OBSERVER is a compact application that does no more and no less than it needs to:

• Set up instrument control and parameters for real-time Mw and Rg measurement in inline or online mode.
• In online mode, an HPLC pump is controlled by OBSERVER to pull a constant flow from the main process stream or container, with optional dilution. Supported pumps are: Agilent 1100 series, 1200 series and 1200 Infinity and 1290 Infinity II quaternary pumps.
• Set up trigger conditions: permissible ranges or end point bands for molar mass or size. Band tolerances and required duration of trigger conditions may also be set.
• Click the ‘Play’ button to begin monitoring. Data are saved for later review in OBSERVER and may transferred via OPC to a central processor.
• End the process when end point is reached or deviation identified, via analog voltage output or OPC.
• Alarms are generated if the hardware diagnostics indicate loss of communication, a pump leak or other improper operation conditions.

Specific, Reversible Interactions – Protein Binding Studies

The CALYPSO software uses CG-MALS data to calculate the affinity and stoichiometry of specific, reversible complexes formed in solution, such as antibody-antigen interactions and protein dimerization. The software enables quick data fitting to simple models of dimer formation, 1:n stoichiometries, and infinite self-association. Alternatively, the highly versatile analysis tools allow you to choose and design more complex binding schemes, including simultaneous self- and hetero-association, cooperativity, and meta-complex formation.

Nonspecific Interactions –Virial Coefficients

The second virial coefficient (A2) is used to quantify nonspecific attractive and repulsive interactions for proteins, polymers, and colloids. Batch light scattering data collected with the CALYPSO software can be analyzed using a Zimm formalism, Debye formalism, or effective hard sphere approximation. Higher virial coefficients may be determined as well.

Time-Dependent MALS – Association and Dissociation Kinetics

Some protein association or dissociation reactions can take seconds or minutes to reach completion. The CALYPSO software gives you the power to set the time for allowing each composition to come to equilibrium—from a few seconds to several hours. In the example pictured below, different concentrations of a small molecule inhibitor were used to disrupt the dimerization of a protein; the light scattering decrease corresponds to the dissociation of dimers into monomers over time.

Light Scattering and Concentration Detection

Maximum versatility of experimentation is provided by the real time display of your light scattering and concentration data with a method timeline showing you exactly how far your experiment has run. Analyze data as it is being collected to monitor and evaluate progress of your experiment. Pause and modify your method at any time to optimize and change parameters on the fly, such as increasing measurement times to accommodate long kinetics.

Simultaneous collection with ASTRA

Light scattering using either the DAWN® or miniDAWN® and concentration data from a differential refractive index detector (such as the Optilab®) or a UV detector can be collected simultaneously with ASTRA®.

Flexible Concentration Detection

Use either UV or RI concentration detectors or nominal concentrations based on the calculated dilutions. These values are automatically computed and saved with your data. Concentration detection modes can be easily selected post-acquisition.

Calypso Pump Controls

The CALYPSO software allows you to customize every aspect of your CG-MALS experiment, giving you full control of your interacting system. Power on degassers, set maximum allowable flow rates and pressures, and perform priming and wash steps to prepare your hardware for an experiment. Flow and wash parameters can be programmed into an experiment method for complete, unattended experiments.

Useful Accessories

External devices may be readily integrated with the Calypso. The Calypso can control external valves to simplify system maintenance. Use an Orbit Solvent Recycler to keep clean solvent pumping through your system when not in use. Switch between two wash buffers using an external two-position valve for thorough washing after an experiment. Both functions can be automated within a method for quick and easy cleanup.

Method Design

Design your own composition gradients or use one of the software’s built-in methods to investigate different macromolecular interactions. Any number of composition gradients may be run in a single experiment, repeat runs can be saved to their own data files, and the system washed and ready for the next use in the morning all from a single method page. In addition to the provided example methods, user-designed methods can be saved for later use.

Simulations

Whether you’ve already performed an experiment and like to optimize parameters or whether you like to hit the ground running designing your first Calypso Experiment, the CALYPSO software provides vital simulation capabilities to aid in experiment design. Input parameters, such as molecular weights, virial coefficients, association stoichiometries, and equilibrium association constants (KA), and the Simulation tool plots predicted light scattering data for up to 5 models. This information lets you determine appropriate solution concentrations for your experiment as well as the optimal composition gradient steps.

