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1
Christoph Johann
Wyatt Technology Europe GmbH
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
Analytica Conference 2012
3Analytica Conference 2012
Evolution of Eclipse Instruments
2002
2004
2006 Eclipse 2
.
2009 Eclipse 3+
5 generations of Eclipse instruments since 2002
2011 Eclipse DUALTEC
FFF Focus Day - ILSC 2011 4
molar mass vs. time
BSA - Standard__20070314__203_01gfedcb
time (min)4.0 6.0 8.0 10.0 12.0 14.0 16.0
mol
ar m
ass
(g/m
ol)
51.0x10
miniDAWN 199622 µg BSA
90° LS
Improvement of Signal Qualitymolar mass vs. time
02 BSA 10ug PLC10 350WSC Vc10 Vx30gfedcb BSA - Standard__20070314__203_01.MDFgfedcb
time (min)4.0 6.0 8.0 10.0 12.0 14.0 16.0
mo
lar
mass (
g/m
ol)
51.0x10
HELEOS II 20088.8 µg BSA
90° LS
miniDAWN 199622 µg BSA
.
Improved sensitivity: less sample required
Improved separation: more precise information
5Analytica Conference 2012
Separation takes place in the channel, the different steps of the process will be shown in detail
How Flow-FFF Separation Works
6Analytica Conference 2012
How FFF Separation Works II
FOCUS Step prepares the channel for injection of the sample
7Analytica Conference 2012
Injection of the sample in FOCUS+INJECT mode
How Flow-FFF Separation Works
8Analytica Conference 2012
How Flow-FFF Separation Works
Sample injected
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Sample injected – by interaction with the cross-flow it is concentrated against the porous bottom wall
How Flow-FFF Separation Works
10Analytica Conference 2012
Sample injected – the Focusing – Relaxation process takes place
How Flow-FFF Separation Works
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Transport in the channel and separation in ELUTION mode
How Flow-FFF Separation Works
12Analytica Conference 2012
How Flow-FFF Separation Works
Elution mode – the sample is transported towards the outlet of the channel
13Analytica Conference 2012
How Flow-FFF Separation Works
Elution mode – the sample components start to separate
14Analytica Conference 2012
How Flow-FFF Separation Works
Elution mode – the sample components separate more
15Analytica Conference 2012
How Flow-FFF Separation Works
Elution mode – the sample components stay close to the membrane throughout the elution process
16Analytica Conference 2012
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
FFF Focus Day - ILSC 2011 17
Method Optimization
Simulation of FFF separation of two particles 3 and 8 nm in a laminar flow profile
Separation example.avi
Animation of a Flow-FFF Experiment in the Eclipse
Animation of the separation in 3-D
FFF Focus Day - ILSC 2011 18
AF4 - Retention Equation
tr retention timew channel thicknessFCR cross-flow rateFOUT flow rate to the detectorDi diffusion coefficient
[1] R. N. Qureshi, Wim Th. Kok, LCGC Europe Jan 2010
ln [1]
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
AF4 Zone Broadening
st standard deviation of the peak in time units
uCR cross-flow velocity (cross-flow rate divided by channel area
[1]
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
Optimization between improvement of resolution and minimization of TREQ and peak dilution is necessary
TREQ = 839 DILF = 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*
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
ln
TREQ = 839 DILF =
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 24
ExampleSample BSA monomer and dimer, 30 µg injected
Fractograms of BSA separation demonstrating that the TREQ is independent on channel length and spacer height, calculation taken from ISIS
SC 350 µm
LC 250 µm
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) 4126-4131
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
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
Hollow-Fiber Flow-FFF (HF5) Principle
r
zVinrf
Cross-flow
Vout
Hollow Fiber
Channel tube
Cross-flow outlet
Inlet connection
(from injector)
Outlet connection (to
detector) .
A single fiber is sealed inside a PET housing, no glue is used. The sealing mechanism works for fibers of different diameters. (German patent 10 2010 043 877.4)
HF5 Channel Design
Analytica Conference 2012
10 2010 043 877.4
FFF Focus Day - ILSC 2011 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 Equation
Retention R in Flow-FFF is a function of the mean layer thickness l
AF4 R = 6 lHF5 R = 4 l
In HF5 the same retention is achievedwith 33% lower concentration at themembrane.
This leads to reduced overloading and aggregation effects.
c0
c = ½ c0
l
.….
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 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
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. 10 2010 041 222.8.
D etector
F lowContro ller
F low M eter
O utlet Port
In le t Port
M ain Pum p
W aste1
2
3 4
5
6
FocusNeedle Valve
PressureSensor
PressureSensor
C rossflowAutosam pler
Only one pump is needed to generate the inject flow and cross-flow
FFF Focus Day - ILSC 2011 36
Focusing Visualization
Visual inspection is not necessary any more with the DUALTEC innovation.
.
