FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Metodi separativi...
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FACOLTA’ DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Metodi separativi Metodi separativi • Elettroforesi • Cromatografia idrodinamica ( HDC HDC) • Frazionamento in Campo e Flusso ( FFF FFF)
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Transcript of FACOLTA DI SCIENZE FF MM NN – ANALISI DI NANO E MICROSISTEMI DISPERSI Metodi separativi...
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Metodi separativiMetodi separativi
• Elettroforesi
• Cromatografia idrodinamica (HDCHDC)
• Frazionamento in Campo e Flusso (FFFFFF)
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Meccanismo separativo in HDCMeccanismo separativo in HDC
La tecnica FFF posside alcune similitudini con la HDCLa tecnica FFF posside alcune similitudini con la HDCma non e’ presente alcuna fase stazionariama non e’ presente alcuna fase stazionaria
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External field
Flow
Detector
External field
Parabolic flow
v1
v2
v3
Sample
Th
ickn
ess
Thickness
Bread
th
The The FFF FFF conceptconceptThe The FFF FFF conceptconcept
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FFF working range
molecules particulate
1 nm
1E0
Radius
Molar Mass
Atom Molecule
Polymers, Aggregates, Micro-Gels, Particles
SolidsProteins, Viruses, DNA, ...
10 nm
1E2 1E4 1E6 1E8 1E10 1E12
100 nm 1 µm
Micelles, Liposomes, EmulsionsViruses, Bacteria, Cells
Proteins, Protein-Complexes, DNAPolymers
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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 F4)
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 F4)Symmetrical Cross Flow FFF (S F4)
Asymmetrical Cross Flow FFF (A F4)Hollow-Fiber Flow FFF (HF F4)
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MALSMALS
Incident light
photodiodes
Transmitted light
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Molar mass and radius
rg < 10 nm isotropic scatter
rg > 10 nm
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Angular dependence of light scattering
detector at 0°scattered light in phase
detector at scattered lightout-of-phase
Intramolecular interference leads to a reduction in scattering intensity as thescattering angle increases
2
scattered Pdcdn
McI
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How light scattering measures rg
To calculate the angular distribution of scattered light, integrate over phase shifts from extended particle
Integrating over extended particle involves integrating over massdistribution
M
mrr iig
2
2
...2
sin3
161 22
20
20
2
gr
nP
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Interpretation of rg
hollow sphere:
solid sphere:
22 arg
2532 arg
M
mrr iig
2
2
Random coil polymer with average end-to-end length L:
6
22 Lrg
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Conformation Plot
Plot log rg vs. log M to determine molecular conformation
rod (slope = 1)
coil(slope near 0.5-0.6)
sphere(slope = 1/3)
log rg
log M
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Radius Results: Light Scattering & FlFFF
Rg or RMS radius – mass average (root mean square) distance of each point in a molecule from the molecule’s 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
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What’s the hydrodynamic radius?
Rh Rh
H2O
H2O
H2O
H2O
H2O
+
+
+_
Rh
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RRhh from FlFFF retention from FlFFF retention
hr
kTD
6
fUF Hydrodynamic fieldHydrodynamic field
C
z
x
DUx
CxC xexp)( 0
l
Axial flow Axial flow
V rhr
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I Conformation: rh vs. rg3-arm star polymer
4.1h
g
r
r
solid sphere
77.0h
g
r
r
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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
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+
AF4 Eclipse (Wyatt Techn. Europe) MALS DAWN (Wyatt Techn. US)
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Viruses: example /1
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0
50
100
150
200
250
300
8.0 12.0 16.0 20.0
R.M.
