Solid Phase Extraction (SPE) Method by Retention Mechanism
Sample Preparation Tip
Level: Basic
Each SPE mechanism/phase has a general set of solvent conditions under which SPE may be performed. Use the solvents/pH conditions listed below, volumes as determined in Method and Sorbent Volume Selection.
Reversed-Phase SPE Method
SDB-L, C18, C8, PH, CN, X, XL Sorbents
Analyte Properties
Low to moderate polarity (or nonpolar) Hydrophobic Neutralized/uncharged
Pharmaceuticals, Pesticides, Herbicides
Sample/Matrix
Aqueous, diluted with buffer
Biological fluids, Water
Conditioning Step
1.Solvation – polar organic solvents, 2.Equilibration – aqueous, buffers
1. Methanol, 2. Water or buffer
Wash Step
Aqueous buffers with 5 to 50 % polar organic solvent
Methanol: Water (5:95)
Elution Step
Polar or non-polar organic solvent(s) with or without water, buffer and/or strong acid or base
Methanol: Acetonitrile (50:50)
Normal Phase SPE Method
Silica, Florisil®, NH2, CN
Analyte Properties
Moderate to high polarity compounds (neutralized/uncharged)
Pesticides
Sample/Matrix
Non-polar organic solvents or moderately polar organic solvents
Hexane, chloroform, petroleum ether, toluene or methylene chloride
Conditioning Step
1.Solvation – polar organic solvents (optional), 2.Solvation – polar organic solvents (optional) Equilibration – sample/matrix solvent
1.Methanol (optional), 2.Hexane or chloroform
Wash Step
Non-polar organic solvents with a low concentration (1 to 5 %) of moderate to low polarity organic solvents
Hexane with 1 % THF, ethyl acetate, acetone, acetonitrile or IPA)
Elution Step
Non-polar organic solvents containing higher concentrations (5 to 50 %) of moderate to high polarity organic solvents
Hexane with 10 % THF, ethyl acetate, acetone, acetonitrile or IPA
Strong Ion-Exchange SPE Method
Anion exchange: Screen-A, SAX, X-A, X-AW, XL-A, XL-AW
Cation exchange: Screen-C, SCX, X-C, X-CW, XL-C, XL-CW
Analyte Properties
Aqueous; Low ionic strength buffers ( > 30 mM), pH adjusted
Biological fluids plus buffer
Sample/Matrix
Non-polar organic solvents or moderately polar organic solvents
Hexane, chloroform, petroleum ether, toluene or methylene chloride
Conditioning Step
1.Conditioning – polar organic solvents, 2.Equilibration – low ionic strength buffers, pH adjusted
1.Methanol , 2. 25 mM Tris-OAc, pH 7
Wash Step
Aqueous buffers of low salt concentrations with or without organic solvent
Anion exchange: Buffer pH 7: Methanol (50:50) Cation exchange:
1.Buffer pH 6 , 2. 1 M acetic acid, 3.Methanol
Elution Step
1.Neutralize the charge on the weak anion or cation, 2.Increase the ionic strength and counter ion concentration, 3.Add a strong counter ion displacer
1.Anion exchange: Hexane: ethyl acetate (75:25) +1 % glacial acetic acid, 2.Cation exchange: Methanol + 5 % NH3
NOTE: These are general starting conditions. Please contact your Phenomenex technical consultant for more assistance.
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