The LCGC Blog: Basics, Applications, and Innovations in Solid-Phase Extraction

May 08, 2014
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In the realm of traditional sample preparation, you will often hear people speak of liquid–liquid extraction (LLE), solid-phase extraction (SPE), and protein precipitation. Of these three, SPE offers the highest specificity for removal of matrix interferences; it is less “quick and dirty” and requires the most effort, but for the return in investment of time and resources, SPE is versatile (many formats) and reliable (many standard methods specify SPE). It also remains fairly simple. I have had high school and undergraduate students in my laboratory regularly begin their “interaction” with analytical chemistry by performing some solid-phase extractions or fractionations to support various projects. However, for its simplicity, there are many aspects that need to be optimized to achieve optimal performance. Learning SPE is sort of like learning the English language — it is easy to become proficient in its use, but it takes a lot of time and practice to master. Here, I want to briefly touch on some of the basics of SPE, its applications, and some new frontiers. Much of this is inspired from a recent educational workshop I attended at The University of Texas at Arlington, cosponsored and delivered by Horizon Technologies, Inc., in conjunction with the Shimadzu Institute for Research Technologies at U.T. Arlington. It is also worth mentioning that LCGC’s “Sample Prep Perspectives” columns authored by Ron Majors throughout the years are excellent resources for exploring the basics of various techniques, and what is new in the world of sample preparation (1).

SPE Basics
Most probably know that SPE has several operational modes, and this aids in its general utility. It is most commonly used for sample cleanup, sample concentration, or both. Because analytes in an applied large volume (up to liters) can be easily eluted into a small volume (a couple of milliliters), they can be concentrated by a few orders of magnitude. Depending on the relative chemistry of the analytes, matrix components, and the solid-phase material, one can devise different strategies for segregating compounds of interest from potential interferences. For example, desired analytes can be retained while interferences are eluted and discarded, or alternatively, interferences can be retained — and left behind to be discarded with the phase — while the analytes are eluted first (essentially filtering the sample). SPE can also be used for strategies such as step fractionation of complex samples and buffer exchange. SPE is generally carried out in four steps:

• Condition and clean the cartridge with a strong solvent
• Equilibrate the phase with a weak solvent
• Load the sample
• Elute the sample components in the desired order with solvents of variable solvent strength, working from weak to strong.

Despite the inherent simplicity of the technique, there are several variables that need to be considered and optimized. For general method development, you must first determine the structure and stability of your analytes and possible interferences in the sample. Then you need to select the proper sorbent type and amount. A good rule of thumb is that for a given amount of sorbent, about 5% of that weight can be retained on the packed bed. Of course, this can be checked experimentally with breakthrough studies and recovery tests, but 100 mg of sorbent can retain approximately 5 mg of chemical compounds. Thus, the sample load volume and concentration is another factor to be optimized, and then the various wash and elution volumes and solvent strengths. Sorbent and solvent choices are consistent with and as widely varied as available modes of chromatography (for example, reversed phase, normal phase, and ion exchange). One clear aspect is that, done manually, it can be very tedious and time consuming — if not impossible — to explore the full variable space. Therefore, the use of an automated SPE system becomes quite attractive. In fact, it is likely the case that most “standard” methods that feature SPE have probably been developed manually and may not even be fully optimized. I do not think that anyone would dispute that there is often room for further improvement of published standard methods; they are provided mostly to encourage consistency among users, and many new methods even allow room for modification of some steps to increase performance. For general utility, Table I provides some standard sizes and operational considerations for SPE cartridge development. For a cartridge containing 500 mg of stationary phase, steps are generally taken in 2.5-mL increments, which is convenient given that this amount of phase contained in a cartridge with a 3-mL solvent capacity is quite common.

Table I: General flow conditions for cartridge SPE

SPE cartridge volume (mL)

6

3

1

Condition, equilibration flow rates (mL/min)

10.0

8.0

5.0

Sample load flow rate (mL/min)

5.0–7.0

2.0–3.0

1.0

Wash flow rate (mL/min)

5.0–10.0

4.0–6.0

1.0–1.5

Elution flow rate (mL/min)

3.0–5.0

2.0–3.0

1.0–2.0

SPE Applications and Formats
Modern applications of SPE are as widely varied as operational choices and variables. In our laboratory, we have used SPE for a variety of means. We are currently working on streamlining EPA Method 521 in our laboratory for the determination of nitrosamines in drinking water. As one can see, quite a few EPA methods feature the use of SPE (2). We have also used SPE for semipreparative step fractionation of natural product extracts, in pursuit of the development of new mass spectrometry (MS)–based methods for antibacterial drug discovery (3). A lot of methods that feature SPE can be found in the literature for the determination of pesticides in foods and beverages, which is of great interest across the world. In general, an SPE method can be envisioned for virtually any liquid sample (or liquid extract) containing semivolatile or nonvolatile compounds. Formats can also be extremely variable, from on-line approaches (4) to extraction phases incorporated into micropipette tips (5). The latter can be useful for desalting proteins and peptides before MS analysis.

