The Postnova Characterization Platform...Viscometer Detector (Visc) – Intrinsic Viscosity (IV) of the sample (inverse density) Multi-Angle Light Scattering (MALS) – Molecular weight

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The Postnova Characterization Platform Characterization of Polymers, Proteins and Particles by FFF and SEC with Advanced

Detection

Paul Clarke

Postnova Analytics UK

International Symposium on GPC/SEC

and Related Techniques

Amsterdam, September 26th-29th, 2016

http://www.postnova.com/

www.postnova.com . www.postnova.com .

Agenda

• Introduction to Field Flow Fractionation

• The Postnova characterization approach

• Separation modules

• Detection modules

• Application examples

2




Field Flow Fractionation

FFF is a complementary technique to GPC/SEC

It can separate things SEC cannot (particles, gels, emulsions etc.)

Repeatability good but SEC still better with ‘easy’ soluble polymers

High-molecular weight polymers are always a challenge!

- Sample preparation (but FFF slightly less demanding than SEC)

- Method development

FFF for polymers

3




FFF Separation Principle

• Separation in a narrow ribbon-like channel

• Laminar flow inside the channel

• External field perpendicular to the solvent flow • Flow

• Centrifugal

• Thermal

• Gravitational

• Electrical

De

tecto

r S

ignal (m

V)

Elution Volume (ml)

4



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What technique to use? Comparison of chromatogram (SEC) and fractogram (FFF):

SEC + FFF

SEC

Mo

lecu

lar

/ P

art

icle

siz

e

Elution volume

AF4

Additional

information

from AF4!

Additional

information

from SEC!



• Hydraulic pressure gradient field (cross-flow) for separation

• Separation based on size

Asymmetric Flow FFF (AF4)

6



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• Focusing of sample

• Cleaning / washing of sample

• Higher resolution / reduced band broadening

• Continuous detector flow

• Increased recovery rate

Asymmetric Flow FFF (AF4)




CF2000 - Centrifugal FFF (CF3)

Centrifugal FFF Channel

• Gravity Separation Field up to 2500 g • Separation Range: 7 nm – 50 µm • Separation based on Size and Density

Applications

• Nanoparticle

• Microparticle

• Latices

• Gold / Silver Nano

• Liposomes

• Emulsions

8




TF2000 - Thermal FFF (TF3)

Principle

Applications

• Rubbers Polymers

• Gels / Latexes

• Cross-linked

Polymers

• Thermal gradient up to Δ120°C

• Separation 103 Da up to >107 Da

• Separation depends on D and DT

(size, chemical composition, micro structure)

9




The Postnova Characterization Platform

TF2000 Thermal FFF (TF3)

AF2000 Flow FFF (AF4)

Light Scattering

Detector

MALS 21

RI detector

Concentration

DLS

Malvern

Zetasizer

Agilent 7900 ICP-MS

CF2000 Centri FFF (CF3)

Size Separation

UV detector

DAD detector

Fluorescence detector

ELS detector

…

SC2000 SEC

Viscometer detector

10



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What information do the detectors contribute?

11

Refractive Index (RI) detector – Concentration of the sample

Ultra Violet (UV) detector – Concentration of sample with a given chromophore

Viscometer Detector (Visc) – Intrinsic Viscosity (IV) of the sample (inverse density)

Multi-Angle Light Scattering (MALS) – Molecular weight (MW) and molecular size (Rg)

By combining the detectors (RI, UV, MALS, Visc), we can enhance the data further:

MALS+Visc = Rh, Rg(ff), Mark Houwink data (structure), Branching

RI+UV = protein conjugate ratio or copolymer content

Polymer characterization by FFF/SEC



Application Examples

12




1. SEC Applications

13




PLGA polymers for nanoparticles

Mark-Houwink plot showing

structure difference in solution

PEG-PLGA PLGA

NovaSEC 2000 System

THF @ 1ml/min



www.postnova.com .

