Field Flow Fractionation Feldflußfraktionierung...Field-Flow Fractionation was invented in 1966 by...

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Field Flow Fractionation

Feldflußfraktionierung

Albena LedererPolymer Separation Group

Institute of Macromolecular Chemistry

Leibniz-Institut für Polymerforschung Dresden e.V.

Polymer Science

Stellenbosch University

Macromolecular

distribution

according to molar mass, branching, composition

Structure distributions

Molmassenverteilungen

Hängt von Reaktionsmechanismus der Polymeraufbaureaktion und die gewählten Bedingungen (zB p, T)

Polydispersität und Mechanismus der Polymerisation

Polymertyp Reaktion Mw/Mn

Lebende

Polymere

Anionisch

Group-transfer

1.0…1.05

Kondensationspolymere Stufenreaktion

Bifunktionale Monomere

~ 2

Additionspolymere Radikaladdition

Kationische Addition

Koordinationpolymerisation

(Metallorganische Komplexe)

2 – 10

2 – 30

Verzweigte

Polymere

Radikalisch 2 – 50

Vernetzte

Polymere

Sufenreaktion von tri-,

tetrafunktionellen

Monomeren

∞

am Gelpunkt

Zahlenmittlere Molmasse

Gewichtsmittlere Molmasse

z-mittlere Molmasse

MMD (Polydispersität) wird mit

Mw / Mn beschrieben

Für monodisperse Proben Mw / Mn = 1

Polymere sind NICHT monodispers, sie

besitzen einen Mw / Mn >1

Molmasse bei Polymeren

Size Exclusion Chromatography (SEC) = molar mass determination based on separation

according to size

Molar mass characterization SEC

Polymer sample

Solvent Column material

Separation

Types of liquid chromatographic separations

mo

lar

ma

ss

elution volume [ml]

SEC

LAC

LCCC

mo

lar

ma

ss

elution volume [ml]

SEC

Size Exclusion Chromatography (SEC)Liquid Chromatography under Critical Condiitons (LCCC)Liquid Adsorption Chromatography (LAC)


according to size


elution time (elution volume)

I

(RI, UV,

LS, Visco)


according to size



MPeak


according to size



Mpeak

I


according to size


molar mass

I

Mn Mw Mz


according to size


Molar mass! Elution volume!

I

Mz Mw Mn


according to size


Separation of HB aromatic polyester with OH end groups

HO

O

C

O

n, hvz

6 8 100,00

0,05

0,10

0,15

0,20

0,25

0,30 RI

LS 90°

RI,

LS

[V

]

Volume [ml]

103

104

105

106

107

108

Molar mass

Mola

r m

ass [

g/m

ol]

Mw = 26 000 g/mol

Mw/Mn = 2,0

Zn = approx. 85

Solvent: THF

PLgel mixed B column

dn/dc = 0,25 ml/g

column-polymer interactions

mo

lar

ma

ss

elution volume [ml]

SEC

LAC

Polymer 2009, 50, 3431

J Polym Sci A 2009, 47, 5158

Problem 3: High number of functioanlities lead to strong interactions!

4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0

1

10

100

1.000

10.000

100.000

1.000.000

Mol

mas

se [g

/mol

]

Elutionsvolumen (ml)

Sepration of hyperbranched aromatic polyester with OH end groups

HO

O

C

O

n, hvz

Solvent: DMAc+ LiCl (2g/L)

PLgel mixed B column

dn/dc = -0,275 ml/g

NO column-polymer interactions

up to 30 000 g/mol

Mw = 26 000 g/mol

Mw/Mn = 2,0

Zn = approx. 85

Asymmetric Flow Field Flow Fractionation (A4F)

Asymmetric Flow Field Flow Fractionation (A4F)


I

(detection:

RI, UV,

LS, Visco)

Mn Mw Mz

8 10 12 14 16

0,0

0,1

0,2

0,3

0,4

RI

LS 90°

RI,

LS

sig

nal [V

]

Volume [ml]

103

104

105

106

Molar mass

Mola

r m

ass [

g/m

ol]


Solvent: THF

A4F channel

dn/dc = 0,25 ml/g

Membrane cut-off 5 kDa

Mw = 42 300 g/mol

Mw/Mn = 2,0 Zn = approx. 140

Mw = 161 000 g/mol


0 5 10 15 20 25 30 35

0

1

2

3

4

5

Elution

Inject/Focus

Flo

w (

ml/m

in)

elution time (min)

cross/focus flow

0,0

0,2

0,4

0,6

0,8

inject flowHO

O

C

O

n, hvz

5 10 15 20

0,0

0,1

0,2

0,3

0,4

0,5

RI

LS 90°

RI,

LS

90°

[V]

Volume [ml]

102

103

104

105

106

107

Mo

lar

ma

ss [

g/m

ol]

Molar mass

5 10 15 20

0,0

0,1

0,2

0,3

0,4

0,5

RI

LS 90°

RI,

LS

90°

[V]

