Shedding Light on Nanoparticle Characterization

71884_LM_Malvern Panalytical_Zetasizer_LeadGen_JL (8e8ea11d-a384-46bc-9c3f-ef056899cbef) Decoding Nanoparticle Characterizing Using Dynamic Light Scattering Insights on Malvern Panalytical's Zetasizer Advance Series for deeper insights in nanoparticle characterization Table of Contents 3Dynamic Light Scattering Method Development Using the Zetasizer Advance Series13Pushing the Limits of DLS: Measuring Large Particles on the Zetasizer Ultra with the Low-Volume Sizing Cell19Measuring the Size of Gold Nanoparticles Using Multi-Angle Dynamic Light Scattering (MADLS) Introduction Shedding Light on Nanoparticle Characterization Malvern Panalytical's Zetasiser Advance Series helps researchers achieve precise, real-time nanoparticle insights with advanced DLS technology Nanoparticle characterization allows researchers to determine critical attributes such as size, shape, surface charge, and distribution, that influence a nanoparticle's functionality, properties, stability, and interactions with biological systems or other materials. This is important as nanoparticles find prominence in applications such as drug delivery systems and advanced materials fabrication. Among the various techniques available for characterizing nanoparticles, Dynamic Light Scattering (DLS) allows researchers to quickly and accurately measure the hydrodynamic sizes of particles in suspension. A non-invasive technique, DLS analyzes the fluctuations in light scattering due to the Brownian motion of particles. The translational diffusion coefficient obtained from these fluctuations is used to calculate the hydrodynamic diameter of the particles. Offering rapid and accurate measurements, including high sensitivity to small particles, the ability to analyze a wide range of sizes, and minimal sample preparation, DLS is an indispensable tool for researchers and manufacturers to obtain reliable, real-time insights on nanoparticle dispersions. Malvern Panalytical's Zetasizer Advance Series offers researchers advanced features for DLS method development to enhance the accuracy, efficiency, and flexibility of nanoparticle characterization workflows. Equipped with innovative tools including adaptive correlation, multi-angle dynamic light scattering (MADLS), and depolarized dynamic light scattering (DDLS). Reinforced by robust software that simplifies method development, the Zetasizer Advance Series sets the stage for users to optimize parameters for different samples and improve the repeatability and reliability of their analyses. This e-book discusses the key factors for DLS method development including instrument verification, cell selection, sample preparation, measurement setup, and analysis techniques to obtain reliable particle size measurements. Readers will learn how DLS enables the exploration of a greater size range of nanoparticles without compromising the integrity of the sample dispersant. Corresponding case studies on the Zetasizer's Low Volume Disposable Sizing Cell and the use of MADLS on the Zetasizer Ultra demonstrate to researchers how to offset errors introduced by sedimentation, thermal currents, and number fluctuations, that may lead to inaccurate size analysis, and identification of off-spec samples, reducing reliance on TEM, and saving time and costs. Using the DLS technique, reinforce and enhance your nanoparticle characterization workflow. DYNAMIC LIGHT SCATTERING METHOD DEVELOPMENT USING THE ZETASIZER ADVANCE SERIES‌ Introduction The technique ofDynamic Light Scattering(DLS) is commonly used to measure particle size and is available in the Zetasizer Advance series of instruments (Figure 1). DLS is a non-invasive technique suitable for the size characterization of nanoparticles. The technique measures the time- dependent fluctuations in the intensity of scattered light that occur due to the random movement of the particles or molecules undergoing Brownian motion. The velocity of this Brownian motion is measured and is called the translational diffusion coefficient (D) which can be converted into a hydrodynamic diameter (DH) using the Stokes-Einstein equation [1,2]. A summary video of the DLS technique can be viewed here. This technical note discusses method development and highlights various factors that need to be considered, from instrument verification, sample preparation, measurement set up and result analysis. Instrument Verification Dynamic light scattering is an absolute technique that uses first principles in its measurement protocol i.e. it cannot be calibrated but should be verified that it is working correctly by measuring a sample with a known particle size value. The frequency of instrument verification should be defined by the user. Polystyrene latex spheres are monodisperse and spherical and are commonly used to verify correct instrument performance. The sphere is the only three-dimensional shape whose size can be unambiguously described by a single figure (i.e. diameter). Polystyrene latex samples have other benefits such as having a density similar to water, so particles less than 1 micron will