ASTS-2014 Postnova Integration Field Flow Fractionation ... · Interfacing Postnova FFF systems PN...

www.postnova.com .www.postnova.com . 02.06.2014 1

Integration of Postnova Field Flow Fractionation Systems with Agilent’sICP-MS 7900 and 8800

Trevor [email protected]

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

FFF Principle

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Separation Mechanism

• Separation in a narrow ribbon-like channel• Laminar flow inside the channel• External field perpendicular to the solvent flow


There are Multiple versions of FFF

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RJ2

Slide 3

RJ2 Rainer Jünger - Postnova Analytics, 12/14/2012


FFF Size of Analyte

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Separation Range

Online Detection: Agilent 7900-8800 ICP-MS & Postnova Light Scattering

www.postnova.com .

The Postnova FFF Platform

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AF4

Asymmetrical Flow Field Flow Fractionation

Universal Separator


Asymmetric Flow FFF

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

Polymers

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AF2000 AT / MT Series

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


Flow FFF

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Asymmetric Flow FFF Principle

• Hydraulic Pressure Gradient Field (Cross-Flow) for Separation• Separation based on Size• Channel at Ambient, Mid or High Temperature


Patented Cross Flow Controller

Cross Flow Controller will always deliver the exact cross flow every run

Hollow FiberMicro ChannelAnalytical ChannelPreparative Channel

No Valves!

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www.postnova.com .

Asymmetric Flow FFFStep 1• Sample is injected directly into the

flow stream• A second pump provides focus flow• Cross flow is achieved by a precise

syringe pump• Constant flow to the detectors, no valves

Step 2• Sample is focused to narrow band• Improved resolution and sample washing• Very low concentrations can be enriched

Step 3 • Focus flow stops and main pump elutes

the sample• Cross flow syringe pump varies• Detector flow stays constant

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Asymmetric Flow FFF

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Smart Stream Splitting

• Major portion of flow in the channel is containing no sample• Minor portions of the flow is carrying the sample above the membrane• Splitting of those two stream via 2 outlets at the channel end• Only the sample containing sub-stream is guided to the detector• Sample dilution is reduced at channel outlet• Increase of sensitivity by factor 5 or more depending on conditions


HDL Lipoprotein Preparation for Mass Spectroscopy

Smart Stream Splitting

Enrichment of HDL by a factor of 2 using Smart Stream Splitting


AF2000 MultiFlow FFF Channels

Asymmetric Flow FFF - Principle


Capabilities of FFF-ICP-MS

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FFF:• MS enables to detect even very low

concentrations• Various elements can be traced

simultaneously• Element concentration & ratio distribution

ICP-MS:• Size distribution of particles or

macromolecules• Size information by Light scattering, from

FFF-Theory• Separation of size fractions for further

analysis

FFF-ICP-MS allows size-based elemental distributions


FFF Coupled with ICP-MS

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Size Separation – Particle Size – Element CompositionSize Separation – Particle Size – Element Composition


BSA – Bovine Serum Albumin – 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?

AF2000 – UV


AF4-Separation of Aquatic Colloids

Element distribution across size distribution from AF4-ICP-MS • Varying distributions of the

different elements were found

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ENVIRO: Hasselhöv et. al., Göteborg, Sweden


AF4-ICP-MS Analysis of Nano Products

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FOOD/COSMETIC: Laborda et. al., Zaragozza, Spain• Silver Nanoparticles of different size and silver-content can be characterized

by AF4-ICP-MS


4 Nanoparticle Mix:Peak 1: Rg = 6.3 nmPeak 2: Rg = 17 nmPeak 3: Rg = 33 nmPeak 4: Rg = 136 nm

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Latex Nanoparticle Separation in 0.01 % SDS

• System: AF2000 MT coupled with PN3621 MALS• Conditions: 25°C, 20 μL injection volume, c = 1,2 mg/mL (total conc.

of mix)

AF2000 – MALS


AF2000 - UV

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Polystyrene Latex Nanoparticle Mix

UV S

ignal

254 n

m

Retention Time [min]

www.postnova.com . 21

The CHALLENGE: Making polystyrene particles visible to ICP-MS

• Field-Flow Fractionation system can fractionate particles according to their size (hydrodynamic diameter)

