Absolute molar mass and size
Not all macromolecules behave alike. Your samples may have a different conformation than molecular standards or exhibit unexpected column interactions. As an absolute technique for determining molar mass and size in solution, SEC-MALS - the combination of size-exclusion chromatography with multi-angle light scattering – offers an advanced characterization technique and overcomes the many limitations of column calibration.
Even though Peak 1 elutes earliest, MALS shows that it does not have the highest molar mass for this example of protein aggregates and fragments.
What is SEC-MALS?
SEC-MALS combines multi-angle light scattering with size-exclusion chromatography. It is a powerful technique for absolute characterization of macromolecules in terms of:
- Molar mass
- Conjugation ratio
In SEC-MALS, the SEC column serves to separate molecules by hydrodynamic volume; retention time is not used to determine molecular weight. After exiting the column of the HPLC or FPLC system, the molecules pass through a MALS detector and are probed by a laser beam.
The MALS signals, along with UV absorbance and/or differential refractive index (dRI) signals, are analyzed to quantify the analyte’s physical properties. In polymer analysis, a differential viscometer is often added to measure intrinsic viscosity. In protein and nanoparticle analysis, an online dynamic light scattering detector is often added to measure hydrodynamic radius.
A SEC-MALS system includes a standard HPLC or FPLC setup incorporating pump and SEC or gel permeation column (left). The LC system’s UV detector, degasser and autosampler may also be utilized. The primary detectors are added downstream of the LC (right): MALS - DAWN®, dRI – Optilab®, and IV – ViscoStar®.
Assumptions of analytical SEC
Size-exclusion chromatography separate molecules by size (hydrodynamic radius). However, conventional analytical SEC relates molar mass with retention time by assuming that the analyte elutes in accord with a column calibration curve.
For column calibration to work correctly, these assumptions must all be true:
- Same molecular conformation
- Protein samples are perfectly globular, like the globular protein standards
- Polymer samples have the same architecture as the polymer standard, e.g. linear random coils
- Same density / specific volume
- Column interactions
- Enthalpic interactions between analyte and stationary phase, such as electrostatic or hydrophobic interactions, are negligible
- Separation takes place according to hydrodynamic size only
In addition, SEC columns age over time, requiring frequent re-calibration.
SEC column calibration is invalid when the analytes differ from the standards in composition, conformation or non-ideal column interactions.
SEC-MALS is absolute
SEC-MALS provides first-principles analysis that is independent of the assumptions inherent in the column calibration curve.
- Basic physical equations connect the molar mass with scattered light intensity (measured by MALS) and concentration (measured by UV or dRI).
- The analysis does not depend on molecular shape/conformation and is not impacted by non-ideal column interactions.
- The molar mass, and (if the molecule is large enough its rms radius as well), are measured at each elution volume, typically once per second.
SEC-MALS determines molecular weight from 200 g/mol to 1 billion g/mol. It can also determine molecular size – the rms radius, or radius of gyration Rg – from 10 nm to 500 nm and beyond. By combining molar mass and size, it can also assess molecular conformation. And MALS detectors only require annual calibration.
SEC-MALS provides both molar mass and size, independently of reference standards.
SEC-MALS characterizes non-standard analytes
Complex macromolecules may not be accessible at all to standard SEC analysis. SEC-MALS readily characterizes many of these, providing information beyond molar mass and size:
- Conjugated molecules (glycoproteins, PEGylated proteins, copolymers) – conjugation ratio, molar mass of each component in binary conjugates
- Branched polymers – branching ratio
- Non-globular proteins including inherently disordered and denatured proteins, extended oligomers, and surfactant-solubilized membrane proteins
- Oligomers, protein-protein and protein-nucleic acid complexes – absolute stoichiometry and native oligomeric state
Who uses SEC-MALS?
Molar mass, size and conformation are essential physical properties of macromolecules. Scientists who will not settle for ‘an answer’, but require ‘the answer’ to their characterization challenges, look to Wyatt MALS instrumentation and software for reliability and confidence.
In academic, government and other labs carrying out fundamental research, SEC-MALS is used extensively in basic protein and polymer characterization.
In the biopharmaceutical and advanced materials industries, SEC-MALS is used in R&D and analytical labs for evaluation of solution properties, stability testing, process development, regulatory filing and quality control.
Get much more out of SEC
Even if you are already using size-exclusion chromatography to characterize your proteins, nucleic acids or polymers, much more information can be extracted by simply adding a MALS instrument. Wyatt’s online detectors can be combined with most industry-standard SEC and FPLC systems.
If you don’t currently have an HPLC system, Wyatt can provide a complete setup, or consult on which are suitable for SEC-MALS. Wyatt also provides MALS-approved SEC columns for proteins.
How does SEC-MALS work?
