The Field-Flow Fractionation principle Thickness Externalfield Sample introduction Flow Detector...
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The Field-Flow Fractionation principleThe Field-Flow Fractionation principle
Thickness
External fieldSample introduction
Flow
Detector
Length
Breadth
Parabolic flow
Th
ickn
ess
Sample
SOME APPLICATIONS OF FFFSOME APPLICATIONS OF FFFPolymeric latexes
Inorganic colloids
Pigments, carbon black
Viruses
Liposomes
DNA, RNA, Ribosomes
Inorganic colloids
Water soluble polymers
Polymeric latexes
Proteins, protein complexes
Viruses
Linear polystyrenes
Lipophilic polymers
Crude oil and asphaltenes
Proteins
Chromatographic silica
Human cells, bloodGFFF
ElFFF
ThFFF
FlFFF
SdFFF
2 6 10 14 Log M
Starch
Cells, blood, yeast
Bacteria, cells
Polymeric beads
Polymeric latexesPolymeric latexes
Inorganic colloidsInorganic colloids
Pigments, carbon blackPigments, carbon black
VirusesViruses
LiposomesLiposomes
DNA, RNA, RibosomesDNA, RNA, Ribosomes
Inorganic colloidsInorganic colloids
Water soluble polymersWater soluble polymers
Polymeric latexesPolymeric latexes
Proteins, protein complexesProteins, protein complexes
VirusesViruses
Linear polystyrenesLinear polystyrenes
Lipophilic polymersLipophilic polymers
Crude oil and asphaltenesCrude oil and asphaltenes
Proteins
Chromatographic silica
Human cells, blood
Bacteria, yeast
GFFF
ElFFF
ThFFF
SdFFF
2 6 10 14 Log M
Starch
Cells, blood, yeastCells, blood, yeast
Bacteria, cellsBacteria, cells
Polymeric beadsPolymeric beads
Polymeric latexesPolymeric latexes
Inorganic colloidsInorganic colloids
Pigments, carbon blackPigments, carbon black
VirusesViruses
LiposomesLiposomes
DNA, RNA, RibosomesDNA, RNA, Ribosomes
Inorganic colloidsInorganic colloids
Water soluble polymersWater soluble polymers
Polymeric latexesPolymeric latexes
Proteins, protein complexesProteins, protein complexes
VirusesViruses
Linear polystyrenesLinear polystyrenes
Lipophilic polymersLipophilic polymers
Crude oil and asphaltenesCrude oil and asphaltenes
Proteins
Chromatographic silica
Human cells, bloodGFFF
ElFFF
ThFFF
FlFFF
SdFFF
2 6 10 14 Log M
Starch
Cells, blood, yeastCells, blood, yeast
Bacteria, cellsBacteria, cells
Polymeric beadsPolymeric beads
Polymeric latexesPolymeric latexes
Inorganic colloidsInorganic colloids
Pigments, carbon blackPigments, carbon black
VirusesViruses
LiposomesLiposomes
DNA, RNA, RibosomesDNA, RNA, Ribosomes
Inorganic colloidsInorganic colloids
Water soluble polymersWater soluble polymers
Polymeric latexesPolymeric latexes
Proteins, protein complexesProteins, protein complexes
VirusesViruses
Linear polystyrenesLinear polystyrenes
Lipophilic polymersLipophilic polymers
Crude oil and asphaltenesCrude oil and asphaltenes
Proteins
Chromatographic silica
Human cells, blood
Bacteria, yeast
GFFF
ElFFF
ThFFF
SdFFF
2 6 10 14 Log M
