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FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Metodi separativi Elettroforesi HDCCromatografia idrodinamica (HDC) FFFFrazionamento.

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Presentation on theme: "FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Metodi separativi Elettroforesi HDCCromatografia idrodinamica (HDC) FFFFrazionamento."— Presentation transcript:

1 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Metodi separativi Elettroforesi HDCCromatografia idrodinamica (HDC) FFFFrazionamento in Campo e Flusso (FFF)

2 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Meccanismo separativo in HDC La tecnica FFF posside alcune similitudini con la HDC ma non e presente alcuna fase stazionaria

3 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI External field Flow Detector External field Parabolic flow v1v1 v2v2 v3v3 Sample Thickness Breadth The FFF concept

4 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI FFF working range moleculesparticulate 1 nm 1E0 Radius Molar Mass AtomMolecule Polymers, Aggregates, Micro-Gels, Particles Solids Proteins, Viruses, DNA,... 10 nm 1E21E41E61E81E101E12 100 nm1 µm Micelles, Liposomes, Emulsions Viruses, Bacteria, Cells Proteins, Protein-Complexes, DNA Polymers

5 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Possible field of force Temperature gradient (Th FFF) Sedimentation field (Sd FFF) Electrical field (El FFF) Magnetic field (Mg FFF) Cross flow (Fl FFF or F 4 ) FFF fields & techniques Current FFF techniques Thermal Field Flow Fractionation (Th FFF) Sedimentation Field Flow Fractionation (Sd FFF) Cross Flow Field Flow Fractionation (Fl FFF or F 4 ) Symmetrical Cross Flow FFF (S F 4 ) Asymmetrical Cross Flow FFF (A F 4 ) Hollow-Fiber Flow FFF (HF F 4 )

6 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI MALS Incident light photodiodes Transmitted light

7 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Molar mass and radius r g < 10 nm isotropic scatter r g > 10 nm

8 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Angular dependence of light scattering detector at 0° scattered light in phase detector at scattered light out-of-phase Intramolecular interference leads to a reduction in scattering intensity as the scattering angle increases

9 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI How light scattering measures r g To calculate the angular distribution of scattered light, integrate over phase shifts from extended particle Integrating over extended particle involves integrating over mass distribution

10 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Interpretation of r g hollow sphere: solid sphere: Random coil polymer with average end-to-end length L :

11 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Conformation Plot Plot log r g vs. log M to determine molecular conformation rod (slope = 1) coil (slope near 0.5-0.6) sphere (slope = 1/3) log r g log M

12 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Radius Results: Light Scattering & FlFFF Rg or RMS radius – mass average (root mean square) distance of each point in a molecule from the molecules center of gravity. lower limit 10nm Rh or Hydrodynamic radius – radius of a sphere with the same diffusion coefficient or viscosity as our sample. lower limit 1nm

13 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Whats the hydrodynamic radius? Rh H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O + + + _

14 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI R h from FlFFF retention Hydrodynamic field C z x D Ux CxC x exp)( 0 l Axial flow Vrhrh r

15 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Conformation: r h vs. r g 3-arm star polymer solid sphere

16 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Why Light Scattering ? absolute molar mass (MM) and size without reference to standards and without assuming conformation better understanding of molecules (MM, conformation, branching determined directly from the data) and particles greater product differentiation (aggregate detection and identification) more reliable and robust than calibration techniques in chromatography, very easy and fast to use

17 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI + AF4 Eclipse (Wyatt Techn. Europe) MALS DAWN (Wyatt Techn. US)

18 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Viruses: example /1

19 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Viruses: example /2

20 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Deactivated influenza virus /1 Light Scattering (90°) signal and radius values radii virusaggregates radii virus molecules UV (280 nm) signal and radius values

21 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Light scattering (90° ) signal and radius values radii virus aggregates radii virus molecules UV (280 nm) signal and radius values Deactivated influenza virus /2

22 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI radii virus aggregates Light scattering (90° ) signal and radius values radii virus aggregates UV (280 nm) signal and radius values Deactivated influenza virus /3

