VISION 3
Intelligent design, operation and
analysis for eld-ow fractionation
VISION
TM
Look ahead, see more
VISION, the brains behind FFF
FFF-MALS—eld-ow
fractionation with online
multi-angle light scattering
detection—is a powerful tool
for characterizing all types of
macromolecules and nanoparticles in solu-
tion or suspension. VISION software is the
intelligent human interface to an FFF-MALS
system built on Wyatt Technology’s Eclipse
™
FFF controller and DAWN
®
MALS instrument.
VISION comprises two
primary modules:
VISION DESIGN
™
carries out physics-
based FFF simulations for in silico
method design and optimization.
The physics engine also calculates
diusion coecients and zeta
potential from FFF and EAF4 measurements.
VISION RUN
™
is comprehensive
software for running FFF methods.
It seamlessly coordinates the pump
and autosampler front-end with the
Eclipse, detectors and ASTRA
®
,
and records FFF, electrical and UV signals for
diagnostics and analysis in VISION DESIGN.
VISION interfaces with ASTRA, the industry-leading light scattering analysis software for
determination of molar mass, size, conformation, composition, particle concentration and more.
ASTRA
VISION
Channel Pressure
Cross Flow Pressure
Inlet Pressure
8.86 bar
11.88 bar
15.12 bar
DCM Pressure 0.54 bar
Current
Voltage
pH
-6.0 V
-1.0 mA
8.0
Conductivity 0.011 S/m
Stop
Flow
Channel Status (mL/min)
1.15 0.00 0.80 0.20
Inlet Inject DCM Outlet
FFF Method
0.15
Cross
-1.0 mA
0
0.01
0.02
0.03
0.04
0
0.1
0.2
0.3
0.4
0.5
0 10 20 30
UV A Signal (AU)
Cross Flow (mL/min)
Time (min)
DASHBOARD HISTORY SETTINGS
Noise Wander Dri
LowLowLow
µAU/minµAUµAU
0.040.280.24
Inj: 25
2
3
Integrated Workflow
From method development to nal results
VISION oers a seamless workow in a few simple steps, starting with developing and optimizing the ow program
in silico, then running a sample sequence, followed by data processing, rening the separation method, reporting
and consolidating the data les in projects.
DESIGN METHOD
• Estimate particle size in sample
• Select spacers and flow profiles
• Use FFF theory to simulate
and predict separation
RUN EXPERIMENTS
• Acquire flow, electrical and
UV data
• Collect fractions
• Monitor and diagnose system
Transfer optimized method
Launch and synchronize ASTRA
• Collate project files
• Estimate particle size from
retention time
• Refine method
• Determine zeta potential
ANALYZE AND REPORT
• Basic and advanced MALS-
DLS-UV-RI characterization
• EASI Graph overlays
• EASI Table consolidated results
• Customized reports
FFF Method Development
Let your PC do the work
Have a new sample to analyze? VISION
DESIGN eliminates the need to spend
inordinate amounts of time running
a matrix of experiments to develop an
FFF method. Just list the estimated
particle sizes, select your FFF channel,
membrane and spacer, and specify
a cross-ow gradient.
VISION DESIGN immediately displays the
predicted fractogram using fundamental
FFF theory to calculate retention times.
It only takes a few minutes to explore
the eect of dierent cross ow rates and
timings to improve resolution and nd
the best method - all from the comfort
of your desk.
Rene the method
The nal design can be transferred in a
click to VISION RUN to test your method.
Your initial guess of sample and
channel parameters may have been
inaccurate, but have no concern —
results of a physical experiment can be
fed back into VISION DESIGN to further
rene the method. Usually a single test
run suces to give VISION DESIGN
the information it needs to help you
fully optimize your method.
4
5
Enter the cockpit
VISION RUN is a sophisticated control center for the
Eclipse FFF system. The dashboard shows the system
status in real time with direct access to instrument
congurations and sample sequences. Status
information from the Eclipse instrument’s Health
Indicators are displayed; should any warnings come
up, you will receive actionable recommendations.
Expand your horizons
VISION RUN uses the ICF instrument control
framework for Agilent
®
modules from the 1260
series. The complete feature sets of autosamplers,
isocratic or quaternary pumps, diode array
detectors, uorescence detectors and fraction
collectors are supported to extract the maximum
from FFF experiments.