In SEC-MALS, the function of the SEC column is to separate solution components by hydrodynamic size (due to column interactions, some components may elute in the opposite order, and components of the same size may elute at different times).
As each component passes through the detectors, its concentration and light scattering properties are measured every second or so. In UHP-SEC-MALS, the peaks are much narrower, so data are collected at roughly 10 times per second.
The MALS detector incorporates between 3 and 18 photodiodes positioned at different angles θ relative to the laser beam to measure the scattered light function R(θ). At each data point:
- The plot of scattered intensity vs. angle is fit to determine R(0) (the y-intersect at angle θ = 0) and the slope.
- Molar mass, M, is calculated from the ratio of R(0) and the concentration
- Radius of gyration, Rg, is calculated from the slope.
Most of the constants used to determine M and Rg are related to system optical properties such as laser wavelength and solvent (mobile phase) refractive index. In addition, the analyte’s dn/dc value (specific refractive index increment) in the mobile phase must be known or measured (this is easier than it sounds!). For more details on the theory of MALS, please see our MALS Theory page.
When SEC-MALS is insufficient
SEC-MALS is only effective if good separation is achieved on the SEC column. When that does not work, either due to analyte-column interactions or simply analytes beyond the useful size range of SEC, MALS can be coupled to a different separation technique, field-flow fractionation. See the FFF-MALS page to learn more.
MALS can also be combined with ion-exchange chromatography (IEX-MALS) and reverse-phase chromatography (RPC-MALS), which—unlike SEC—cannot be calibrated to assign a molar mass to any specific retention time. Online MALS analysis give instant identification of peaks, with no need to collect and analyze offline. Learn more about these and other types of MALS measurements on the Other Techniques Application Notes page.
Multi-angle light scattering analyzes the amount of light scattered by the analyte into detectors positioned at various angles relative to the illuminating beam. The extrapolation to zero angle gives molar mass and the angular variation provides the size.
What constitutes a complete SEC-MALS system?
For basic SEC-MALS, all you need are a standard HPLC or FPLC system including a concentration detector (UV for proteins, dRI for polymers), SEC column(s), a MALS instrument and a computer with ASTRA® software for data acquisition and analysis.
- HP/FPLC-SEC-MALS uses a DAWN® or miniDAWN® MALS instrument; UHP-SEC-MALS requires a microDAWN®.
- DAWN has 18 angular detectors; it covers a molar mass range of 200 to 109 g/mol and a size range of 10 to 500 nm with maximum sensitivity.
- miniDAWN and microDAWN have 3 angular detectors; they cover a molar mass range of 200 to 107 g/mol (106 g/mol for linear polymers), and a size range of 10 to 50 nm.
- MALS is plumbed downstream of the UV detector or upstream of the dRI detector
- Sample injection and ASTRA data collection are synchronized via analog auto-inject signal, and concentration data are acquired via analog output from the concentration detector. Native HPLC software or a hand-held controller is used to control the HPLC system.
miniDAWN and Optilab are often combined with popular FPLC systems in protein research labs.
Most SEC-MALS users find it beneficial to include an Optilab® dRI detector, or microOptilab™ for UHPLC.
- Protein analysis - including an Optilab is very useful since nearly all proteins have the same dRI response (dn/dc) - it is not necessary to know the extinction coefficient of each peak.
- Bioconjugates - MALS, UV and dRI are combined for analysis of glycoproteins, AAVs, protein/DNA complexes, membrane proteins embedded in surfactants or lipids, and other dual-component biomacromolecules. The same analysis is suitable for many copolymers.
- Polymer analysis - replacing the HPLC’s dRI detector with an Optilab affords many benefits including direct digital data acquisition, matched MALS and RI wavelengths for more accurate molar masses, and concentration range up to ~ 20 mg/mL for convenient and accurate dn/dc measurements.
Full system control
ASTRA software can directly control select HPLC instruments, eliminating the need to synchronize the ASTRA method with native HPLC software. Please contact email@example.com for the current list of supported HPLC and UHPLC components.
Combining MALS, UV and RI enables analysis of conjugated molecules. Here the concentration of empty and full AAVs, determined by SEC-MALS-UV-RI at each eluting fraction, is shown.
Additional Wyatt products enable extended characterization capabilities:
- Proteins and nanoparticles – most proteins and some nanoparticles fall below the lower size limit for Rg (this does not prevent molar mass determination). A WyattQELS dynamic light scattering module is embedded in the MALS detector to determine hydrodynamic radii down to 0.5 nm, ideal for most proteins and peptides. Alternatively, a NanoStar or Mobius may be connected to the MALS flow cell by optical fiber to serve as the online DLS detector.
