Transcript Eclipse DUALTEC

Christoph Johann
Wyatt Technology Europe GmbH
Analytica Conference 2012
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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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Evolution of Eclipse Instruments
5 generations of Eclipse instruments since 2002
2002
2004
2006
Eclipse 2
2009 Eclipse 3+
2011 Eclipse DUALTEC
.
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Improvement of Signal Quality
miniDAWN
miniDAWN 1996
1996
22
22 µg
µg BSA
BSA
HELEOS II 2008
8.8 µg BSA
90° LS
LS
90°
.
Improved sensitivity: less sample required
Improved separation: more precise information
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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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How FFF Separation Works II
FOCUS Step prepares the channel for injection of the
sample
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How Flow-FFF Separation Works
Injection of the sample in FOCUS+INJECT mode
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How Flow-FFF Separation Works
Sample injected
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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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How Flow-FFF Separation Works
Sample injected – the Focusing – Relaxation process
takes place
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How Flow-FFF Separation Works
Transport in the channel and separation in ELUTION mode
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How Flow-FFF Separation Works
Elution mode – the sample is transported towards the
outlet of the channel
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How Flow-FFF Separation Works
Elution mode – the sample components start to
separate
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How Flow-FFF Separation Works
Elution mode – the sample components separate more
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How Flow-FFF Separation Works
Elution mode – the sample components stay close to
the membrane throughout the elution process
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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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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
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AF4 - Retention Equation
𝑡𝑅 =
tr
w
FCR
FOUT
Di
𝑤2
6𝐷𝑖
ln 1 +
𝐹𝐶𝑅
𝐹𝑂𝑈𝑇
[1]
retention time
channel thickness
cross-flow rate
flow rate to the detector
diffusion coefficient
[1] R. N. Qureshi, Wim Th. Kok, LCGC Europe Jan 2010
Retention depends on the flow rate ratio only, not the
length or width of the channel
(with given Di and channel height)
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AF4 Zone Broadening
𝜎𝑡 =
st
uCR
𝑤
0.82
𝑢𝐶𝑅
ln( 1 +
𝐹𝐶𝑅
)
𝐹𝑂𝑈𝑇
[1]
standard deviation of the peak in time units
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
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Peak dilution and analysis time
TREQ = 839
𝐷1
𝑢2 𝐶𝑅
DILF =
𝑤 2 . 𝑢2 𝐶𝑅
6𝐷2
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
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Separation example.avi
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How to come up with a method
TREQ = 839
𝜎𝑡 =
𝑢2
𝐶𝑅
𝑤
0.82
𝑢𝐶𝑅
𝑡𝑅 =
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𝐷1
𝑤2
6𝐷𝑖
DILF =
ln( 1 +
ln 1 +
𝑤 2 . 𝑢2 𝐶𝑅
6𝐷2
𝐹𝐶𝑅
)
𝐹𝑂𝑈𝑇
𝐹𝐶𝑅
𝐹𝑂𝑈𝑇
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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)
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Example
Sample BSA monomer and dimer, 30 µg injected
LC 250 µm
SC 350 µm
Fractograms of BSA separation demonstrating that the
TREQ is independent on channel length and spacer height,
calculation taken from ISIS
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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.
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Hollow-Fiber Flow-FFF (HF5)
J. Chromatogr. A, 1218 (2011) 4126-4131
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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
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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
Cross-flow
outlet
Channel
tube
Hollow Fiber
Vin
rf
r
z
Vou
t
Cross-flow
.
Inlet
connection
(from
injector)
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Outlet
connection (to
detector)
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HF5 Channel Design
10 2010 043 877.4
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)
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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.
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HF5 –AF4 Retention Equation
Retention R in Flow-FFF is a function of the mean
layer thickness l
AF4
HF5
R=6l
R=4l
c = ½ c0
In HF5 the same retention is achieved
with 33% lower concentration at the
membrane.
l
c0
This leads to reduced overloading and aggregation
effects.
.….
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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
....
.
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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.
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HF5 Flow Schematic

Autosampler
Inlet Port
Crossflow
Pressure
Sensor

Flow
Controller

The position of the focus
point is determined by
measuring the focus flow
rate entering the channel
Outlet Port
Flow Meter
Only one pump is needed
to generate the inject flow
and cross-flow

