Dynamic Light Scattering (DLS)
Material
DLS requires a liquid sample, either dispersed in solvent or emulsions. Solid or dried samples are not directly compatible with this measurement. A 1 – 2 mL volume is recommended; however, for volume limited samples, 100 – 200 µL may be acceptable. The choice of cuvette will greatly impact what volume may be used.
As DLS measures particulate size, dust is a common interferant. Pure solvents (ACS grade or better) are highly recommended. Additionally, all solvents should be filtered prior to combination with sample to ensure no dust or contamination is introduced to solution. Be sure all other materials—such as sample containers, transferring pipettes, cuvettes, surfactant additives, salt additives, etc.—are kept capped, cleaned, and/or filtered prior to use to minimize the possibility of interfering contaminates. Something as simple as a puff of compressed air can quickly and easily blow out residual dust and/or solvent prior to beginning sample preparation.
Best practice is to filter solutions before (i.e., solvent) and after sample addition. It is especially important to filter prepared solvents, such as after surfactant or salt has been added to the solvent. At the very least, sample solutions should be filtered after a suitable incubation time. Be sure to choose a filter size appropriate for your particle of interest. A filter pore size approximately 3x the largest anticipated particle size is best. Generally, a 0.2–0.45 µm filter pore size is suitable for most applications. Be sure to rinse the filter before introducing your sample.
Solvents
Solvent must be non-reactive with the sample. Solvent should not dissolve the materials of interest. The material should remain suspended in the solution. Pure solvents (ACS or HPLC grade), free of possibly interfering contaminates and/or dust, are preferred. DI water is not recommended. Impure solvents can increase the measurement noise, particularly for small or weakly scattering sample particles. Some solvents, such as toluene and DMSO, contribute to light scattering themselves and should be avoided. These and similar solvents increase the measurement background noise and can impede particulate movement as a function of temperature.
The most common solvents are:
- Water
- Methanol
- Ethanol
- Toluene
- Glycerol
Surfactant
Surfactant might be useful to aid dispersion, especially for samples suspended in water. However, surfactant should only be added in minute proportions. A dilute solution of 1-10% surfactant should first be prepared and filtered. Add 1–3 DROPS of diluted surfactant solution to the solid sample, which is then added to water.
Common non-ionic surfactants:
- Triton X-100
- Igepal CA-630
- TWEEN 20 or TWEEN 80
- Span 20 or Span 80
DLS Sample Preparation, Entegris technical note.
Salt
For charged particle samples or aqueous suspensions, salts can reduce interfering particle interactions. Salt concentrations range between 0.1-10 mM, with 10 mM being the most common concentration. Salt choices are based on suspension solvent:
- Aqueous: KBr, NaBr, KNO3
- Nonhalogenated organics (e.g., THF, DMF, DMA): LiBr
- Halogenated organics (e.g., CHCl3, CH2Cl2, o-dichlorobenzene): NBut4Br
Concentration
Dilute suspensions are necessary for DLS measurements. Generally, 0.1 – 1% or 1 – 10 mg/mL sample concentrations are most common. Sample suspensions should be clear or mildly hazy. There should be no sample precipitation; precipitation is indicative of an overly concentrated sample or sample aggregation. If the sample is too concentrated, it should be diluted further. The addition of a dispersion agent, such as surfactant or salt, can reduce sample particulate aggregation.
Suspensions should be well dispersed and homogenous. Polymers, proteins, and other fragile sample types should be gently mixed. Allow 12 – 24 hours dissolution time. Swirling or pipette mixing can be used as a mixing aid. Do not sonicate. For sturdy samples, vortexing or sonication (bath only) for 15 minutes can assist homogenous distribution.
Colored samples may be acceptable, provided no sample absorption occurs. In general, colored or fluorescing samples are harder to measure and should be avoided, if possible. For white, darkly colored, or very hazy samples, additional dilution will be necessary. To determine if coloration is from over-concentration or natural coloring, try reading text through the sample. If text can still be read, coloration will likely not affect measurements (provided their is no light absorption).
