1. Wyatt Technology Europe GmbH
A new instrument to improve
operation and performance in
Flow Field-Flow Fractionation
Christoph Johann
Wyatt Technology Europe GmbH
Changes made by CJ:
V 12: DualTec and HF5 added to FFF section
V13: Frit-Inlet AF4 added
Analytica Conference 2012

2. A new instrument to improve operation and performance in Flow Field-Flow Fractionation
Introduction
Flow-FFF Theory
Hollow-Fiber Flow-FFF and the Eclipse DUALTEC
Application Examples
Summary
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3. Evolution of Eclipse Instruments
5 generations of Eclipse instruments since 2002
2002
2004
2006 Eclipse 2
Rougly every 10 years a new generation of light scattering instruments was developed. Each generation had exiting new features and offered a significant increase in signal to noise.
2009 Eclipse 3+
2011 Eclipse DUALTEC .
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4. Improvement of Signal Quality
HELEOS II 2008
8.8 µg BSA
90° LS
miniDAWN 1996
22 µg BSA
miniDAWN 1996
22 µg BSA
90° LS
This figure compares two BSA measurements, one from a miniDAWN built 1996 to a current HELEOS II. The DAWN DSP could not be used for protein work, because it needed very high injected amounts. The HELEOS results were achieved with only 1/3 of the injected amount of the miniDAWN. There is a dramatic increase of signal to noise but that is not all. The new instruments need significant less user maintenance, user expertise and are more tolerant to problems stemming from chromatographic conditions. They are almost as easy to use as a UV Detector.
Improved sensitivity: less sample required
Improved separation: more precise information .
FFF Focus Day - ILSC 2011

5. How Flow-FFF Separation Works
Separation takes place in the channel, the different steps of the process will be shown in detail
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6. How FFF Separation Works II
FOCUS Step prepares the channel for injection of the sample
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7. How Flow-FFF Separation Works
Injection of the sample in FOCUS+INJECT mode
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8. How Flow-FFF Separation Works
Sample injected
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9. How Flow-FFF Separation Works
Sample injected – by interaction with the cross-flow it is concentrated against the porous bottom wall
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10. How Flow-FFF Separation Works
Sample injected – the Focusing – Relaxation process takes place
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11. How Flow-FFF Separation Works
Transport in the channel and separation in ELUTION mode
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12. How Flow-FFF Separation Works
Elution mode – the sample is transported towards the outlet of the channel
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13. How Flow-FFF Separation Works
Elution mode – the sample components start to separate
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14. How Flow-FFF Separation Works
Elution mode – the sample components separate more
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15. How Flow-FFF Separation Works
Elution mode – the sample components stay close to the membrane throughout the elution process
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16. Eclipse FFF Field-Flow Fractionation (FFF) Principle
Simulation of FFF separation of two particles 3 and 6 nm radius
Separation example.avi
3D-Animation of the separation in an AF4 channel
3D example.avi
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17. Method Optimization
Simulation of FFF separation of two particles 3 and 8 nm in a laminar flow profile
Separation example.avi
Animation of the separation in 3-D
Animation of a Flow-FFF Experiment in the Eclipse
FFF Focus Day - ILSC 2011

18. AF4 - Retention Equation
𝑡 𝑅 = 𝑤 2 6 𝐷 𝑖 ln 1+ 𝐹 𝐶𝑅 𝐹 𝑂𝑈𝑇
[1]
tr retention time
w channel thickness
FCR cross-flow rate
FOUT flow rate to the detector
Di diffusion coefficient
[1] R. N. Qureshi, Wim Th. Kok, LCGC Europe Jan 2010
One equation we show: retention is proportional to stokes diameter d and we can tune the retention easily by controlling flow rates, possibility to extract stokes diameter from retention time
Retention depends on the flow rate ratio only, not the length or width of the channel (with given Di and channel height)
FFF Focus Day - ILSC 2011

19. 𝜎 𝑡 =0.82 𝑤 𝑢 𝐶𝑅 ln ( 1+ 𝐹 𝐶𝑅 𝐹 𝑂𝑈𝑇 ) AF4 Zone Broadening
[1]
st standard deviation of the peak in time units uCR cross-flow velocity (cross-flow rate divided by channel area
Zone broadening depends only on instrumental parameters and not only on the ratio of flows, but also on the magnitude of the cross-flow rate
FFF Focus Day - ILSC 2011