FFF Instrument Control
Fly on autopilot
Once your sample sequence table is dened in
VISION RUN, including FFF operation and MALS
analysis methods, it’s time to take ight. Sit back and
trust VISION to run on autopilot, orchestrating the
entire FFF-MALS measurement.
Should the need arise, system health indicators
will alert you to intervene and prevent wasted runs.
Decided to add more samples to a running sequence?
Simply create another line in the table and VISION RUN
will incorporate the additional samples.
Automated Sample Sequences
• Administrator, researcher, technician
and guest access levels
• Full audit trails
• Secure, joint SQL database
for VISION and ASTRA data
• Local or remote database connectivity
• Data integrity validation
• Full IQ/OQ procedures
and documentation validation
Regulatory Compliance
VISION oers an optional 21 CFR Part 11 compliance package, including IQ/OQ documents and procedures.
6
7
Distributions and moments,
percent aggregation
Molar Mass and Size
Absorption Correction
Glycoproteins, pegylated proteins,
membrane proteins,
protein-nucleic acid complexes
Protein Conjugation
Branching ratio and
number of branches
Polymer Branching
Custom Plots
From RI concentration
UV Extinction
Coefficient
MALS and chromatography
quality assessment
Diagnostics
Plate count, resolution,
asymmetry, tailing
Peak Statistics
Column calibration,
universal calibration
Traditional SEC
Correct for non-linear baseline changes
Baseline Subtraction
rms radius, sphere radius,
rod length, hydrodynamic radius
Particle Size
Total number of particles
Particle Concentration
Radius or intrinsic viscosity
vs. molar mass
Conformation
Total viral titer,
empty:full ratio (C
p
/V
g
)
Viral Vector
Refractive Index Increments
Particle Shape
Copolymer Analysis
Results Fitting
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ASTRA
Comprehensive characterization of FFF-MALS data
Building on over 40 years of light scattering research and development, ASTRA includes
an unrivaled range of analysis features to characterize macromolecules and nanoparticles.
Learn more about ASTRA’s capabilities at www.wyatt.com/astra-benets
FFF Data Analysis
Particle size distributions
Even without light scattering, VISION
DESIGN can read in FFF data collected
by VISION RUN to determine the hydro-
dynamic radius (R
h
) and its distribution in
the sample based on FFF retention time.
Choose from a calculation based on
fundamental FFF theory or calibration
with reference standards. The detailed,
high-resolution R
h
distribution comple-
ments FFF-MALS, batch dynamic light
scattering (DLS) and nanoparticle tracking
analysis (NTA).
Electrophoretic mobility
and zeta potential
VISION calculates zeta potential and
electrophoretic mobility from EAF4
measurements made with dierent
applied electric elds. Data treatment
is fully automated, including baseline
and peak selection plus determination
of peak retention time and its shift with
the electrical eld.
VISION compares hydrodynamic radius distributions from FFF theory (red)
with geometric radius distributions from MALS (green).
8
6330 Hollister Avenue | Santa Barbara, CA 93117 | Tel: +1 (805) 681-9009 | Email: [email protected]
Left to Right
Geofrey K. Wyatt, Chief Executive Ocer
Dr. Philip J. Wyatt, Chairman of the Board
Cliord D. Wyatt, President
Wyatt Technology provides
absolute macromolecular and
nanoparticle characterization
solutions by developing the nest
instrumentation and services
to chemical, petrochemical,
pharmaceutical, biotechnological
and academic laboratories
worldwide. We delight our
customers with unparalleled levels
of service and support, facilitating
their cutting-edge research and
development eorts.
VISION is one of many tools in
Wyatt’s Light Scattering Toolkit used
to characterize proteins, polymers
and nanoparticles in solution.
Learn more at www.wyatt.com
Project and Data
Management
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© Wyatt Technology | W2000D
Learn more about FFF-MALS: www.wyatt.com/f-mals
System data traces
An invaluable function of VISION
DESIGN is the overlay of system
data traces for experiments within a
sequence or project. Deviations in
ow rate, pressure, or any other data
trace are readily identied to validate
results and facilitate troubleshooting.
Smart project
administration
VISION organizes all FFF and MALS
data as projects, for convenient
review and replication, as well as
comparison and reporting. Projects
can be merged and experiments
added or deleted at will, making
this a powerful and exible way to
handle large sets of FFF experiments.