Multi-Angle Light Scattering (MALS)

Multi-angle light scattering (MALS) is the preferred analytical technique for determining absolute molar masses and sizes of all types of macromolecules and particles in solution. 

MALS is a straightforward, absolute method and provides results that are not dependent on so-called standards or measurements that someone else made in another laboratory. In the limit of low concentration, the amount of light scattered by a suspension of molecules is directly proportional to the product of their weight-average molar mass times the concentration. The angular variation of the scattering (as a function of angle) reveals the molecules’ root mean square radius (RMS or Radius of Gyration). If you want to determine molar masses and sizes over the greatest possible range, a MALS instrument is the ideal solution. MALS can be applied to macromolecules such as proteins, pharmaceuticals, biopolymers, polymers and particles such as liposomes, micelles, and encapsulated proteins. Measurements can be made in one of two modes: either in continuous flow mode (with SEC, GPC, HPLC or any other flow fractionation method, such as AF4 or FFF) or in batch mode (un-fractionated).

Continuous flow experiments can be used to study material eluting from virtually any source. Most conventionally, the detectors are coupled to a variety of different chromatographic separation systems, such as GPC, SEC, HPLC or Field Flow Fractionation (FFF or AF4). The ability to determine the mass and size of the materials eluting then combines the advantage of the separation system with an absolute measurement of the mass and size of the species eluting.

Batch mode experiments can be performed either by injecting a sample into a flow cell with a syringe or with the use of discrete vials (scintillation vials or microCuvettes where sample volume requirements are low). These measurements are most often used to measure timed events like antibody-antigen reactions or protein assembly. Batch mode measurements can also be used to determine the second virial coefficient (A2), a value that gives a measure of the likelihood of crystallisation or aggregation in a given solvent (useful for protein crystallography).

How does MALS work?

MALS measurements work by calculating the amount of light scattered at each angle detected. This process overcomes the problems associated with low angle detectors (typically there is around ten times the noise at an angle of 11° or below compared to 90°) and allows a reliable and accurate measure of the light scattered. The higher the number of detectors, the better the accuracy of the experiment. The amount of light scattered is then related to the molar mass.
For a full explanation of light scattering theory, please see our theory section.

The molar mass and size are calculated by producing a Debye plot for each data slice:

• The intercept gives molecular weight Mw for that slice
• The initial slope gives the root mean square
  radius (RMS or Rg: radius of gyration) for that
  slice.

Why Use Multi-Angle Light Scattering?

The addition of a MALS detector coupled downstream to a chromatographic system allows the utility of SEC or similar separation combined with the advantage of an absolute detection method. The light scattering data is purely dependent on the light scattering signal times the concentration; the elution time is irrelevant and the separation can be changed for different samples without recalibration. In addition, a non-size separation method such as HPLC or IC can also be used. As the light scattering detector is mass dependent, it becomes more sensitive as the molar mass increases. Thus it is an excellent tool for detecting aggregation. The higher the aggregation number, the more sensitive the detector becomes.

As mentioned above, the MALS detector can also provide information about the size of the molecule. This information is the Root Mean Square radius of the molecule (RMS or Rg). This is different from the Hydrodynamic Radius (Rh), in that it is not affected by the hydration layer and is purely the root mean square of all the radii making up the molecule multiplied by the mass at that radius. This would seem like a strange parameter to measure but in fact this is very useful as it is sensitive to changes in shape of the molecule. If you consider adding a side group to a large molecule (branching), this would hardly affect the hydrodynamic radius (Rh) but would have a significant impact on the Radius of Gyration (Rg). In addition, if you plot the Log Rg vs. Log M then the conformation of the molecule can be derived. If there are any shape changes which are associated with changes in molar mass, they will be identified.

What are the advantages of using MALS?