20. Peak dilution and analysis time
TREQ = 𝐷 1 𝑢 2 𝐶𝑅
DILF = 𝑤 𝑢 2 𝐶𝑅 6 𝐷 2
TREQ Minimum time required to obtain baseline separation between two species that differ in Mw by a factor of 2
DILF Dilution factor between the concentration at the membrane c0 and the concentration in the detector c*
Optimization between improvement of resolution and minimization of TREQ and peak dilution is necessary
R. N. Qureshi, Wim Th. Kok, LCGC Europe Jan 2010
Separation example.avi
FFF Focus Day - ILSC 2011

21. How to come up with a method
DILF = 𝑤 𝑢 2 𝐶𝑅 6 𝐷 2
TREQ = 𝐷 1 𝑢 2 𝐶𝑅
𝜎 𝑡 =0.82 𝑤 𝑢 𝐶𝑅 ln ( 1+ 𝐹 𝐶𝑅 𝐹 𝑂𝑈𝑇 )
𝑡 𝑅 = 𝑤 2 6 𝐷 𝑖 ln 1+ 𝐹 𝐶𝑅 𝐹 𝑂𝑈𝑇
FFF Focus Day - ILSC 2011

22. General conclusion
For high efficiency the preferred method should have
High cross-flow density
Small channel thickness w
Limiting factor is c0, the concentration at the membrane and detectability
Efficiency has to be sacrificed to avoid those problems These facts can be illustrated using a simulation software based on FFF theory (called ISIS)
FFF Focus Day - ILSC 2011

23. Example FCR uCR FOUT Sample BSA monomer and dimer, 30 µg injected TREQ
(ml/min)
uCR
(105 m/s)
FOUT
TREQ
(min)
DILF
Peak Width
SC 350 µm 3
2.34
4.7
1.81
1420
0.28
LC 250 µm
4.5
2.8
895
0.31
FFF Focus Day - ILSC 2011

24. Example Sample BSA monomer and dimer, 30 µg injected
SC 350 µm
LC 250 µm
Fractograms of BSA separation demonstrating that the TREQ is independent on channel length and spacer height, calculation taken from ISIS
FFF Focus Day - ILSC 2011

25. Hollow-Fiber Flow-FFF (HF5)
Wyatt Technology introduces a new Flow-FFF instrument, the Eclipse DUALTEC.
Eclipse DUALTEC allows to apply HF5 and AF4 in one instrument.
FFF Focus Day - ILSC 2011

26. Hollow-Fiber Flow-FFF (HF5)
J. Chromatogr. A, 1218 (2011)
FFF Focus Day - ILSC 2011

27. Intoduction to HF5
HF5 has been pioneered in 1974 by Lee et al and further developed by Carlshaf and Jönsson in 1988, 1989 and Wim Kok et al.
Advantages:
high efficiency (high plate numbers) and high sensitivity because of low peak dilution
low-cost channel, possibly disposable
Problem:
Channel construction used to be tedious, no commercial products were available
FFF Focus Day - ILSC 2011

28. Hollow-Fiber Flow-FFF (HF5) Principle
The HF5 cross-flow is generated by the elution flow, which splits into a longitudinal and a radial direction: no depletion wall, only accumulation wall r z
Vin rf
Cross-flow
Vout
Hollow Fiber
Channel tube
Cross-flow outlet
Inlet connection (from injector)
Outlet connection (to detector)
….so, we can use hollow-fibers as microcolumn channels for FlFFF. The idea dates back to the early 70’s. Only recently, however, the availability of proper hollow-fibers has put the idea into real systems.
The prototype channel developed by Prof. Min and Prof. Moon is made up of a piece of hollow fiber, which is about 20 cm long and with ID of 0.8 mm.
With respect to classic, commercial FlFFF, HF FlFFF is low-cost and channels can be disposable, the channel volume is much reduced (about 100 uL) and so does sample dilution and analysis time.
This channel is made of a piece of… .
FFF Focus Day - ILSC 2011

29. HF5 Channel Design
A single fiber is sealed inside a PET housing, no glue is used. The sealing mechanism works for fibers of different diameters. (German patent )
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30. Hollow Fiber Membranes
Hollow Fibers are a high-tech product, they are very reproducible and robust
Hollow Fiber membranes have ideal properties for FFF application in terms of flux, stability and lack of interaction with the sample, e.g. antibodies.
FFF Focus Day - ILSC 2011

31. HF5 –AF4 Retention Equationc0
c = ½ c0 l
Retention R in Flow-FFF is a function of the mean layer thickness l
AF4 R = 6 l
HF5 R = 4 l
In HF5 the same retention is achieved with 33% lower concentration at the membrane.
This leads to reduced overloading and aggregation effects. …. .
FFF Focus Day - ILSC 2011