Calculator

Calculators are included in the CALYPSO software to aid in experiment design and evaluation of results: Use the A2 Calculator to predict the excluded volume A2 and select an appropriate concentration range. The dn/dc Calculator helps you estimate the change in your molecule’s dn/dc in buffers of varying refractive index. The KD Calculator calculates the equilibrium dissociation constant (KD) for one or two binding sites and helps identify cooperativity.

VISION CSH

VISION Chromatography Software Hub serves as FFF Central Station on your lab PC. Open VISION to launch any and all of the primary Wyatt software packages for FFF-MALS:

• SCOUT DPS: Combines simplified, rationalized FFF method development with FFF data analysis to determine distributions of hydrodynamic radii based on first-principles physics.
• VOYAGER CDS: Complete, integrated solution for FFF experimental runs. Instrument control and data acquisition for Eclipse and Agilent HPLC components.
• ASTRA: Wyatt’s flagship software for characterization of macromolecules and nanoparticles by SEC/FFF-MALS. Acquires, analyzes and reports measurements from multi-angle light scattering, dynamic light scattering, UV/Vis, dRI and differential viscometry detectors. Determines molar mass, size, concentration, intrinsic viscosity, conformation, shape, particle concentration (number density) and more.

SCOUT DPS

The flexibility of FFF allows users to achieve excellent separations for most applications, but in some cases finding the optimal method for the separation run can require tedious trial-and-error.

SCOUT DPS is an indispensable tool for FFF method development which utilizes in-silico, “virtual” experiments to eliminate most of the time and effort required for FFF method development.

Typical method optimization in SCOUT combines the results of one or two real FFF runs with an unlimited number of virtual FFF runs. A proprietary simulation algorithm quickly calculates the effect of changing parameters such as:

• Channel flow rate
• Cross-flow rate gradients
• Spacer height
• Membrane porosity

From virtual to physical

SCOUT makes use of first-principles FFF physics to simulate the separation process. The results are shown immediately, including the effect of band broadening and dilution of the sample at the channel outlet.

The example to the right shows the process for optimizing a nanoparticle separation.

• The blue experiment is the result of a default method for separating a sample containing three nanoparticle sizes. The dashed blue line shows the cross-flow gradient used, and the solid blue line is the measured concentration signal.
• The red experiment is the result of a series of simulations tested to improve the resolution. With a modified cross-flow gradient, the peaks are expected to exhibit baseline resolution.

The final measurement shows that the optimized method performed well, with enhanced separation between the three peaks.

Added value: Rh

An additional benefit of SCOUT is the analysis of fractograms to determine size distributions according to basic FFF physical theory.

This graph shows the size distributions determined for the nanoparticles in terms of hydrodynamic radius.

SCOUT DPS and the VOYAGER CDS software for performing FFF experiments exchange methods and data seamlessly.

VOYAGER CDS

VOYAGER CDS is a complete chromatography data system that incorporates everything you need for maximum productivity in FFF-MALS with an Eclipse and Agilent HPLC components:

• Eclipse: Complete control over all flows plus readings of pressures and flow rates
• Agilent® HPLC: Control and digital data acquisition from Agilent HPLC components including pumps, degassers, autosamplers, fraction collectors and detectors
• ASTRA control: Digital synchronization of multi-sample sequences with ASTRA

Extra benefits

VOYAGER CDS offers additional flexibility beyond OpenLab or ChemStation, such as storing all data files (Eclipse method, Agilent data, ASTRA) in one project folder.

A primary benefit is the ability to exchange files with SCOUT DPS:

• Read in FFF methods designed in SCOUT DPS
• Export HPLC detector data to SCOUT DPS for method feedback and size distribution analysis

ECLIPSE plug-ins

An ECLIPSE plug-in module controls the Eclipse DualTec and Eclipse AF4 Field-Flow Fractionation (FFF) systems. Coupled with ASTRA for data collection and analysis, an ECLIPSE plug-in provides users with a basic turn-key solution for macromolecular characterization.

ECLIPSE modules are available for these chromatography software packages:

• Agilent
• Dionex
• Shimadzu

Plug-ins benefits:

• Familiarity: The Eclipse instrument and the associated HPLC components are all operated from a single, familiar interface.
• Simple configuration: The Eclipse instrument is configured using the standard configuration dialog.
• Simple setup: Setup of all flow modes and flow rates is controlled by standard software menus within the HPLC software.

ASTRA Features and Capabilities

Browse these tabs to learn about the many capabilities and features in ASTRA which make it is the most powerful and versatile software available for the characterization of macromolecules and nanoparticles via multi-angle and dynamic light scattering.

