Kontrolle über die Struktureigenschaften

Transkript

1 8. Trennmethoden

2 Kontrolle über die Struktureigenschaften Elutionsfraktionierung von hochverzweigten (hyperbranched, B) Polymeren Polyetheramide = n,hb Elutionsgradient Fraktionen Polymer auf Glassubstrat Probe: 2g hochverzweigtes Polymer Substrat: Glaskugeln, 0,1-0,2mm Gutes LM: DMAc Schlechts LM: TF

3 Intensity UV 280nm [a.u.] Kontrolle über die Struktureigenschaften Elutionsfraktionierung von hochverzweigten (hyperbranched, B) Polymeren Polyetheramide = n,hb ,18 0,16 PEA 5 Fraktionen 8 9,6 elution volume [ml] 0,14 0,12 Probe: 2g hochverzweigtes Polymer Substrat: Glaskugeln, 0,1-0,2mm Gutes LM: DMAc Schlechts LM: TF m i 0,10 0,08 0,06 0,04 0,02 0, fraction

4 Controlling the structure properties Degree of branching of the fractions Polyetheramide D = L n,hb T fraction 0,18 0,16 DB Fréchet DB Frey Aryl- - Aryl- - F6 0,54 0,56 0,45 0,48 F9 0,54 0,57 0,47 0,50 F11 0,55 0,57 0,51 0,53 F13 0,56 0,56 0,52 0,53 F15 0,56 0,56 0,52 0,54 0,14 1 -MR DLT Aryl- 2L 2D 7.0 1D 2T 1L 1T ((ppm) DB Fréchet = DB Frey = T+D T+L+D 2D 2D+L m i 0,12 0,10 0,08 0,06 0,04 0,02 0, fraction

5 Molar mass determination Universal calibration vs. light scattering fractions [ h ] PS - Standard calibration universal calibration light scattering detection dl/g M w M p M w M lin < M B V h, lin = V h,b F6 0, F9 0, F11 0, F13 0, F15 0, ,18 0,16 0,14 0,12 [h] lin.m lin = [h] hb.m hb V e =const. m i 0,10 0,08 0,06 0,04 0,02 0, fraction

6 Ultrazentrifuge Introduction to Analytical Ultracentrifugation Greg Ralston

7 Feld-Fluss-Fraktionierung FFF

8 Feldflussfraktionierung (FFF) Separierung und Charakterisierung von Polymeren, Proteinen und Partikeln Standardmethode zur Polymercharakterisierung: Größenausschlusschromatographie (SEC) Alternative zur SEC: Feldflussfraktionierung schnelle, schonende und hoch auflösende Trennung von 10 3 bis g/mol Feldflussfraktionierung Eingang Kanalfluss Kraftfeld Ausgang Kanalfluss Fluss FFF Elektrische FFF Thermische FFF symmetrische FFFF allg. Prinzip der FFF asymmetrische FFFF Flüssigchromatographie ohne stationäre Phase Trennung in einem dünnen, langförmigen Kanal verschiedene Subtechniken

9 Inventor of FFF 1966 Invention of Field-Flow Fractionation by Prof. Giddings Field-Flow Fractionation was invented in 1966 by Prof. Calvin Giddings [1] at University of Utah, Salt Lake City, USA. e developed and published the complete theory of Field-Flow Fractionation and was issued several patents in the area. e authored or co-authored more than 400 publications and edited 32 books [2], [3] and was executive editor of the journal Separation Science and Technology. e founded the Field-Flow Fractionation Research Center at University of Utah in FFFresearch Center University Utah Prof. Giddings was twice nominated for the obel-price in 1984 and In 1986 he founded FFFractionation, the worlds first company which started to commercialize the FFF technology. [1] J.C. Giddings, ew separation concept based on a coupling of concentration and flow non-uniformities, Sep. Sci., 1, (1966) [2] J.C. Giddings, Dynamics of Chromatography, (1965) [3] J.C. Giddings, Unified Separation Science, (1992)

10 FFF Principle Separation Mechanism Separation in a narrow ribbon-like channel Laminar flow inside the channel External field perpendicular to the solvent flow

11 FFF Principle - Detail Separation Mechanism Flow Field Thermal Field Centrifugal Field Gravitational Field

12 Log M FFF Principle General FFF Elution rder Flow FFF: V E ~ 1/D ~ R h Elution Volume

13 FFF Methods FFF Family most important Techniques Different Separation Forces but Same Separation Principle

14 FFF Size of Analyte Separation Range / Centrifugal FFF

15 FFF Versions

16 Flow FFF Flow FFF Principle

17 Flow FFF Asymmetric Flow FFF Principle ydraulic Pressure Gradient Field (Cross-Flow) for Separation Separation based on Size Channel at Ambient, Mid or igh Temperature