remain in suspension during measurement. Dispersions can be stored at room temperature and have storage lifetimes of months or years. A wide range of monodisperse polystyrene lattices are available from a variety of manufacturers. However, not all are supplied with an individual calibration certificate. Some size standards are supplied with their own calibration certificate, measured by transmission electron microscopy (TEM) and are traceable to the National Institute of Standards and Technology (NIST) [3]. The certificate provided also includes a hydrodynamic diameter measured by dynamic light scattering (DLS). The result quoted on the standard bottle is the certified TEM result. The DLS result (i.e. the hydrodynamic size) is not a certified value. The samples should be prepared in 10mM NaCl [2] to suppress the electrical double layer. Dilution of the standard in deionized water will give an extended double layer and result in an artificially increased size which may be out of specification. The pass/fail criteria for sizing standards are defined in ISO22412 [2]. In summary, the mean value of the first 5 repeat measurements must fall within the range specified on the Certified Reference Material (CRM) certificate (hydrodynamic diameter). The polydispersity index (PdI) value must be less than 0.1 for each of the repeat measurements. In addition, there must be no measurement bias as assessed by verifying that the difference between the mean value measured and the certified mean sample size stated on the CRM certificate (TEM size value) should be less than the combined uncertainty from the CRM stated mean size and the measured mean uncertainty, as described on page 13 of ISO22412 [2]. The relative standard deviation of the first 5 repeat measurements must be <2%. Cell Selection TheZetasizer Advance range of instruments has various cuvettes/cells options available for the measurement of particle size (Table 1). The choice of cell will be dependent upon the application (Table 2). Zeta potential cells are also capable of measuring particle size for certain Zetasizer Advance models. Sample Preparation Sample Concentration Each sample has its own ideal concentration range for optimal measurements. The minimum and maximum required sample concentration should be experimentally determined. Data Quality Guidance in the Zetasizer Advance series helps determine whether the sample concentration is appropriate. If the sample concentration is too low, there may not be enough light scattered to make a measurement. This is unlikely to occur with the Zetasizer except in extreme circumstances. If the sample is too concentrated, then light scattered by one particle will itself be scattered by another (this is known as multiple scattering). Non-Invasive Backscatter (NIBS) optics are designed to minimise multiple scattering issues [7]. Consistency of the measurement position between samples can confirm that the concentrations are the same or comparable. The upper limit of the concentration is also governed by the point at which the particles no longer freely diffuse, because of particle interactions. The size of the particles is an important factor in determining the onset of restricted diffusion and particle-particle interactions. Sample Dilution If a sample must be diluted, it should be done carefully to preserve the existing state of the particle surface. Results obtained from DLS should be independent of sample concentration [2]. Measurement Set Up This section discusses the setup of DLS measurements in ZS Xplorer software. To set up a measurement, the following steps need to be completed (Figure 2) and these parameters are discussed in Table 3: Sample Parameters Instrument Settings The measurement properties, data processing options and post-analysis settings are shown in Figure 3 and discussed in Table 4 with a discussion of the size analysis models available in Table 5. Advanced Settings In this box (Figure 4), additional measurement settings can be altered, and their influences are discussed in detail in Table 6 below. For a rapid, routine measurement, there might be no need to alter these settings. DLS users often enquire as to which instrument parameters are important when transferring a measurement method from Research & Development to Quality Control for the same material. Table 6 lists the key parameters that need to be considered. Note that these could vary when the measurements are transferred from Research and Development to Quality Control. Whenever the sample is being measured for a comparative evaluation, as in a Quality Control environment, it is advised that the parameters mentioned above i.e. Angle of Detection, Positioning Method, Attenuation and Measurement Process be 'fixed' in a Method. Result Analysis Various parameters and reports are available in ZS Xplorer which will aid the interpretation of the results. The precision (repeatability) of the size results obtained is a key parameter in the analysis of the data. For monodisperse samples with diameters between 50nm and 200nm, the repeatability of the average particle size should be lower