Starch
Cells, blood, yeastCells, blood, yeast
Bacteria, cellsBacteria, cells
Polymeric beadsPolymeric beadsPolymeric beadsPolymeric beads
Polymeric latexes
Inorganic colloids
Pigments, carbon black
Viruses
Liposomes
DNA, RNA, Ribosomes
Inorganic colloids
Water soluble polymers
Polymeric latexes
Proteins, protein complexes
Viruses
Linear polystyrenes
Lipophilic polymers
Crude oil and asphaltenes
Proteins
Chromatographic silica
Human cells, bloodGFFF
ElFFF
ThFFF
FlFFF
SdFFF
2 6 10 14 Log M
Starch
Cells, blood, yeast
Bacteria, cells
Polymeric beads
Polymeric latexesPolymeric latexes
Inorganic colloidsInorganic colloids
Pigments, carbon blackPigments, carbon black
VirusesViruses
LiposomesLiposomes
DNA, RNA, RibosomesDNA, RNA, Ribosomes
Inorganic colloidsInorganic colloids
Water soluble polymersWater soluble polymers
Polymeric latexesPolymeric latexes
Proteins, protein complexesProteins, protein complexes
VirusesViruses
Linear polystyrenesLinear polystyrenes
Lipophilic polymersLipophilic polymers
Crude oil and asphaltenesCrude oil and asphaltenes
Proteins
Chromatographic silica
Human cells, blood
Bacteria, yeast
GFFF
ElFFF
ThFFF
SdFFF
2 6 10 14 Log M
Starch
Cells, blood, yeastCells, blood, yeast
Bacteria, cellsBacteria, cells
Polymeric beadsPolymeric beads
Polymeric latexesPolymeric latexes
Inorganic colloidsInorganic colloids
Pigments, carbon blackPigments, carbon black
VirusesViruses
LiposomesLiposomes
DNA, RNA, RibosomesDNA, RNA, Ribosomes
Inorganic colloidsInorganic colloids
Water soluble polymersWater soluble polymers
Polymeric latexesPolymeric latexes
Proteins, protein complexesProteins, protein complexes
VirusesViruses
Linear polystyrenesLinear polystyrenes
Lipophilic polymersLipophilic polymers
Crude oil and asphaltenesCrude oil and asphaltenes
Proteins
Chromatographic silica
Human cells, bloodGFFF
ElFFF
ThFFF
FlFFF
SdFFF
2 6 10 14 Log M
Starch
Cells, blood, yeastCells, blood, yeast
Bacteria, cellsBacteria, cells
Polymeric beadsPolymeric beads
Polymeric latexesPolymeric latexes
Inorganic colloidsInorganic colloids
Pigments, carbon blackPigments, carbon black
VirusesViruses
LiposomesLiposomes
DNA, RNA, RibosomesDNA, RNA, Ribosomes
Inorganic colloidsInorganic colloids
Water soluble polymersWater soluble polymers
Polymeric latexesPolymeric latexes
Proteins, protein complexesProteins, protein complexes
VirusesViruses
Linear polystyrenesLinear polystyrenes
Lipophilic polymersLipophilic polymers
Crude oil and asphaltenesCrude oil and asphaltenes
Proteins
Chromatographic silica
Human cells, blood
Bacteria, yeast
GFFF
ElFFF
ThFFF
SdFFF
2 6 10 14 Log M
Starch
Cells, blood, yeastCells, blood, yeast
Bacteria, cellsBacteria, cells
Polymeric beadsPolymeric beadsPolymeric beadsPolymeric beads
FFF retention in normal modeFFF retention in normal mode
w
x
C(x) C0
l<v>
FieldField
FieldField