Multi-angle light scattering coupled to SEC or other fractionation methods can provide an absolute means for measuring the molar mass, size, and distribution of polymers of all sorts. Wyatt Technology’s MALS also enables the elucidation of additional data such as branching, conformation, and eluent behaviour. The same instrumentation also allows observation of molecular interactions in a real-time environment. As MALS is an absolute mass and size measuring technique it is insensitive to the separation mechanism and as such is ideal for both highly variable research applications as well as routine quality assurance of previously characterised samples.

Wyatt Technology Instruments:

Wyatt Technology offers the world's most advanced Multi-Angle Light Scattering with its unique and patented DAWN® detectors:

Additional and complementary technologies can be integrated and combined with Wyatt Technology's MALS detectors:

Do you have any questions? We would be delighted to hear from you! Contact us 


MALS — Application Notes:

Below, please find a selection of MALS application notes or select your application of interest: Proteins, Biopolymers, Polymers, Particles

#	Description	Download
1	Aggregation of proteins
2	Reverse-phase protein characterization
3	Wheat proteins by reversed phase chromatography
4	Protein modifications using PEGylation to change the drug-delivery properties of a protein.
5	PEGylated protein characterization with the number of PEG attachments!
6	Micro-batch characterization of proteins
7	Aggregation as a function of concentration of proteins.
8	Peptides and proteins showing the power of the miniDAWN for low molar mass characterization of biopolymers.
9	Hemoglobin--One of the building blocks of life! This note examines the elution properties of this protein and how different they are from more "conventional" proteins.
10	Lens proteins--a complex protein which exhibits conformational changes across the molar mass distribution.
11	Gelatins--Used in everything from food thickening agents to photographic film substrates, gelatins can be analyzed successfully and absolutely with SEC-MALS, as this note shows.
12	Protein-Protein Interactions--Scientists working at Pfizer's Central Research Laboratory show that the DAWN is an invaluable tool for the development of a scale-up purification protocol.
13	How antibodies that are developed to be powerful drugs can bind specifically to a target molecule or cell.
14	This note concerns the use of an infrared (IR) laser in characterizing a protein that fluoresces at the red wavelength, but did not fluoresce at the longer IR wavelength.
15	It discusses the application of MALS-QELS interfacing to enable absolute molar mass and size determinations from 1nm to 500nm. In this note we use BSA, as well as a glycoprotein sample to show the range of masses and sizes we can characterize by both static and dynamic light scattering.
16	Proteins can elute at radically different times depending on their structure, yet simple column chromatography misses this completely. By adding a MALS with the WyattQELS accessory, the subtleties of proteins may be studied.
17	To obtain absolute molar masses of heat shock proteins at different temperatures, multi-angle light scattering (MALS) detection was used following SEC.
18	Membrane protein is generally soluble only in the presence of micelles; thus, it is very difficult to characterize the oligomerization state of the membrane protein in a lipid-containing solvent. In this application note we demonstrate the use of multi-angle light scattering (MALS) detection in combination with UV absorption and differential refractive index (DRI) detection to determine the molar masses (MM) of both the core protein and the entire protein-lipid complex.
19	Calcium Storage Protein Calsequestrin
20	Hemoglobin Unfolding & Aggregation
21	Refractive index based determination of detergent concentration and its application to the study of membrane proteins
22	Ribosomal Subunits in Protein Biosynthesis
23	Binding Stoichiometry of an Antibody Fragment
24	Kinase Fragment
25	Automated, Online Second Virial Coefficient (A2) Measurements
26	Stoichiometry of Lysin Holoenzyme
27	Low Molecular Weight Lens Peptides and Changes in the Oligomeric Organization of αB-crystallin
28	Molecular weight distribution of gluten proteins analysed by MALS
29	Characterization of Spongiform Encephalopathies
30	Multi-Angle and Dynamic Light Scattering - Role Of Oligomeric Size In Chaperone Function Of αB-Crystallin
31	Soluble Protein-Protein Associations
32	Biophysical Properties of Crystallin Aggregates
33	Novel Malaria Vaccine Candidates
34	Protein Aggregation States
35	Purification of the Rab11-FIP2 Complex