than 2 % [2]. For samples where the repeatability of the results is poor, improved results could be achieved by averaging the records. This should increase the quality of the underlying raw data and subsequently improve result repeatability. In ZS Xplorer software, automatic averaging can be set up as part of the measurement (Figure 3) or, results can be averaged once they have completed by either selecting the required records, right-mouse clicking and selecting the Create Average Result option (Figure 5) or clicking on the Create Average Result button (Figure 6). Conclusions This technical note has discussed DLS method development with the Zetasizer Advance series and highlighted the various factors that need to be considered. These include cell selection, instrument verification, sample preparation, measurement set up and results analysis. References PUSHING THE LIMITS OF DLS:‌ MEASURING LARGE PARTICLES ON THE ZETASIZER ULTRA WITH THE LOW-VOLUME SIZING CELL Introduction Dynamic Light Scattering (DLS) allows the hydrodynamic size of particles in dispersion to be measured by quantifying their diffusive motion. At particle sizes approaching the upper size range for DLS, sedimentation, thermal currents and number fluctuations can mean that the detected scattering no longer accurately describes purely diffusive motion, and particle size analysis becomes less accurate. This can be seen in measurements of monodispersed samples that exhibit artefacts in the correlation function baseline, Figure 1. This may be mitigated by adjusting dispersant, either density or viscosity, however both offer a less than ideal remedy as we are no longer measuring the original system of interest. Here we will discuss how the Low Volume Disposable Sizing Cell can improve data quality at large particle sizes and allow the entire size range of DLS to be explored without modification of the dispersant system. Figure 1: Example auto-correlation data from 1.0 µm and 3.0 µm latex in water. Measurements from both a 1.0 mm capillary and a 10.0 mm cuvette are shown. Despite these samples being mono-disperse, results for these particles in the 10 mm cuvette show an additional mode of decay in the measured auto-correlation function. Sedimentation As particle size increases, thermal Brownian motion is no longer sufficient to keep particles in suspension, and samples may sediment over time, meaning that the motion of the particles is no longer random. We can however rule out sedimentation as the dominant factor in the skewing of reported particle size at higher sizes by considering the time for particles to transit the incident laser beam during a DLS measurement. Figure 2 below shows that the times scales associated with sedimentation are much greater than the correlation times used to capture data in a typical DLS measurement, even when considering differences in material density. Figure 2: Calculated settling time as a function of particle size for polystyrene latex (ρ = 1050 kg/m3) and silica (ρ = 2650 kg/m3) particles dispersed in water at 25oC (η = 89 x 10-3 Pa s). Thermal effects Measurements of particles over 1 micron in size may show some difference in variability as a function of temperature, suggesting that thermal effects may influence the artefacts seen in the measured correlation functions. Thermal modelling of a 10mm cuvette, with temperature controlled by contact with the cell holder of the instrument, shows that the geometry of the cuvette supports the formation of convection currents, Figure 3. Modelling of a capillary however, with a similarly controlled temperature shows that convection currents are not supported due to the constrains imposed by the narrow cross section. This modelling does not look at the impact of particle size on the significance of these convection effects, however if we consider the rate of diffusion as a function of particle size, smaller particles diffuse more rapidly and thus the diffusive motion of the particles is the dominant transport property. At larger particle sizes, diffusion is slower and the increased cross section of the particles mean that they are more readily influenced by these additional currents. Figure 3: Thermal models of a 10 mm cuvette and a 1 mm capillary, calculated using ANSYS. The thermally driven velocity gradients shown represent the steady state condition after 120s of equilibration with a Peltier device at the bottom of each image controlling the system. The red cross-hair indicates the position of the detection volume where the DLS measurements are performed. Results and Discussion To demonstrate the improvement of measurement accuracy at larger particle sizes facilitated by the capillary, a series of differently sizes polystyrene latex particles were prepared in a 10mM NaCl solution and measured in both the low volume disposable sizing cell and a standard 10 mm cuvette. Mean and standard deviation particle size is shown in Figure 4, showing that without modification of the dispersant, the measurements performed in a cuvette were reported outside of their specified range at around 1 μm, whereas measurements