Generic SPE Method by Retention Mechanism

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

Sorbent - SDB-L, C18, C8, PH, CN, X, XL

• 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
  • Solvation – polar organic solvents – Methanol
  • Equilibration – aqueous, buffers – 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

Sorbent - 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
  • Solvation – polar organic solvents (optional) – Methanol (optional)
  • Equilibration – sample/matrix solvent – 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

Sorbet: 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: Ionized/charged compounds – Anion exchange: Acidic analytes
  Cation exchange: Basic drugs
• Sample/Matrix: Aqueous; Low ionic strength buffers (>30 mM), pH adjusted – Biological fluids plus buffer
• Conditioning Step
  • Conditioning – polar organic solvents – Methanol
  • Equilibration – low ionic strength buffers, pH adjusted – 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:
    • Buffer pH 6
    • 1 M acetic acid
    • 3. Methanol
• Elution Step:
  • Neutralize the charge on the weak anion or cation
  • Increase the ionic strength and counter ion concentration
  • Add a strong counter ion displacer
    • Anion exchange: Hexane:ethyl acetate (75:25) + 1 % glacial acetic acid
    • Cation exchange: Methanol + 5 % NH₃

NOTE: These are general starting conditions. Please contact your Phenomenex technical consultant for more assistance.