LevelBasic
On-line SPE-LC consists of three steps:
- Sample fractionation
- Sample transfer
- Separation and detection
On-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
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.
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:- Size-Exclusion Chromatography (SEC). HMW sample components are eluted to waste in dead volume of SPE-column due to restricted access
- 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 materials
Boos K-S, Grimm C-H, TrAC (1999)
Cassiano NM et al., Anal Bioanal Chem (2006)
Topochemistry
Boos K.-S., Grimm C.-H., TrAC 18 (1999) 175-180
Characteristics:
- 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 Hardware
Supplier: www.merck.de or www.vwr.com
LiChrospher® ADS: Special SPE Column Sealing
Special in-line Filter
Supplier: RECIPE Chemicals + Instruments GmbH, Munich, Germany
On-line SPE-LC System: “Home-made”
On-line SPE-LC System: Ultimate 3000 
In cooperation with Dionex Softron GmbH
On-line SPE-LC System: Symbiosis™ Pharma
On-line SPE Workstation: High SPEed™
In cooperation with CTC Analytics