in the capillary were reliable and accurate up to 10 μm. Figure 4: Discrepancy in the measured particle size, derived from cumulants analysis, and associated error, compared to the specified nominal size of a range of NIST traceable polystyrene latex particles, measured in both a 10 mm cuvette and 1 mm capillary. As a demonstration of improved measurements at extended size range with a polydisperse sample, an arbitrary sample of UK soil was dispersed in filtered ultra-pure water and measured both in a 10 mm cuvette and the low volume disposable sizing cell. The characterization of soil is critical to the understanding the incursion of nano- and micro-particles in the environment, however the error bars for the cuvette-based measurements in Figure 5 would suggest that DLS is not a suitable technique in this case. The same measurements performed with sample loaded into a capillary, however, show much better resolution and improved repeatability as denoted by the narrower error bars, despite the sample being polydisperse in size from around 100 nm to over 1 μm. Figure 5: Intensity weighted particle size distributions for a polydisperse sample of soil, measured both in a 10 mm cuvette (left), and a 1 mm capillary. The distribution represents the average of 10 measurements and corresponding standard deviations. The measurements in the capillary show better repeatability shown by the narrower error bars, and a better resolved result. Conclusion We have shown that at the upper measurable size range of dynamic light scattering, thermal currents are the dominating phenomena that lead to artefacts in the correlation function and therefore deterioration in measurement accuracy. The geometry of the 1mm capillary used in the low volume disposable sizing cell does not support the formation of these convection currents and thus accurate measurements can be performed without modification of the sample dispersant over the entire measurable size range for DLS, and repeatability for polydisperse samples is improved over comparable measurements in a standard cuvette. MEASURING THE SIZE OF GOLD NANOPARTICLES USING MULTI- ANGLE DYNAMIC LIGHT SCATTERING (MADLS)‌ Reducing the need for time-consuming and expensive TEM analysis with Zetasizer Ultra Introduction When producing particles of a known size, the monodispersity of the samples in terms of both size and shape is important and often requires a high-resolution technique such as Transmission Electron Microscopy (TEM) to look at these sample properties. For example, Particle Works, a brand of Blacktrace Holdings Ltd, produce gold nanoparticles which must be highly monodisperse and have high batch-to-batch consistency, with CV values as low as 5% for the size distribution and 2.5% for the batch consistency. Currently, Particle Works use TEM as the primary technique for characterizing their samples for QC. However, TEM is a time consuming and expensive technique that requires an experienced user to perform analysis. Particle Works were therefore keen to explore whether the Zetasizer Ultra could be used to reduce the amount of TEM analysis required. The Zetasizer Ultra has multiple features that help to reduce the time taken for measurements to be carried out while providing much more detail on sample properties. These include Adaptive Correlation, Multi-Angle Dynamic Light Scattering (MADLS) and Depolarized Dynamic Light Scattering (DDLS). In this study, samples of Particle Works gold nanoparticles were measured using both TEM and Multi-Angle Dynamic Light Scattering (MADLS)with the Zetasizer Ultra to determine whether the samples were of high enough quality to be sold. Experimental The samples tested in this study were gold nanoparticle samples synthesized by Particle Works during the early development of the Ultraspherical Gold Nanoparticle product range. Target sizes of 10, 15, 20 and 50 nm were measured on the Zetasizer Ultra using MADLS in DTS0012 plastic cuvettes. These samples were also measured using TEM with at least 100 particles being measured for each sample. Details of the samples measured are shown in Table 1, including their target size. Table 1: Sample details including target size Results For all of the samples, the Peak by Intensity values as measured by the Zetasizer are higher than those measured by TEM due to the effects of stabilising ligands, ionic surfactants and hydration layers, which increase the hydrodynamic size of the dispersed gold. Zetasizer measures the hydrodynamic diameter of the gold, while TEM only measures the core particle diameter, therefore this is the primary reason for the discrepancy. Measuring the first sample, Au-2-016-7, with DLS showed the size populations to be much larger than the target size of 10 nm, to the point that the 10 nm peak does not appear in the distribution shown in Figure 1. Therefore, this sample has failed to meet the specifications. The TEM image also shows that there are multiple large particles but also shows the expected 10 nm particles. It is likely that these are being masked in the MADLS measurement by the scattering of the larger