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Introduction to Dynamic Light Scattering Technology
Author: Malvern Instruments Nano particle and molecular identification, product marketing manager Stephen Ball
Dynamic Light Scattering ( DLS ) is a valuable particle size measurement technique for proteins, colloids and dispersions that can be easily extended to below 1 nm . In this article, Stephen Ball , Product Marketing Manager at Malvern Instruments, will introduce you to the workings of DLS and provide you with some professional advice on the issues of concern when purchasing a light scattering system.
By observing the scattered light, the characteristics of the particle dispersion or molecular solution such as particle size, molecular weight and zeta potential can be determined. The light scattering system fully exploits the correlation between these features and has been refined over the last few decades to provide highly automated inspections for routine laboratory applications. Detection using light scattering instruments is fast and efficient and can be used to characterize dispersions, colloids and proteins.
In theory, the various techniques used in light scattering instruments may look similar, but their functions and test results vary widely in practical applications, which has a significant impact on the lifetime value of the instrument. Differences in components and design in light scattering systems can also result in large differences in data quality and instrument range. For example, some light scattering systems can measure protein charge and particle size by measuring protein electrophoretic mobility, making it an efficient choice for biopharmaceutical applications.
The purpose of this article is to provide an introductory guide for readers considering the use of dynamic light scattering DLS technology. This article will examine the main uses and application areas of DLS, especially focusing on the importance of specific performance in system design, thus providing background information and theoretical support for those who are concerned about DLS technology for their own needs.
Understand the basics
When we begin to evaluate a new analytical technique, the first important step is to understand its basic workings. One of the advantages of DLS is that it is very simple to operate, and this is directly due to its measurement principle.
Due to thermal energy, the solvent molecules move continuously and collide with the suspended particles, causing the particles in the dispersion or solution to undergo random Brownian motion. The velocity of the Brownian motion of the particle can be obtained by observing the volatility of the scattered light over time. This technique is called photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS), but is now commonly referred to as dynamic light scattering ( DLS).
The Stokes-Einstein equation defines the relationship between particle Brownian velocity and particle size:
Where D = diffusion rate, k = Boltzmann constant, T = absolute temperature, h = viscosity, D H = hydrodynamic diameter
The above relationship clearly shows how the particle size is determined according to the diffusion rate in the case where the sample temperature and the continuous phase viscosity are known. Although it is necessary to control the temperature of the test, many commercial instruments measure the temperature; for many dispersants, especially water, the viscosity is known. In many cases, the additional information required for a DLS experiment is also just a viscosity measurement.
Advantages of DLS
The inherent ease of operation of the DLS means that operators do not need to have the expertise to get detailed and useful data. This advantage is particularly evident in the latest highly automated systems – the average analysis takes only a few seconds And the choice of dispersant is relatively large, whether aqueous or non-aqueous, as long as they are transparent and not too viscous, they can be used. The amount of sample required for this test method is also small, requiring only a few microliters at a minimum, which is very attractive for early studies involving valuable samples.
In fact, the DLS method is excellent for measuring particle sizes ranging from 0.1 nm to 10 μm. Its ability to measure small particles is particularly prominent, providing accurate and repeatable data of 2nm and above for most systems under test. Theoretically, detecting the particle size of low-density molecules is only limited by the sensitivity of the instrument, but for dense particles, sedimentation is a potential problem that may lead to inaccurate analysis. For example, for particles having a density of 10 g/ml, the maximum detected particle size is usually limited to within about 100 nm.
Both Dilution and Turbid samples can be measured using the DLS method, with concentrations ranging from 0.1 ppm up to 40% w/v. However, since the sample concentration greatly affects its apparent size, preparation of the sample requires extra care when the particle content is high.
The above-mentioned suitable particle size and concentration range and the high reproducibility of the measurement technique (up to +/- 0.1 nm at a particle diameter of 20 nm) make the DLS measurement method have wide applicability. For example, it is particularly suitable for detecting subtle changes in the average particle size, which may reflect the stability of the colloidal sample; it can also detect the presence of small amounts of aggregates. These phenomena are likely to be precursors to the disintegration of certain samples. When used for protein research in drugs, the occurrence of such conditions may have adverse or even harmful effects on drug performance.
