LS Instruments

Experimental Guidelines - DLS Sample Preparation

I) Sample

The measurement principle of Dynamic Light Scattering (DLS) is based on the Brownian motion of particles. This restricts the range of samples that can be measured with this method.

DLS allows measurement of colloids, such as:

Solid particles, polymers, and proteins dispersed in a solvent
Emulsions

DLS does not allow measurement of:

Dry powders (these can be measured only after homogenous dispersion in the solvent)

II) Solvent

Samples used for DLS should be, in general, dispersed in a liquid phase. After production or synthesis and purification if prepared in a research lab, in most cases samples should be already in dispersed form. In the case of dry samples (powders), solvent addition is necessary prior to measurement. The most common solvents are:

Deionized or MiliQ water
Toluene
Methanol
Ethanol
Glycerol

In order to choose a solvent, two main requirements should be taken into account. Firstly, the solvent should disperse the particles well. In some cases, it might be necessary to use dispersing agents such as steric or ionic surfactants. Ensure that the chosen solvent, along with the dispersing agent, yields sufficient colloidal stability over a time span that allows for DLS measurements. Secondly, the solvent should not alter the properties of the samples, e.g. the solvent should not dissolve the particles.

If multiple solvents meet the criteria above, the solvent that yields the largest contrast between sample and solvent should be chosen. This means that the difference between refractive indices should be the largest. This will yield the best signal at a given sample concentration. If the sample cannot be concentrated and the signal is not sufficient for a reliable measurement, the user should adopt a setup equipped with a more powerful laser.

Make sure that the solvent used is of high purity and does not contain large molar mass impurities (e.g. dust). In the case of small, weakly scattering particles, the absence of large molar mass impurities is of great importance as their scattering signal could "shadow" the signal of the particle being measured. Large molar mass species can be removed by means of multiple filtrations through suitable filters. If possible, use filters with a pore size of 0.1 µm. Another possibility is distillation and centrifugation. Vials and containers should also be rinsed with high purity solvent before being used for the sample preparation.

For samples obtained in a suspension form, filtration might be used as well to remove undesired large molar mass species. In this case, however, as-synthesized aggregates might also be removed, meaning that the overall nature of the sample might be altered. Choose a filter with an appropriate pore size (to ensure that particles intended for measurement are not removed). Note that the sample concentration might change during the filtration, due to the adsorption of particles onto the filter. Another possibility to exclude agglomerates is to let them sediment or apply gentle centrifugation.

Good laboratory practice includes work in very clean conditions (ideally in a clean room, a laminar-flow hood, or a well-ventilated fume hood). The risk of dust contamination will be minimized by closing solvents, pipettes, vials, and samples tightly. Additionally, wearing gloves is advisable not only due to the possible contamination of samples but also due to safety reasons. As for sample concentrations, the lower the concentration, the higher the probable influence from large molar mass impurities.

III) Concentration

Measurable sample concentrations mainly depend on the specific DLS measuring technique adopted. Excessively concentrated samples should be diluted with the same solvent used to suspend your particles. Overly diluted samples can be concentrated by centrifugation, removal of the solvent, and subsequent re-dispersion.

Traditional DLS is performed in standard 2D geometry (auto mode) and requires highly diluted suspensions. This is necessary in order to minimize undesired multiple scattering effects and, furthermore, to minimize interparticle interactions, as both invalidate the DLS data treatment. Excessively concentrated samples will result in systematic errors in the determination of hydrodynamic radius (see Application Note Particle Sizing in Highly Scattering Samples). On the other hand, overly diluted samples might yield an insufficient signal. Visually, the suspension should be transparent or faintly opaque. A good practice is to prepare several dilutions per experiment to determine the optimal concentrations.