FlowFlow
FlowFlow
2
6w
x
w
xvV
lxecxc /0
Basic theory in normal FFFBasic theory in normal FFF
Mean layer thickness Retention
VFwV
kTr 0
6
PotentiallyPotentiallyABSOLUTEABSOLUTE
TECHNIQUETECHNIQUE
lxecxc /0
Uw
D
w
l
r
kTD 6
Fw
kT
fUF
2
6w
x
w
xvV
RV
V
V
vo
r
6
1
22 6
coth
w
x
Lift forcesd
FFF retention in reversed modeFFF retention in reversed mode
FieldField
FlowFlow
w
a
w
aR 16 S
V
ddr
log
logRtt
dr
Sd 0
1
Selectivity
Sd V
d Mr ln
ln
Resolution
RN R
Rs 4
RN d V
d M
M
Msr
4
ln
ln
Efficiencyk
k
'
'1K
KSelectivity
k
k
'
'1K
K333 555 777 999 111111 131313 151515 171717
St/Hyp modeSt/Hyp modeSt/Hyp modeNormal modeNormal modeNormal mode
Log MWLog MW
1.01.01.0
0.50.50.5
000
Sel
ecti
vity
(dl
nV
R/d
lnM
)S
elec
tivi
ty (
dln
VR
/dln
M)
Transit.zone
Transit.zone
ThFFFFlFFF
ThFFFFlFFF
Sd FFFSd FFF
SECSEC
Sd= 0.9Sd= 0.9
S= 0.6S= 0.6
S= 1S= 1
S= 0.1S= 0.1
333 555 777 999 111111 131313 151515 171717
St/Hyp modeSt/Hyp modeSt/Hyp modeNormal modeNormal modeNormal mode
Log MWLog MW
1.01.01.0
0.50.50.5
000
Sel
ecti
vity
(dl
nV
R/d
lnM
)S
elec
tivi
ty (
dln
VR/d
lnM
)Transit.
zone Transit.
zone
ThFFFFlFFFThFFFFlFFF
GrFFF
Sd FFFSd FFF
SECSEC
Sd
= 0.9Sd= 0.9
S= 0.6S= 0.6
S= 1S= 1
S= 0.1S= 0.1
Selectivity and Resolution in FFF Selectivity and Resolution in FFF
Gravitational FFF Gravitational FFF
Sample outlet
Clampingframe
Spacer
Channelwall
Injection
valve
Sample
GrFFF of GrFFF of E.coliE.coli
Sample: CS5 0398 (fimbriated) Number of cells/mL in the 1:1 sample = 4.44 107
Sample: CS5 001101-5 (non-fimbriated)Number of cells/mL in the 1:1 sample = 7.25 106
Carrier: 80% 0.05% w/v SDS, 0.01% w/v NaN3
20% MeOH
Detection: UV-Vis detector @ 600 nm; 260 nm
Injection volume: 20 µLInjection flow rate: 0.2 mL/minInjection time: 25 sStop flow time: 30-40 minElution flow rate: 0.2-0.3 mL/min
CS5 0398 (fimbriated)
CS5 001101-5 (non-fimbriated)
0 2 4 6 8 10 12 14
(1)
(2)
Det
ecto
r Res
pons
e
1/R
0 5 10 15 20
(1)
(2)
De
tec
tor
Re
sp
on
se
Retention time (min)
Fimbriated CS5 0398 (1) 0.44 104 cells(2) 2.22 104 cells
0 10 20 30 40
(c)(b)
(a)
Det
ecto
r R
esp
on
se
Retention time (min)
E.coli E.coli sorting by GrFFF sorting by GrFFFElution flow rate = 0.3 mL min-1
Stop-flow time= 15 min
(a) Fimbriated CS5 0398 (b) Non fimbriated XC113A2 (c) Non fimbriated CS5 001101-5
Repeated runs reported for each case
Non fimbriated 001101-5 (1) 0.3 mL min-1 (2) 0.6 mL min-1
Chemiluminescence detection in GrFFFChemiluminescence detection in GrFFF
• High signal/mass ratio• Long-lasting emission (glow kinetics)
Horseradish peroxidase (HRP)Horseradish peroxidase (HRP)
Alkaline phosphatase (AP)Alkaline phosphatase (AP)
luminol
NH2
NHNH
O
O
H2O2, OH-
HRPHRPNH2
COOH
COO-
NH2
COOH
COO-
+ light
1,2-dioxetane
O OOMe
OPO3--
APAPO O
OMe
O-
O
O-
COOMe
+ lightHPO4
--
O-
COOMe
Enzyme-coated particlesEnzyme-coated particlesWhere is CL localized?
