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Troubleshooting SPE Cleanliness

Previously we have looked at troubleshooting and resolving the problems of poor recovery and reproducibility in solid phase extraction (SPE).1,2 Another common problem we see with SPE is poor cleanliness which will be the subject of this article.

Symptoms of a sample which needs further clean-up are poor reproducibility, column failure (high backpressure, changing retention times), or incorrect quantitation.
For LC-MS users the insidious problem of ion-suppression can also be caused by poor extract cleanliness. Suppression is the result of co-eluting sample and analyte components entering the ion source, the sample components inhibit ionization of the analyte reducing sensitivity and affecting quantitation (Figure 1, screen 2).

Ion-suppression will not be evident in the chromatogram (i.e. peak shapes will not be affected) but will be apparent in analyte quantitation. To resolve the problem of insufficient extract cleanliness either modify the wash protocols, use another sorbent which utilizes the same extraction mechanism as the original, or change the extraction method completely. These three choices are listed in order of experimental priority.

The wash solvent should have the strongest elution strength for the mechanism employed such that it elutes the maximum number of interferences but not the analyte (Figure 1, screen 3). The wash step can also be improved by using a solvent in which the analyte is insoluble, for example, in a non-polar extraction water-miscible solvents that are weaker than the elution solvent are normally employed, however, dramatic improvements in cleanliness may be achieved by using very non-polar water-immiscible solvents such as, dichloromethane, ethyl acetate, or hexane.

These solvents exhibit very strong elution characteristics for the non-polar mechanism (i.e. they will elute many interferences), yet analyte insolubility means that it will be retained on the sorbent surface. The elution solvent can also be manipulated; a weaker solvent may result in cleaner extracts, however, analyte recovery should be monitored (Figure 2).

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Figure 1: Problems resulting from poor extract cleanliness.

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Figure 2: Diagnosing SPE method reproducibility problems.

If altering wash solvents does not provide sufficient additional clean-up, changing the sorbent may help.  For example, if the method uses a C8 sorbent for a non-polar extraction a less retentive C4 or C2 sorbent may be employed as they tend to retain fewer matrix components, therefore, more interferences will pass through the SPE sorbent during sample loading (Figure 3, screen 1).  Of course the analyte itself must be retained on the less retentive sorbent. 

The final step to improve sample cleanliness is to change the extraction mechanism entirely.  Appropriate alternatives may be identified by reviewing the information gained during method development regarding potential usable mechanisms.  One especially effective technique is to move from a single extraction mechanism to a mixed-mode mechanism (Figure 3, screen 2).  This works best for analytes which contain both non-polar and ionizable functional groups. 

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Figure 3: Optimizing sorbent retentivity.

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Previous SPE troubleshooting articles

Investigating SPE Recovery Problems »

Troubleshooting SPE Reproducibility Problems »

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What Can You Learn in 5 Minutes - SPE? »

SPE Method Development Interactive Learning Tool »

Webcasts & Tutorials

Troubleshooting Sample Preparation »

Understanding and Improving SPE »


Solid Phase Extraction - Method Development »

SPE Mechanisms »

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Dr. Dawn Watson

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

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