ASTRA 7 increases productivity for routine analyses

Running a SEC-MALS analyses has never been easier. ASTRA’s One-Click MW feature complete an entire method at the click of a button.

Click on Run Default and ASTRA will:

• Synchronize data collection with your HPLC
• Autoset parameters to determine MW and Rg
• Generate custom reports and graphs
• Prepare for the next run
• run-default-button

Method Builder

ASTRA’s Method Builder simplifies the specification of methods for the most common protein and polymer analyses, in three intuitive steps:

• Choose an analysis profile
• Specify the instruments
• Enter additional parameters

Then save the method, load the sample(s), click Run Default and return later to find out the MW results in the final report.

Freedom to Explore

With ASTRA, you are never locked in to default methods or standard procedures. You always* have access to each parameter and processing step to review, validate and improve.

*Except network accounts where access is limited under the 21CFR(11) compliant Security Pack.

Ahead of the Curve

As you build expertise and confidence, and move on to complex analyses, ASTRA allows you to refine the results of automated processing with fine adjustments to peak selection, baseline specification, detector filtering and other settings.

HPLC Control

Full digital synchronization between your HPLC system, Wyatt detectors and ASTRA 7:

• Minimizes user errors
• Runs SEC-MALS from a single, familiar environment
• Saves you time and money

ASTRA 7’s HPLC control modules works with standard Agilent hardware including pumps, autosamplers, column ovens and UV/Vis detectors.*

*Control of additional HPLC instruments coming soon

Integrated HPLC control

Let ASTRA take control of your HPLC stack for effortless synchronization between injections and data collection. ASTRA 7 eliminates the need for separate HPLC and light scattering software. HPLC modules are listed together with Wyatt detectors in a common experiment configuration, and key parameters such as vial position, injection volume and flow rate specified in ASTRA’s sequences.

HPLC Manager

The HPLC Manager app controls and monitors HPLC modules including pumps, column ovens, UV detectors and autosamplers, right from the desktop. Included free with the HPLC Control module.

High Throughput Processing

Built from the ground up to handle with ease large sets of data, ASTRA has the tools you need to process and report on sequences containing many samples, including replicates. “One-to-Many” processing interface lets you focus on your goals, and leave the drudgery to the computer.

EASI Table

Quickly compare multiple data runs with the EASI Table feature to assess compound stability and consistency. The table data can be exported in a variety of formats, or copied and pasted into your favorite word processing software.

Specify which data should be displayed in EASI Table, as well as the format and precision used to display the values. Choose from percent or numerical uncertainties, or omit them entirely.

Sequence Processing

Sequences are used to specify and control the automated collection of data as well as system functions. A sequence can be configured with the default method or a different method can be specified for each sample. Sequences also perform post-collection maintenance, such as running the COMET flow cell cleaner or switching the Orbit to solvent recycle mode.

In the Samples view, it is possible to modify sample parameters on-the-fly, adding samples, changing collection times, or skipping samples.

Let your computer do the work.

ASTRA allows you to process an experiment as you wish, then instruct the computer to apply the same settings and analysis steps on any number of additional data files. You can quickly and easily specify which elements of your ‘source’ experiment should be incorporated in the target experiments.

Once the processing is finished, your resulting data files are fully analyzed and ready to be reviewed.

Unmatched Analysis

Building on over 40 years of light scattering research and development, ASTRA 7 includes an unrivaled range of analysis features. ASTRA takes full advantage of modern multicore computers, processing data at blazing speeds and with superior accuracy.

Molar Mass Determination

Determine molar mass and size.

Molar Mass via Light Scattering

Light scattering stands alone in its ability to measure the molar mass of macromolecules in solution both accurately and quickly.

Molar Mass via Conventional and Universal Calibration

Build a bridge between legacy conventional and universal calibration results and modern, accurate light scattering analysis.

Protein Conjugate and Copolymer Analysis

If you have an RI detector, a UV detector, and a light scattering instrument, you can directly measure the molar mass, size, and relative fractions of copolymers and protein conjugates.

Particles and Number Density

It is possible to measure the size and number density of a sample with just a light scattering instrument. ASTRA supports several sophisticated models to determine the size of a macromolecule based solely on the angular variation in the light scattering intensity.

On-Line QELS

With the support of a QELS instrument module, you can extend your data analysis to determine the translational diffusion and corresponding hydrodynamic radius of your samples.

QELS batch Regularization and Cumulants

Determine translational diffusion and corresponding hydrodynamic radius of your unfractionated samples.

