Sample preparation is the under-appreciated workhorse of the analytical chemistry lab. In fact, 60-80% of an analyst’s time is devoted to this! Selecting the most appropriate sample preparation tools for analysis and understanding the proper techniques in addition to common challenges are well worth an analyst’s investment of time.

Solid-phase extraction (SPE) is a highly selective sample preparation technique widely used to:

  • Purify and extract the sample of interest from complex matrices for better chromatographic analysis (HPLC or GC), longer column lifetime, and reduced system maintenance.
  • Concentrate the sample of interest for improved sensitivity and reproducibility.
  • Reduce ion suppression for mass spectrometric application.
  • Fractionate samples to allow analysis by class.

This article will help analysts get the most from this powerful technique. We’ll examine some of the most common challenges in SPE and address how to overcome them.

This technique is, in fact, governed by the same principles as liquid chromatography. Consequently, the basics are familiar to most analytical chemists. [For a refresher on solid-phase extraction principles, process, and sorbent types, see “Understanding and Improving Solid-Phase Extraction.”]

SPE utilizes differences in affinity between the analyte and the interferent in the matrix for the solid phase (or sorbent). It is valued for its high selectivity, reproducibility, ease of use, and rapid extractions — typically 30 minutes or less. Other advantages include good quantitative recovery, low solvent requirements, and ease of automation.

SPE is used to extract specific analytes of interest – unlike liquid–liquid extraction, which targets broader classes of compounds. Available sorbent chemistries and advances in technology make SPE a versatile technique used in pharmaceutical, biomedical, clinical and environmental applications, among others.

Now let’s explore some common SPE challenges.

Common Problems in Solid-Phase Extraction

The common challenges with SPE fall into four main categories:

  1. poor recovery
  2. Lack of reproducibility (inconsistent extractions)
  3. Impure extractions (interferences eluting with the analyte)
  4. Slow flow rates

The main factors that affect the SPE process are:

  1. sorbent choice
  2. Sample pH
  3. Sample volume
  4. Sorbent mass
  5. Elution solvent strength
  6. Elution solvent volume

Prior to troubleshooting SPE issues, you should (unsurprisingly!) first take steps to ensure that your analytical equipment is in good operating condition and producing accurate results. You can do this by running pure standards to diagnose system issues (see Three Common SPE Problems at LCGC).

1. Poor Recovery (Recovery Less Than 100%)

To determine where analytes are being lost, begin by collecting and analyzing fractions at each step in the process. Poor recovery in SPE can imply that the:

  • Analyte is in the loading fraction.
  • Analyte is eluting in the wash fraction.
  • Analyte is stuck on the sorbent.

If the analyte is in the loading fraction, it indicates that the analyte binding is insufficient. You’ll want to ensure that the recommended conditioning or pre-equilibration steps were followed for the selected stationary phase and that the correct solvent is being used. Try alternative solvents and be sure to wet the entire solvent bed.

If the analyte is being lost during loading, the analyte may have a greater affinity for the loading solvent than for the sorbent. In this case, some options to explore are:

  • Trying a sorbent with greater affinity.
  • Adjusting the pH of the sample to increase the affinity of the analyte for the sorbent.
  • Changing the polarity of the loading solvent to reduce its affinity with the analyte.
  • Diluting the sample with a weaker solvent.
  • Decreasing flow rate during sample loading to increase the time the analyte interacts with the sorbent.

If the sample volume is too large relative to the SPE cartridge, or the sample concentration too high, the analyte may wash out during loading. This is called overload. In this case, select a sorbent with a higher loading capacity or use a bigger SPE cartridge.

If the analyte is eluting in the wash fraction, you may need to modify your wash procedure by decreasing the volume or the strength of the wash solvent. Be sure to dry the column completely prior to the wash step. The wash solvent used should have the maximum strength to elute the impurities but not the analyte.