CL emission takes place in solutionCL emission takes place in solution:: the CL signal is localized the CL signal is localized on the enzyme-coated beads if the lifetime of the excited, on the enzyme-coated beads if the lifetime of the excited,
CL-emitting product is shorter compared to its diffusion rateCL-emitting product is shorter compared to its diffusion rate
HRP-catalyzed oxidation of luminolHRP-catalyzed oxidation of luminol
H2O
luminol
intermediate products
excited3-aminophthalate
3-aminophthalate + lightlight
2H2OHRPHRP
PSPS
In situ In situ GrFFF-CLGrFFF-CLInstrumental layout
CCDSlow-scan, ultrasensitive cooled CCD camera for sequential image acquisition
Mobile phase with Mobile phase with CL substrateCL substrate
Injection Injection valvevalve
PumpPump
0.750.75
GFFF channelGFFF channel
CL images collection: visualization of the fractionation of free/particle-bonded analytes
CL substrates in the mobile phase to reduce extra-column band broadening
In situ In situ GrFFF-CLGrFFF-CLReal-time imaging of the analyte fractionation
Direct evaluation ofDirect evaluation of
sample recovery and sample recovery and
particle wall-interactionsparticle wall-interactions
Direct evaluation ofDirect evaluation of
sample recovery and sample recovery and
particle wall-interactionsparticle wall-interactions
Information on theInformation on the
fractionation processfractionation process
from band profiles from band profiles optimizationoptimization
and kinetic studiesand kinetic studies
Information on theInformation on the
fractionation processfractionation process
from band profiles from band profiles optimizationoptimization
and kinetic studiesand kinetic studies
Direct analysis of Direct analysis of
sample relaxationsample relaxation
Direct analysis of Direct analysis of
sample relaxationsample relaxation
Direct analysis of Direct analysis of
non-ideality effects non-ideality effects
Direct analysis of Direct analysis of
non-ideality effects non-ideality effects
ElutionElution
Channel inletChannel inlet
InjectionInjection
Sample: 1.25 ng HRP e 7.5 Sample: 1.25 ng HRP e 7.5 g PS/HRP 6 g PS/HRP 6 mm
In situIn situ GrFFF-CL GrFFF-CLReal-time imaging of free HRP + PS/HRP fractionation
FFF a campo idrodinamico (FlFFF)FFF a campo idrodinamico (FlFFF)Flusso in ingresso
nel canale(iniezione del campione)
Flusso trasversale
Flusso in uscitadal canale (al rivelatore)
Plexiglass
Frit poroso
Membrana
Plexiglass
Flusso trasversale
Spacer
Frit poroso
opzionaleopzionale
Can
ale
Fl
FFF
Can
ale
Fl
FFF
Sistema FlFFFSistema FlFFF
Meccanismo FlFFFMeccanismo FlFFFflusso di
campionein entrata
flusso di campionein uscita
flussotrasversale
flussotrasversale
flusso trasversalein uscita
parete di accumulazione(membrana opzionale)
parete di deplezione
profiloparabolico
A B C
Ritenzione in FlFFFRitenzione in FlFFF
V dr La ritenzione dipende La ritenzione dipende solo dalle dimensionisolo dalle dimensioni
FlFFF in modo normaleFlFFF in modo normaleSeparazione di 3 lattici PSSeparazione di 3 lattici PS
Condizioni sperimentali
V
V
wc
198
0 70
0 02
. /
. /
.
