Beginner's Guide to SPE

What is Solid-Phase Extraction (SPE)?

Don't be confused by the term solid-phase extraction [SPE]. A typical SPE device has 50 times more separation power than a simple, single liquid-liquid extraction. SPE is actually column liquid-solid chromatography. Since SPE is liquid chromatography [LC], its practice is governed by LC principles. A sample is introduced into a column or a cartridge device containing a bed of appropriate particles, or other form, of a chromatographic packing material [stationary phase]. Solvent [mobile phase] flows through the bed. By choosing an appropriate combination of mobile and stationary phases, sample components may pass directly through the column bed, or they may be selectively retained.

Individual compounds in the sample each typically appear to travel at different speeds through the device. Using a weaker solvent causes them to move slowly and/or be strongly retained. A stronger solvent speeds up their passage through the bed and elutes the analyte(s) in a more concentrated volume. Elution from an SPE device is usually done by increasing the strength of the mobile phase in a series of discrete, rather than continuous, steps during which selected analytes or interferences are either fully retained or rapidly eluted-this variation of gradient elution called a step gradient.

Most commonly, SPE is practiced using miniature column or cartridge devices. An example is shown here. A mixture of three dyes is loaded onto the cartridge in a weak solvent, causing strong sample retention in a narrow band that appears black at the column inlet. Subsequent gradient steps, each with a successively stronger solvent, are used to elute the dyes individually [yellow, red, then blue].

Typical SPE cartridges are low-pressure devices-constructed of solvent-resistant plastic or glass-filled with particles ≥30 µm in diameter. Suitable flow rates may be achieved by gravity or with the assistance of vacuum or low positive pressure. [The latter requires putting a cap on the open inlet of a column or using a sealed device with inlet and outlet fittings.]

Importance of Sample Preparation

In the last two decades, dramatic advances in analytical instrumentation and laboratory information management systems shifted the analyst's predominant tasks from assay measurements to sample preparation and data processing. As the stringency of requirements for higher sensitivity, selectivity, accuracy, precision, and number of samples to be processed has escalated, the corresponding increases in speed and sophistication of analysis and data collection have outpaced improvements in the many traditional techniques of sample collection and preparation. By some estimates, 75 to 80% of the work activity and operating cost in a contemporary analytical lab is spent processing and preparing samples for introduction or injection into an analytical separation and/or measurement device. Clearly, efforts directed and products designed to streamline sample preparation protocols are essential to future progress in analytical science.

Goals of Sample Preparation

Successful sample preparation for most analytical techniques [HPLC, GC, spectrophotometry, RIA, etc.] has a threefold objective: namely, to provide the sample component of interest

• in solution
• free from interfering matrix elements
• at a concentration appropriate for detection or measurement

To accomplish these goals, a sample, or a representative portion thereof [not always easy to obtain], is prepared via traditional methods of dissolution, homogenization, extraction [liquid- or solid-phase], filtration, concentration, evaporation, separation, chemical derivatization, standardization [internal or external], etc.

Usually such methods are used in combinations of multiple steps, which form a sample prep protocol. The fewer steps and methods used in any given protocol, the simpler, more convenient, cost effective, and less time consuming it is. Simpler protocols lend themselves more readily to automation and also lead to increased accuracy, reliability, reproducibility, and safety.

Innovation in Sample Preparation Methods

There are many ways to combine standard tools and techniques to accomplish the goals of sample prep. However, it is best to seek innovative means to streamline sample prep protocols:

• to combine the functions of several steps, if possible, into one operation;
• to eliminate needless sample transfers and manipulations;
• to reduce the scale as much as practicable [gaining economies of time, labor, and cost];
• to use new tools in creative ways.

Benefits of Solid-Phase Extraction [SPE] Cartridges

When compared to other sample preparation processes, solid-phase extraction using SPE cartridges offers:

Achieving Sample Preparation Objectives with Solid-Phase Extraction [SPE]

• To remove sample constituents that elute after the analytes of interest or are strongly adsorbed:
  • use solid-phase extraction with sorbent surface chemistry that is the same as that in the analytical HPLC column.
  • tailor the gradient steps to elute analytes selectively.
• To remove sample constituents that coelute with an analyte of interest:
  • use solid-phase extraction with sorbent surface chemistry and/or separation mode different from that in the analytical column.
  • tailor the gradient steps to elute analytes selectively.
• To enrich sample components present in low concentration:
  • tailor the gradient steps to elute analytes selectively.
  • use "large" sample volumes in adsorption-promoting solvent.
  • use "small" collection volume in desorption-promoting solvent.
  • use sorbent chemistry tailored to the analyte, independent of that in analytical column.
  • carefully choose chemistry of solid-phase extraction column so further sample prep will be unnecessary.
• To desalt samples:
  • first, adsorb analytes on reversed-phase sorbent while salt breaks through unretained.
  • then, after using water to wash away residual salt, desorb analytes using water-miscible organic solvent.
• To exchange solvents:
  • adsorb the sample completely onto a strongly retentive sorbent and flush away the original solvent with a weaker eluent.
  • elute the analyte with the desired solvent.
• To fractionate classes of compounds:
  • use a step-gradient sequence to divide a sample-on the basis of hydrophobicity, polarity, or charge-into fractions containing groups of analytes that share common properties.
• To derivatize analytes using solid-phase reagents:
  • adsorb a derivatization reagent on the surface of the sorbent; then, collect the sample (usually a gas) under conditions that favor complete adsorption of the analyte; wait for the reaction to occur and then selectively elute the derivative.