mediated by electrostatic interactions between charged chemical groups on both the solid phase and the analyte of interest. It is typically used to separate large proteins, amino acids, or nucleotides based on their charge. Proteins, for example, can have a variety of positively or negatively charged groups, which are the basis for their separation and elution from the stationary phase. This is achieved by using an ionic exchange packing material for the stationary phase, and a mobile phase with ionic concentrations appropriate for interacting with the analyte of interest. Gradually increasing the pH or concentration of other ions in the mobile phase will elute the analyte selectively.
The type of ion exchange you use depends partly on the analyte, and on the composition of the ion exchanger. For example, strong cation exchangers with sulfuric acid moieties on their surfaces are always ionized with a negative charge, while weaker cation exchangers bearing carboxylic acid groups are ionized at higher pH, but neutral at lower pH. The converse is true for strong (for example, positively charged and bearing ammonium groups) and weak (positive at low pH, or neutral at high pH, and bearing amine groups) anion exchangers.
A combination method of SPE called mixed-mode ion exchange chromatography adds ion exchange and reversed-phase SPE. It is useful for complex mixtures that may contain neutral, acidic, and basic species that can be retained by the column via hydrophobic interactions.
Several instrument manufacturers now offer automated SPE platforms to expedite SPE, speed sample preparation, and free up valuable researcher time. Another benefit of automated systems is that pipetting errors and other human causes of inaccuracies are minimized or avoided.
When perusing automated SPE systems, make sure that your sample types will be compatible with the stationary-phase format used by the system. While the term “column” is often used to refer to the packing material, or sorbent, with which the sample interacts during extraction, the stationary phase in SPE can take several forms, which can differ widely in volume: cartridges that are packed with sorbent, usually in 1-, 3-, or 6-mL volumes and generally in the shape of a syringe; 96-well microplates; flat disks; and the miniature MEPS (microextraction by packed sorbent) format, which is a tiny SPE cartridge inside a syringe through which a sample is injected, using mere microliters of fluid.
Many automated systems use 96-well microplates for sampling and collection, and use industry-standard SPE cartridges for the stationary phase, as, for example, Aurora Biomed’s VERSA™ Automated SPE Workstation (Vancouver, BC, Canada; www.aurorabiomed.com). Zinsser’s Speedy liquid handling system for SPE also uses a 96-well plate format (Zinsser North America, Northridge, CA; www.zinsserna.com). In addition, some systems are designed to extend throughput even further, such as the GX-271 ASPEC™ from Gilson (Middleton, WI; www.gilson.com), which can process up to four samples in parallel, while allowing individual, independent monitoring of each extraction.
Automated SPE systems for more specialized purposes are also emerging. For example, the SPE-DEX 4790® Automated Extraction System is particularly geared toward analyzing samples for trace amounts of organic chemical contamination (Horizon Technology, Salem, NH; www.horizontechinc.com). It includes preprogrammed methods from the U.S. EPA, though it can also be programmed for customized routines.
Conclusion
Whether your needs include a specialized SPE system, or an all-purpose workhorse for a large lab, you can make an informed choice by considering the nature of your analytes and the types of extraction you will need.
For more information on solid phase extraction systems, please visit www.labcompare.com.
Caitlin Smith is a freelance science writer who has a Ph.D. in Neuroscience from Yale University and postdoctoral work in Electrophysiology and Synaptic Plasticity; e-mail: [email protected].
Please check out our Solid Phase Extraction Equipment sections for more information or to find manufacturers that sell these products