cm min
cm min
cm
T = 298 K
3
3
Tur
bidi
tà
tr [min]
0 5 10 15 20
0.00
0.02
0.04
0.06
0.08
102 nm
V0 = 1.11 mL 155 nm
54 nm
t0
Reversed FlFFF-CLReversed FlFFF-CL
Injected sample:Injected sample:mixture of PS/HRP 6/3 mixture of PS/HRP 6/3 m and PS 10/4 m and PS 10/4 mm
Injected sample:Injected sample:5 5 g PS/HRP 6 g PS/HRP 6 m + 5 m + 5 g PS/AP 3 g PS/AP 3 mm
0 50 100 150 200
0
5
10
15
20
0
10
20PS/AP 3 m
PS/HRP 6 m
AP-CLHRP-CLUV/Vis
Retention time (s)
UV
/Vis
sig
nal
(m
V) CL
sign
al (RL
U)
0 50 100 150 200
0
5
10
15
20UV/Vis
0.0
0.5
CLPS/HRP 3 m
PS/HRP 6 m
PS 4 m
PS 10 m
Retention time (s)
UV
/Vis
sig
nal
(m
V) CL
sign
al (RL
U)
Void
Hollow-Fiber FlFFF: miniaturizationHollow-Fiber FlFFF: miniaturization
FlowFlow
FieldField
FlowFlow
1/8” 1/8” PEEK ferrulePEEK ferrule1/8” 1/8”
PEEK ferrulePEEK ferrule
1/8” 1/8” Teflon tubeTeflon tube
cPVC / PSfcPVC / PSfHF membraneHF membrane24x0.08 ID cm24x0.08 ID cm
1/8” 1/8” PE fittingPE fitting
1/8” 1/8” PE TeePE Tee
HF FlFFF in normal modeHF FlFFF in normal mode
vC(z)
C0
lFlowFlow
radV
inV outV
z
FieldField
L
Ux
rrff
HF FlFFF in reversed modeHF FlFFF in reversed mode
LiftLift
radV
inV outV
FlowFlowrrff
FieldField
dd
rrff
ddRR 44 22
4 2f f
a dR
r r 4 2
f f
a dR
r r
HF FlFFF retentionHF FlFFF retention
Reversed modeReversed mode
Normal/reversedNormal/reversed modemodeNormal/reversedNormal/reversed modemode
1
11
0
4
3
dSr
if d
kTtd
r Ut S
1
11
0
4
3
dSr
if d
kTtd
r Ut S
1
1
dSr
r
td
t
1
1
dSr
r
td
t
f f
l D
r Ur
0 44
r f
t DR
t Ur Normal modeNormal mode
HF FlFFF of HF FlFFF of V. cholerae V. cholerae
0 2 4 6 8
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
Membrane: PSf 30,000 Da
Serotypes: Inaba, Ogawa
Injected cells~ 5 106 (in 5 µl)
Mobile phase: FL-70 0.1% NaN3 0.02%
Focusing time: 3 min
Vrad = 51 µl/minVin = 1.5 ml/min
tr (min)
UV
/Vis
sig
nal (
mA
U)
Void
HF FlFFF of HRBCHF FlFFF of HRBC Recovery and Detectability
Membrane: cPVC 30,000 Da
Mobile phase: Isotonic PBS (300 mOsm) Cholic acid 1mMVin = 3.00 ml/minVrad = 0.27 ml/min
Injected cells: 70,000 Area = 8.52 10-3 min Recovery = 68% 7,000 Area = 0.669 10-3 min Recovery = 78%
Void
HF FlFFF of winemaking yeastHF FlFFF of winemaking yeast
Membrane: PSf 30,000 DaMobile phase:FL-70 0.1%NaN3 0.002%TRIS 0.125%
Focusing time: 6 minVin= 3 ml/minVrad= 0.44 ml/min
PS standardsSd=1.50±0.10
S. cerevisiaeS. cerevisiaeHF FlFFF-based size=4.8±0.3 µm
0 1 2 3 4
0
20
40
60
80
100
120
140
PS
4 µ
m
PS
6 µ
m
PS
3 µ
m
UV
/Vis
sig
nal
(m
AU
) @
254
nm
Retention time (min)
Void
S. cerevisiaeS. cerevisiae