Triple Detection Chromatogram

15

Time [min]9080706050403020100

5.60000E-4

5.50000E-4

5.40000E-4

5.30000E-4

5.20000E-4

5.10000E-4

5.00000E-4

4.90000E-4

4.80000E-4

4.70000E-4

4.60000E-4

4.50000E-4

4.40000E-4

4.30000E-4

4.20000E-4

4.10000E-4

4.00000E-4

3.90000E-4

3.80000E-4

3.70000E-4

3.60000E-4

3.50000E-4

3.40000E-4

3.30000E-4

3.20000E-4

3.10000E-4

3.00000E-4

2.90000E-4

2.80000E-4

2.70000E-4

2.60000E-4

2.50000E-4

2.40000E-4

2.30000E-4

2.20000E-4

2.10000E-4

2.00000E-4

1.90000E-4

1.80000E-4

1.70000E-4

1.60000E-4

1.50000E-4

1.40000E-4

1.30000E-4

1.20000E-4

1.10000E-4

1.00000E-4

9.00000E-5

8.00000E-5

7.00000E-5

6.00000E-5

5.00000E-5

4.00000E-5

3.00000E-5

2.00000E-5

1.00000E-5

-7.35225E-19

-1.00000E-5

-2.00000E-5

3.55000E-2

3.50000E-2

3.45000E-2

3.40000E-2

3.35000E-2

3.30000E-2

3.25000E-2

3.20000E-2

3.15000E-2

3.10000E-2

3.05000E-2

3.00000E-2

2.95000E-2

2.90000E-2

2.85000E-2

2.80000E-2

2.75000E-2

2.70000E-2

2.65000E-2

2.60000E-2

2.55000E-2

2.50000E-2

2.45000E-2

2.40000E-2

2.35000E-2

2.30000E-2

2.25000E-2

2.20000E-2

2.15000E-2

2.10000E-2

2.05000E-2

2.00000E-2

1.95000E-2

1.90000E-2

1.85000E-2

1.80000E-2

1.75000E-2

1.70000E-2

1.65000E-2

1.60000E-2

1.55000E-2

1.50000E-2

1.45000E-2

1.40000E-2

1.35000E-2

1.30000E-2

1.25000E-2

1.20000E-2

1.15000E-2

1.10000E-2

1.05000E-2

1.00000E-2

9.50000E-3

9.00000E-3

8.50000E-3

8.00000E-3

7.50000E-3

7.00000E-3

6.50000E-3

6.00000E-3

5.50000E-3

5.00000E-3

4.50000E-3

4.00000E-3

3.50000E-3

3.00000E-3

2.50000E-3

2.00000E-3

1.50000E-3

1.00000E-3

5.00000E-4

-2.42861E-17

-5.00000E-4

-1.00000E-3

-1.50000E-3

-2.00000E-3

4.10000E-2

4.00000E-2

3.90000E-2

3.80000E-2

3.70000E-2

3.60000E-2

3.50000E-2

3.40000E-2

3.30000E-2

3.20000E-2

3.10000E-2

3.00000E-2

2.90000E-2

2.80000E-2

2.70000E-2

2.60000E-2

2.50000E-2

2.40000E-2

2.30000E-2

2.20000E-2

2.10000E-2

2.00000E-2

1.90000E-2

1.80000E-2

1.70000E-2

1.60000E-2

1.50000E-2

1.40000E-2

1.30000E-2

1.20000E-2

1.10000E-2

1.00000E-2

9.00000E-3

8.00000E-3

7.00000E-3

6.00000E-3

5.00000E-3

4.00000E-3

3.00000E-3

2.00000E-3

1.00000E-3

-2.77556E-17

-1.00000E-3

-2.00000E-3

-3.00000E-3

-4.00000E-3

-5.00000E-3

-6.00000E-3

-7.00000E-3

-8.00000E-3

-9.00000E-3

-1.00000E-2

-1.10000E-2

-1.20000E-2

PN3621b 90° RI DP11

-0.11

4.79650E-43.00152E-23.30655E-2

Hyaluronic Acid by SEC

Time [min]

4039.53938.53837.53736.53635.53534.53433.53332.53231.53130.53029.52928.52827.52726.52625.525

