On-line coupling SPE with LC


On-line SPE-LC consists of three steps:

  • Sample fractionation
  • Sample transfer
  • Separation and detection

On-line SPE-LC PlatformOn-line SPE-LC Platform

1 = Pump 2 = Pump 3 = Autosampler   4 = In-line Filter 5 = SPE-Column 6 = Six-port Valve   7 = Analytical column 8 = Detector  

Restricted Access Materials (RAM)

Some columns packings are changing their retention behaviour after a number of injections

Retention (extraction) of a model compound depending on SPE-column packing material and number of injections.Retention (extraction) of a model compound depending on      
SPE-column packing material and number of injections.

Therefore we now use taylor made SPE-columns, e.g. the RAM column (RAM= Restricted Access Materials). Some characteristics of the way in which restriction is done:

  • Defined Exclusion Barrier:
    • Physical (by pore diameter)
    • Chemical (polymer network)
    • Restricted access for HMW sample componenets (e.g. proteins)
  • Biocompatible Outer Surface
    • No irreversible interaction with HMW sample components
  • Adsorptive internal surface
    • Selective interaction with LMW sample components

Underlying Chromatographic Principles of RAM materials:

Sample clean-up relies on the simultaneous performance of  2 chromatographic separation processes:
  1. Size-Exclusion Chromatography (SEC). HMW sample components are eluted to waste in dead volume of SPE-column due to restricted access
  2. Reversed Phase (Partioning) Chromatography (RPC). (Or Ion-Exchange Chromatography (IEC), or Affinity Chromatography (AC). LMW sample components are bound adsorptively or covalently and thus extracted

Classification of RAM materialsClassification of RAM materialsBoos K-S, Grimm C-H, TrAC (1999) Cassiano NM et al., Anal Bioanal Chem (2006)

Example: LiChrospher® ADS

TopochemistryTopochemistryBoos K.-S., Grimm C.-H., TrAC 18 (1999) 175-180


  • Basis Material :  Spherical silica (LiChrospher® )
  • Particle Diameter :  25 µm
  • Pore Diameter :  ~ 6 nm
  • RP-Modification :  C-4 ; C-8 ; C-18
  • Size Exclusion Limit :  ~ 15 x 103 Dalton
  • Column Dimensions :  25 x 4 mm ID, 25 x 2 mm ID
  • Recovery (Protein) :  100 %  ( Human plasma )
  • Column Lifetime :  > 2000 injections of human plasma (50 µL)
  • Supplier :  Merck KGaA, Darmstadt, Germany. VWR International


LiChrospher® ADS : SPE Column HardwareLiChrospher® ADS :  SPE Column HardwareSupplier: www.merck.de or www.vwr.com

LiChrospher® ADS: Special SPE Column SealingLiChrospher® ADS:  Special SPE Column Sealing

Special in-line FilterSpecial in-line FilterSupplier: RECIPE Chemicals + Instruments GmbH, Munich, Germany

On-line SPE-LC System: “Home-made”On-line SPE-LC System:  “Home-made”

On-line SPE-LC System: Ultimate 3000 On-line SPE-LC System:  Ultimate 3000 In cooperation with Dionex Softron GmbH 



On-line SPE-LC System: Symbiosis™ PharmaOn-line SPE-LC System:   Symbiosis™ Pharma


On-line SPE Workstation: High SPEed™On-line SPE Workstation:  High SPEed™In cooperation with CTC Analytics

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