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SPE Procedure Optimization

In recent Quick Guides we have looked at the steps involved in solid phase extraction method development; these include analyte assessment, mechanism selection, and sorbent screening.  The final step is procedure optimization; this optimization process helps to ensure that the final method is robust, rapid, and cost effective.

A robust method is one which can be readily used by multiple extraction chemists, allows an appropriate degree of variability within sorbent surface chemistry and solvent protocols, yet still provides quality extracts in a reproducible manner.  The method must also be usable on a sufficiently wide range of samples of the type for which the procedure is designed.  For example, a protocol for extracting single species plasma will most likely have less variability requirements than a procedure for forensic human blood, since the latter matrix will vary in quality to a much greater extent than the former.  Therefore, during method development, the forensic user must evaluate a much wider range of samples than the clinician.

Too assess method robustness, it is necessary to start from a given step of the procedure and test the boundaries of the parameters within the step; the main parameters being solvent composition, solvent volume, and flow rate.

As a very specific example, assume that a non-polar extraction has been developed that employs 1 mL of 80% acetonitrile/20% water, applied at a flow rate of 1 mL/min., as the elution step.  To test the solvent composition robustness, a weaker solvent such as 70% acetonitrile/30% water should be used to verify adequate elution.  Similarly, a stronger solvent, such as 90%acetonitrile/10% water should be used to verify that the extract is still sufficiently clean, i.e. no retained interferences have been eluted from the SPE sorbent.

Elution volumes both larger and smaller should be tested to identify the minimum required volume as well as the point at which larger volumes offer no additional benefit
(Figure 1, Screen 1). 

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Figure 1: Robustness testing for elution volume (Screen 1) and sorbent quality (Screen 2).

For the flow rate it is recommended that at least 50-100% variance should be tested, with faster flow rate testing being the most important since slower flow rates very rarely have a negative impact in the quality of a protocol.  All of the above tests should be performed using a representative sample of a fixed size.  Once the optimized procedure has been created, it is then recommended to test different sample sizes to verify adequate performance.

Another major factor which affects SPE robustness is the quality of the SPE product.  The user should request multiple sorbent lots for any protocol to be used over extended time periods, since a specific lot of sorbent may only be available over a very short time frame (Figure 1, screen2).

The main factors contributing to the speed of an SPE protocol are the size of the sorbent bed, the volume of solvent used in each protocol step (which is somewhat influenced by the sorbent bed, Figure 2), the flow rate used to apply the solvent, and the number of steps used in the procedure.

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Figure 2: Sorbent mass selection based on sample volume.

To enhance procedure speed, it is desired to use:

a) the smallest sorbent bed possible which gives adequate capacity
b) the smallest solvent volumes possible which accomplish their task
c) the fastest flow rates that do not cause deleterious effects
d) the fewest steps possible. 

Therefore, the user should push the limits of each of the above parameters to evaluate optimum conditions.

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Figure 3: Effect of sorbent bed and flow rate.

Another major factor which influences procedure speed is the degree of sample pre-treatment required prior to the SPE protocol.  For example, if a liquid/liquid extraction (LLE) is required to transfer analytes from an aqueous matrix to a solvent one in order to perform a polar extraction, this will be more time consuming than using a mechanism which can work directly with an aqueous matrix.  On the other hand, there may be cases where a polar extraction provides such superior extract quality than the non-polar extraction, that the additional sample pre-treatment may well be worth the time.

A procedure which has been optimized for speed will most often also be highly cost effective, since small sorbent beds, reduced solvent volumes, fewer protocol steps, and less technician time expended all contribute to cost benefits.


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Top Tips to Improve Reproducibility and Sensitivity in Solid Phase Extraction »

Understanding the Mechanisms of Solid Phase Extraction »

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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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