SPE Method Development Summary

The following table summarizes the foregoing discussion of the modes of SPE:

Summary of Utility and Practice of Principal LC Modes for Solid-Phase Extraction [SPE]

  Reversed Phase Normal Phase Ion Exchange
Analyte Moderate to low polarity

Low to high polarity/neutral


Charged or Ionizable

Separation Mechanism Separation based on hydrophobicity  Separation based on polarity

Separation based on charge

Sample Matrix Aqueous Non-polar organic solvent Aqueous/ Low ionic strength
Condition/ Equilibrate SPE Sorbent

1. Solvate with polar organic

2. Water  

Non-polar organic Low ionic strength buffer
Preliminary Wash Step Aqueous/buffer Non-polar organic Low ionic strength buffer
Elution Steps Increase polar organic content Increase eluotropic strength of organic solvent mixture Stronger buffers - ionic strength or pH to neutralize the charge
[Anion Exchange]
[Cation Exchange]
Sorbent Functionality C18, tC18, C8, tC2, CN, NH2, HLB, RDX, Rxn RP Silica, Alumina, Florisil, Diol, CN, NH2 Accell Plus QMA, NH2, SAX, MAX, WAX Accell Plus CM, SCX, MCX, WCX, Rxn CX 
Sorbent Surface Polarity Low to Medium High to Medium High High
Typical Solvent Polarity Range High to Medium Low to Medium High High
Typical Sample Loading Solvent Water, low strength buffer Hexane, chloroform, methylene chloride Water, low strength buffer Water, low strength buffer
Typical Elution Solvent CH3OH/water, CH3CN/water Ethyl acetate, acetone, CH3CN Buffers, salts with high ionic strength, increase pH Buffers, salts with high ionic strength, decrease pH
Sample Elution Order Most polar sample components first Least polar sample components first Most weakly ionized sample component first Most weakly ionized sample component first
Mobile Phase Solvent Change Required to Elute Compounds Decrease solvent polarity Increase solvent polarity Increase ionic strength or increase pH Increase ionic strength, or lower pH

This has been a brief introduction to sample enrichment and purification using solid-phase extraction [SPE]. The best way to start using SPE is to first learn what others have done with analytes and/or matrices similar to those of interest to you. You will find > 7,700 references to the use of SPE in the Resource Library on waters.com. Fill in the blank with a partial compound or matrix name in the following search phrase:
“Sep-Pak” OR “Oasis” AND ______*

NOTE: Rather than risk a spelling error, use an asterisk [*] with a root name for best results. Using this same search string, even more references [> 60,000] may be found on GOOGLE Scholar.

Further reading:

J.C. Arsenault and P.D. McDonald, Beginners Guide to Liquid Chromatography, Waters [2007]; Order P/N 715001531 on waters.com

P.D. McDonald and E.S.P. Bouvier, A Sample Preparation Primer and Guide to Solid-Phase Extraction Methods Development, Waters [2001] Search for WA20300 on waters.com

Waters, Purity by SPE [2008]; Search for 720001692en on waters.com

U.D. Neue, P.D. McDonald, Topics in Solid-Phase Extraction. Part 1. Ion Suppression in LC/MS Analysis: A Review. Strategies for its elimination by well-designed, multidimensional solid-phase extraction [SPE] protocols and methods for its quantitative assessment [2005]; Search for 720001273en on waters.com

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