S. R
adius
(nm)
Volume (mL)
RMS Radius vs. Volume V31___01V22___02V22___01
VirusViruses: example /2es: example /2
0.0
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0.8
1.0
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Cum
ulativ
e Nu
mbe
r Fra
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R.M.S. Radius (nm)
Cumulative number fraction V31___01V22___02V22___01
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Deactivated influenza virus /1
Light Scattering (90°) signal and radius values
rms radius vs. time/volume
12 10B 2mgmL 10uL 350um Vc15 Vx15g.vafgfedcb
time or volume5.0 10.0 15.0 20.0 25.0 30.0 35.0
rms r
ad
ius (
nm
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1.0
10.0
100.0 radii
virus aggregates
rms radius vs. time/volume
12 10B 2mgmL 10uL 350um Vc15 Vx15g.vafgfedcb
time or volume5.0 10.0 15.0 20.0 25.0 30.0 35.0
rms
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1.0
10.0
100.0radii
virus
molecules
UV (280 nm) signal and radius values
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Light scattering (90° ) signal and radius values
rms radius vs. time/volume
14 66B 01mgmL 100uL 350um Vc15 Vx15g.vafgfedcb
time or volume5.0 10.0 15.0 20.0 25.0 30.0
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10.0
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virus
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rms radius vs. time/volume
14 66B 01mgmL 100uL 350um Vc15 Vx15g.vafgfedcb
time or volume5.0 10.0 15.0 20.0 25.0 30.0
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UV (280 nm) signal and radius values
Deactivated influenza virus /2
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rms radius vs. time/volume
04 Influ 04mgmL 40uL 250um Vc15 Vx15g.vafgfedcb
time or volume5.0 10.0 15.0 20.0 25.0 30.0
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1000.0
radii
virus
aggregates
Light scattering (90° ) signal and radius values
rms radius vs. time/volume
04 Influ 04mgmL 40uL 250um Vc15 Vx15g.vafgfedcb
time or volume5.0 10.0 15.0 20.0 25.0 30.0
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virusaggregates
UV (280 nm) signal and radius values
Deactivated influenza virus /3
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Adenovirus*
35
40
45
50
55
24.0 26.0 28.0 30.0 32.0 34.0
Geom
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nm
)
Time (min)
Geometric Radius vs. Time Adenovirus ATCC Standard...Geometric Radius vs. Time
-2.0
0.0
2.0
4.0
6.0
8.0
0 5 10 15 20 25
Lig
ht
Sca
tte
ring
(vo
lts)
Time (min)
Light Scattering Fractogram
Parameter Actual Measured % Error
Total Particle Count 2.9 X 1010 2.79 X 1010 1.9
Average Radius 43.0 45.0 4.7
*courtesy of MedImmune
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Chitosans
Fractions up to 500 nm radius present
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Elution time (min)
40 80 120 1600
RM
S-
Ra
diu
s (n
m)
0
40
80
120
160
Radius versus elution time for a filled and unfilled liposome sample
unfilled liposomefilled liposome
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I LiposomesEPC: egg phosphatidyl choline
EPC-BPS: 90% of the egg phosphatidyl choline with 10% brain phosphatidyl serine
0
20
40
60
80
100
10 20 30 40 50
Hyd
rody
nam
ic R
adiu
s (n
m)
Volume (mL)
Hydrodynamic Radius vs. Volume 1A____011B____012A____012B____01
EPC
EPC-BPS
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Polystyrene latex beads UV (solid line) and 90° LS (broken line)
chromatograms
01 Mix1 10uL Vc10 Vx04g.vafgfedcb
time or volume0.0 10.0 20.0 30.0 40.0 50.0
rela
tive
sca
le
0.0
0.2
0.4
0.6
0.8
1.0
41 nm
404 nm
199 nm
102 nm
Particles separation by A FParticles separation by A F44
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AF4 of 18-nm Au particleshydrodynamic radius vs. time
Gold 18.vafgfedcb
time (min)10.0 15.0 20.0 25.0 30.0
hyd
rod
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ic r
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0.0
100.0
200.0
300.0
UVLS
rH
Mobile phase: 0.05% SDS
Average hydrodynamic radius of 11 nmMany big-size aggregates: ca. 300 nm
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FlFFF of fluorophore-derivatized silica nanobeadsFlFFF 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
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FlFFF of buckyonionsFlFFF of buckyonions
0 5 10 15 20 25 30 35 40 45
0.00
0.02
0.04
0.06
UV
sig
nal
@ 3
30 n
m (
AU
)
retention time (min)
0 50 100 150 200
hydrodynamic diameter (nm)
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A closer look to particles….A closer look to particles….
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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 1253—1256 (1998).