- Polymers – many polymers are also too small for Rg analysis, even though MALS has no difficulty in determining their molar mass. For analysis of size, conformation and branching of such small molecules, a ViscoStar® differential viscometer is added (microViscoStar® for UHPLC). SEC-MALS-IV determines hydrodynamic radius for conformation analysis and Mark-Houwink-Sakurada parameters.
- Challenging polymers – ViscoStar may be used without MALS, in SEC-IV configuration, for analysis of polymers that are not amenable to light scattering such as highly fluorescent lignins, materials that absorb strongly at the MALS wavelength, or polymer-solvent systems with no scattering (dn/dc = 0).
Combining MALS, viscometry and RI enables analysis of Mark-Houwink-Sakurada parameters for polymers.
Advanced macromolecular analyses
The key application areas of SEC-MALS are biotherapeutics, proteins and other biomacromolecules, and polymers. Below you will find a select set of application for each area. Additional links to application notes, webinars, our searchable bibliography and more are available in the Resources tab. If you find something promising, please contact us by clicking on the Request Information button, and an expert Application Scientist will be in touch to help ascertain if SEC-MALS meets your specific characterization goals.
Target, candidate and reagent proteins
SEC-MALS characterizes proteins and other biomolecules for solution properties, including molar mass of the molecule and its soluble aggregates, regardless of conformation or non-ideal column interactions. This is beneficial in quickly identifying optimal purification conditions and can be a key indicator of the viability of reagent proteins used in various assays.
The importance of SEC-MALS for quality control of ELISA reagents is investigated in AN1606: Assessing ligand-binding assay reagent proteins.
Aggregation, characterization and quality attributes
SEC-MALS is the preferred method for characterizing soluble aggregates of biopharmaceuticals in regulatory filings. It is well-established in comparing lots from different processes as well as comparing biosimilars to originator biologics.
To learn more about the basics of SEC-MALS analysis of proteins, download WP1615: SEC-MALS for absolute biophysical characterization.
SEC-MALS detects aggregated ELISA reagents that lead to poor performance. Figure courtesy J. Lowe, Genentech.
Gene therapy: RNA, VLPs and AAVs
Small viruses such as AAVs, as well as virus-like particles and nucleic acids that are amenable to separation by SEC, may be characterized by SEC-MALS for critical quality attributes such as molar mass, aggregation, physical titer and ratio of nucleic acid-bearing to empty capsids.
View AN1617: AAV critical quality attribute analysis by SEC-MALS and AN1616: SEC-MALS methods for characterizing mRNA for essential SEC-MALS applications in small viral vectors.
Some biologics, such as insulin, are required to form well-defined oligomeric states for optimal stability and efficacy. SEC-MALS unambiguously identifies the oligomers that are present in different formulations.
SEC-MALS analysis of insulin oligomerization in different formulations is explored in AN1605: Identification of insulin oligomeric states.
SEC-MALS determines the ratio of total to full AAVs with high fidelity, along with other critical quality attributes, in a single 30-minute run.
Post-translational modifications such as glycosylation or PEGylation are often applied to proteins or peptides in order to improve stability and increase physiological retention time. Protein-polysaccharide vaccines are another common type of conjugated biotherapeutic. Triple-detection SEC-MALS combines UV, MALS and RI instruments to determine the degree of glycosylation or PEGylation, or the molecular weight distribution of conjugated polysaccharides and the number of proteins per molecule. In some instances SEC-MALS may be used to calculate the drug-antibody ratio (DAR) of antibody-drug conjugates (ADCs).
See AN1612: Protein PEGylation processes characterized by SEC-MALS to read how this technique is used in developing PEGylation processes.
Heparin, hyaluronic acid and other polymers derived from animals can be highly variable and must be characterized carefully by SEC-MALS to establish quality metrics related to molar mass distributions and conformation.
AN6301: Discriminating heparin from chondroitin sulfate by charge:mass ratio explains how SEC-MALS is combined with charge analysis to ensure product identity and quality.
MORE BIOTHERAPEUTIC APPLICATIONS
PEGylated protein is distinguished from unreacted protein and aggregates .
Proteins & Biomacromolecules
Protein identification and quality
Molar mass is the key to identifying proteins, their oligomers or complexes, yet all too many researchers rely on potentially invalid analysis of molecular weight by native PAGE or traditional size exclusion chromatography (SEC). These techniques invoke assumptions of conformation and ideal matrix interactions that may lead researchers to fundamentally inaccurate interpretation of their data for scientific publications. SEC-MALS constitutes a rigorous, first-principles analysis of molar mass that does not rely on retention time or calibration with reference molecules. The only function of the SEC column is to separate molecules by size, while MALS determines molar mass of eluting proteins independently.
To learn more about the basics of SEC-MALS analysis of proteins, download WP1615: SEC-MALS for absolute biophysical characterization.
