As a chemist, I’ve constantly stressed the importance of proper sample preparation. Whether I’m diluting, digesting, preconcentrating, extracting, or performing a combination of these, sample preparation is the key to making my analysis a success, yet it’s often the most challenging part of my workflow.

    Some of my preparation procedures are simply daunting – a series of challenging, time-consuming steps with multiple opportunities for error or cross-contamination. On top of that is the multitude of parameters that must be selected. Questions such as “what should the pH be?”, “which solvents should I use?” and “what should my sample volume be?” are a few of the many, many parameters that must be optimized. When you look at all the opportunities for something to go wrong, sample preparation can seem very overwhelming. While powerful, sample preparation becomes a lot less complicated when you understand the science behind what you’re trying to accomplish with this step.

    Solid-phase extraction is a good example of this. Years of technological advancements have resulted in a wide range of sorbents that allow you to tailor your chemistry for almost any application. It can be overwhelming to select which media is best suited for your extraction – C18, HLB, WAX, SDVB, C8, SCX – to name just a few. The options seem endless.

    The key to selecting the right media is to understand the chemistry behind what you’re trying to achieve. SPE sorbent media can be broken into four general categories, according to their retention mechanism:

    1) Polar
    2) Nonpolar
    3) Ion Exchange
    4) Mixed Mode


    If you’re a chromatographer, you might be familiar with this chemistry – although you probably refer to it as normal phase liquid chromatography. Polar sorbents prefer to interact with polar molecules over nonpolar molecules, which makes the chemistry selective for polar analytes. If you recall your general chemistry lectures on polarity, you remember that polar molecules interact with other polar molecules via their electric dipole moments. If you can’t, don’t or don’t want to remember your general chemistry days, don’t be alarmed. Polar molecules interact via slight surface charges which are created because they have an asymmetric distribution of electric charge. How do you know if a molecule is polar or nonpolar? An oversimplified rule of thumb is to determine whether the molecule consists of different elements.


    • -Hydrogen cyanide (H-C-N)
    • -Hydrogen fluoride (H-F)
    • -Methanol (CH3OH)
    • -Water (H2O) or (H-O-H)

    To achieve this functionality in an SPE sorbent, a polar functional group is usually bonded to a silica-based material. Popular examples include the addition of a diol(-CH2CH(OH)CH2(OH)), cyanopropyl(-CH2CH2CH2CN) or an aminopropyl(-CH2CH2CH2NH2) group.

    When using these sorbents, you’ll maximize the retention of your analytes if they’re in a nonpolar matrix. As your sample passes through your SPE disk or cartridge, your analytes will interact preferentially with the polar sorbent over the nonpolar sample matrix. When it comes time to elute your analytes, use a slightly polar solvent to disrupt the interactions between your analytes and the sorbent material. Elution solvents such as methanol or acetonitrile often work well for this.


    Commonly used in columns for reverse phase liquid chromatography, these sorbents typically consist of a silica-based material with functional groups such as C18, C8, C6 or cyclohexyl groups. Nonpolar sorbents interact with nonpolar molecules preferentially over polar ones utilizing temporary, electric interactions known as van der Waals forces. Contrary to their polar counterparts, nonpolar molecules do not contain a net dipole. In other words, charge is shared equally among all the atoms in the molecule. This occurs either because the molecule contains a single type of element (Ex. H-H, Cl-Cl), or because the molecule is symmetric and all the dipole moments within the molecule cancel each other out (Ex. O-H-O, O=C=O).

    Keep in mind that electrons are always moving within a molecule. As the electrons move, they create temporary (and very short-lived) moments when a net dipole exists. Nonpolar molecules use these temporary dipole moments as the basis for their interactions. As you might imagine, these interactions are relatively weak – the weakest of all molecular interactions, in fact! However, under the right conditions, these interactions are suitable when performing solid-phase extractions.

    These sorbents are best used for retaining nonpolar analytes from a polar sample matrix. In this scenario, the polar molecules in the solvent repel the nonpolar analytes, strengthening the interaction with the sorbent material. To elute these analytes, use a slightly nonpolar solvent to disrupt the interactions with the sorbent. There are many solvents with slightly nonpolar characteristics that work well – methanol, acetonitrile and tetrahydrofuran to name a few. The key is to select a solvent that is less polar than water.

    Ion Exchange

    As the name implies, ion exchange media relies on the exchange of positively charged ions (cations) or negatively charged ions (anions) to retain analytes. These exchangers can be tailored for a wide range of application needs, dictated by the type of chemical media being used. Selectivity can be based on ion size, charge (singly charged versus doubly charged) or chemical structure, among other properties.

    These sorbents are typically classified as being “weak” or “strong” based on the pH range over which the media retains its ionic functionality. Therefore, ion exchange media can be divided into four general categories:

    1) WCX (weak cation exchange)
    2) SCX (strong cation exchange)
    3) WAX (weak anion exchange)
    4) SAX (strong anion exchange)

    This media type is optimal for extracting analytes that can be charged or neutralized by adjusting the pH or ionic strength of your solvent. As the sample passes through the SPE media, your charged analytes exchange with the charged counterions on the media, allowing the counterions to elute with the rest of the sample matrix. To elute your retained analytes, utilize similar ion exchange chemistry with the use of: (1) a buffer with a strong counterion (relative to that of your analytes), (2) a high ionic strength buffer or (3) an acidic/basic solution to neutralize the charge on your analytes.

    Mixed Mode

    To be clear, this isn’t a fourth type of media, rather a mixture of two of the media types described above (nonpolar and ion exchange, for example) mounted onto the same sorbent material. Analytes are retained via 1 of 2 retention mechanisms, based on the two media types used, and is ideal for fractionating multiple classes of compounds from the same sample. The key to a successful extraction with this media is to selectively interrupt each retention mechanism.

    For example, a mixed-mode sorbent containing nonpolar media and ion exchange media could be used for extracting nonpolar and acidic compounds from the same sample. After passing the sample through the sorbent material, pass a slightly nonpolar solvent through the media to selectively elute your nonpolar analytes into your first collection vessel. Remove your first collection vessel (which now contains your nonpolar analytes) and install a clean one under your SPE media. Use a basic solvent to raise the pH and neutralize your acidic analytes, and you’ve collected your acidic compounds in a separate collection vessel.

    Looking Ahead

    The development of SPE sorbents is still an active area of research chemistry. As our extraction challenges continue to increase, so too must the efficiency and selectivity of our media. As the list of available media grows, selecting the proper media (or mix of media) will seem like a more and more overwhelming task. Just keep in mind, your selection becomes simpler once you understand the chemistry behind your extraction – it’s all about the retention mechanism!



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