Viscometry

ASTRA provides several analysis features that take advantage of viscosity data. Use these features to further quantify your macromolecules to determine Mark-Houwink-Sakurada terms, hydrodynamic radius, and more. Learn more…

Molecular Conformation

The shape of molecules can be assessed from the molar mass and rms radius of the sample.

Distributions

Sample properties are determined for individual peaks in a chromatography run. ASTRA also supports visualizing these data in terms of the distribution of these measured quantities. This is extremely powerful when assessing how much of a sample falls with a particular range of molar mass or size.

Second Virial Coefficient (A2) Measurements

The second virial coefficient (A2) is a measure of macromolecular self-association, and is one of the few parameters that can be used to predict the crystallization properties of a sample. ASTRA allows you to measure this important characteristic.

dn/dc and UV extinction coefficient determination. Measure the dn/dc and UV extinction coefficients for your novel samples.

While dn/dc and UV extinction coefficients are documented for many existing compounds, they must be experimentally determined for novel samples. ASTRA provides analysis features to measure these critical sample characteristics.

Band Broadening Correction

While most analysis packages force you to live with the undesirable effects of passing a sample through a series of detectors. ASTRA’s revolutionary band broadening correction accounts for these effects to give you most accurate results possible.

Absorbance Correction

If a sample absorbs light at the MALS laser wavelength, molar mass calculations will be skewed due to less light reaching the scattering volume than expected. ASTRA uses the Forward Monitor detector signal available in all Wyatt MALS instruments to calculate the laser attenuation produced by sample absorption and correct the scattering signal levels. In this manner accurate molecular weights are ensured, even in the presence of absorbtion.

Blank Subtraction

Certain analysis techniques, such as FFF, cause large changes in refractive index and viscosity measurements as flow rates are modified during the run. This can make it difficult to properly analyze the file, as the RI and viscosity changes due to flow and pressure changes mask those caused by the sample. ASTRA provides a mechanism to subtract a data collection run without sample (a ‘blank’) from a sample run to reveal the sample-specific signal changes.

Error Analysis

Wyatt Technology software is written by scientists for scientists. ASTRA was designed from the ground up to properly compute and propagate uncertainties to ensure our results are publication-quality.

Cutting Edge Technology

Wyatt Technology is committed to pushing macromolecular characterization to new levels. Consequently, we have a very strong program to develop intellectual property. Many of the algorithms in ASTRA are on the cutting edge of scientific analysis, and are either patented or have patents pending. ASTRA is protected by:

• U.S. patent numbers: 5,528,366; 6,411,383; 6,651,009; 6,774,994; 7,386,427; 7,911,594;
• German patent Numbers: 694 33 615.7-08; 603 19 078.2-08;
• British patents based on European application numbers: EP 0 665 433; EP 1 510 807;
• Japanese patent Numbers: 3,580,380; 4,439,211; 4,381,914; and other patents pending.

State of the Art Reporting

Take control of your analysis reporting with ASTRA’s custom reporting system. With just a few mouse clicks, adjust which data to display and how it should be formatted. Add your own logo, incorporate some personal notes, or just pare the default reports to focus on what you care about.

Personalize your reports.

In addition to the numerical and content selections provided in the Report Designer, ASTRA allows you to specify a logo image, and where it should be displayed. You can specify the title as well as additional summary information or notes that will appear on the report.

Personalized Data Selection

Control the content and presentation of your reports with a few mouse clicks. Any bit of data generated during analysis is now available for you to incorporate into your report, with just a click of the mouse.

Built from the ground up to comply with 21 CFR Part 11, ASTRA 7 with Security Pack makes compliance complete and intuitive.

• Data Safety and Integrity
• User Security
• Audit Trails
• Electronic Signatures
• Enforcing Permitted Sequences
• Human Readable Records
• Validation

Full Numerical Formatting

Click on a result, and ASTRA presents a simple, intuitive interface for controlling the numerical precision and display format of the result. Quickly and easily change results from fixed point to scientific notation, increase or decrease the number of significant digits to display, and determine if you wish uncertainties to be displayed as numerical values or percentages.

Weight-Average Molar Mass

In addition to equilibrium association constants Kd and absolute stoichiometry of reversible self- and hetero-associations, it can also help determine reaction or aggregation rates, non-specific interaction parameters (virial coefficients) and even automate measurements of weight-average molar mass and dn/dc for polymers.

CALYPSO Software

Included with each Calypso is a copy of CALYPSO software, the most versatile software package available for analysis of biomolecular interactions in solution by light scattering.

