the field-flow fractionation principle thickness externalfield sample introduction flow detector...

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


Water soluble polymersWater soluble polymers


Proteins, protein complexesProteins, protein complexes

VirusesViruses

Linear polystyrenesLinear polystyrenes

Lipophilic polymersLipophilic polymers

Crude oil and asphaltenesCrude oil and asphaltenes

Proteins


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




VirusesViruses

LiposomesLiposomes






VirusesViruses




Proteins



ElFFF

ThFFF

FlFFF

SdFFF

2 6 10 14 Log M

Starch







VirusesViruses

LiposomesLiposomes






VirusesViruses




Proteins


Human cells, blood

Bacteria, yeast

GFFF

ElFFF

ThFFF

SdFFF

2 6 10 14 Log M

Starch



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



ElFFF

ThFFF

FlFFF

SdFFF

2 6 10 14 Log M

Starch

Cells, blood, yeast

Bacteria, cells

Polymeric beads




VirusesViruses

LiposomesLiposomes






VirusesViruses




Proteins


Human cells, blood

Bacteria, yeast

GFFF

ElFFF

ThFFF

SdFFF

2 6 10 14 Log M

Starch







VirusesViruses

LiposomesLiposomes






VirusesViruses




Proteins



ElFFF

ThFFF

FlFFF

SdFFF

2 6 10 14 Log M

Starch







VirusesViruses

LiposomesLiposomes






VirusesViruses




Proteins


Human cells, blood

Bacteria, yeast

GFFF

ElFFF

ThFFF

SdFFF

2 6 10 14 Log M

Starch



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


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


sample relaxationsample relaxation


non-ideality effects non-ideality effects


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


Void

S. cerevisiaeS. cerevisiae

the field-flow fractionation principle thickness externalfield sample introduction flow detector...

Documents

light slide

coth slide

nonfimbriated slide

field field flow flow

pshrp fractionation

applications of fff

reversed flfff

r kt d