Volume [ml]

102

103

104

105

106

107

Mo

lar

ma

ss [

g/m

ol]

Molar mass


Solvent: THF

A4F channel

dn/dc = 0,25 ml/g

Membrane cut-off 5 kDa

Mw = 148 000 g/mol

Mw/Mn = 3,3

Mw = 161 000 g/mol


0 5 10 15 20 25 30 35

0

1

2

3

4

5

Elution

Inject/Focus

Flo

w (

ml/m

in)

elution time (min)

cross/focus flow

0,0

0,2

0,4

0,6

0,8

inject flow

10 20

0,0

0,1

0,2

0,3

0,4

0,5

RI

LS 90°

RI,

LS

90°

(V

)

elution time (min)

102

103

104

105

106

Molar mass

Mol

ar m

ass

[g/m

ol]

0 5 10 15 20 25 30 35

0

1

2

3

4

5

Elution

Inject/Focus

Flo

w (

ml/m

in)

elution time (min)

cross/focus flow

0,0

0,2

0,4

0,6

0,8

inject flow

Feld-Fluss-Fraktionierung

FFF

20.01.2020 www.postnova.com 21

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 103 bis 1010 g/mol

„Flüssigchromatographie“ ohne stationäre Phase

Trennung in einem dünnen, langförmigen Kanal

Eingang

Kanalfluss

Ausgang

KanalflussKraftfeld

verschiedene Subtechniken

allg. Prinzip der FFF

Feldflussfraktionierung

Fluss FFF

symmetrische FFFF

asymmetrische FFFF

Elektrische FFF

Thermische FFF

1966 – Invention of Field-Flow Fractionation by Prof. Giddings

Inventor of FFF

[1] J.C. Giddings, „New separation concept based on a coupling of concentration and flow non-uniformities“, Sep. Sci., 1, 123-125 (1966)[2] J.C. Giddings, „Dynamics of Chromatography“, (1965)[3] J.C. Giddings, „Unified Separation Science“, (1992)

Field-Flow Fractionation was invented in 1966 by Prof. Calvin

Giddings [1] at University of Utah, Salt Lake City, USA.

He developed and published the complete theory of Field-Flow

Fractionation and was issued several patents in the area.

He 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”.

He founded the Field-Flow Fractionation Research Center at

University of Utah in 1972.

Prof. Giddings was twice nominated

for the Nobel-Price in 1984 and 1992.

In 1986 he founded FFFractionation,

the worlds first company which started

to commercialize the FFF technology.

FFFresearch Center

University Utah


24

Separation Mechanism

Separation in a narrow ribbon-like channel

Laminar flow inside the channel

External field perpendicular to the solvent flow

FFF Principle

25

FFF Principle - Detail

Separation Mechanism

Flow Field

Thermal Field

Centrifugal Field

Gravitational Field

Log

M

Elution Volume

General FFF Elution Order

FFF Principle26

Flow FFF:

VE ~ 1/D ~ Rh

27

Different Separation Forces but Same Separation Principle

FFF Methods

FFF Family – most important Techniques

28

Separation Range

FFF Size of Analyte

/ Centrifugal FFF

FFF Versions


20.01.2020www.postnova.com30

Flow FFF

Flow FFF Principle


Asymmetric Flow FFF Principle

Hydraulic Pressure Gradient Field (Cross-Flow) for Separation

Separation based on Size

Channel at Ambient, Mid or High Temperature

Flow FFF

Thermal gradient up to Δ120°C

Separation kDa up to several MDa

Analysis time, 10 – 120min (no upper limit)

Separation depends on D and DT

Separation according to Size and chemical composition

Thermal FFF - Principle32

VE ~ DTΔT/D

Thermal Diffusion Coeffcient

Depends on chemical

composition

• Gravity Separation Field up to 2.500 g• Size Separation Range: Particles 5 nm – 100 µm• Separation based on Size and Density

Centrifugal FFF - Principle20.01.202033www.postnova.com


Hollow Fiber Flow FFF Principle





Channel at Ambient and Mid Temperature


Symmetric Flow FFF Principle

Cross-Flow Field for Separation

Separation based on Hydrodynamic

Size/Molecular Weight

Channel top and bottom made by porous frit





Channel at Ambient, Mid or High Temperature

Cartridge-based Channel design with Holder and exchangeable Membrane

Different Cartridges are available (sizes and materials)

Asymmetric Flow FFF (AF4)

Principle Set-up of an AF4 Channel

20.01.202040www.postnova.com

Cartridge-based Channel design with Holder and exchangeable Membrane

Different Cartridges are available (sizes and materials)

Asymmetric Flow FFF (AF4)