Limitations of the DLS method
Most of the limitations of the DLS method can or have been overcome by improving the experimental procedure or by improving the DLS technique; however, its limitations in distinguishing instrument types, especially for applications that require extreme requirements. Still worthy of our attention. In general, most of the problems encountered during the use of DLS are for the following reasons:
· There are larger particles
Particles that exceed the maximum range of the instrument should be filtered out beforehand. Alternatively, if large particles are present in very small amounts, they can be processed by software.
· Precipitation
This phenomenon is particularly prone to occur in denser particles. Increasing the density of the dispersion is a more effective method of inhibition (such as adding sucrose to the system), but this method is only suitable for sample systems with a density of not more than 1.05 g/ml.
· Low resolution
DLS is not a high resolution technology. When the sample size distribution is very dense and there are more than three particle size distribution differences, DLS will not be able to accurately characterize multiple dispersion samples. In this case, it is recommended to separate the sample before the measurement; in the measurement method, DLS and preparation techniques such as gel permeation or size exclusion chromatography (GPC / SEC) and/or flow are required. Field separation technology (FFF) is used in combination.
· Multiple light scattering
Multiple scattering means that scattered light from one particle is again scattered by other particles before reaching the detector. In dense samples, this phenomenon will affect the accuracy of the particle size calculation. The backscatter detector measures at an angle greater than 90°, which greatly suppresses this phenomenon, thereby expanding the measurement range of the technology.
· Dispersant selection
Although most dispersants are suitable for DLS, if the dispersant viscosity is greater than 100 mPa.s, the reliability of the measurement is often affected, and the absorption of light by the dispersant may also interfere with the detection. For example, the intensity of scattered light of a colored sample may be reduced. A possible solution is to analyze or dilute the sample with different laser wavelengths depending on the sensitivity of the system. Fluorescence in the sample also affects the signal-to-noise ratio, but can be solved by using a narrow-band filter to eliminate the effects of fluorescent stray light.
Defining the characteristics of the DLS detector
The above discussion was developed in the context of testing the defined features of the DLS instrument. Sensitivity is the most basic element for any analysis technique. For DLS systems, this performance is determined by the optical hardware and the corresponding settings. At higher dilutions, systems with superior optical settings can reliably measure smaller particles, but alternatives may be more economical for applications that are less demanding in these functions. The main components of the optical setup include:
· Laser source
Stable laser sources with low noise characteristics are most suitable, such as some helium gas lasers. Some specific solid-state lasers can also be used, but at a much higher price; low-cost solid-state lasers greatly affect the accuracy and reproducibility of measurement results.
· Optical setting
The core of the optical setup is the scattering angle at which the measurements are taken. When the measurement angle is fixed at 90 o , the system is simple, cost-effective and provides the right level of sensitivity for many applications (see Figure 1). Such systems have been widely used.
When the experiment requires higher sensitivity or a higher sample concentration, it is best to choose a larger measurement angle. For example, Malvern Instruments' Zetasizer Nano series of laser particle size analyzers use a non-invasive backscatter detector (NIBS) to adjust the measurement angle to 175 o (see Figure 1), expanding the measurement range of particle size and concentration. Since the incident light does not need to pass through the entire sample, the measurement inaccuracy caused by multiple scattering is significantly reduced, and the influence of large dust particles is also excluded.
Fiber optic collection assemblies are employed in both types of settings, which provide a signal-to-noise ratio that is superior to conventional counterparts, thereby greatly improving data quality.
· Detector
There are two types of detectors: one is a cheaper, less sensitive photomultiplier tube PMT, and the other is a more expensive, better performing avalanche photodiode detector (APD). The latter claims to be as efficient as 65%, far superior to the efficiency of the alternative product PMT 4-20%, maximizing data collection, faster measurement and higher quality.