If the analyte is stuck on the sorbent or has not fully eluted, make sure that the correct elution solvent is being used and increase the solvent strength so that it moves the analyte off the sorbent, or increase the elution solvent volume, or try eluting in two separate half volumes. Changing the solvent pH or polarity can also improve the solvent affinity for the analyte.

A change to a less retentive sorbent may be needed if the analyte has a greater affinity for the sorbent than for the elution solvent (for example, using a C4 cartridge rather than a C8). Secondary interactions between the sorbent and analyte can also be the source of incomplete elution.

Decreasing the flow rate during elution also can be effective in increasing the time needed to move the analyte off the sorbent.

2. Lack of Reproducibility (Inconsistent Extractions)

Variation in SPE extractions can be caused by a number of factors. Before starting troubleshooting, look for errors in your method procedures. If you’re using an automated process, make sure that your instruments are calibrated and operating properly.

The most common reasons for lack of reproducibility – and how to fix them – are:

  • Errors or inconsistency in sample pre-treatment. Follow a consistent sample preparation method and make sure analytes are fully dissolved in the solvent. Use a stronger solvent or additional solvent if needed.
  • Cartridge needs conditioning. Follow the recommended cartridge conditioning process and do not allow the sorbent to dry before the sample is loaded.
  • Improper flow rate. Use a flow rate for sample loading that is slow enough to permit the solution to thoroughly soak into the cartridge before completing the extraction. Typical flow rates are generally around 1 mL/min.
  • Not using soak steps for solvent conditioning. In the soak step, the solvent remains in the sorbent long enough to allow solvent–sorbent equilibration. Soaking steps are used during solvent loading or elution and typically require 1-5 minutes.
  • Cartridge is overloaded. Decrease the sample volume so that it is appropriate for the cartridge capacity.
  • Solvent used for washing is too strong. Using a solvent that is too strong to remove interferences for these steps may cause too much of your sample to elute from the cartridge.
  • Cartridge is not fully dry following the wash steps. Drying times range from 5-20 minutes and are especially important for aqueous samples used with water-immiscible solvents. Polar sorbents typically require longer drying times, and water-miscible solvents may not require drying.

3. Impure Extractions (Interferences Eluting with the Analyte)

Take the following steps to help ensure pure recovery of your analyte:

  • Choose the proper strength of wash solvent and elution solvent. The wash solvent strength should be selected to elute weaker impurities, but not the analyte. The elution solvent should have a strong affinity with the analyte, but not be so strong that it will elute the stronger impurities (that should remain on the cartridge).
  • Pretreat your sample, if needed. Proteins that bind to analytes may require pH adjustments, precipitation, or ultrafiltration. Oils and lipids can be removed using liquid–liquid extraction (LLE). Inorganic salts can be removed using ion-exchange, desalting, or by using a nonpolar sorbent.
  • Choose a sorbent that is more selective for the analyte than the impurities. You can also use mixed-mode cartridges, or 2 different cartridges in a row.
  • Wash the cartridge with the elution solvent before conditioning to avoid interference from leaching substances.
  • Avoid cross-contamination from other samples (SPE cartridges are single-use).

4. Flow Rate Issues

Slow or uneven flow rates and flow blockage are common problems in solid-phase extraction. Similar to practice in liquid chromatography, the factors you should consider are:

  • cartridge quality (ensure that the manufacturer’s quality control is robust)
  • cartridge clogging due to particulate matter
  • vacuum problems
  • using a sample solution that is too viscous.

SPE Adoption Driven by Excellent Recovery, High Accuracy

Sample preparation can seem like a tedious step in the analysis process, but the demands for increased sophistication in analysis (higher selectivity, accuracy, precision, and fast sample throughput) necessitate efficient sample preparation. Solid-phase extraction is used universally in analytical labs due to its excellent recovery and accuracy capabilities, speed, ease of use, and low cost. Although it’s a relatively simple technique, an investment in learning the nuances of SPE is valuable for any analytical chemist.

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