CROMATOGRAFIA LIQUIDA (L.C.) Gel filtrazione (Size-exclusion chromatography) Scambio ionico (Ion exchange chromatography) Affinità (Affinity chromatography) Separa sulla base delle dimensioni molecolari Utilizzo nelle fasi tardive di una purificazione La risoluzione dipende: -dalla lunghezza della colonna -dalle proprietà della resina - materiale (silice, gel) - granulometria - porosità - resistenza (rigidità) - dal volume del campione caricato Requisiti strumentali da semplici a sofisticati (per caduta, FPLC, HPLC) Sulla base di affinità molecolari nelle fasi precoci La risoluzione dipende: - dalla “capacità” della resina (porosità) - dalla sua affinità Requisiti strumentali semplici o nessuno (per caduta, batch) Sulla base della carica netta (+ o - ) nelle fasi precoci, ma non solo La risoluzione dipende: - dalla “capacità della resina (porosità) - dal pH dell’eluente - dal pI delle proteine da separare Requisiti strumentali da semplici a sofisticati (per caduta, FPLC e sue evoluzioni)

Micro 3μL/min. (Eldex MicroPro™) Test Valve Sample Loop (10 nL for Peptide Separation) Hot Sleeve™ Col. Heater (50 °C) ABI 783 UV Detector 3-nl Dionex LCP flow cell (220 nm) 0.3mm x 150mm C18 (Higgins Analytical) Instrumentation/Test Set-up (Peptide Analysis) 20μm ID Fused Silica Used For All Connections

Columns Usually stainless steel (to withstand pressure), mm diameter, ~5  m packing. Very efficient but limited length (cf. GC) because of pressure drop. Packing - usually silica Stationary phase - could be involatile liquid (like those in packed-column GC). More usual now to use similar chemical species actually bonded to the silica (longer column life), e.g. R can be non-polar (C 8 or C 18 hydrocarbon chain) or polar (amine, nitrile etc.).

Rheodyne

Stationary phase polar, mobile phase non-polar = NORMAL PHASE CHROMATOGRAPHY. Stationary phase non-polar, mobile phase polar = REVERSED PHASE CHROMATOGRAPHY. In normal phase, least polar analyte elutes first. In reversed phase, most polar analyte elutes first.

Detectors In both GC, HPLC – great effort to separate analytes. When the separated analytes leave the column, we need to detect them. What are the criteria for an ideal detector? It should be: UNIVERSAL (i.e. detects everything) SENSITIVE (i.e. detects a very small amount of everything) It should have a LINEAR RESPONSE (i.e. linear relationship between intensity of response and amount of analyte). It should give STRUCTURAL INFORMATION (i.e. tell you what the analyte is, even if you didn’t know beforehand).

Selective Detectors Very many possibilities - some of the more common:- ¶ - thermionic detection (mainly for N, P) ¶ - fluorescence ¶ - light-scattering ¶ - electron-capture detection (ECD) - especially for elements with high electron affinities (e.g. halogens) ¶ - UV - single wavelength or scanning ¶ - FTIR ¶ - mass spectrometry