Another basic requirement for accurate DLS measurements is that temperature must be well controlled. Like the viscosity of the dispersant, the Brownian motion of the particles is also directly related to temperature, so the effect of poor temperature control is very severe. For example, in an aqueous system at ambient temperature measurement, 1 o C temperature error will result in the detection result of variation of 2.4% over ISO13321 [1] standard or +/- 2% of the updated ISO 22412 standard [2] The scope. For all types of cuvettes used, the reasonable target for temperature control of the DLS instrument is +/- 0.2 o C.
The built-in temperature controller is more convenient to use than the water bath unit connected to the outside of the detector, and is also preferable in terms of measurement accuracy, stability and reproducibility. In addition, instruments with high-performance control systems allow for rapid system warm-up and rapid temperature adjustment to study the effects of temperature changes, such as protein thermal instability.
Other measurement evaluation functions
In addition to being widely used in particle size measurement, the combination of DLS system components with a high degree of specialization is also suitable for the following measurements:
- Absolute molecular weight
- Zeta potential
- Protein charge
- Microfluidic properties
If these features are of value in the application (or potential application) selected by the system, then their choice of instrument will also play a significant role.
Based on static light scattering (SLS) data, the absolute molecular weight of the sample can be measured. This technique relates the single-molecule scattered light to its molecular weight by the Rayleigh equation, which is discussed in detail in Document 3.
In addition to particle size, zeta potential is one of the most widely used measurement parameters for characterizing colloidal systems. The Zeta potential quantifies the extent of electrostatic repulsion between particles. This quantification is not directed to the repulsive force between the surfaces of the particles, but to a point within the boundary layer of the particle, beyond which the particles no longer affect the surrounding solvent molecules (see Figure 2).
Zeta potential measurements provide an effective reference for the inherent electrostatic stability of dispersions, allowing formulators to understand and control stability. For example, in emulsion formulations, zeta potential data helps to screen for stability candidate samples and use them for water treatment flocculation process control, which helps to reduce the dosage level of the additive [4] .
The Zeta potential is measured by electrophoresis. By applying a voltage across the capillary sample cell, the velocity of the particle moving toward the electrode can be measured. Therefore, as with particle size measurement, the most important point of zeta potential measurement is that it can measure particle motion with high precision. Similarly, electrophoretic mobility measurements are similar, and their measurement depends on the rate at which the particles travel through the electric field. This type of measurement also measures protein charge, thereby increasing the value of light scattering systems for protein measurements.
Finally, let's discuss the novel emerging technology of microrheological measurement. In the traditional DLS experiment, the Stokes-Einstein equation is mainly to measure the particle size by measuring the particle's Brownian motion velocity and viscosity. In contrast, microrheology discusses the rheological properties of a solution environment around a particle by measuring particle motion. The novelty here is that in order to examine the viscoelastic properties of the sample, the mathematical description required is far beyond the scope of the Stokes-Einstein equation.
Microrheology offers the opportunity to finely and non-destructively detect the rheological properties of weak structural materials with minimal samples, extending rheological technology to areas that are not accessible to mechanical rheometers.
Daily use
When selecting an instrument, it is especially important to evaluate the overall performance characteristics. However, the inconvenience caused by using a system that does not meet the operational requirements every day can be very annoying, and you don't even want to use it anymore. Therefore, when it is necessary to choose between the final several alternative instruments, the following questions are worth considering:
· What is my most important need: speed or accuracy?
· What is the range of my sample size?
· What type of sample do I want to measure, such as toxic? Or is it particularly corrosive?
· Will the operator of the instrument be an expert or a newbie in the future? How much knowledge do they have about light scattering?
Speed ​​and accuracy
DLS measurements are usually performed in batches, and the samples are usually different and smaller. The measurement time is generally set according to the level of repeatability that can be achieved, but it is generally not more than a few minutes. However, analytical efficiency may vary depending on sample preparation and system cleaning requirements, and the ease of use of different systems may vary. If the DLS system is used as a GPC/SEC detector, the system will be set to the fluid mode of operation. As the sample flows through the instrument, the measurement must be completed in just a few seconds to achieve the necessary accuracy.