Elution order of these proteins does not necessarily correspond to their molar masses, but SEC-MALS provides absolute molecular weight determination. It also indicates heterogeneity or homogeneity across the peak.
Membrane proteins and glycoproteins
Membrane proteins solubilized with detergent are particularly difficult to analyze by traditional techniques or even by mass spectroscopy because of the surfactant micelle surrounding the protein. Denaturing SDS-PAGE dissociates native oligomers and precludes their identification, while cross-linked mass spectroscopy can create oligomers that do not exist in solution.
Heavily glycosylated proteins cannot be represented by reference standards or common models for globular proteins, and so are not amenable to analysis by traditional techniques.
These challenges are met by SEC-MALS which can distinguish between a protein and its associated detergent or carbohydrate by combining data from three detectors: UV, MALS and dRI. ASTRA's Conjugate Analysis algorithm calculates the molar masses of both the proteinaceous component and the conjugated or micellar component. The true oligomeric or complexed state of the protein, as well as the degree of glycosylation, are determined unambiguously.
View AN1602: Characterizing lipid membrane protein complexes by SEC-UV-MALS-dRI for an example of analysis of the protein and total content of a complex containing a membrane protein and lipids that solubilize it.
SEC-MALS provides, at each elution time, the molar mass of the core protein as well as the total molar mass of a lipid membrane protein complex.
Purification & aggregates
Scientists carrying out detailed mechanistic studies of proteins and their biological function can't afford to work with poor quality material. SEC-MALS is a fundamental means of assessing the quality and purity of protein samples. It performs true separations with absolute molar mass measurements in order to understand just which proteins and degradants are present in solution.
SDS-PAGE can be quite misleading as to protein quality, but SEC-MALS provided the true picture in the application note AN1603: Protein Reagent Quality for Drug Discovery.
Oligomerization and complex formation
Most biological oligomers are in a dynamic equilibrium with the monomer. The degree of oligomerization depends on the concentration as well as buffer pH and ionic strength, therefore proper identification of a native oligomer must be performed wholly in solution.
SEC-MALS provides an initial diagnosis of oligomerization when a protein's molar mass differs significantly from monomer sequence weight, or the mass varies over the eluting peak according to concentration. Verification may be obtained by a few additional SEC-MALS measurements consisting of different starting concentrations.
View these webinars for an in-depth understanding of how SEC-MALS analyzes protein oligomers and complexes:
Understanding Absolute Stoichiometry of Oligomeric Protein Complexes Using SEC-MALS
Applications of Analytical Light Scattering in a Biophysics Core Facility
Characterizing Protein-Nucleic Acid Interactions by Light Scattering
Applications of Light Scattering to HIV Integrase Structural Biology and Drug Discovery
Absolute Characterization of Glycoproteins and their Interactions with Proteins and Antibodies by Light Scattering
Different insulin formulations produce different levels of monomers and oligomers, as well as monomer-dimer equilibrium.
Long-chain branching is one of the most important characteristics that impacts the material performance of polymers. SEC-MALS and SEC-MALS-IV are most appropriate for small and intermediate polymers, while FFF-MALS provides superior separation and characterization of large and highly branched macromolecules.
The theory and applications of light scattering and differential viscometry in branching analysis are examined in WP1003: Branching revealed: Characterizing molecular structure in synthetic polymers by multi-angle light scattering and in the webinar Branching Revealed: Characterizing Molecular Structure in Synthetic and Natural Polymers by Multi-Angle Light Scattering.
Long- and short-chain branching affect material properties in fundamentally different ways. They may be distinguished and characterized independently using SEC-MALS and SEC-MALS-IV.
Download the application note AN1005: Identifying short-chain branched polymers with conformational analysis.
SEC-MALS determines absolute molar mass of branched polymers. A linear analog is used in the analysis of the number of branching units per molecule.
Lignin and lignosulfonate
Lignin and lignosulfonate analysis are among the most demanding applications of SEC-MALS and FFF-MALS. Their absolute molar mass distributions are characterized successfully via FFF-MALS, aided by the advanced technical features of the DAWN and the Eclipse.
Applications of MALS for lignin and lignosulfonate are reviewed in WP2303: Lignin and lignosulfonate characterization with SEC-MALS and FFF-MALS while applications to lignocellulosic material are explored in the webinar Pulp Non-Fiction: Absolute Macromolecular and Nanoparticle Characterization of Lignocellulosic Materials.
Branching is used to adjust the pharmaco-kinetic, biodegradable and mechanical properties of PLGA. Characterization of branched PLGA by conventional size-exclusion chromatography (SEC) suffers from the lack of suitable calibration standards, but this is not an obstacle for SEC-MALS.
