agilent meeting spring 2014 · . agilent meeting spring 2014 applications of field flow...

www.postnova.com .

Agilent Meeting Spring 2014Applications of Field Flow Fractionation Part 2

02.06.2014 1

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

Company Customers

Academics, Government, Food, Agro, Petrochem, Polymer, Biopharma, Enviro, Nanotechnology

02.06.2014 2

World-wide Customer Base

NANOTECHEuropean Commission, BEEuropean Commission, ITLGC Teddington, UKNational Nano Initiative, USFed.Material Res. BAM, DEAdv.Inst. Science Tech, JPNat. Inst. Metrology, CN

BIOPHARMBoehringer, DEGenentech, USNovartis, ITAbbott, USRoche, CHBayer, DEEli Lilly, USSanofi, FR US FDA, USUS CDC, USUS NIH, US

POLYMERDow Chemical, USBridgestone, JPSaudi Aramco, KSAGoodyear, USBASF, DEBayer, DEKumho, KRMichelin, FRKao Corp, JP

ENVIRO-FOODUniv. of Vienna, ATUniv. Of Munich, DEUniv. Gotebörg, SELGC Teddington, UKUniv. Southampton, UKKIT Karlsruhe, DEEPFL Lausanne, CHMonash Univ., AUSUS ARMY COE, USUS EPA, USA

www.postnova.com .

About Postnova

02.06.2014 3

2001 Acquisition of FFFractionation - Product Portfolio and Patents

1997 Founding Postnova, Munich, Germany / launching first commercial AF4

2007 Launching TF2000 Thermal FFF

2008 Launching AF2000 Flow FFF

2010 Launching CF2000 Centrifugal FFF

2012 Agilent VAR Partner ICP-MS

1966 Invention and FFF Patents by Prof. Giddings in Salt Lake City, USA

1986 Invention and FFF Patents by Prof. Giddings in Salt Lake City, USA


System Information

► Injector

► Separator

► Channel

► Detection

► Fraction.

► Eluent

► Flow Rate

PN5300 Auto Injector

CF2000 Centrifugal FFF System (CF3)

CF2000 Channel250 µm Thickness

PN3211 UV – AbsorbancePN3621 MALS – Static Light Scattering, 532 nmPN3704 DLS - Dynamic LSICP-MS Agilent 7700

PN8050 – Fraction Collector

0.05 % NovaChem100Eluent filtered by 0.1 µm filter

0.5 mL/min

02.06.2014 - C O N F I D E N T I A L - 4

Postnova CF2000 Centrifugal FFF


0.075

0.175

0.275

0.375

0.194

0.214

0.234

0.254

0.274

10 30 50 70 90

UV

Detector S

ignal [V]LS

Det

ecto

r Sig

nal [

V]

Time [min]

__ LS 90° __ UV

Sample TiO2

02.06.2014 - C O N F I D E N T I A L - 5

Raw Data Fractogram of CF3 - MALS and UV

System• PN5300 Auto Injector• CF2000 Centrifugal FFF System• PN3211 UV Detector 254 nm• PN3621 MALS Detector• PN3704 DLS Detector

Conditions• Injection Volume: 20 µL• LS 90°(red trace)• UV 254 nm (blue trace)


0.00

0.02

0.04

0.06

0.08

10

100

1,000

30 40 50 60 70 80 90

Detector S

ignal [V]

Rad

ius

[nm

]

Time [min]

Radius / LS 90°

Sample TiO2

02.06.2014 - C O N F I D E N T I A L - 6

Overlay: Radius of Gyration and LS Signal• The Radius of Gyration was calculated from MALS angular data

Conditions• Radius of Gyration (red dots)• LS Signal (blue trace)• Fitting by Random Coil Model

Rg [nm]

n-Average 39

w-Average 49

z-Average 88


Radius

Radius [nm]200180160140120100806040200

Diff

eren

tial

0.045

0.04

0.035

0.03

0.025

0.02

0.015

0.01

0.005

0

Cum

ulative

1.0

0.0

Sample TiO2

02.06.2014 - C O N F I D E N T I A L - 7

Particle Size Distribution

• Differential Particle Size Distribution (blue trace)• Cumulative Particle Size Distribution (red trace)


