Biomolecular Science: How to Measure Molecular Weight and Size Using Light Scattering Detectors

Light scattering detectors measure the size and determine the structure molecules through a variety of techniques and equations. However, with so many systems of measuring available, it’s important to know how they work, and which instrument is most beneficial to your application.

What is molecular weight and size?

Molecular weight is the mass of an individual molecule, specifically the mass of material required to make a single mole. Molecular size relates to the physical size of an individual molecule. When used in light scattering techniques we refer to the ‘radius of gyration’ or Rg. Measurements are in nanometers.

Why is measuring molecule weight and size important?

In industrial application, there are a range of reasons for measuring the weight and size of molecules, such as:

• improved performance in strength and durability of plastics and polymers
• controlling the release rate and degradation of polymers used in medicine delivery
• improving mouth-feel of polysaccharides in food products

For both consumer industries and industrial design and manufacturing, knowing the molecular size and weight of materials can have a profound affect on the design and manufacturing of new and existing products.

How is light scattering related to molecular size and weight

The equation that describes this relationship is called the Rayleigh equation. In basic terms, it tells us how the intensity and angle of scattered light is related to the size and weight of a molecule.

Molecules that are larger in size or have a higher molecular weight will scatter more light than lighter or smaller molecules. Furthermore, there is a linear relationship with the intensity of the light scattered and the increase in molecular weight, but a non-linear relationship to the size of a molecule.

Therefore, mathematically speaking, if we know all the other aspects of the Rayleigh equation, we can measure scattered light intensity to calculate a sample’s molecular weight.

How does static light scattering work?

Static light scattering (SLS) measures the intensity of scattered light in an environment where other variables are already known to measure the size and weight of a sample of molecules.

Chromatography systems

Using a chromatography system with static light scattering greatly reduces the number of issues that come about when purifying and preparing samples. SLS measurements can be made in a cuvette or in a Gel-permeation chromatography (GPC) system also known as Size-exclusion chromatography (SEC).

In SEC/GPC molecules are separated according to their size or hydrodynamic radius as they enter and exit the pores of a porous gel packing matrix in the column. In addition to separation, GPC/ SEC systems can be coupled to multiple detectors (Ultraviolet absorbance detector (UV-Vis), Refractive index detector (RI), Viscometer and Light scattering) delivering a complete set of data for each sample.

In a standard GPC / SEC system you’ll find the following process:

1. The sample passes through the degasser and pump into the injection loop, or
   1. an autosampler places samples directly into the injection loop.
2. Molecule samples are separated in the columns and oven.
3. A light scattering detector measures absolute molecular weight.
4. A refractive index or UV detector collects further data and measures concentration.
5. A viscometer measures the intrinsic viscosity (IV) to investigate molecular structure and branching.

Understanding angular dependance

For higher sized molecules, the intensity of light scatter will vary with the measurement angle. Accounting for this is called ‘angular dependence’, and is crucial for measuring larger molecules. Remember ‘radius of gyration’, this plays a keep part in determining which sized molecules are affected by angular dependence. Generally speaking:

• Molecules with an Rg < ≈15 nm are isotropic and have little to no angular dependence.
• Molecules with an Rg > ≈15 nm are anisotropic and the intensity of light which they scatter will vary at different angles.

As molecular size increases with respect to the laser wavelength, the scattered photons no longer scatter independently, but start to interfere with each other. Different types of light scattering devices attempt to overcome this in different ways.

Types of Static Light Scattering instruments

SLS instrumentation comes in 4 major variants:

• Right angle light scattering (RALS)
• Low-angle light scattering (LALS)
• RALS / LALS hybrid detectors
• Multi-angle light scattering (MALS)

Right angle light scattering (RALS)

RALS instruments are the simplest device for measuring light scattering, but they can be amongst the best.

Advantages of a RALS system

• Straightforward without complicated optics.
• Have great signal to noise ratio and sensitivity. As the light passing through the liquid interface is at 90 degrees, the change by flare or noise in the refractive index is minimised.
• Great for measuring the molecular weight of proteins.
• Has small flow cells.

Disadvantages of a RALS system

• For an accurate 90 degree measurement, the assumption is that scattering is isotropic (i.e. equal at all angles). For molecules large enough to display angular dependence, this is not true. So RALS can’t accurately measure the molecular weight of molecules with an Rg > ≈15nm.

Since proteins are almost always smaller than 15nm radius, RALS detectors are excellent for measuring protein molecular weight.

Low angle light scattering

LALS instruments measure the scattering of light as close as possible to 0 degrees, giving a closer measurement of the intensity of the light scattering. To be considered a LALS system, the measurement must be below 10 degrees, with most LALS devices settling on 7 degree measurements. LALS are typically used for larger molecules and anisotropic scattering from synthetic and natural polymers

Advantages of LALS systems

• By measuring close to the axis of the Zimm plot, LALS provide high accuracy on molecular weight.
• Can measure the molecular weight of any molecule.
• Accommodating a single measurement angle, only a small flow cell is required.

Disadvantages of a LALS system

• LALS systems are traditionally more prone to noise from large particles scattering in a forward direction. Modern techniques reduce this noise.
• Since scattered light is being measured at a single angle, a LALS system can’t measure Rg.
• Due to the proximity of the scattered light and laser light, production of LALS are more difficult than RALS.

RALS / LALS hybrids

Hybrid RALS / LALS detectors combine the effectiveness of both right angle light scattering and low angle light scattering to produce highly complementary results.

• RALS measurements provide accuracy on weak scattering smaller molecules.
• LALS measurements give better readings on larger anisotropic scattering molecules.

Combining the readings of RALS and LALS systems into a single computation can be used to estimate the radius of gyration.

Multi-angle light scattering

MALS instruments measure light scattering at multiple points to increase accuracy in data for molecules with an Rg > ≈15 nm. Different angles can be removed to reduce noise, however complexity in design means increased costs as well as lower signal to noise readings.

Advantages of MALS systems

• Ideal for measuring large, anisotropic light scattering molecules.
• Provides insight into angular dependance.
• Can also be used on isotropic, smaller molecules that don’t exhibit angular dependence.
• Some angles can be removed from readings if they produce too much noise.

Disadvantages of MALS systems

• Produce more noise than other systems.
• Require a larger flow cell resulting in increased peak broadening.
• Expensive to produce.
• It is not always clear which extrapolation fit and model gives the right answer, as exact shape and structure of the molecule are not known.

Which system is right for you?

Light scattering systems have a number of beneficial uses across many industries, however choosing the right instrument can be difficult. Talk to ATA Scientific about which GPC / SEC system is right for your needs, and find out how our instruments can help you gain further insights for your research today.