Waste
Pressure
Sensor
Focus
Needle Valve
1


6
2
5
3

4
Detector
Technical specification of the flow
schematic has been filed for patent in
Germany No. 10 2010 041 222.8.
Main Pump
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Focusing Visualization
Visual inspection is not necessary any more with the DUALTEC
innovation.
.
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DUALTEC Autofocus
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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
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Benefits of the Eclipse DUALTEC
Sensitivity
Productivity
Flexibility
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Benefits of the Eclipse DUALTEC
Sensitivity
Productivity
Flexibility
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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
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Sensitivity comparison between HF5 and AF4
UV peak heights at 280 nm as a function of injection amount
25
y = 4,165x - 0,516
R2 = 0,9999
HF ID 0.8 mm
HF calculated
20
AF4 SC calculated
Peak height [mAU]
AF4 LC calculated
15
y = 1,9701x + 5E-15
10
5
y = 1,1493x + 2E-15
0
0
1
2
3
4
5
6
Injection amount [µg]
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
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Sensitivity with respect to light scattering signals
Light scattering signal at 90 ° as a function of injection amount
3,5
Light scattering signal at 90° [mV]
3
y = 0,6102x + 0,2298
R2 = 0,9956
2,5
2
y = 0,0994x + 0,0182
2
R = 0,9998
1,5
1
0,5
HF ID 0.8 mm
flat channel 490 µm
0
0
5
10
15
20
25
Injection am ount [µg]
Peak height of the 90° LS signal for BSA monomer separated on HF5 and AF4
channel SC with 490 µm spacer – experimental data
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Separation of protein mix on HF5– peak
identification
2
2
V W D 1 A , W a ve le n g th = 2 1 5 n m (P R O T E IN M IX \1 7 1 1 2 0 1 0 \D E F _ L C 2 0 1 0 -1 1 -1 7 1 8 -5 0 -1 0 \1 7 1 1 2 0 1 0 _ P M _ 0 1 3 .D )
m AU
1.
2.
3.
4.
5.
6.
7.
30
25
1
1
20
Carbonic anhydrase (1mer)
BSA (1mer) + Carbonic anhydrase (2mer)
Carbonic anhydrase (3mer)
BSA (2mer)
Apoferritin (1mer)
Thyroglobulin (1mer) + Apoferritin (2mer)
Apoferritin (3mer) + Thyroglobulin (2mer)
5
5
15
6
6
10
5
33
44
77
0
0
5
10
15
20
25
30
35
m in
Total protein amount injected was 0.5 µg, longitudinal flow rate 0.2 ml/min, crossflow rate 0.85 ml/min, UV signal 215 nm
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Separation with narrow-bore fiber (250 µm radius)
c hro m a to g ra m s
2 1 2 0 1 1 - 0 8 - 1 8 W te a b v c 0 2 2 v x 0 4 5 f f 0 2 2 0 1 5 µg 5 0 mM PB S 1 5 0 mM Na Cl p H 7 ,2
R elative Scale
1.0
UV
Peak half width 100 µl
Retention time
3 minutes
Injection amount 150 ng
0.5
0.0
0.0
0.5
1.0
1.5
vo lu m e (m L )
Sample is a monoclonal antibody for therapeutic use
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Monomer-Dimer Separation
c hro m a to g ra m s
2 1 2 0 1 1 - 0 8 - 1 8 W te a b v c 0 2 2 v x 0 4 5 f f 0 2 2 0 1 5 µg 5 0 mM PB S 1 5 0 mM Na Cl p H 7 ,2
UV
0.020
R elative Scale
0.015
0.010
0.005
0.000
0.4
0.6
0.8
1.0
1.2
1.4
vo lu m e (m L )
Sample is a monoclonal antibody for therapeutic use - dimer amount < 1% or < 2 ng
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Benefits of the Eclipse DUALTEC
Sensitivity
Productivity
Flexibility
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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.
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Benefits of the Eclipse DUALTEC
Sensitivity
Productivity
Flexibility
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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
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Applications
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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.
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MAb IgG1 separated on HF5
Vial #2 (2 µg)
77,3% Recovery
18,9 % Recovery
3,95 % Recovery
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Comparison of HF5 and AF4
(28/01/2011)
(21/01/2011)
2μg
5 μg
10 μg
280
UV absorbance @ 215 nm(mAU)
180
260
240
220
Vial # 2
UV absorbance @ 215 nm (mAu)
300
200
180
160
140
120
100
80
60
40
Vial # 2
1μg
2 μg
5 μg
7 μg
160
140
120
100
80
60
40
20
20
0
0
0
5
10
Time (min)
HF5
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0
5
10
15
Time (min)
AF4
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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.
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Particle Characterization
geometric radius vs. time
b
c
d
e
f
g
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% SDS
geometric radius (nm)
UV
100.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
0.0
5.0
10.0
15.0
20.0
time (min)
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)
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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