Usually, concentrations that range between 1-10 mg/mL should provide a good starting point for the determination of the proper sample concentration. The optimum concentration is found in the range of concentrations exhibiting the plateau in the measured hydrodynamic radius. In other words, if a series of dilutions provide different results, samples should be further diluted. On the other hand, at very low concentrations, the signal-to-noise ratio is very low. Furthermore, overly concentrated samples might also result in substantial multiple scattering, resulting in an underestimation of the hydrodynamic radius.

In the case of 3D measurements, highly concentrated (opaque, almost milky) samples can be measured, as this technology suppresses the multiple scattering signal. Note, however, that the 3D cross-correlation technique results in decreasing signal-to-noise ratio as the sample concentration is increased. This means that, despite the absence of systematic errors, very concentrated samples might be practically unmeasurable. In this case, modulated 3D cross-correlation technology (if available) considerably extends the measurable concentration range as it drastically improves the signal-to-noise ratio (see Application Note Exploring Sample Turbidity Limits).

IV) Homogeneity

Samples should be sufficiently well dispersed and suspension should be homogenous. This can be achieved by bath sonication (for example 15 minutes on the highest power) or multiple resuspension with the pipette (in case of fragile samples).

In the case of charged particle systems, long-range double layer interactions start to play a role. The addition of a small amount of salt or dilution with salt solutions can help to reduce such interaction while preserving sample stability. Usually, the salt concentration necessary to screen charge interactions varies in the range between 0.1 mM and 10 mM. As to the choice of the salt, it depends on the solvent used:

Aqueous solutions: KBr, NaBr, NaCl, KNO₃
Nonhalogenated solvents (THF, DMF, DMA): LiBr
Halogenated solvents (CHCl₃, CH₂Cl₂, o-dichlorobenzene): NBut₄Br

V) Cuvette

Make sure that measurement cuvettes are clean and dust-free. You can clean them with Hellmanex III from Helma upon hot sonication, rinse multiple times with pure solvent (for example MiliQ water or absolute ethanol), dry in a dust-free environment (laminar-flow hoods might be advisable when available). For faster drying, an oven at 60°C under vacuum or N₂can be used. In this case, hold the cuvettes upside down to avoid any contamination.

While pouring suspension into the cuvette, make sure that no air bubbles are introduced. If this happens, sonicate the cuvette or resuspend the suspension with the pipette. To avoid bubble formation, it is good practice to pour the suspension onto the wall of the cuvette.

The amount of suspension in the cuvette should be optimized. This is due to the fact that for small sample volumes the laser beam will not pass through the sample. On the other hand, for large volumes, a part of the suspension will be above the heating decalin bath, which will result in temperature gradients and subsequent solvent convective motion influencing the Brownian motion of particles.

Depending on the instrumental setup, different types of cuvettes can be used. In the case of LS Spectrometer, the sample is embedded in an index matching fluid and typically cylindrical cuvettes are used. In some special cases, square cuvettes might be required.

Do not use markers to write notes on cuvettes (i.e. no ink on the cuvette). Ink will dissolve in decalin solution and contaminate the thermal bath (this will obviously change the optical properties of decalin and influence the measurement).

In the case of NanoLab 3D, three types of glass square cuvettes are offered (3 x 3 mm, 5 x 5 mm as well as 10 x 10 mm). Additionally, in cases where the glass cuvette cannot be used, disposable plastic cuvettes (10 x 10 mm) serve a convenient alternative. Since the temperature of NanoLab 3D cuvettes is controlled by the Peltier module, the use of markers is allowed (only make sure that you do not write on the wall through which the beam traverses).

Before inserting the cuvette into the sample holder, clean it again with a tissue. It is important to use low lint, non-scratching tissues designed for lab glassware cleaning. Do not leave fingerprints on the cuvette, and make sure that cuvette external walls are dry.

VI) Equilibration

When you insert the cuvette with the sample into the sample holder, wait 10-15 minutes so that the temperature of the sample is the same as the temperature of the thermal bath.

Now, the sample is ready. The next steps include the choice of measurement parameters, measurement, and data analysis.