18 Thermal FFF - Principle Thermal gradient up to Δ120 C Separation kda up to several MDa Analysis time, min (no upper limit) TF2000: Separation Thermal Diffusion Coeffcient Separation depends on D and T Separation according V E to ~ Size Dand T ΔT/D chemical composition Depends on chemical composition Content FFF Principle Systems Applications Summary

19 Centrifugal FFF - Principle Separation Principle Gravity Separation Field up to g Size Separation Range: Particles 5 nm 100 µm Separation based on Size and Density Content FFF Principle Systems Applications Summary

20 ollow Fiber Flow FFF Principle

21 ollow Fiber Flow FFF Principle

22 ollow Fiber Flow FFF Principle ydraulic Pressure Gradient Field (Cross-Flow) for Separation Separation based on Size Channel at Ambient and Mid Temperature

23 Symmetric Flow FFF Principle Cross-Flow Field for Separation Separation based on ydrodynamic Size/Molecular Weight Channel top and bottom made by porous frit Cross Flow perpendicular to main channel flow

24 Asymmetric Flow FFF Principle

25 Asymmetric Flow FFF Principle ydraulic Pressure Gradient Field (Cross-Flow) for Separation Separation based on Size Channel at Ambient, Mid or igh Temperature

26 Asymmetric Flow FFF (AF4) Principle Set-up of an AF4 Channel Cartridge-based Channel design with older and exchangeable Membrane Different Cartridges are available (sizes and materials)

27 Asymmetric Flow FFF (AF4) Details - Principle Set-up of an AF4 Channel Cartridge-based Channel design with older and exchangeable Membrane Different Cartridges are available (sizes and materials) older Flow In Foc In Membrane older Slot ut Flow ut Top Plate Spacer Low Plate -Ring

28 Asymmetric Flow FFF (AF4) AF2000 Multi Flow FFF Channels

29 Vergleich SEC/AF4 SEC AF4 AF4 Trennung nach Einflussmöglichkeiten Molmassenbereich [g/mol] hydrodynamisches Volumen Säulenmaterial, Partikelgröße, Diffusionskoeffizient 10 3 bis bis >10 10 Scherbeanspruchung hoch gering Beeinträchtigungen Limitierung Degradation, Adsorption, Aggregate Verzweigungen, Funktionalität der Polymere Co-Elution Querfluss, Membran, Kanalhöhe,- größe, Temperatur, Flussrate, Lösungsmittel Membrandurchlässigkeit Membrandurchlässigkeit schnelle, schonende, hochauflösende Trennung variable Trennbedingungen Abwesenheit von Scherkräften keine Zerstörung von Aggregaten keine stationäre Phase keine WW der Probenbestandteile im Trennkanal

30 Einleitung Was ist AF4? Querfluss Eingang Injektion Ausgang zu Detektoren (RI, UV, MALLS Spacer Ultrafiltrations- Membran Querfluss Ausgang Stahlfritte

31 Einleitung Prinzip Prinzip der A4F: 1. Phase: Injektion 2. Phase: Fokussierung 3. Phase: Elution Einlass Fokus-Fluss Injektion Einlass Fokus-Fluss Stahlfritte Membran 8

32 Einleitung Prinzip Prinzip der A4F: 1. Phase: Injektion 2. Phase: Fokussierung 3. Phase: Elution Einlass Fokus-Fluss Einlass Fokus-Fluss Stahlfritte Membran 8

33 Einleitung Prinzip Prinzip der A4F: 1. Phase: Injektion 2. Phase: Fokussierung 3. Phase: Elution Einlass Querfluss Ausgang zum Detektor Stahlfritte Membran 8

34 Flow FFF Applications Proteins BSA Refractometer Signal Separation of BSA with high concentration using the PREP Channel The separation shows nearly the same performance for all runs Channel usable for separation of higher sample amounts without artificial effects! Possible aggregation study if the concentration is excessively increased Possible quantitative fractionation of aggregates for further investigation

35 AF4 - Steps of Separation Slot-utlet: Separation Injection and and Focussing Elution Carrier-Flow (I) Focus off Focus-Flow (I) utlet Slot open Semipermeable Membrane = Cross-Flow (UT) Diffusion Flow I = Flow UT Carrier-Flow + Focus-Flow = Cross-Flow + Detector-Flow (const.) Diffusion

36 Detectors RI Refractive Index Detection MALS Multi-Angle Light Scattering DLS Dynamic Light Scattering MS Mass Spectrometry