Read how PLGA samples were analyzed by SEC-MALS-IV to determine absolute molar mass distributions and conformations in AN1301: Characterization of PLGA using SEC-MALS-IV. To learn about the characterization of PLGA as a generic drug for FDA approval, view the webinar Using SEC-MALS-IV to Characterize PLGA for FDA Generic Drug Approval.
Conformation plots of three silicone polymers with different degrees of short-chain branching but overall similar conformation.
The physiochemical behavior of hyaluronic acid (HA) is tied closely to material characteristics such as the weight-average molecular weight, polydispersity, intrinsic viscosity, and molecular conformation. Differential viscometry is used in combination with SEC-MALS to analyze HA from a variety of sources, yielding detailed understanding of these properties and why sample-averaged literature values are so different.
View AN1302: Characterization of hyaluronic acid with online multi-angle light scattering and differential viscometry.
Characterization of polymers with light scattering and UHP‐SEC
UHP‐SEC offers multiple benefits for synthetic polymer characterization, but is more sensitive to column calibration errors and drift than traditional HP‐SEC. In conjunction with MALS, UHP-SEC accurately and reliably determines the molecular weight, size and conformation of polymers ranging from hundreds to millions of g/mol, while maintaining high resolution and sensitivity and low solvent consumption.
The benefits of UHP-SEC-MALS are explained in WP1004: Absolute characterization of polymers with light scattering and UHP‐SEC. A novel online microviscometer for UHP-SEC analysis is described in the webinar Development of a New Online Micro-Viscometer for APC and UHPLC Applications.
MORE SYNTHETIC POLYMER APPLICATIONS
At each elution time, SEC-MALS-IV determines molar mass, rms radius Rg, and hydrodynamic radius Rh (derived from intrinsic viscosity) for heterogeneous polymers such as this hyaluronic acid.
One small step for SEC, one giant leap for your lab
If you already analyze your samples by analytical SEC, you have probably overcome the most challenging part of SEC-MALS: finding the right column, mobile phase and other conditions that ensure good separation and recovery. Now you are ready to add MALS for absolute characterization and deeper understanding.
As shown in the example on the right, getting the correct molar mass with MALS is independent of molecular conformation and elution volume.
How do I use SEC-MALS?
Running a SEC-MALS measurement is quite similar to standard SEC. It involves
- Sample prep (minimal)
- Column equilibration
- Loading the sample(s) into an injection loop or autosampler
- Injecting the sample and collecting data until the included column volume is complete
- Analyzing the data
If you have an autosampler, sequences may be set up for dozens of automated injections that can proceed throughout the workday and overnight.
The are two main differences in workflow relative to standard SEC: the use of ASTRA for data analysis, and some extra precautions taken to minimize particulates in the system for high-quality light scattering measurements.
Sample and system preparation
Light scattering is especially sensitive to particulates, which are usually the main source of noise in the MALS signal but are not visible in UV and dRI signals. Eliminating particles from samples and solvents is not difficult, but it does require some diligence and adaptation relative to standard HPLC/FPLC protocols.
- Mobile phase should be filtered to 0.1 µm, though acceptable results might be obtained with the more common 0.22 µm filters. In addition, a 0.1 µm filter is added to the system between the pump and injection loop. In UHP-SEC-MALS the level of cleanliness required for UHPLC systems is usually sufficient for good MALS results and additional filtration is not needed.
- Columns need to be maintained well and a column with minimum shedding should be selected. Wyatt offers SEC columns, optimized for light scattering, which minimize particle shedding for fast equilibration and low baseline noise.
- Samples should be filtered to the smallest pore size that does not remove the analyte of interest.
Is my system clean?
After flushing and equilibrating the SEC column, it may not be obvious if the system is sufficiently particle-free for high-quality MALS measurements. The System Health Monitor on the front panels of the DAWN, miniDAWN and microDAWN will let you know if the noise level is low enough to produce good data.
The Dashboard on the DAWN, miniDAWN and microDAWN includes health indicators to let you know if the noise is within an acceptable level.
ASTRA software is built in many ways like typical chromatography software. However, since it acquires and analyzes MALS data rather than retention time, there are important differences in the procedures and calculations.
- Calibration – whereas standard SEC requires frequent calibration with analyte-specific standards, the response of the MALS detector is usually calibrated annually, using only pure toluene. The Optilab and ViscoStar require no calibration.
- Band broadening correction and normalization – these procedures determine certain system parameters that are required for multi-detector SEC and light scattering. The parameters are determined simultaneously by running any known monodisperse sample with Rg < 10 nm, preferably with a known Rg value; the molar mass and concentration are not needed. These procedures are typically run every week or two if the mobile phase is the same (most aqueous solvents can be considered the same for this purpose).
- Bridge balancing – the ViscoStar’s capillary bridge is balanced using mobile phase alone. Balancing is fully automated and takes only a few minutes; typically it is done daily or weekly.