Creating composition gradients…

CG-MALS employs a Calypso connected to a MALS instrument and optional concentration detector. The Calypso performs sample preparation and delivery, combining up to three different solution in precise mixing ratios and injecting into the detectors.

Accurate compositions result from setting the Calypso’s pumps to appropriate flow rate ratios, all controlled from the CALYPSO software through an intuitive GUI. Mixing is achieved by dispensing the solutions simultaneously from all three pumps via a static mixer.

…that work with MALS detectors

Reliable light scattering measurement require the samples to be degassed and filtered. The Calypso incorporates built-in, low-volume degassers and filter housings, connected via PEEK tubing and finger-tight fittings. Even the filter housing is finger-tight, making use of a proprietary design for maximum convenience.

Calypso features

• Biocompatible wetted materials.
• Flexibility to re-organize fluid paths and utilize different wetted materials as needed.
• Convenient loading of sample and diluent solutions via conical tubes: 5, 15 or 50 mL.
• Autoinject Out contact closure signal, to trigger data acquisition by ASTRA® or DYNAMICS® software for data acquisition and analyses not supported by the CALYPSO software (e.g., DLS acquisition).
• Built-in Wash port, allowing the pumps to draw on reservoirs of wash solutions for post-experimental clean-up. Automation of up to two sequential wash solutions, e.g. buffer (to clean out proteins) followed by water (to clean out buffer salts) or detergent followed by water, is possible when an Orbit solvent selector valve is added to the setup. These operations are appended to the Method for unattended operation.

CG-MALS

Composition-Gradient Multi-Angle Light Scattering (CG-MALS) employs a series of unfractionated samples of different composition or concentration in order to characterize a wide range of macromolecular interactions. No special modifications (e.g., sample tagging or immobilization procedures) are necessary: samples are unlabeled and entirely in solution.

Addressing a host of biomolecular phenomena

The primary analysis techniques supported by Calypso hardware and software are:

• Dynamic equilibria: specific binding and complex assemblies of proteins, oligonucleotides and other biomolecules; KD (equilibrium dissociation constant) from picomolars to millimolars; absolute molecular stoichiometry of associating complexes; self and/or heteroassociations
• Non-specific macromolecular interactions: self- and cross-virial coefficients
• Stop flow kinetics: aggregation, dissociation and other time-dependent reactions: equilibration time from seconds to hours
• Zimm plots: concentration gradients for determining solution-average molar mass MW, size Rg, and second virial coefficients
• Refractive index increment: dn/dc

Calypso’s automation enhances productivity while the CALYPSO software provides an unparalleled selection of interaction models. Relative to other techniques for characterizing protein interactions, such as surface plasmon resonance, sedimentation equilibrium, kinetic exclusion or isothermal titration calorimetry – as well as manual CG-MALS measurements – the Calypso provides fast and accurate results.

Applications

Drug Discovery

• Quantify binding affinity and stoichiometry of enzyme/inhibitor or antibody/antigen interactions, including complex multi-valent and multi-protein complex formation
• Study the impact of small molecules on protein-protein interactions

Process Improvement

• Determine second virial coefficient and adjust buffer parameters to improve formulation stability and viscosity
• Determine cross virial coefficients to optimize antibody purification and understand the effects of large excipients on formulations

Self-assembly / Aggregation

• Quantify impact of solvent ionic strength, pH, or excipients on polymerization or protein associations
• Measure kinetics of self-assembly and aggregation via rate of change of molar mass and radius of gyration, and hydrodynamic radius (with a WyattQELS module or NanoStar and parallel analysis in ASTRA or DYNAMICS)

Biotech R&D

• Characterize macromolecular binding affinity and associated complex stoichiometry over a wide range of buffer compositions, time, and temperature scales

Highly Sensitive

A highly sensitive, on-line differential viscometer used with SEC-MALS-IV to determine the size and conformation of polymers, proteins and peptides separated by UHPLC.

Another path to polymer and protein characterization: SEC-MALS-IV

When coupled with size-exclusion chromatography and a concentration detector such as the Optilab, the ViscoStar derives the intrinsic viscosity (IV) of polymers. An inherently valuable physico-chemical property in itself, IV is best utilized together with molar mass information from multi-angle light scattering (SEC-MALS-IV) to derive the hydrodynamic radius, conformation, and branching ratio of a macromolecule in solution.