4.20000E-6

4.00000E-6

3.80000E-6

3.60000E-6

3.40000E-6

3.20000E-6

3.00000E-6

2.80000E-6

2.60000E-6

2.40000E-6

2.20000E-6

2.00000E-6

1.80000E-6

1.60000E-6

1.40000E-6

1.20000E-6

1.00000E-6

8.00000E-7

6.00000E-7

4.00000E-7

2.00000E-7

-5.82335E-22

PN3621b 12° PN3621b 20° PN3621b 28° PN3621b 36° PN3621b 44° PN3621b 52° PN3621b 60° PN3621b 68°

PN3621b 76° PN3621b 84° PN3621b 90° PN3621b 100° PN3621b 108° PN3621b 116° PN3621b 124° PN3621b 132°

PN3621b 140° PN3621b 148° PN3621b 156° PN3621b 164°

40.00

1.58587E-7

Angular Dependence – MALS View

Volume [ml]

24.023.022.021.020.019.018.017.016.015.014.013.012.0

Sig

nal [V

]

4.0e-02

3.8e-02

3.6e-02

3.4e-02

3.2e-02

3.0e-02

2.8e-02

2.6e-02

2.4e-02

2.2e-02

2.0e-02

1.8e-02

1.6e-02

1.4e-02

1.2e-02

1.0e-02

8.0e-03

6.0e-03

4.0e-03

2.0e-03

-2.1e-17

-2.0e-03

MP013 1-2 MP013 2-2 MP014 1-2 MP014 2-2

log(M)

7.576.565.554.543.532.5

log

(IV

)

2.6

2.4

2.2

2

1.8

1.6

1.4

1.2

1

0.8

0.6

0.4

0.2

0

-0.2

-0.4

MP013 1-2 MP013 2-2 MP014 1-2 MP014 2-2

HA sample of ~2 MDa reduced to

<0.5 MDa on sterilization.

RI overlay shows effect of

sterilization on distribution. Note

also MH slope change



2. AF4 Applications

(Flow FFF)

16




Polystyrene Latex Nanoparticle Mix

UV

Sig

nal 2

54

nm

Retention Time [min]

AF2000 – Flow FFF (AF4)

Polystyrene Standards Mix

Same instrument, different

flow method!




Separation and MW of Hyaluronic Acid

0.0

0.2

0.4

0.6

0.8

1.0

1,000,000 10,000,000 100,000,000

Molar Mass [Da]

Rela

tive A

mou

nt

Hyaluronic AcidMolar Mass Distribution

Mw = 7.4 MDa

Cumulative Curve

AF4 - High MW Polymers



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Crosslinked HA Sample by FFF

19

AF4 – MALS/RI Fractogram Molecular weight data

Crosslinked HA showed <10% recovery on SEC but >80% recovery on FFF


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Dextran Coated Magnetic Nanoparticles

Raw Data Fractogram of AF4 - MALS and UV

Rg/Rh ratio indicates

aggregation which is

confirmed by TEM

20



Polymersome structure

Radius of Gyration (MALS) and Hydrodynamic Radius (DLS) rh (DLS) rg (LS) Concentration

Time [min]

3836343230282624

r[nm

]

350

300

250

200

150

100

50

0

Concentra

tion

8.5E-4

8.0E-4

7.5E-4

7.0E-4

6.5E-4

6.0E-4

5.5E-4

5.0E-4

4.5E-4

4.0E-4

3.5E-4

3.0E-4

2.5E-4

Ratio rg/rh Concentration

Time [min]353025

Ratio r

g/r

h3.0E+0

2.0E+0

1.0E+0

Concentra

tion

6.0E-4

5.0E-4

4.0E-4

3.0E-4

2.0E-4

1.0E-4

21

Data indicates mixture of

structures (spheres and

tubes) also seen on TEM




SiO2 Sample AF4-MALS-ICP-MS

22

SiO2 Standard, Conc. ~2 mg/L

90° LS Signal (red trace) and Si 28 (blue trace)

Conditions • Injection Volume: 2000 µL

• Concentration:~ 2 mg/L

• LS 90°(red trace)

• Si 28 Signal (blue trace)

Overlay: Radius of Gyration and Si 28

trace • Calculated from MALS angular data • Broad distribution starting from 7 nm up to large

particles (998 nm).