In daily operation, all that remains is to set baselines, select peaks and report the results. Baselines and peak selection can even be automated by ASTRA, relieving users of this effort when multiple files are to be analyzed. ASTRA will also gather key user-specified results from multiple runs in tables and graphs for convenient reporting. The final report may be customized to meet different needs.
For those in GMP/GLP-regulated environments, ASTRA’s Security Pack add-on enables 21 CFR Part 11 compliance including full audit trails and electronic signatures.
ASTRA supports chromatographers with peak statistics calculations, comparison with conventional calibration methods and chromatography quality assessment.
In addition, ASTRA offers many advanced analyses to support a variety of applications such as protein conjugate, branching, viral vector and particle concentration analysis.
Wyatt Technology provides several avenues for supporting novice and advanced users, including a full set of tutorials, training materials and technical notes in the online Wyatt Support Center, phone support and Light Scattering University. Site visits may be arranged for IQ/OQ, service and preventive maintenance as well as group training.
The gold standard for SEC-MALS
More than 45 years ago, Wyatt Technology's scientists invented the very first commercial light scattering instruments incorporating lasers as their light source. We've been defining and redefining the state-of-the-art for laser light scattering hardware, software, training and services to meet customer needs for over three and a half decades.
Founded in 1982 by Dr. Philip Wyatt (center), Wyatt Technology is still family-owned and operated and maintains its core values of quality and personal service.
Multi-Angle Light Scattering Instruments
DAWN® - The most sensitive MALS detector available, anywhere. Incorporates detectors at 18 angles to determine molar masses from 200 Da to 1 GDa and radii from 10 to 500 nm.
- Standard option: ambient temperature
- Heated/cooled option: -15 °C to +150 °C
- High-temperature option: ambient to +210 °C
The DAWN offers special options to handle fluorescent samples: fluorescence-blocking filters and an infrared, 785 nm laser. Learn more.
miniDAWN® - Second only to the DAWN in sensitivity, designed for smaller macromolecules. Incorporates detectors at 3 angles to determine molar masses from 200 Da to 10 MDa and radii from 10 to 50 nm. Ambient only. Learn more.
microDAWN™ - The first MALS detector for UHPLC, with interdetector dispersion as low as 1.5 µL. Incorporates detectors at 3 angles to determine molar masses from 200 Da to 20 MDa and radii from 10 to 50 nm. Ambient only. Learn more.
Refractive Index Detectors
Optilab® - A unique on-line differential refractometer for measuring concentration of any macromolecule, regardless of chromophores. Temperature controlled from 4 °C to 65 °C. The high-concentration option accommodates protein concentration up to 180 mg/mL. Learn more.
microOptilab™ - The first RI detector specifically designed for use with all UHPLC systems. Learn more.
ViscoStar® - A highly sensitive, on-line differential viscometer used in conjunction with SEC-MALS to determine the size and conformation of all types of biopolymers, synthetic polymers and even proteins and peptides.
The ViscoStar incorporates multiple novel technologies to provide the highest sensitivity, stability and solvent compatibility of any available viscometer for GPC. Its ease-of-use and serviceability make it the perfect companion for Wyatt's DAWN® light scattering and Optilab® refractive index detectors. Temperature controlled from 4 °C to 70 °C. Learn more.
microViscoStar™ - Similar to the ViscoStar but designed specifically for use with UHPLC/APC. Learn more.
Online Dynamic Light Scattering Instruments
WyattQELS - Dynamic light scattering module that can be embedded in any Wyatt MALS instrument for on-line DLS.
DynaPro® NanoStar® - Stand-alone dynamic light scattering detector, may be connected via optical fiber to the flow cell of any Wyatt MALS instrument for on-line DLS.
Mobius® – Stand-alone dynamic and electrophoretic light scattering detector, may be connected via optical fiber to the flow cell of any Wyatt MALS instrument for on-line DLS.
ASTRA® – Comprehensive analysis of SEC-MALS measurements to determine distributions of molar mass, size, and conformation. Also analyzes online DLS and intrinsic viscosity (IV) data. Among the complete set of ASTRA analyses, some of the special features included are:
- Protein conjugate analysis – analyzed binary conjugates and complexes to determine the molar mass of each component.
- AAV CQAs – determines aggregation, physical titer and empty:full ratio of adeno-associated viruses used for gene therapy.
- Polymer branching – utilizes size - molar mass relations of the sample and a linear analog to assess branching ratio.
- Mark-Houwink-Sakurada analysis – determines the MHS parameters from simultaneous MALS, dRI and IV signals.
- Particle number density – determines the instantaneous particle concentration (particles/mL) at each data slice.
The Security Pack add-on enables 21 CFR Part 11 compliance, including full audit trails and multiple signatures.