Sample TiO2

Ti measured by ICP/MS after CF3 separation

02.06.2014 8


Sample TiO2

02.06.2014 - C O N F I D E N T I A L - 9

Hydrodynamic SizeThe Hydrodynamic Radius (Rh) was measured by Zetasizer DLS

Rt [min] Rh [nm]

32 – 85 57 ± 17

0

200

400

600

800

1000

10 20 30 40 50 60 70 80 90

Inte

nsity

(kcp

s)

Time (mins)

10

100

1000

Z-Average (r.nm)

Flow trace v Time


0.106

0.111

0.116

0.121

0.126

0.131

0.136

0 10 20 30 40 50 60 70 80

Detector S

igna

l [V]

Time [min]

‐ Au Mix high resolution ‐ Au Mix short method

Sample Au 10nm, 30 nm, 60 nm (NIST)

02.06.2014 - C O N F I D E N T I A L - 10

Raw Data Fractogram of CF3 – UV 530 nm

System• PN5300 Auto Injector• CF2000 Centrifugal FFF System• PN3211 UV Detector 530 nm• PN3621 MALS Detector• PN3704 DLS Detector

Conditions• Injection Volume: 20 µL• UV 530 nm

• Au Mix separated using method with high resolution (black trace)• Au Mix separated using short method (green trace)


Sample Au 10nm, 30 nm, 60 nm (NIST)Au measured by ICP/MS after CF3 separation

02.06.2014 11

• Au Mix separated using method with high resolution (black trace)• Au Mix separated using short method (green trace)


Asymmetric Flow FFF

Applications• Peptides• Proteins• Antibodies• Virus• Liposomes• Latex Bead• Nano Particles• Synthetic / natural

Polymers

02.06.2014 12

AF2000 AT / MT Series

• Universal Separator for Proteins, Polymers and Nanoparticles ambient to mid temp


AF4-ICP-MS Analysis of BSA Protein

• Elements in are associated with Protein species

• Monomer, Dimer, Trimer visible in different element traces

• Adsorption-/association tendency is element-specific

02.06.2014 13

BIOPHARM: Fellner, et. al., Agilent, Waldbronn, DE / Postnova, LL

www.postnova.com .

AF4 -ICP-MS fractogram obtained for mixtures of HSA and transferrin

1 g L-1 of each protein, 350 micron channel spacer

02.06.2014 14Heidi Goenaga-Infante, LGC et al, Trevor Havard, Postnova.

www.postnova.com .

AF4 -ICP-MS fractogram obtained for mixtures of HSA and transferrin

1 g L-1 of each protein, 500 micron channel spacer

02.06.2014 15Heidi Goenaga-Infante, LGC et al, Trevor Havard, Postnova.

www.postnova.com . 02.06.2014 www.postnova.com 16

BSA and Aggregates

0

1

2

3

4

0 2 4 6 8 10 12 14 16Retention Time [min]

Det

ecto

r Res

pons

e [U

V/m

V]

BSA monomer

Dimer

TrimerTetramer?

Analysis Conditions / System Set-upSolvent : 0.1 M PBS Buffer, pH 7.4 - Channel Thickness : 250 μmInjection Volume : 5 μL - Membrane : NovaRC PLGC 10 KDSample Concentr. : 4 mg/mL - Channel Flow : 0.32 mL/minCross Flow : 5.8 mL/min - Tip Flow : 0.05 mL/min

Focus Time : 3.5 minFFF System : Postnova AF-2.000 Focus Series - Asymmetric Flow Field-Flow FractionationLS Detector : Postnova PN3000SLS/DLS - Static/Dynamic Laser Light Scattering DetectorUV Detector : Postnova PN3240 UV/Vis - 4-Channel UV/Vis Detector (Wavelength used: 280 nm)

www.postnova.com .www.postnova.com . 02.06.2014 17

Protein Virus Mix

90° SLSUV abs .