Key Benefits

• High sensitivity, equivalent to 100 ng of 100 kDa polystyrene in THF at 1.0 mL/min injected onto a standard GPC column
• High dynamic range of over 135,000:1. The full measurement range corresponds to over 13 mg/mL of 100 kDa polystyrene in THF
• Extended solvent compatibility with all-316 stainless steel transducers
• Temperature range from 4°C to 70°C for polymers or proteins that require a range of temperatures
• Minimal band broadening thanks to fast pressure transducers
• Pump pulse suppression to below 4 Pa (hardware alone) or 1 Pa (hardware + software) without loss of chromatographic resolution
• Automated capillary bridge tuning with no moving parts, to guarantee a perfectly balanced bridge, every run
• Enhanced thermal stability with drift of <1.25 Pa/hour

SEC-IV without MALS?

Yes, as much as we love MALS for optimal polymer analysis, it’s true that there are occasions when it just won’t fit the bill.

Universal calibration is a parallel technique to SEC-MALS for determining molecular weight, commonly used to characterize linear polymers. While universal calibration overcomes some of the problematic issues associated with column calibration by reference molecules, it does not account for non-ideal column behavior caused by chemical solute-column interactions or hyperbranching.

Universal calibration is particularly-well suited to replace MALS for:

• Strongly fluorescent polymers
• Weakly-scattering polymer/solvent systems with very low dn/dc values

The Mark-Houwink-Sakurada relationship makes use of empirical relationships between intrinsic viscosity and molar mass, specific to each polymer and solvent, to estimate molar mass from SEC-IV measurements. Of course, the best way to determine MHS coefficients is to measure them using SEC-MALS-IV and ASTRA!

Highly Sensitive

A highly sensitive, on-line differential viscometer used in conjunction with SEC-MALS-IV to determine the size and conformation of all types of biopolymers, synthetic polymers and even proteins and peptides.

Another path to polymer and protein characterization: SEC-MALS-IV

When coupled with size-exclusion chromatography and a concentration detector such as the Optilab, the ViscoStar derives the intrinsic viscosity (IV) of polymers. An inherently valuable physico-chemical property in itself, IV is best utilized together with molar mass information from multi-angle light scattering (SEC-MALS-IV) to derive the hydrodynamic radius, conformation, and branching ratio of a macromolecule in solution.

Key Benefits

• High sensitivity, equivalent to 100 ng of 100 kDa polystyrene in THF at 1.0 mL/min injected onto a standard GPC column
• High dynamic range of over 135,000:1. The full measurement range corresponds to over 13 mg/mL of 100 kDa polystyrene in THF
• Extended solvent compatibility with all-316 stainless steel transducers
• Temperature range from 4°C to 70°C for polymers or proteins that require a range of temperatures
• Minimal band broadening thanks to fast pressure transducers
• Pump pulse suppression to below 4 Pa (hardware alone) or 1 Pa (hardware + software) without loss of chromatographic resolution
• Automated capillary bridge tuning with no moving parts, to guarantee a perfectly balanced bridge, every run
• Enhanced thermal stability with drift of <2.5 Pa/hour

SEC-IV without MALS?

Yes, as much as we love MALS for optimal polymer analysis, it’s true that there are occasions when it just won’t fit the bill.

Universal calibration is a parallel technique to SEC-MALS for determining molecular weight, commonly used to characterize linear polymers. While universal calibration overcomes some of the problematic issues associated with column calibration by reference molecules, it does not account for non-ideal column behavior caused by chemical solute-column interactions or hyperbranching.

Universal calibration is particularly-well suited to replace MALS for:

• Strongly fluorescent polymers, in particular those not adequately addressed by a DAWN with long-wavelength (785 nm) laser or a standard DAWN (685 nm) with fluorescence-blocking filters.
• Weakly-scattering polymer/solvent systems with very low dn/dc values

The Mark-Houwink-Sakurada relationship makes use of empirical relationships between intrinsic viscosity and molar mass, specific to each polymer and solvent, to estimate molar mass from SEC-IV measurements. Of course, the best way to determine MHS coefficients is to measure them using SEC-MALS-IV and ASTRA!

Independent of Chromaphores

Independent of chromaphores or fluorophores, differential Refractive Index (dRI) detectors measure concentrations of all types of samples in almost all solvents. The microOptilab brings the universal detection capabilities, unsurpassed concentration range, and superb sensitivity and linearity – familiar from the Optilab – to the domain of UHPLC.

SEC-MALS with UHPLC

Like standard SEC, UHPLC-SEC analysis relying on column calibration with molecular standards is unreliable when the standards do not accurately reflect your samples in terms of conformation, non-ideal (e.g., hydrophobic) column interactions or reversible association. SEC-MALS employs a microDAWN MALS detector downstream of the SEC column and UV detector, to determine molar mass / molecular weight distributions of macromolecules independently of column calibration.