• Coupled to ICP-MS can provide element/size distributions• Most size calibrants are polymers

• Polystyrene spheres often used• Don’t contain metal ions and cannot be detected with ICP-MS

• Therefore other detectors (UV, MALS, DLS) need to be coupled before the ICP-MS to obtain both size and elemental distribution


Detection of Sulfur-Coated PolystereneParticles by FFF-ICP-MS

• Polystyrene (PS) spheres can agglomerate and surface-charged particles are often produced to prevent it

• Surface Sulfonated-PS particles used as calibrants

• S present on the surface of nanoparticles

• The nanoparticles are in µg/g range and S is in low ng/g

• S not detectable in nanoparticles (surface) with Agilent 7700 (Xemode) due to much poorer LODs in comparison with those of the 8800 ICP-MS (Reaction gas: O2)


FFF-ICP- 8800 MS detection of S-coated PS nanoparticles (100 and 200 nm) via S detection

0

1

2

3

4

5

6

7

8

9

0 20 40 60 80 100

Time [min]

Norm

aliz

ed U

V si

gnal

0

10

20

30

40

50

60

70

80

ICP-

MS

sign

al [k

cps]

UV 254 nm UV 236 nm S32 -> 48 S34 -> 50

H2 + O2 mode: 4.5 ml/min H2 and 45% of O2

Heidi Goenag Infante -LGCTeddington,Middlesex TW11 0LY, UK


Centrifugal Field Flow Fractionation

Nanoparticle Separator


Centrifugal FFF – Principle

Content• FFF Principle• Systems• Applications• Summary

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Separation Principle

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


GIT Innovation Award

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CF2000 has been the winning instrument at Analytical Instrumentation and Software categoryCF2000 has been the winning instrument at Analytical Instrumentation and Software category

RJ1

Slide 26

RJ1 Rainer Jünger - Postnova Analytics, 12/14/2012


CF2000 – Centrifugal FFF

• Postnova Centrifugal FFF• Eluent 0,05 % NovaChem100• Power Decay Gradient 3500 rpm• Postnova PN3000 MALS• Sample Concentration:• Relaxation Time:

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Polystyrene Nano-Micro Particles


0

2

4

6

8

10

12

14

0 10 20 30 40 50 60 70 80

DetectorS

igna

l [V]

Retention time [min]

__ UV

10 nm

30 nm60 nm


• Postnova Centrifugal FFF• Eluent 0,05% NovaChem100• Power Decay Gradient 3500 rpm• Postnova PN3211 UV @ 530 nm• Sample Concentration:• Relaxation Time:

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Gold Nanoparticle High Resolution Separation



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Gold Nano Rods Separation• 0,05% NovaChem100• 90° LS Signal MALS • Concentration:• Relax. Time:

Axial = 10 nmLength = 35 nm

Peak 1 – „Rods“ Peak 2 – „Eggs“Axial = 25 nmLength = 35 nm

Peak 1 „Rods“

Peak 2 „Eggs“



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Gold / Silver Nanoparticles

2 0 2 5 3 0 3 5 4 00 . 0

0 . 2

0 . 4

0 . 6

0 . 8

1 . 0

A u D = 2 0 n m

A g D = 2 0 n m

Inte

nsity

R e t e n t i o n T i m e [ m i n ]

9 0 ° L ig h t S c a t t e r i n g S ig n a l U V @ 5 3 0 n m

Ag

Au

• Centrifugal FFF allows separation of particles with the same size according to differences in density



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Silica Coated Gold Nanoparticles• 0,05% NovaChem100• 90° LS Signal MALS • Concentration:• Relax. Time:

Fraction Size (nm)1 98-1082 154-1683 123-1294 173-1875 277-364

www.postnova.com .

Interfacing Postnova FFF systemsPN 9050 Interface box, will provide and automated system with the

capability to divert flow from the plasma in the event the plasma goes out.

Acidifying dosing pumps are available and can be fully integrated

PN9020 provides all triggers and data collection required to unify the two systems

AF2000 is completely metal free.

Postnova and Agilent are in a VAR partnership

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