Delve deeper to learn how SEC-MALS works and performs comprehensive macromolecular characterization. These resources are all available on our web site.
On-demand webinars presenting the theory and applications of SEC-MALS are available for unrestricted viewing on the SEC-MALS Webinars page. Listed below are some of the most popular:
- Quantifying Viral Vector Attributes with Light Scattering
- Vaccines Illuminated: Biophysical characterization, PAT, and quality control via light scattering techniques
- Latest Advancements in Absolute Characterization of Proteins, Polymers and Nanoparticles
Protein research and life sciences
- Understanding Absolute Stoichiometry of Oligomeric Protein Complexes Using SEC-MALS
- Applications of Analytical Light Scattering in a Biophysics Core Facility
- Characterizing Protein-Nucleic Acid Interactions by Light Scattering
- Applications of Light Scattering to HIV Integrase Structural Biology and Drug Discovery
- Using SEC-MALS-IV to Characterize PLGA for FDA Generic Drug Approval
- Absolute Molar Mass Analysis of Medical Device and Pharmaceutical Polymers
- Structural Characterization of Biopolymers by Analytical Separation Techniques with Advanced Detectors
- Separation and Characterization of Complex (Bio)macromolecular Architectures
- Absolute Characterization of Polymeric Excipients with Size Exclusion Chromatography and Light Scattering
Application notes highlighting the use of SEC-MALS are available for unrestricted viewing on the SEC-MALS Application Notes page. Listed below are some of the most popular:
- WP9007: Characterizing vaccines with light scattering
- AN1605: Identification of Insulin Oligomeric States
- AN1617: AAV critical quality attribute analysis by SEC-MALS
- AN1616: SEC-MALS method for characterizing mRNA
- AN3002: Understanding Antibody and Viral Glycoprotein Interactions
Protein research and life sciences
- WP1615: SEC-MALS for absolute biophysical characterization
- WP3001: Characterizing protein-DNA interactions
- AN1607: Kinase Fragment Oligomerization
- AN1610: Stoichiometry of Intrinsically-Disordered Protein Complexes
- AN1614: Multi-protein complexes identified by SEC-MALS
- E-book: Beyond GPC Light Scattering for Absolute Polymer Characterization
- eBook: Advancing HPLC/ UHPLC Analysis with Multi-Angle Light Scattering Technology
- AN1002: Copolymer analysis: poly(styrene-co-acrylic acid)
- WP1003: Analyses of Polymer Branching
- AN1005: Identifying Polymer Short-Chain Branching
- WP2303: SEC-MALS and FFF-MALS characterization of lignin and lignosulfonates
- AN1310: SEC-MALS Analysis of Fluorescent Lignosulfonate Polymers
- AN1312: Analysis of PLGA using SEC-MALS with APC
- AN1305: Irradiation-induced degradation of sodium alginate by SEC-MALS
Please see the following pages for details of SEC-MALS theory:
- Understanding Multi-Angle Static Light Scattering
- Understanding Light Scattering and Chromatography Mode
These pages provide additional information on specific analyses performed in SEC-MALS:
An extensive searchable bibliography of publications citing Wyatt SEC-MALS instruments is available at www.wyatt.com/Bibliography. Just open the Advanced Search and click the Multi-Angle Light Scattering box, then enter your search terms below.
Wyatt offers its customers comprehensive online support via the Wyatt Support Center, including many technical notes that can help make the most of SEC-MALS experiments. If you are a customer and do not have access, please request an account on the Support Log-in page.
Some of the most popular SEC-MALS technical notes are:
- TN1001 SEC-MALS Branching Analysis using the Radius Method
- TN1002 SEC-MALS Branching Analysis using the Viscosity Method
- TN1006 Performing Protein Conjugate Analysis in ASTRA
- TN1012 Using the Baseline Correction Procedure in ASTRA
- TN1018 Instrument Connection Guide for ASTRA
- TN3501 SEC-MALS Noise Assessment Guide
- TN3505 SEC-MALS with AKTA systems
- TN3507 Moments and Averages Reported by ASTRA
- TN3600 Agilent HPLC Connection Guide for Wyatt Instrumentation
- TN3610 ACQUITY UPLC Connection Guide for Wyatt Instrumentation
- TN9100 Self-Associating Macromolecules: Analysis by SEC-MALS, DLS, and CG-MALS
- TN9101 Hetero-Associating Macromolecules: Analysis by SEC-MALS, DLS, and CG-MALS
At Wyatt, it’s personal
Your success is ours! From the time you purchase a Wyatt instrument, we are there for you every step of the way and give you more than just an instrument warranty. Wyatt provides a full suite of support offerings to help you make the most of your investment. Most importantly, you get unrivaled personal attention to ensure success and productivity.