Atomic force microscopy (AFM)
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FlFFF of functionalized, water-soluble MWNT FlFFF of functionalized, water-soluble MWNT
I.D. =20-30nm
N
NO O
NH3+
+H3NO O
1,3-dipolar azometine addition to
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SampleSample outlet
Clampingframe
Spacer
Channelwall
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/1-G/Sd FFFSd FFF: Gravitational FFF (GrFFF): Gravitational FFF (GrFFF)
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15 cm
On-channelinjection
Il nostro canale GrFFF….Il nostro canale GrFFF….
Rheodyneinjection
Pum
p
UV/Visdetector
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T-lymphocytes 90 - 95%T-lymphocytes 90 - 95%smooth cellssmooth cellsB-lymphocytes 5-10%B-lymphocytes 5-10%villous cellsvillous cells
Mean diameter:Mean diameter: 8 8 mm
Morphology:Morphology:dense large nucleusdense large nucleusthin cytoplasmthin cytoplasm
T-lymphocytes B-lymphocytes
CD45+/CD45+/CD19+CD19+CD45+/CD19-CD45+/CD19-
GrFFF of hGrFFF of human lymphocytesuman lymphocytes
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Mean diameterMean diameter8 8 mm
MorphologyMorphologydense large nucleusdense large nucleusthin cytoplasmthin cytoplasmT: smooth membraneT: smooth membraneB: villous membraneB: villous membrane
Burkitt lymphoma B cells
CD45+/CD19+CD45+/CD19+
2 m
/2/2 GrFFF of hGrFFF of human uman lymphocyteslymphocytes
CD45+/CD45+/CD19-CD19-
TT
Healthy lymphocytes
2 m
CD45+/CD19+CD45+/CD19+
BB
MorphologyMorphologylarger than B-lymphocyteslarger than B-lymphocytes
lower nucleus densitylower nucleus density
Mean diameterMean diameter14 14 mm
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FC map
20% (cell/cell) from Burkitt lymphoma B cell line
80% (cell/cell) from healthy donor (T+B lymphocytes)
/3/3 M Mixture of living human lymphocytesixture of living human lymphocytes
B
T
B: B-cells (healthy+Burkitt)T: T-cells (healthy)
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44
T cells
B cells
2 B cells
T cells
/4/4
0 5 10 15 20
0.00
0.05
0.10
0.15
0.20
42
UV
/vis
sig
na
l (6
40 n
m;
mA
U)
Retention time (s)
GrFFF of GrFFF of human lymphocyteshuman lymphocytes
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T and healthy B
CD
45
FS
CD
45
FS
4
T and healthy B
0 5 10 15 20
0.00
0.05
0.10
0.15
0.20
42
UV
/vis
sig
na
l (6
40 n
m;
mA
U)
Retention time (s)
Neoplastic B
T and healthy B
B-cell, size-based sortingB-cell, size-based sorting
CD
45+
/CD
19+
CD
45+
/CD
19+
Neoplastic BNeoplastic B
/5/5
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GrFFF of human GrFFF of human leukocytesleukocytes
CD 34+: haemopoietic precursor cells
2.6% 10%
Leukocytes collected from pheripheral blood of patient
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58.3%4.7%
0 4 8 12 16 20 24
0
40
80
120
160
2UV
/vis
sig
nal
(60
0 n
m;
mA
U)
Retention time (min)
GrFFF of GrFFF of leukocytes from patientleukocytes from patient/2 /2
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9.3%29.2%
0 4 8 12 16 20 24
0
40
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3UV
/vis
sig
nal
(60
0 n
m;
mA
U)
Retention time (min)
/3 /3 GrFFF of GrFFF of leukocytes from patientleukocytes from patient
FA
CO
LT
A’
DI
SC
IEN
ZE
FF
MM
NN
– A
NA
LIS
I D
I N
AN
O E
MIC
RO
SIS
TE
MI
DIS
PE
RS
I
7.3% 41.1%
0 5 10 15 20 25
0
20
40
60
4UV
/vis
sig
nal
(60
0 n
m, m
AU
)Retention time (min)
/4 /4 GrFFF of GrFFF of leukocytes from healthy donorleukocytes from healthy donor