particles. Figure 1: Intensity size distribution (left) as measured by MADLS and TEM (right) of Au-2-016-7 Measuring the second sample, Au-2-017-6, with MADLS would suggest that this sample is on specification as a narrow distribution is measured (Figure 2). However, the TEM measurements disagree, as while the majority of the populations fall within the expected range, there is a tail of particles below the target size which means it does not meet specifications. The MADLS measurements was unable to pick this up because the scattering of the sample is much more skewed towards the larger sizes masking that of the tail of smaller particles. This means that while the MADLS measurements are well suited to detecting particles that are larger than the majority of the sample, they are less able to identify a sample that contains particles which fall under the specification. Figure 2: Intensity size distribution as measured by MADLS (left), TEM image (centre) and the TEM size distribution (right) of Au-2-017-6 For the third sample, the TEM image, as shown in Figure 3 shows a uniform size distribution that falls within specifications for the particles themselves. However, there are rings around many of the particles and it is unclear if these are a true reflection of the particle size when dispersed or if this is a result of the drying process. By measuring the sample using MADLS, it can be seen the distribution is narrow and within specifications. If the rings were around the particles when dispersed then the DLS distribution would be much wider and higher in size. Therefore, it is likely during the drying process some of the surfactant in the dispersion has dried around the particles causing these visible rings. By identifying that these rings are not representative of the sample means that this sample can be sold. Figure 3: Intensity size distribution as measured by MADLS (left), TEM image (centre) and the TEM size distribution (right) of Au-2-037-4 The fourth sample, Au-2-041-3, was measured using MADLS, shown in Figure 4, and while the distribution was mostly uniform, there was an unexpected small peak below 10 nm. Figure 4: Intensity size distribution as measured by MADLS of Au-2-041-3 With an unexpected peak such as this it can be useful to use the DDLS capabilities on the Zetasizer Ultra. By removing the vertically-polarized scattered light, leaving just the horizontal-polarized scattered light, it can be determined whether this unexpected peak is due to translational diffusion of particles and therefore another population present in the sample. Alternatively, this peak could be due to the rotational diffusion of a non-spherical particle, if so, then the <10 nm peak would be expected to increase in relative intensity compared to the larger size peak when using horizontal polarisation. Measurements using both polarizations (a typical backscatter measurement), vertically-polarized light and horizontally- polarized light were made as shown below in Figure 5. As can be seen while the vertically-polarized backscatter measurement is similar to the typical measurement, the horizontally-polarized measurement has a much higher relative intensity for the smaller size peak than the higher size peak. This means that the small size peak is not due to the translational diffusion of particle but instead the rotational, therefore, some of the particles are not spherical. Figure 5: Intensity size distributions of Au-2-041-3 in backscatter using all polarizations (blue), vertical polarization (green) and horizontal polarization (red) The sample was also measured on TEM, shown below in Figure 6, which confirms the conclusion from using MADLS. This shows that many of the particles are faceted rather than spherical and some are even rod like. These rod-like particles are most likely those that cause the <10 nm peak seen when using DLS. Figure 6: TEM image of Au-2-041-3 The results from measuring the final sample with MADLS show it to be a sample with monomodal, narrow distribution, shown in Figure 7. This suggests this sample meets the specification which is confirmed by the TEM measurements which also show the sample contains a uniform size. These data confirm that this sample is a production quality sample suitable for sale to customers. of this information and we shall not be liable for errors contained herein or for damages in connection with the use of this material. Malvern Panalytical reserves the right to change the content in this material at any time without notice. Figure 7: Intensity size distribution as measured by MADLS (left), TEM image (centre) and the TEM size distribution (right) of Au-2-016-13 Conclusion The Zetasizer Ultra has been shown to be a useful tool for QC for both R&D and manufacturing. Its speed and ease of use means that multiple samples can be quickly measured to decide on if they are within specifications. In this study, the use of the Zetasizer has meant that fewer samples need to be measured using TEM as many off spec samples have been identified using the Zetasizer Ultra alone. 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