On-site installation and training
With every instrument purchase, we offer an on-site installation and familiarization visit at your laboratory to ensure proper instrument set-up and function. We also offer customized on-site training tailored to your specific needs. These additional services may be especially desirable if a large group of staff members wants to receive instruction.
First-year service and support
All new Wyatt instruments come with a full year of unlimited telephone and e-mail support as well as our standard full-coverage warranty during the first year. Our congenial staff of Ph.D. scientists and expert technicians provides both technical and application assistance. We will show you how to analyze your data or set up instrument communications by remotely accessing your PC.
After the first year, annual service plans are available for continuing, unlimited telephone support, e-mail support and instrument service. Benefits of the service plans include annual preventive maintenance, on-site calibration, re-qualification, and discounts on parts and labor for repairs. Learn more about service plans.
The specifics of first-year service as well as continuing support may vary by region. Please contact your local Wyatt representative for more information.
IQ/OQ and compliance
Wyatt Technology offers a complete compliance program including documentation and on-site validation for all of its instruments. We provide the resources and tools necessary to ensure compliance. We’ve worked extensively with our partners in compliant environments to build up a robust set of documents and services. Wyatt instruments and software are used in GMP environments at pharmaceutical, biotech, and other regulated industries around the world. Learn more about our Compliance program.
Light Scattering University
Our flagship training program Light Scattering University® (LSU) is included with every purchase of a light scattering instrument. In the 2, 3 or 4 day course—depending on which system is purchased—LSU students discover advanced data processing methods and alternative analytical tools they may not be familiar with. The students also learn how their MALS and DLS data complements information from other techniques they are using in the lab. Advanced classes are offered for LSU graduates on more detailed techniques and topics.
During LSU our students meet Dr. Philip Wyatt, Founder and Chairman of the Board who gives an acclaimed Light Scattering Instrument Museum tour. Students also meet those who invent, build, service and support the instruments, so they know the team that will support them when back in the lab.
An LSU credit, inclusive of airfare, accommodations and most meals, is included for North American customers who purchase one of our light scattering instruments. While you are here, we work you hard but feed you well—at a variety of Santa Barbara restaurants!
Learn more here.
Service and Support Plans
At Wyatt, we understand that downtime and loss of productivity is critical and have developed comprehensive service offerings — our Platinum, Gold and Silver Service Plans — specifically designed to make preventative maintenance and instrument repair requests quick, smooth and hassle-free. These plans are available on an annual basis after the first-year instrument warranty expires and offer priority phone and e-mail support in addition to instrument service.
The Platinum and Gold Service Plans offer comprehensive on-site preventive maintenance and repair services. A highly trained Wyatt Field Service Engineer will come to your facility and perform annual or semi-annual preventative maintenance, calibration and optional re-qualification checks. We also offer loaner units should an instrument require factory repair. With our Silver Service Plan, you can expect full instrument calibration and quality control testing, priority service including parts, labor, shipping, as well as the same comprehensive, first-priority technical and application support by phone, email, and screen sharing sessions as offered with the Platinum and Gold Service Plans.
Our Software Support Plan provides all software upgrades, priority support and increased productivity through continual enhancements. With a 3, 5 or 10 year Software Support Plan, the savings grow exponentially and your cost of software ownership is greatly reduced.
Learn more here.
Our Customer Support Center contains a wealth of useful resources on everything related to your Wyatt light scattering instruments, software and applications:
- Software updates and bug fixes
- Technical Notes on how to connect and work with your instruments and software
- Video tutorials, software tutorials and educational webinars
- Learning Resource section with New User Guides
- Reference materials such as User's Manuals, Certificates of Analysis for Wyatt-supplied standards, CE and TUV declarations
Register or log in to the Support Center here.
We're Here to Help
Phone number +1 805-681-9009 option 4
Business hours: M-F 8 a.m. - 5 p.m. Pacific
By special arrangement, we can provide extended on-site consulting services by an Applications Scientist to set up methods and run samples on your instruments in your lab. This avoids the need to send samples off-site for analysis and allows for intimate, real-time consultations with the best experts available. Availability may be limited to certain geographical regions.
If you would like to have our lab staff analyze samples for you and provide consultations, explore Wyatt's in-house Sample Analysis services.
Wyatt Technology Store
With our online store, world-wide customers can search for parts, consumables and accessories and view product images and part numbers. The store is categorized by Wyatt product families making it easy to know which parts are compatible with your instrument. US and Canadian customers can register for an account and order parts and accessories with either a purchase order or a credit card.
Browse our most popular consumables, review your order history and easily place repeat orders online. If you have questions on which Wyatt protein SEC column is best for your application, check out the friendly SEC Columns Guide which will interactively guide you to the recommended column. In addition to ordering parts and accessories, you can also inquire about our training courses, IQ/OQ validation, and service plans.
Visit our online store here.