FFF Focus Day - ILSC 2011
DUALTEC Autofocus
382011 Copyright Wyatt Technology Europe GmbH – All Rights reserved
The Eclipse DUALTEC
Integration with Agilent 1260 and ChemStation ® as well as with Dionex Ultimate andChromeleon®
FFF Focus Day - ILSC 2011 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
concentration vs. time
2 2010-11-08 CAH single vc 035 vx 85 ff 85 1 ul lHF10 50 mM NH4HCO3]gfedcb 4 2010-11-08 CAH single vc 035 vx 85 ff 85 2 ul lHF10 50 mM NH4HCO3]gfedcb6 2010-11-08 CAH single vc 035 vx 85 ff 85 3 ul lHF10 50 mM NH4HCO3]gfedcb 8 2010-11-08 CAH single vc 035 vx 85 ff 85 4 ul lHF10 50 mM NH4HCO3]gfedcb10 2010-11-08 CAH single vc 035 vx 85 ff 85 5 ul lHF10 50 mM NH4HCO3]gfedcb
time (min)8.0 10.0 12.0 14.0 16.0
con
cen
trat
ion
(g
/mL
)
0.0
-65.0x10
-51.0x10
-51.5x10
-52.0x10
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
UV peak heights at 280 nm as a function of injection amount
y = 4,165x - 0,516
R2 = 0,9999
y = 1,9701x + 5E-15
y = 1,1493x + 2E-15
0
5
10
15
20
25
0 1 2 3 4 5 6
Injection amount [µg]
Pe
ak
he
igh
t [m
AU
]
HF ID 0.8 mm
HF calculated
AF4 SC calculated
AF4 LC calculated
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
Light scattering signal at 90 ° as a function of injection amount
y = 0,6102x + 0,2298
R2 = 0,9956
y = 0,0994x + 0,0182
R2 = 0,9998
0
0,5
1
1,5
2
2,5
3
3,5
0 5 10 15 20 25
Injection amount [µg]
Lig
ht
scat
teri
ng
sig
nal
at
90°
[mV
]
HF ID 0.8 mm
flat channel 490 µm
FFF Focus Day - ILSC 2011 45
Separation of protein mix on HF5– peak identification
1
2
34
5
6
7
min0 5 10 15 20 25 30 35
mAU
0
5
10
15
20
25
30
VW D 1 A , W avelength=215 nm (PR O TEIN M IX\17112010\D EF_LC 2010-11-17 18-50-10\17112010_PM _013.D )
1
2
34
5
6
7
1. Carbonic anhydrase (1mer)2. BSA (1mer) + Carbonic anhydrase (2mer)3. Carbonic anhydrase (3mer)4. BSA (2mer)5. Apoferritin (1mer)6. Thyroglobulin (1mer) + Apoferritin (2mer)7. Apoferritin (3mer) + Thyroglobulin (2mer)
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)chromatograms
21 2011-08-18 Wte ab vc 022 vx 045 ff 022 015µg 50 mM PBS 150 mM NaCl pH 7,2
volume (mL)0.0 0.5 1.0 1.5
Rela
tive S
cale
0.0
0.5
1.0 UV
Peak half width 100 µlRetention time 3 minutesInjection amount 150 ng
Sample is a monoclonal antibody for therapeutic use
FFF Focus Day - ILSC 2011 47
Monomer-Dimer Separation
chromatograms
21 2011-08-18 Wte ab vc 022 vx 045 ff 022 015µg 50 mM PBS 150 mM NaCl pH 7,2
volume (mL)0.4 0.6 0.8 1.0 1.2 1.4
Rela
tive S
cale
0.000
0.005
0.010
0.015
0.020UV
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 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 56
MAb IgG1 separated on HF5
Vial #2 (2 µg)
77,3% Recovery
18,9 % Recovery
3,95 % Recovery
FFF Focus Day - ILSC 2011 57
HF5 AF4
0 5 10 15
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
UV
abs
orba
nce
@ 2
15 n
m(m
AU
)
Time (min)
2μg 5 μg 10 μg
Vial # 2
0 5 10 15
0
20
40
60
80
100
120
140
160
180
UV
abs
orba
nce
@ 2
15 n
m (m
Au
)
Time (min)
1μg 2 μg 5 μg 7 μg
Vial # 2
(21/01/2011) (28/01/2011)
Comparison of HF5 and AF4
FFF Focus Day - ILSC 2011 58
Analysis of oligomers in IgG1
Although prepared from the same stock, two samples of the four aliquotes have got much higher amount of dimer and trimer.
Vial # 2&3
Vial # 1&4
zoom
FFF Focus Day - ILSC 2011
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)
Particle Characterizationgeometric radius vs. time
1 2011-03-08 latex 25 50 100 nm vc 065 vx 035__035 18 min 006 ff 035 4 min gr foc 5 min2 ul lhf2 02% SDSgfedcb
time (min)0.0 5.0 10.0 15.0 20.0 25.0
ge
om
etr
ic r
ad
ius
(n
m)
30.0
40.0
50.0
60.0
70.0
80.090.0
100.0
UV
A new instrument for Hollow-Fiber- (HF5) and asymmetrical Flow-FFF (AF4) is introducedHF5 is characterized by
High sensitivity coming from high plate numbers and low peak dilutionImproved 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 sampleThe new development makes Flow-FFF accessible for every chromatography user
Summary
Analytica Conference 2012
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