32. HF5 – AF4 Sensitivity and Efficiency
In Flow-FFF efficiency (maximum Plate Numbers N) depends mainly on cross flow velocity and retention time.
The HF5 fiber material is polysulfone, which has a very high flux (maximum cross flow rate per unit area). Therefore high cross flow rates are possible.
Peak dilution is proportional to detector flow rate, which can be smaller in HF5 because of low channel volume.
High plate number + low dilution = high sensitivity
.... .
FFF Focus Day - ILSC 2011

33. HF5 – AF4 Channel Comparison
Yellow: cross flow rate at constant flow density
Green: maximum feasible cross flow density
FFF Focus Day - ILSC 2011

34. HF5 Limitations HF5 requires low sample load.
HF5 is not as flexible in terms of channel height and membrane material.
HF5 and AF4 complement each other, therefore we developed the DUALTEC principle.
FFF Focus Day - ILSC 2011

35. HF5 Flow Schematic
Only one pump is needed to generate the inject flow and cross-flow
The position of the focus point is determined by measuring the focus flow rate entering the channel
Technical specification of the flow schematic has been filed for patent in Germany No
FFF Focus Day - ILSC 2011

36. Focusing Visualization
Visual inspection is not necessary any more with the DUALTEC innovation. .
FFF Focus Day - ILSC 2011

37. DUALTEC Autofocus
FFF Focus Day - ILSC 2011

38. The Eclipse DUALTEC
Integration with Agilent 1260 and ChemStation ® as well as with Dionex Ultimate and Chromeleon®
2011 Copyright Wyatt Technology Europe GmbH – All Rights reserved

39. Benefits of the Eclipse DUALTEC
Sensitivity
Productivity
Flexibility
FFF Focus Day - ILSC 2011

40. Benefits of the Eclipse DUALTEC
Sensitivity
Productivity
Flexibility
FFF Focus Day - ILSC 2011

41. Example – Carbonic Anhydrase
Separation of carbonic anhydrase, (1 mg/ml) with hollow fiber cartridge, injection amounts increase from 1 µg to 5 µg, UV signal at 280 nm
FFF Focus Day - ILSC 2011

42. Carbonic Anhydrase Separation Results
* Recovery of the SEC separation on a Superdex column was 70%, indicating the protein purity of the standard is 70%. Recovery was calculated using the UV detector response at 280 nm wavelength with an extinction coefficient of 1600 ml/(g cm).
FFF Focus Day - ILSC 2011

43. Sensitivity comparison between HF5 and AF4
Carbonic anhydrase, peak height as a function of injection amount compared to calculated peak heights on AF4 channels SC and LC with 350 µm channel height
FFF Focus Day - ILSC 2011

44. Sensitivity with respect to light scattering signals
Peak height of the 90° LS signal for BSA monomer separated on HF5 and AF4 channel SC with 490 µm spacer – experimental data
FFF Focus Day - ILSC 2011

45. Separation of protein mix on HF5– peak identification2 1 2 3 4 5 6 7
Carbonic anhydrase (1mer)
BSA (1mer) + Carbonic anhydrase (2mer)
Carbonic anhydrase (3mer)
BSA (2mer)
5. Apoferritin (1mer)
6. Thyroglobulin (1mer) + Apoferritin (2mer)
7. Apoferritin (3mer) + Thyroglobulin (2mer) 1 5 6 4 7 3
Total protein amount injected was 0.5 µg, longitudinal flow rate 0.2 ml/min, cross-flow rate 0.85 ml/min, UV signal 215 nm
FFF Focus Day - ILSC 2011

46. Separation with narrow-bore fiber (250 µm radius)
Peak half width 100 µl
Retention time 3 minutes
Injection amount 150 ng
Sample is a monoclonal antibody for therapeutic use
FFF Focus Day - ILSC 2011

47. Monomer-Dimer Separation
Sample is a monoclonal antibody for therapeutic use - dimer amount < 1% or < 2 ng
FFF Focus Day - ILSC 2011

48. Benefits of the Eclipse DUALTEC
Sensitivity
Productivity
Flexibility
FFF Focus Day - ILSC 2011

49. Eclipse DUALTEC - Productivity
Disposable channel cartridge for HF5 allows to install a new channel with minimal downtime. No operator skill is needed (in contrast to replacing a membrane).
AutoFocus places the focusing zone at the user defined place without manual intervention or visualization experiments.
Short run time is possible.
Low consumption of sample and solvent, which is important in method development
HF5 is ideal for temperature controlled operation.
FFF Focus Day - ILSC 2011