Key Benefits

• Very low band broadening, < 4 µL, to maintain the resolution of very narrow UHPLC peaks
• Extraordinary dRI measurement range, -4.7 x 10-3 to +4.7 x 10-3 RIU (comparable to 25 mg/mL protein in PBS)
• Highly sensitive, ± 1.5 x 10-9 RIU across the entire measurement range
• Measures solvent refractive index, require for MALS calculations
• Measures at the same wavelength as the MALS detector, for maximum accuracy in MALS calculations
• Temperature range 4°C to 65°C for proteins and polymers that require a range of temperatures
• High-resolution, touch-screen front panel displays data graphs and diagnostics, even with the host computer turned off

Unparalleled Features

Considering the extremely small peak volumes of UHPLC, band broadening in the microOptilab is kept to the absolute minimum, at less than 4 µL, as compared to 20 – 40 µL or more for standard RI detectors. As a result, the superior resolution from UHPLC columns and instruments will not be compromised with the microOptilab RI detector.

Even though the microOptilab offers a dRI measurement range orders of magnitude beyond that of other UHPLC dRI detectors, unlike other detectors there are no range or gain settings in the microOptilab. The full range of instrument detection is always present, and the full sensitivity exists over the entire range. The small flow cell and proprietary temperature regulation enable stable RI baselines and signals which further enhance the detector’s sensitivity.

Unlike all other dRI detectors, the microOptilab also measures absolute refractive index in order to determine solvent RI, necessary in light scattering calculations and useful in assessing the accuracy of mixing of solvent gradients.

The microOptilab can be operated below ambient temperatures as easily as above ambient. No hassle temperature control can be programmed down to 4°C or as high as 65°C.

The microOptilab is compatible with all UHPLC systems.

Independent of Chromaphores

Independent of chromaphores or fluorophores, differential Refractive Index (dRI) detectors serve as accurate and versatile concentration detectors in all types of solvents. dRI is part of standard instrumentation for a variety of macromolecular characterization techniques including standard chromatography, SEC-MALS, FFF-MALS and CG-MALS.

Unique dRI Detector

The Optilab® is a unique dRI detector for standard HPLC/GPC with 256 times the detection power and 50 times the dynamic range of every other RI detector for chromatography in existence today, making it a valuable addition to any MALS setup.

Key Benefits

• Extraordinary dRI measurement range, -0.0047 to +0.0047 RIU (comparable to 25 mg/mL protein in PBS)
• Highly sensitive, ±7.5 x 10-10 RIU across the entire measurement range
• Measures solvent absolute refractive index, required for MALS calculations
• Measures at the same wavelength as the MALS detector, for maximum accuracy in MALS calculations
• Band broadening < 20 µL preserves SEC resolution
• Temperature range 4°C to 65°C for proteins and polymers that require a range of temperatures
• High-resolution, touch-screen front panel displays data graphs and diagnostics, even with the host computer turned off

Unparalleled Features

Even though the Optilab offers a dRI measurement range orders of magnitude beyond that of other dRI detectors, unlike other detectors there are no range or gain settings in the Optilab. The full range of instrument detection is always present, and the full sensitivity exists over the entire range.

The Optilab can also do something no other on-line refractive index detector can: it can measure the absolute refractive index of a solution. In addition, it can measure the dn/dc of a solvent at the same wavelength of light as the light scattering instrument. And just to make sure it can’t be matched, you can replace the light source in the Optilab for other wavelengths (e.g., 690, 633, 488, etc.) in just minutes!

The Optilab can go below ambient temperatures as easily as above ambient. No hassle temperature control can be programmed from 4°C to 65°C. Compatible with all HPLC systems.

Uniquely Versatile

Flow Field-Flow Fractionation is a uniquely versatile means of size-based separations, capable of fractionating and characterizing proteins and aggregates, liposomes, emulsions, viral particles, polysaccharides, nanoparticles, polymer latex particles, colloidal soil suspensions, chemical mechanical polishing slurries, and many more – with no stationary phase and almost no shear.

AF4 Techniques

The Eclipse AF4 combines with a DAWN MALS detector and Optilab dRI detector to solve the most difficult separation, fractionation and sizing tasks in the macromolecular and colloidal arenas including polymers, biotechnology, pharmaceuticals, nanotechnology and environmental analysis.