Instruments with good test speed and accuracy are usually expensive, but the cost of life is more important. Considering the time and cost of repetitive experiments due to failure to meet repeatability standards, and the reduced efficiency of analysis due to the inability of the equipment to meet routine laboratory requirements, more expensive systems may be more versatile. Value.
Cuvettes for a variety of sample types
Most light scattering systems use a variety of cuvette cells or cuvettes to hold samples during batch sample analysis. They are usually made of plastic (usually polystyrene), glass or quartz, but vary in size. The minimum amount of sample depends on the optical setting, typically 2-3 ml. However, if you do not consider any sample recovery requirements, there are some system measurements that require only 2 μl of sample usage.
Disposable plastic cuvettes do not require cleaning, eliminating the risk of cross-contamination, especially for toxic materials; some cuvettes are only 50 μL in size. The use of cuvettes avoids the problem of inaccurate measurements due to incomplete cleaning due to the 'non-cuvette' system (ie, placing the sample directly on the glass slide). Quartz cuvettes have better measurement quality, especially for low or small particle sizes, because quartz materials have excellent optical properties and scratch resistance.
Reduce the burden of analysis
Light scattering is usually just one of many techniques that many researchers routinely use in the lab. The instrument operator may not be an expert in light scattering, so the ease of operation of the instrument is helpful.
Some DLS systems evaluate data during the data collection process, eliminating the consequences of contamination due to the presence of large particles. Such systems help to increase the speed and tolerance of sample preparation. Particles larger than 10 microns in particle size primarily undergo forward scattering, so instruments containing backscatter detectors are less sensitive to the presence of these particles. Measuring a wide range of concentrations minimizes the need for sample dilution and further increases measurement efficiency.
Most modern measurement systems require no operator intervention during data acquisition, reducing analyst workload and increasing measurement repeatability. However, some of the more complex samples may require special methods for measurement, so these special methods should be included in standard operating procedures (SOPs) to ensure application standardization.
Although automatic measurements are now commonplace, there are significant differences between instruments in terms of the level of built-in data analysis support. If it's a light scattering measurement system for non-professionals, advanced software with built-in data analysis and expert advice will be extremely valuable, just like having a reliable, living expert on the other end of the phone.
to sum up
DLS is a relatively mature technology for particle size and molecular size measurement of various types of samples. Therefore, when selecting an instrument, the system capabilities must be closely tied to the user's requirements to match the two. The light scattering system can measure the molecular weight, the molecular charge, the zeta potential, and even the microrheological measurement function.
Sensitivity varies widely between systems, as can measurements at high concentrations, as well as effective measurements of particles or molecules of various sizes. Compared to those detectors 90 o degrees, backscatter instrument has very practical advantages.
In addition to performance, there are other factors that can affect the life of the instrument, including ease of cleaning; support available and a friendly user software interface. Regardless of the specification of the instrument, the best advice is to test before buying to see if you can easily get useful data. DLS has been around for many years, so no matter what your purpose is, you can expect to have a targeted, productive and easy to use measurement system.
End
references:
[1] ISO 13321 (1996) Particle size analysis - Photon correlation spectroscopy.
[2] ISO 22412 (2008) Particle Size Analysis - Dynamic Light Scattering
[3] GPC / SEC Static Light Scattering Technique Description, (Malvern Instruments White Paper). Download URL:
[4]
image
Figure 1: Key components of the DLS system include (1) laser, (2) measurement unit, (3) detector, (4) attenuator, (5) correlator, and (6) data processing PC. The detector can be placed at an angle of 90° or greater, such as the NIBS detector shown here set at 175°.
Figure 2: Zeta potential is used to quantify the repulsion between particles in suspension stability studies
Laser
Attenuator: attenuator
Detector: detector
Digital signal processor
Correlator: correlator
Electrical double layer: double layer
Stern layer: tight potential layer
Diffuse layer: diffusion layer
Negatively charged particle: negatively charged particles
Slipping plane: sliding surface
Surface potential
Zeta potential: Zeta potential
Distance from particle surface: the distance from the surface of the particle