Optimal concentration is at the hydrodynamic radius plateau or constant region (see figure below). To determine if the sample is suitably dilute for accurate DLS size measurements, sample should be diluted and measured until no change in hydrodynamic radius is observed. Highly concentrated samples may result in underestimation of hydrodynamic radius from multiple scattering. After the DLS measurement, dilute the sample 50% and re-measure. If the resultant size is the same and the count rate is 1/2 as previous, the first measurement (previous concentration) was acceptable.
Zeta potential (ZP)
Also called the electrokinetic potential. A measure potential difference between electrophoretically mobile particles and the dispersant [surrounding those particles] at the slipping plane. As the electrophoretic particle motion also scatters incident light, both particle size and zeta potential can be measured/calculated simultaneously via DLS.
As electrokinetic potential is highly dependent on sample environment, ZP samples should be carefully diluted with the original sample solvent. If possible, procure supernatant from the original, concentrated sample, either by filtering or centrifuging, and use the supernatant for further dilution. Allow the concentrated sample to naturally sediment and measure the resulting supernatant. Attempt to recreate the original medium as closely as possible, accounting for pH, total ionic concentration, concentration of any known additives, etc. If diluting ZP samples in polar or non-polar solvents, the lack of ions can lead to over-estimation of the measured ZP.
Like size measurements, it is not recommended to prepare ZP samples in DI water. DI water, without salt additives, has low conductivity, making the electrical double layer difficult to measure. This can result in poor reproducibly and the resultant measurement is difficult o interpret. Although, the addition of salts, surfactants, or other dispersing agents can aid in typical DLS size measurements, these additives will alter the solution’s chemistry compared to the original sample. This chemistry alteration will result in a ZP measurement that is different from the ZP of the original sample.
Cuvette
A variety of cuvette types are suitable for DLS and/or ZP measurements. Some cuvette types are only suitable for DLS size measurements while some can also be used for ZP measurements. Sample volume and solvent compatibility should be considered when determining which cuvette is most suitable for your experiment. Several examples are given in the table below and are compatible with our DLS instrument. Most of these cuvettes are available for use.
Always first inspect the cuvettes for damage prior to use. DLS measures light scattering from several angles; it is recommended to use fully transparent (4 side transparent windows) cuvettes so frosted sides do not impede incident or detected light. It is exceptionally important cuvettes are cleaned before (and possibly also after) use. Rinse the cuvettes with water to remove any residual solvent and/or dust and/or blow out the cuvette with compressed air before filling with your sample. Do not insert tips, especially metal tips, into the cuvette as this can scatch the cuvette interior. Ensure there are no bubbles when filling the cuvette. Wipe all 4 cuvette sides with an optical tissue prior to your measurement. Covering the cuvette with a suitable cuvette lid ensures no dust or debris contaminates the prepared sample between preparation and measurement.
| Cuvette type/material | Temperature range | Solvent compatibility | Experiment compatibility |
| Square, polystyrene (disposable) | 0 – 70 °C | Aqueous, weak polar | DLS only |
| Micro-cuvette, plastic | 0 – 70 °C | Aqueous, acetone, benzaldehyde, butanone, dioxane, DMF, ethyl acetate, isopropanol, various acids and bases | DLS: 40 µL min. |
| Micro-cuvette, quartz | 0 – 120 °C | any solvent | DLS: 12 – 45 µL |
| Square, glass | 0 – 120 °C | any solvent | DLS only |
| Folded capillary zeta cell, disposable/plastic | 0 – 70 °C | aqueous, weak polar | DLS; pH titration; Zeta Potential |
| High concentration zeta potential cell | 0 – 70 °C | aqueous, weak polar | DLS; pH titration; Zeta Potential: high concentration (e.g., ceramics, cosmetics, emulsions), low volume samples |
| Universal ‘Dip’ cell | 0 – 70 °C | aqueous, non-polar* | Zeta potential |