Time [min]

14012010080604020

Dete

cto

r S

ignals

[V

]

0.1790

0.1788

0.1786

0.1784

0.1782

0.1780

0.1778

0.1776

0.1774

0.1772

0.1770

0.1768

Dete

cto

r Sig

nals

[V]

7.0E+4

6.5E+4

6.0E+4

5.5E+4

5.0E+4

4.5E+4

4.0E+4

3.5E+4

3.0E+4

2.5E+4

2.0E+4

1.5E+4

1.0E+4

5.0E+3

Radius / Si28

Time [min]

100908070605040

Radiu

s [

nm

]

1

10

100

1 000

10 000

Dete

cto

r Sig

nal[V

]

6E+04

5E+04

4E+04

3E+04

2E+04

1E+04



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AF4-UV-ICP-MS – Tattoo ink analysis

23

Cu63

Ti47

Al27 UV



3. CF3 Applications

(Centrifugal FFF)

24




CF3-DLS – Detection of PEG-PLGA-Nanoparticles in cell medium

Nanoparticle drug delivery

Kohl Y., et al, Nanosafety Conference 2013, Biopolymer Nanoparticles for Therapeutic Applications: Synthesis,

Characterization and Assessment of Biocompatibility..

• Incubation of the

nanoparticles in cell media

was performed at 98 °F.

• Detected NP size

increased over time

• The decreasing overall

particle numbers observed

after prolonged incubation

shows agglomeration/

aggregation of

nanoparticles

25




CF2000-ICP-MS

Mass Discrimination: Au- and Ag Nanoparticles (d = 20 nm)

26

• Density-based

separation (CF3) +

information about

the chemical

composition of the

particles (ICP-MS)

in one single run

Tadjiki and Klein, The Column, 2014, 10(14), 11-16.

RS = 0.8

Au

Ag




CF2000-UV + TEM

Fractionation of Au Nano Rods from Au Nano Eggs

27

Original Fraction 1

Fraction 2 Fraction 3

Tadjiki and Klein, The Column, 2014, 10(14), 11-16.

Rods

Eggs Spheres

Rods

Thickness = 10 nm

Length = 35 nm

Aspect Ratio: 3.5

Eggs

Thickness = 25 nm

Length = 35 nm

Aspect Ratio: 1.4

• Separation according to mass and hydrodynamic diameter




4. TF3 Applications

(Thermal FFF)

28




TFFF Applications – PS

System • TF2000 FFF System

• PN3150 RI Detector

Conditions • Injection Volume: 20 µL

• Concentration: 2 mg/mL

• Temp. grad. T = 90°K to 0°K

Separation of PS standards

29



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TF2000 Applications

Separation of PS and PMMA-Standards with same Rh

SEC in THF (SDV Columns) vs. Thermal FFF in THF

Separation by Hydrodynamic Size and Chemical Composition

30



0.025

0.125

0.225

0.202

0.206

0.21

0 10 20 30 40 50 60 70

ELS

D D

ete

cto

r Sig

nal [V

]

LS

Dete

cto

r S

ignal [V

]

Time [min]

__ LS 92° __ ELSD

Natural Rubber Sample

31

TF2000 Applications

See Gerhard Heinzmann talk tomorrow at 14:00 !

ELSD

MALS



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Summary

FFF and GPC/SEC are both well-established techniques for the

characterization of macromolecules, with FFF also a valuable

tool for separation and characterization of (nano)particles, gels,

aggregates and larger complexes.

The overlap between the GPC/SEC and FFF application areas

and technique capabilities can now be fully exploited in a

single, highly flexible modular

instrument platform from Postnova.

32



Thank you!

[email protected]

33



Documents

The Postnova Characterization Platform...Viscometer Detector (Visc) – Intrinsic Viscosity (IV) of the sample (inverse density) Multi-Angle Light Scattering (MALS) – Molecular weight