50. Benefits of the Eclipse DUALTEC
Sensitivity
Productivity
Flexibility
FFF Focus Day - ILSC 2011

51. Eclipse DUALTEC - Flexibility
Automatic switching between two separation modes is a standard feature.
Scale-up of separation in terms of sample load can be done without additional experiments by transferring the method from HF5 to AF4 using ISIS (Intelligent Separation Improvement System).
Possible combinations:
HF5 – AF4: method development vs. higher sample load
HF5 – HF5: doubles lifetime of the channel before manual replacement)
AF4 – AF4: two channel geometries can be compared
FFF – SEC: comparison to column separation
FFF Focus Day - ILSC 2011

52. Eclipse Flow-FFF Overview
3 inlet ports to the channel
Central down stream injection (CDI) AF4
Semi-Preperative Channel
Frit-Inlet AF4 (FI-AF4)
Metal free system
2 inlet ports to the channel
Tip-Injection AF4
Hollow-Fiber Flow-FFF (HF5)
Software controllable selection between two separation modes
FFF Focus Day - ILSC 2011

53. Applications
FFF Focus Day - ILSC 2011

54. Analysis of MAb Aggregation
Characterization of MAb aggregation is of vital importance.
Separation of aggregates and their quantification is difficult and results may depend on the method applied (SEC, AF4).
HF5 results presented here on an MAb indicate that HF5 is well suited for aggregate quantification.
FFF Focus Day - ILSC 2011

55. Aggregation behavior of MAb IgG1
Sample is a monoclonal antibody for therapeutic use currently on the market.
A stock sample with 38 mg/ml was taken thawed from -80°C, diluted 1:100 in mobile phase (PBS 10 mM, NaCl 25 mM) and again frozen at - 20 °C and aliquots analyzed after different storage time.
Vial 4°C analyzed the same day
Vial -20°C for 4 days
Vial -20°C for 8 days
Vial -20°C for 11 days
FFF Focus Day - ILSC 2011

56. MAb IgG1 separated on HF5 Vial #2 (2 µg) 77,3% Recovery
FFF Focus Day - ILSC 2011

57. Comparison of HF5 and AF4 HF5 AF4 (28/01/2011) (21/01/2011)
2μg 5 μg 10 μg
Vial # 2
1μg 2 μg 5 μg 7 μg
Vial # 2
HF5
AF4
FFF Focus Day - ILSC 2011

58. Analysis of oligomers in IgG1
Vial # 2&3
Vial # 1&4
zoom
Although prepared from the same stock, two samples of the four aliquotes have got much higher amount of dimer and trimer.
FFF Focus Day - ILSC 2011

59. Comparison of IgG1 oligomer concentration with time
Measurements performed on the same sample (Vial # 2), 2μg
After three hours
UV 215 nm
First day
The amounts of 2mer and 3mer progressively descrease with elapsing time while the 1mer increases.
Reversible aggregates ? (2mer& 3mer)
After three days
FFF Focus Day - ILSC 2011

60. Eclipse Frit-Inlet AF4
Frit-Inlet AF4 is an alternative way to achieve separation with AF4 without a focusing step
A frit is placed at the inlet in the upper plate
The sample is injected through the tip of the channel
A flow stream equivalent to the cross-flow is introduced through the top-frit
Relaxation is achieved „on the fly“ without stopped-flow focusing
FFF Focus Day - ILSC 2011

61. FI-AF4 – Relaxation Principle
Relaxation is achieved while the sample is transported throught the first section of the AF4 channel with the frit inlet flow coming from the top
FFF Focus Day - ILSC 2011

62. Resolution of FI-AF4
Separation efficiency is a little less compared to focusing AF4, shown is a separation of BSA monomer and dimer
FFF Focus Day - ILSC 2011

63. Possible problem of sample aggregation in AF4
Conventional AF4 can lead to sample aggregation. Example of increasing molar mass with higher sample load of hyaluranic acid (data taken from poster presented at the ILSC 2011)
FFF Focus Day - ILSC 2011

64. Benefit of FI-AF4
FI-AF4 minimizes the problem of sample aggregation. Hyaluranic acid sample does not increase in molar mass with higher sample load (data taken from poster presented at the ILSC 2011)
FFF Focus Day - ILSC 2011

65. Particle Characterization
Separation of a mix of 3 latex samples, with 35, 50 and 100 nm radius with the HF5 channel, particle size determined with Multi-Angle Light Scattering (MALS)
FFF Focus Day - ILSC 2011

66. Summary
A new instrument for Hollow-Fiber- (HF5) and asymmetrical Flow-FFF (AF4) is introduced
HF5 is characterized by
High sensitivity coming from high plate numbers and low peak dilution
Improved productivity with a disposable channel, short analysis time and low eluent consumption
The Eclipse DUALTEC has the flexibility to switch between two separation modes, allowing specifically to combine FFF and SEC separation experiments for the same sample
The new development makes Flow-FFF accessible for every chromatography user
Analytica Conference 2012