One could go on for pages talking about various applications, but I would rather also speak a little to some recent innovations in SPE before wrapping up. Continuous and multidimensional SPE is an interesting concept. Featuring multiple iterations of sequential SPE conducted on one sample, continuous SPE can provide enhanced preconcentration of analytes and removal of interferences from highly complex samples. The incorporation of molecularly imprinted polymers (MIPs) into SPE formats also offers potential for enhanced selectivity for targeted analytes. Because MIP phases can even be templated to be specific enough even for enantioselective separations (6), the isolation of a desired analyte from the matrix background can be very efficient. However, it may not be practical to incorporate MIP phases across the board, since you would need such a wide variety of phase types to cover analyte variations — of course, phase manufacturers might like to sell you a different phase for each analyte you are targeting. On a related note, we recently reviewed MIPs and their use in the context of restricted access media (7).

As another interesting development, I would like to say a couple of words about SPE disks. In this format, a bed of SPE sorbent material is confined within a polymeric membrane about 5 cm in diameter. The look and thickness of the disk is similar to that of a circular piece of filter paper. With all of the variety of sorbent types and operation modes capable for cartridge formats, a disk has some significant advantages. Multiple disks and prefilters can be combined to accommodate very dirty samples (consider extraction of polyaromatic hydrocarbons from seawater or sludge). Whereas flow rate is an important consideration in cartridge SPE, it is less important with the disk format because it allows for higher flow rates: The surface area exposed to the sample on the disk is much higher than that on the head of the cartridge packing. Greater sample throughput can be accommodated. Table II compares some operational characteristics of disks versus cartridges, and also versus LLE. It is not difficult to discern the advantages of a disk format. SPE disks can be used with standard vacuum filtration formats (think of a vacuum filter funnel). More-elaborate enclosures for on-line analysis or field sampling can also be envisioned.

Table II: Comparison of sample preparation parameters for SPE disk, SPE cartridge, and LLE

 

SPE Disk

SPE Cartridge

LLE

Solvent usage (mL)

15–25

5–15

5–500

Operation

Filtration

Filtration

Shaking

Flow rate (mL/min)

85–125

5–10

N/A

Emulsions?

No

No

Yes

Particles in sample?

Yes

Limited

Limited

Sample size (mL)

Up to 8000

0.5–1000

5–500

Time for extraction (min)

20

Depends on volume

10–120

Closing Thoughts
I would like to end this blog entry with one thought that arose during discussions at the educational workshop. The thought centers around the concept that sample preparation is really important! It often takes the most time, and it has the potential to introduce the most error in an analytical determination. When I was in graduate school, I had a whole semester-long class on sample preparation. This was clearly because of the traditional analytical focus of the professors that were at Virginia Tech at the time (Go Hokies!). Who would not want to learn supercritical fluid extraction from Prof. Larry Taylor and solid-phase extraction and microextraction from Prof. Harold M. McNair? But nowadays, many universities have moved away from employing traditional analytical chemists. Some think analytical chemistry is just something that any chemist does. I think that is a shame, because very likely students at such places are not getting proper instruction in analytical fundamentals for techniques such as SPE, much less sample preparation in general. What would a new B.S. chemist likely do the most of, when they enter into a quality assurance position in industry? Probably, extractions. I count myself lucky to be among some great analytical chemists here at U.T. Arlington, and I am happy to say that our students get considerable exposure to basics of sample preparation in the classroom, in the laboratory, and through great workshops. Thanks again to Horizon Technologies, Inc., especially Toni Hofhine and Chad Schewe, for the inspiration to write this article, and for providing some of the information included above.

References
(1) http://www.chromatographyonline.com/majors
(2) United States Environmental Protection Agency, Drinking Water Methods Developed by EPA's Exposure Research Program. http://www.epa.gov/nerlcwww/ordmeth.htm (Accessed May 3, 2014).
(3) K.A. Schug, E. Wang, S. Shen, S. Rao, S. Crader, L. Hunt, and L.D. Mydlarz, Anal. Chim. Acta 713, 103–110 (2012).
(4) S. Rodriguez-Mozaz and M.J. Lopez de Alba, J. Chromatogr. A 1152, 97–115 (2007).
(5) J. Rappsilber, Y. Ishihama, and M. Mann, Anal. Chem. 75, 663–670 (2003).
(6) N.M. Maier and W. Lindner, Anal. Bioanal. Chem. 389, 377–397 (2007).
(7) S.H. Yang, H. Fan, R.J. Classon, and K.A. Schug, J. Sep. Sci. 36, 2922–2938 (2013).

Previous blog entries from Kevin Schug:

The LCGC Blog: My Own March Madness

The LCGC Blog: A View of Separation Science Research at a Czech Conference

The LCGC Blog: What is the Optimal Training to Provide Students Interested in a Career in Industry?

The LCGC Blog: Flow Injection Analysis Can Be Used to Create Temporal Compositional Analyte Gradients for Mass Spectrometry-Based Quantitative Analysis

The LCGC Blog: A Closer Look at Temperature Programming in Gas Chromatography

The LCGC Blog: Back to Basics: The Role of Thermodynamics in Chromatographic Separations

The LCGC Blog: The Dimensionality of Separations: Mass Spectrometry Is Separation Science

The LCGC Blog: What Can Analytical Chemists Do for Chemical Oceanographers, and Vice Versa?

The LCGC Blog: Do Not Forget to Assess Potential Matrix Effects in Your LC-ESI-MS Trace Quantitative Analysis Method from Biological Fluids

The LCGC Blog: Derivatization

The LCGC Blog: Restricted-Access Media for Biomonitoring Applications: A Solution That Deserves More Attention