Macromolecules

The advantage of AF4 over traditional SEC column chromatography is the ability to separate both soluble and colloidal components over a wide size range as well as sensitive and “sticky” samples, since there is no stationary phase. AF4 even overcomes the non-ideal behavior exhibited by large, branched polymers that elute abnormally on GPC columns. The Eclipse provides resolution similar to that of analytical centrifugation (AUC) but surpasses even this benchmark tool when one considers its flexibility, speed, and ease of use.

Nanoparticles

Common solution-based methods for determining the size and size distributions of nanoparticles, such as batch dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA), cannot offer the resolution, statistical confidence and absolute accuracy of a true separation-based technique that incorporates MALS and DLS downstream detection.

FFF-MALS and FFF-DLS performed with an Eclipse AF4, DAWN and an embedded WyattQELS DLS detection module offer excellent resolution and accuracy in analysis of size distributions. Particle counts and sizes of viruses and virus-like particles estimated by FFF-MALS match closely TEM-based counts.

Key benefits of FFF with a Wyatt Eclipse

• Robust separation of macromolecules and nanoparticles from 1 nm – 1000 nm with excellent resolution
• No stationary phase – open channel separation with minimal shear and surface interactions
• Versatile choice of separation channels, permitting sample loads from nL to mL, including semi-preparative FFF
• Optimize separation methods to enhance resolution or size range simply by adjusting flow ratios, even programmatically during a run
• Fractions can be collected and used for off-line analysis (with electron microscopy, MS, ICP-MS, ELISA, etc.)

Eclipse AF4

• Typical analysis times 10 – 30 minutes
• Utilize the fritless column for aggregation-prone samples
• Make use of the highest quality HPLC pumps and autosamplers from Agilent, Shimadzu, Thermo, or Waters
• Integrate with your favorite HPLC software from Agilent, Dionex or Shimadzu
• Couple to ICP-MS for elemental analysis of nanoparticles per size bin

The Eclipse is the world’s most advanced AF4 system

• Fully automated, software controlled system with only one pump to generate detector flow, crossflow and injection flow. This innovative concept to generate all three flow streams from one main flow has been a breakthrough for the AF4 method!
• One chassis contains all necessary electronic and fluid components, designed to stack with Wyatt Technology detectors
• Chassis can be ordered in two versions: for aqueous solvents (biocompatible) and for organic solvents
• The Eclipse integrates into various market-leading HPLC packages, such as Agilent, Dionex and Shimadzu
• Independent, continuous measurement of the crossflow and injection flow rate, which allows software controlled check on the system performance
• Motor-driven, software controlled needle valves for focus balance and injection flow rate control
• Proprietary pressure sensor without dead-volume provides continuous read-out of channel pressure
• High performance electronic controller inside the chassis (PLC programmable logic controller) with TCP/IP communication to PC
• Optimized layout of fluid connections minimizes tubing length and ensures easier serviceability
• Eclipse chassis and electronics are protected against fluid damage through customized drip-pan with leak sensor
• LCD displays system status information
• CE classification surpasses laboratory standards for safety and emission and reaches industry grade
• The Eclipse is certified by TÜV Rheinland

Eclipse AF4 Channel Design

The Eclipse AF4 offers the most versatile separation channel technology. There are four different channel types available to accommodate different requirements for sample load, analysis time, and resolution, as well as a wide choice of spacers and pre-cut membranes for all channel types.

All channels except the frit-inlet channel have a metal-free flow path, making them inert even in harsh conditions. In the frit-inlet channel, the frit supporting the membrane is made from stainless steel, which is the most robust choice. As an option a metal free ceramic frit can be installed.

All channels are easy to maintain. Replacing a membrane or cleaning the channel parts is straightforward, with just a few screws to tighten during the reassembly process.

• Short Channel (SC 145 mm length)
• Long Channel (LC, 275 mm length)
• Semi-Preparative Channel (SP, 45 mm width, 275 mm length)
• Frit-inlet Channel (Fl-AF4)

Integration with Wyatt Technology instruments and market-leading HPLC packages

• The Eclipse separation system is designed to integrate with Wyatt Technology’s range of unique light scattering and dRI detectors, ensuring highest sensitivity and optimum baseline quality.
• The Eclipse operates in conjunction with Agilent, Shimadzu, or Dionex chromatography components. You can stay with your current HPLC software platform and run the Eclipse with the familiar interface you already know.
• Only a singe HPLC pump is needed to generate detector flow, crossflow, and injector flow, accomplished by splitting the main flow delivered through the HPLC pump. Using only one pump results in a much improved signal-to-noise ratio because pump pulsations are reduced and balanced between flows, so operation is much easier.