90° DLS

100

0

200

300

400

500

600

700

0

20 25 3010 155Retention time [min]

Hydrodynamic diam

eter (DLS) [nm

]

virusparticlesproteins

DLS- signal

UV/S

LS si

gnal

Inte

nsity

[a.u

.]

AF2000 – UV-MALS-DLS


Membranes Suitability for Protein Separation's

Fractograms of five consecutiveinjections of protein mixture for thefollowing membranes

Regenerated Cellulose (RC)Polyethysulfone (PES),Cellulose triacetate (CTA)

Proteins analyzed: 1) Lysozyme 14 kDa,2) BSA 66 kDa (M)3) BSA 132 kDa (D)4) g-Globulin 150 kDa (M)5) g-Globulin 300 kD, (D)6) Thyroglobulin 660 kDa (M)7) Thyroglobulin 1,320 kDa (D)

02.06.2014 18

Postnova Constantly Stock Membranes

RC

1

4

2

53

7

6



02.06.2014 19


PES4

2

53

7

6






02.06.2014 20


PES4

2

53

7

6





Reproducibility and Recovery of Protein

02.06.2014 21

Regenerated Cellulose (RC)

Example of the recovery information for the membrane under the conditions used

Inj.

Mean retention time (min)Total Area1 2 3 4 5 6 7

1 7.66 12.13 15.6 18.81 22.58 26.53 31.51 1.38E+07

2 7.66 12.10 15.6 18.70 22.38 26.40 31.53 1.36E+07

3 7.58 11.97 15.59 18.44 22.58 26.12 31.46 1.35E+07

4 7.63 12.04 15.59 18.60 22.43 26.27 31.62 1.40E+07

5 7.57 11.98 15.59 18.51 22.41 26.20 31.69 1.38E+07

Average 7.62 12.04 15.594 18.61 22.48 26.30 31.56 1.37E+07

s 0.04 0.07 0.005 0.15 0.10 0.16 0.10 1.94E+05

%RSD 0.56 0.59 0.035 0.79 0.43 0.62 0.29 1.42


Asymmetrical Flow FFF planar and Tubular (HF) channels

02.06.2014 22

Separation forms


Planar RC-amph

02.06.2014 23

• Fractograms of consecutive injections of BSA using the planar channel with thePolyethersulfone (PES) and Regenerated Cellulose amphiphilic (RC-amph)membranes, and a PES tubular channel (hollow fiber).


Planar PES

02.06.2014 24



Tubular PES

02.06.2014 25


Tubular Asymmetrical flow field-flow fractionation channel. Thechannel is made of a semi-permeable hollow fibercartridge. the cross flow exitsradially through the fiber walland the channel flow is flowingaxially along the fiber length.

www.postnova.com .

Comparison of resolution and reproducibility of planar and tubular channels

02.06.2014 26

Channel Mean retention time (min) Standard deviation (min) %RSD Resolution Number of plates

BSA monomer BSA dimer BSA monomer BSA dimer BSA monomer BSA dimer

Planar PES

14.43 19.66 0.45 0.71 3.10 3.60 2.18 935.00

PlanarRC-

amph

13.44 17.99 0.43 0.68 3.17 3.80 1.90 1000.00

Tubular PES

8.46 11.17 0.06 0.09 0.71 0.80 1.35 157.00


Size Exclusion Separation of Protein mixture

Retention time (min)

Det

ecto

r re

spon

se

0 2 4 6 8 10 12 14 16 18 20

Thyr m

IgG d

Thyr d

IgG m

HSA

90o SLS

UV


AF4 Separtion of Protien mixture

0

5

10

15

20

25

0 5 10 15 20

IgG mHSA

IgG dThyr d

Thyr m


UV

resp

onse


Comparing SEC with AF4

0 2 4 6 8 10 12 14 16 18 20

90o SLS

UV

0

5

10

15

20

25

0 5 10 15 20


Det

ecto

r re

spon

se

AF4 is a SelectiveMechanism andProvides GreaterResolution


FFF Detectors


PN3150 RI – Detector

Specifications• Concentration Detector for FFF• For Molar Mass determination

02.06.2014 31


PN3621 MALS – Detector

Specifications• Molar Mass Range:

10E3 to 10E9 Da• Particle Size Range:

Rg from ca. 8 to 500 nm• Laser Power:

2,5 to 50 mW at 532 nm• Angular Range:

21 Angles from 7° to 164°• Lowest Angles:

7°, 12°, 20°, 28°, 36°, 44°• Ideal Application Range:

ultra-high molar mass,complex macro structures,aggregates and particles

02.06.2014 32


FFF Detection

02.06.2014 33

FFF – Online DLS CouplingChange over between Batch and Flow Mode is possible in seconds

AF2000 Multi Flow FFF Online / Offline DLS


Dextrane Coated Ironoxide ParticlesRadius

Time [min]50484644424038363432302826242220181614121086

Rad

ius

[nm

]

200

180

160

140

120

100

80

60

40

20

0

Detector Signal[V]

2.0

1.0

0.0

Radius

Radius [nm]1201101009080706050403020100

Diff

eren

tial

0.05

0.045

0.04

0.035

0.03

0.025

0.02

0.015

0.01

0.005

0

Cum

ultative

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

Ferrofluid Particle Size [nm] Polydispersity Indexn-Average 7

1,9w-Average 13z-Average 28

AF2000 – MALS


AF2000FFF – Zetasizer DLS

02.06.2014 35

Magnetite

Analysis of the poly(acrylicacid)-conjugated magnetite particles using hollow fiber FFF system hyphenated with MALS and on-line DLS detections. The average radius of gyration (Rg) and hydrodynamic radius (Rh) were calculated as 28.9 nm and 22.6 nm respectively. The particles exhibit a shape factor (Rg/Rh) of 1.28 which is between the values for coil and rod shapes

Time [min]

Intensity [k cps]

Rh

[nm

]

1

10

100

1000

0 10 20 30 40 50 600

100

200

300

400

Channel flowrate 0.75 mL/min

Cross flowrate 1.5 mL/minLinear decay in 60 min

Injection flowrate 0.2 mL/minInjection volume 20 µLFocusing time 4 minSample concentration 1,0 mg/mL

Hollowfiber 10 kDa PESCarrier 0.2 % NovaChem

www.postnova.com .

Worldwide Support Network

• Postnova Analytics EU; Max-Planck-Str. 14, 86899 Landsberg, GER, Tel: +49 8191 985 688-0• Postnova Analytics USA; 230S., 500E., #120, Salt Lake City, UT-84102, USA, Tel: +1 801 521 2004• Postnova North Europe; P.O. Box 44, Vantaa, Helsinki, 01721, FIN, Tel: +358 9 854 551-0• Web: www.postnova.com, Email: [email protected]

02.06.2014 36

Postnova North AmericaSalt Lake City, Utah, USAPostnova North AmericaSalt Lake City, Utah, USA

Postnova Central EULandsberg, GermanyPostnova Central EULandsberg, Germany

Postnova Norther EuropeHelsinki , FinnlandPostnova Norther EuropeHelsinki , Finnland

DistributorsLocations

www.postnova.com .

Any Questions

Thank you for your attentionTrevor [email protected]

617-314-7351

02.06.2014 37


Summary

• Fast ,Gentle and Flexible Separation in various Solvents • Ultra High Resolution / Separation (+/- 1 nm)• Open Flow Channel without Stationary Phase – No Shear /

Interaction• Broad Separation Range: ca. 10 nm - 100 µm• Direct Injection of complex Matrices without Filtration• Separation of small, medium and larges Species• Physical Collection of Particle Size Fractions• Hyphenation of FFF with other Detection Systems• On-line Coupling: DLS, MALS, ICP-MS, RI, UV, FLD, ESI-MS, …• Off-line Coupling: EM, AFM, NMR, MALDI, …• Correct Determination of Particles Size, Structure

02.06.2014 38

agilent meeting spring 2014 · . agilent meeting spring 2014 applications of field flow...

Documents