SPE has been around for decades, and for good reason.  When scientists want to remove background components from their samples, they are faced with the challenge of doing so without reducing their ability to accurately and precisely determine the presence and quantity of their compound of interest. SPE is one technique that scientists often use to help prepare their samples for the sensitive instrumentation used for quantitative analysis.  SPE is robust, works for a broad array of sample types, and new SPE products and methods continue to be developed.  At the heart of developing those methods is an appreciation that even though the word “chromatography” doesn’t appear in the technique’s name, SPE is nonetheless a form of chromatographic separation.

SPE: The Silent Chromatography

There’s an old saying “if a tree falls in a forest, and no one is around to hear it, does it still make a sound?”  That saying reminds us of SPE.  That might seem strange to say, but when we think of SPE, the question becomes “if a separation takes place and there’s no detector there to record it, did chromatography really happen?”  In the case of SPE, the answer is a resounding “yes!”   When developing or troubleshooting a SPE method, it can very helpful to remember that SPE is just chromatography without the chromatogram.  When you think about it, wasn’t Mikhail Tsvet, known as “the father of chromatography,” doing what we would call “SPE” today?  When he separated his mixtures of plant pigments by letting gravity carry them, dissolved in a solvent, through a bed of ground up chalk, was it that much different than a modern SPE method?

Understanding Your Sample

Since SPE is based on chromatographic principles, at the heart of every good SPE method is the relationship between the analytes, the matrix, the stationary phase (the SPE sorbent), and the mobile phase (the solvents used to wash or elute the sample).

Understanding the nature of your sample as much as possible is the best place to start if you have to develop or troubleshoot a SPE method.  To avoid unnecessary trial and error during method development, descriptions of the physical and chemical properties of both your analytes and the matrix is very helpful.  Once you know about your sample, you will be in a better position to match that sample with an appropriate SPE product.  For instance, knowing the relative polarity of the analytes compared to each other and the matrix can help you decide if using polarity to separate analytes from the matrix is the right approach.  Knowing whether your analytes are neutral or can exist in charged states can also help direct you to SPE products that specialize in retaining or eluting neutrals, positively charged, or negatively charged species.  These two concepts represent two of the most commonly used analyte properties to leverage when developing SPE methods and selecting SPE products.  If you can describe your analytes and the prominent matrix components in these terms, you are on your way to picking a good direction for your SPE method development.

Separation by Affinity

The principles that define the separations that occur within an LC column, for instance, are at play in an SPE separation.  The foundation of any chromatographic separation is establishing a system that has varying degrees of interaction between the components of the sample and the two phases present in column or SPE cartridge, the mobile phase and the stationary phase.

One of the first steps towards feeling comfortable with SPE method development is to have a familiarity with the two most commonly encountered types of interactions employed in SPE separation: polarity and/or charge state.

Polarity

If you are going to use polarity to clean up your sample, one of the first choices you have to make is to decide what “mode” is best.  It is best to work with a relatively polar SPE medium and a relatively nonpolar mobile phase (i.e. normal mode) or the opposite, a relatively nonpolar SPE medium coupled with a relatively polar mobile phase (i.e. reversed mode, so named just because it’s the opposite of the initially established “normal mode”).

As you explore SPE products, you will find that SPE phases exist in a range of polarities. Moreover, the choice of mobile phase solvent also offers a wide range of polarities, often very tunable through the use of blends of solvents, buffers, or other additives.  There is a great degree of finesse possible when using polarity differences as the key characteristic to exploit to separate your analytes from matrix interferences (or from each other).

Just keep in mind the old chemistry adage “like dissolves like” when you are considering polarity as the driver for separation.  The more alike a compound is to the polarity of a mobile or stationary phase, the more likely it is to interact more strongly.  Stronger interactions with the stationary phase lead to longer retentions on the SPE medium.  Strong interactions with the mobile phase lead to less retention and earlier elution.

Charge State

If the analytes of interest either always exist in a charged state or are able to be put in a charged state by the conditions of the solution they are dissolved in (e.g. pH), then another powerful means of separating them from the matrix (or each other) is through the use of SPE media that can attract them with a charge of their own.

In this case, classic electrostatic attraction rules apply.  Unlike separations that rely on polarity characteristics and the “like dissolves like” model of interactions, charged state interactions operate on the rule of “opposites attract.”  For example, you may have an SPE medium that has a positive charge on its surface.  To balance that positively charged surface, there is typically a negatively charged species (an anion) initially bound to it.  If your negatively charged analyte is introduced into the system, it has the capability of displacing the initially bound anion and interacting with the positively charged SPE surface.  This results in retention of the analyte on the SPE phase.  This swapping of anions is called “Anion Exchange” and is just one example of the broader category of “Ion Exchange” SPE products.  In this example, positively charged species would have a strong incentive to stay in the mobile phase and not interact with the positively charged SPE surface, so they would not be retained.  And, unless the SPE surface had other characteristics in addition to its ion exchange properties, neutral species would also be minimally retained (although, such blended SPE products do exist, allowing you to utilize ion exchange and reversed phase retention mechanisms in the same SPE medium).

An important distinction to keep in mind when employing ion exchange mechanisms is the nature of the charge state of the analyte.  If the analyte is always charged, regardless of the pH of the solution it is in, it is considered a “strong” species.  If the analyte is only charged under certain pH conditions, it is considered a “weak” species.  That is an important characteristic to understand about your analytes because it will determine which type of SPE media to use.  In general terms, thinking about opposites going together will help out here.  It is advisable to pair a weak ion exchange SPE sorbent with a “strong” species and a strong ion exchange sorbent with a “weak” analyte.