Details - Principle Set-up of an AF4 Channel

20.01.202041www.postnova.com

Flow In Flow Out

Slot Out

Foc In

Membrane

Top Plate

Spacer

Low Plate

Holder

Holder

O-Ring

Asymmetric Flow FFF (AF4)20.01.202042www.postnova.com

AF2000 Multi Flow FFF Channels

Vergleich SEC/AF4

schnelle, schonende, hochauflösende Trennung

variable Trennbedingungen

Abwesenheit von Scherkräften keine Zerstörung von Aggregaten

keine stationäre Phase keine WW der Probenbestandteile im Trennkanal

SEC AF4

Trennung nach hydrodynamisches

Volumen

Diffusionskoeffizient

Einflussmöglichkeiten Säulenmaterial,

Partikelgröße,…

Querfluss, Membran,

Kanalhöhe,- größe, Temperatur,

Flussrate, Lösungsmittel

Molmassenbereich

[g/mol]

103 bis 107 104 bis >1010

Scherbeanspruchung hoch gering

Beeinträchtigungen Degradation, Adsorption,

Aggregate

Membrandurchlässigkeit

Limitierung Verzweigungen,

Funktionalität der

Polymere Co-Elution

Membrandurchlässigkeit

AF4

ΔT

DP2 DP1

Hot wall

P1P2

Cold wall

DT, P2DT, P1

Separation force fieldby T

ThFFF principle

Separation force fieldby cross-flow

AF4 principle

DP2

DP1

P1P2

From diffusion coefficient to size?

kB – Boltzmann’s constant

T – temperature (Kelvin)

– viscosity of solvent

Dt – diffusion coefficient

Rh – hydrodynamic radius

Stokes - Einstein Relation

bh

D

TkR

p6=

ΔT

DP2 DP1

Hot wall

P1P2

Cold wall

DT, P2DT, P1


ThFFF principle


AF4 principle

DP2

DP1

P1P2

Separation according to translational diffusion coefficient (D)

Ve ~ 1/D

Beispiel AF4.pptx

1

Encapsulation - state of the art

In situ loading of polymersome

in situ i)

ii) self assembly

Block copolymer

hydrophilic block hydrophobic block

pH < 6

DpH

2

Encapsulation - state of the art

In situ loading for

Doxorubicin delivery

Yassin et al. Small 2015, DOI: 10.1002/smll.201402581

3

Esterase and Myoglobin

Encapsulation of proteins studies by AF4

Mw 360 kg/molÐ ~ 2

Ø 10 nm

Mw 17 kg/molÐ ~ 1

Ø 5 nm

Gumz et al. Advanced Science 2019, doi.org/10.1002/advs.201801299

4

Encapsulation of proteins studies by AF4

Gumz et al. Advanced Science 2019, doi.org/10.1002/advs.201801299

5Gumz et al. Advanced Science 2019, doi.org/10.1002/advs.201801299

Proteins Polymersome & Protein

Size difference between polymersomes and proteins

15 20 25 30 35 40 45

0,0

0,2

0,4

0,6

0,8

1,0

UV

de

tec

tor

inte

ns

ity

(a

.u.)

Elution time (min)

Esterase:Polymersome (29:1) post

Myoglobin:Polymersome (112:1) post

12 14 16 18 20 22 24

0.0

0.2

0.4

0.6

0.8

1.0

UV

de

tec

tor

inte

ns

ity

(a

.u.)

Elution time (min)

Myoglobin

Esterase


Esterase Myoglobin

Loading efficiency: Encapsulation quantification

15 20 25 30 35 40

0.00

0.05

0.10

0.15

0.20

0.25

UV

de

tec

tor

inte

ns

ity

(a

.u.)

Elution time (min)

Myoglobin

Polymersome

Myoglobin:Polymersome

(112:1) post

15 20 25 30 35 40 45

0.0

0.2

0.4

0.6

0.8

1.0

UV

de

tec

tor

inte

ns

ity

(a

.u.)

Elution time (min)

Esterase

Polymersome

Esterase:Polymersome

(29:1) post

0.00 0.01 0.02 0.03 0.04

0

10

20

30

40

50

Es

tera

se

(µ

g)

UV signals peak area (a.u.)

y = 1413.8x - 0.602

R² = 0.997

0 50 100 150

0

10

20

30

40

50

60

y = 0.40775x + 0.486

R² = 0.986

My

og

lob

in (

µg

)

UV signal peak area (a.u.)



0 50 100150 200 250 300350 400 600750 1000

0

50

100

150

Enzyme per polymersome in batch

En

zy

me

pe

r p

oly

me

rso

me

Esterase

post

Myoglobin

in situ

Myoglobin

post

Esterase

in situ

ΔT

DP2 DP1

Hot wall

P1P2

Cold wall

DT, P2DT, P1


ThFFF principle


AF4 principle

DP2

DP1

P1P2

Separation according to translational (D) & thermal diffusion coefficient (DT)

Ve ~ DT T/D

Field Flow Fractionation Feldflußfraktionierung...Field-Flow Fractionation was invented in 1966 by...

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