Dynamic light scattering
Batch dynamic light scattering: a few microlitres in a cuvette or plate well, correlated for a minute or two, giving a Z-average, a PDI and an intensity size distribution. The everyday aggregation check for purified proteins and particles.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
DLS
Batch dynamic light scattering: a few microlitres in a cuvette or plate well, correlated for a minute or two, giving a Z-average, a PDI and an intensity size distribution. The everyday aggregation check for purified proteins and particles.
How it differs from the standard method
- Spin or filter the sample and use a clean cuvette; dust ruins more measurements than anything else.
- Enter the buffer's viscosity and refractive index at the measurement temperature.
- Report the Z-average and PDI, and the intensity distribution, with the concentration measured.
- Plate-based instruments trade some sensitivity for throughput in screening.
1Size from flickering light
Particles in solution are never still: they are pushed about by collisions with solvent molecules, and small particles move faster than large ones. When a laser shines through the sample, the light scattered by all the particles interferes, and because they keep moving the interference pattern, and so the intensity at the detector, flickers.
2What the instrument reports
- Z-average
- The mean hydrodynamic size from a cumulant fit to the correlogram. It is an intensity-weighted harmonic mean, and the most robust number DLS produces; many instruments report it as a diameter (d.nm).
- Polydispersity index (PDI)
- The relative width of the distribution of decay rates from the same fit. Below about 0.1 is a near-monodisperse protein; above 0.2–0.3 the sample is a mixture and the Z-average describes nothing in particular.
- Intensity distribution
- A size distribution from a regularised inversion of the correlogram, weighted by how much light each size scatters. Converting it to volume or number weightings makes large particles look smaller in share but also amplifies noise.
- Intercept
- The value of g₂ − 1 at the shortest lags. A high intercept (0.8–0.95) means the signal is well above the solvent; a low one means the sample barely outscatters the buffer or the optics are misaligned.
3Why a trace of aggregate dominates
Scattered intensity from particles much smaller than the wavelength grows with the square of their mass (roughly the sixth power of their radius). A 60 nm aggregate scatters hundreds of thousands of times more light than a 3.5 nm protein, so 0.1–0.2% of the protein by mass in aggregates can carry more than half of the light.
4Zeta potential by electrophoretic light scattering
The same instruments usually measure zeta potential. An alternating electric field is applied across a capillary cell, charged particles drift towards an electrode, and their velocity Doppler-shifts the scattered light.
Key terms
Practise these as flashcards →Radius of a sphere that diffuses at the same rate as the particle, including its bound solvent; larger than the anhydrous radius and larger again for elongated or flexible molecules.
D = kT / (6πηRh), the link between diffusion coefficient and size, through temperature and solvent viscosity.
Fitting the logarithm of the correlogram with a polynomial in lag time; the first term gives the Z-average, the second the PDI (ISO 22412).
Slope of D against concentration, D = D₀(1 + kD·c). Positive for net repulsion, negative for attraction; used in formulation to rank colloidal stability.
Particle velocity per unit field strength, measured by electrophoretic light scattering and converted to a zeta potential.
Thickness of the ionic atmosphere around a charged surface: about 3 nm in 10 mM monovalent salt and under 1 nm in physiological buffer.
When to use it
- A quick check that a purified protein is monodisperse and free of aggregates before crystallisation, cryo-EM, SAXS or a binding assay.
- Screening buffers, formulations or temperature ramps for aggregation, and ranking colloidal stability by kD.
- Sizing nanoparticles, liposomes and lipid nanoparticles, and checking their surface charge by zeta potential.
Compared with alternatives
Separates species first and gives an absolute mass for each; use it to say how much of each oligomer there is, which DLS cannot.
Resolves monomer, dimer and aggregates and quantifies them in solution without a column; slower, but the reference method for heterogeneity.
Gives Rg, mass and shape information as well as size, but needs more sample and beam time.
Where DLS and zeta potential are routine release tests for liposomes and lipid nanoparticles alongside encapsulation measurements.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Batch dynamic light scattering: a few microlitres in a cuvette or plate well, correlated for a minute or two, giving a Z-average, a PDI and an intensity size distribution. The everyday aggregation check for purified proteins and particles.
- Spin or filter the sample and use a clean cuvette; dust ruins more measurements than anything else.
- Enter the buffer's viscosity and refractive index at the measurement temperature.
- Report the Z-average and PDI, and the intensity distribution, with the concentration measured.
- Plate-based instruments trade some sensitivity for throughput in screening.