23 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Adenovirus* Geometric Radius vs. Time Light Scattering Fractogram ParameterActualMeasured% Error Total Particle Count2.9 X 10 10 2.79 X 10 10 1.9 Average Radius43.045.04.7 *courtesy of MedImmune

24 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Chitosans Fractions up to 500 nm radius present

25 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Liposomes

26 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Liposomes EPC: egg phosphatidyl choline EPC-BPS: 90% of the egg phosphatidyl choline with 10% brain phosphatidyl serine EPC EPC-BPS

27 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Polystyrene latex beads UV (solid line) and 90° LS (broken line) 41 nm 404 nm 199 nm 102 nm Particles separation by A F 4

28 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI AF 4 of 18-nm Au particles UV LS rHrH Mobile phase: 0.05% SDS Average hydrodynamic radius of 11 nm Many big-size aggregates: ca. 300 nm

29 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI FlFFF of fluorophore-derivatized silica nanobeads Nanobeads as 3D scaffold for the fluorophore Energy transfer between fluorophore units FRET nanosensors Unbound and silica-bound fluorophore separation Particle size sorting

30 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI FlFFF of buckyonions 051015202530354045 0.00 0.02 0.04 0.06 UV signal @ 330 nm (AU) retention time (min) 050100150200 hydrodynamic diameter (nm)

31 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI A closer look to particles….

32 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Carbon nanotubes Liu, J., A.G. Rinzler, H.Dai, J.H. Hafner, R.K. Bradley, P.J. Boul, A. Lu, T. Iverson, K. Shelimov, C.B. Huffman, F. Rodriguez-Macias, Y.-S. Shon, T.R. Lee, D.T. Colbert and R.E. Smalley, Fullerene Pipes, Science 280 12531256 (1998). Atomic force microscopy (AFM)

33 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI FlFFF of functionalized, water-soluble MWNT I.D. =20-30nm 1,3-dipolar azometine addition to

34 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Sample outlet Clamping frame Spacer Channel wall Injection valve Low cost Potentially disposable channel Easy maintenance and simple sterility issues Autoclavable, no membranes, no moving or metal parts Easy implementation in flow systems 1-G/Sd FFF: Gravitational FFF (GrFFF) 1-G/Sd FFF: Gravitational FFF (GrFFF)

35 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI 15 cm On-channel injection Il nostro canale GrFFF…. Rheodyne injection Pump UV/Vis detector

36 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI T-lymphocytes 90 - 95% smooth cells B-lymphocytes 5-10% villous cells Mean diameter: 8 m Morphology: dense large nucleus thin cytoplasm T-lymphocytes B-lymphocytes CD45+/CD19+ CD45+/CD19- GrFFF of human lymphocytes

37 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Mean diameter 8 m Morphology dense large nucleus thin cytoplasm T: smooth membrane B: villous membrane Burkitt lymphoma B cellsCD45+/CD19+ 2 m /2 GrFFF of human lymphocytes CD45+/CD19- T Healthy lymphocytes 2 m CD45+/CD19+ B Morphology larger than B-lymphocytes lower nucleus density Mean diameter 14 m

38 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI FC map 20% (cell/cell) from Burkitt lymphoma B cell line 80% (cell/cell) from healthy donor (T+B lymphocytes) /3 Mixture of living human lymphocytes B T B: B-cells (healthy+Burkitt) T: T-cells (healthy)

39 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI 4 T cells B cells 2 T cells /4 GrFFF of human lymphocytes

40 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI 2 T and healthy B CD45 FS CD45 FS 4 T and healthy B Neoplastic B T and healthy B B-cell, size-based sorting CD45+/CD19+ Neoplastic B /5

41 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI GrFFF of human leukocytes CD 34+: haemopoietic precursor cells 2.6% 10% Leukocytes collected from pheripheral blood of patient

42 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI 58.3% 4.7% GrFFF of leukocytes from patient /2

43 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI 9.3% 29.2% /3 GrFFF of leukocytes from patient

44 FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI 7.3% 41.1% /4 GrFFF of leukocytes from healthy donor


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