A Basic Guide to Particle Characteristics Analysis-4

Technique 2: Dynamic Light Scattering 

  Dynamic Light Scattering (DLS), also known as Photon Correlation Spectroscopy (PCS) or Quasi-Elastic Light Scattering (QELS), is a well-established non-invasive technique for measuring the size of particles and polymers ranging from sub-micron to 1 nanometer or less.

  This technique can be used to measure samples consisting of particles suspended in liquids such as proteins, polymers, micelles, carbohydrates, nanoparticles, colloidal dispersions, and emulsions.

Key Advantages:
      • Ideal size range for nano-size and biomaterials
      • Requires a small sample size
      • Fast analysis and high throughput
      • Non-invasive technique allows for complete sample recovery

Principle

  Suspended particles undergo Brownian motion due to thermally induced collisions between the suspended particles and solvent molecules.

  When a laser illuminates the particles, the intensity of the scattered light fluctuates rapidly on a short time scale, which depends on the size of the particles. Smaller particles are moved further and faster by the solvent molecules. Analyzing these intensity fluctuations yields the speed of Brownian motion, which is used to determine size via the Stokes-Einstein relationship.

  The diameter measured by DLS is called the hydrodynamic diameter, referring to the way a particle diffuses in the fluid. The diameter obtained refers to a spherical diameter with the same translational diffusion coefficient as the measured particle. 

 

  An illustration of the hydrodynamic diameter recorded using DLS that is larger than the ‘core’ diameter, where the translational diffusion coefficient is affected not only by the size of the ‘core’ but also by any surface structure, as well as the concentration and type of medium ions. This means the size is larger than that measured by electron microscopy, signifying, for example, that the particles are removed from their original environment.
  DLS yields an intensity-weighted particle size distribution, indicating that large particles can dominate the particle size result, and recognizing this is important.

Equipment

  Conventional DLS equipment consists of a laser light source focused on the sample using lenses.

  Light is scattered by the particles at all angles, and a single detector placed typically at 90° to the laser beam collects the intensity of scattered light.

  The intensity fluctuations of scattered light are converted into electrical pulses fed into a digital correlator. This generates an autocorrelation function from which particle size is calculated.

NIBS

  In modern equipment, Non-Invasive Backscatter (NIBS) technology extends the range of measurable particle size and sample concentration.

  The size measuring capability of these instruments detects backscattered light at an angle of 173°, known as backscatter detection. Additionally, the optics do not contact the sample, thereby making the detection non-invasive.

  There are many benefits of using non-invasive backscatter detection. improved sensitivity and the ability to measure a broader range of sample concentrations.
      • Sample preparation is simplified. 

      • Sample preparation is simplified. 

(a)  For small particles or low concentration samples, it is an advantage to maximize the amount of scattered light from the sample. When the laser penetrates the cuvette wall, the difference in refractive index between the air and the cuvette material causes ‘flare’. This flare can interfere with the signal from the scattered particles. Moving the measurement position from the cuvette wall to the center of the cuvette will eliminate this effect.

(b)  Large particles or high concentration samples scatter more light. Measuring closer to the cuvette wall minimizes the path length through which scattered light must travel, reducing multiple scattering effects. 

Technique 3: Automated Imaging

  Automated imaging is a high-resolution method for analyzing the properties of particles ranging from approximately 1 micron to a few millimeters in size.

  Individual particle images are captured from the dispersion and are analyzed to determine particle size, shape, and other physical properties. By measuring tens to thousands of particles in a single measurement, a statistically representative distribution can be constructed.

  In static imaging systems, the dispersed sample is stationary, whereas in dynamic imaging systems, the sample flows past the imaging optics. This technique is often used in conjunction with ensemble-based particle size measurement methods like laser diffraction to verify ensemble-based measurements or to gain a deeper understanding of the sample. Typical applications include:

      •  Measuring differences in particle shape that cannot be distinguished by size alone
      •  Detecting and/or enumerating aggregates, large particles or contaminated particles
      •  Measuring non-spherical particle size, such as needle-shaped crystals
      •  Verifying ensemble-based particle size measurements like laser diffraction

Equipment

  A typical automated imaging system consists of three main elements.

1. Sample Presentation and Dispersion
      This step is crucial to achieving good results, i.e., spatial separation of individual particles and aggregates in the field of view.
 

      Various sample presentation methods can vary depending on the sample type and measurement method used. Dynamic imaging measurements use a flow cell through which the sample passes during measurement. Static imaging measurements use flat surfaces such as microscope slides, glass plates, or filter membranes. An automated dispersion method is preferred to avoid potential operator variability.
 

 

 

  

2. Image Capture Optics

      Images of individual particles are captured using appropriate optical lenses and digital CCD cameras suitable for the sample being measured.
 

      Static imaging systems provide greater flexibility in terms of sample illumination, such as reflective (episcopic) illumination, transmission (diascopic) illumination, and darkfield illumination, whereas in dynamic imaging systems, the sample is typically illuminated from behind.
 

      For birefringent materials such as crystals, polarizing optics can be used. The most advanced dynamic imaging systems employ hydrodynamic sheath flow mechanisms to maintain a constant focus even for very fine particles.
  

 

3. Data Analysis Software

      General equipment measures and records various morphological properties for each particle.
 

      The most advanced equipment includes software with graphing and data classification options, allowing related data to be extracted as simply as possible from measurements using an intuitive visual interface.
 

      Grayscale images stored individually for each particle provide qualitative verification of the quantitative results.
 

 

 

Technique 4: Electrophoretic Light Scattering (ELS)

 Electrophoretic Light Scattering (ELS) is a technique used to measure the electrophoretic mobility of particles or molecules in a solution or dispersion. This mobility is often converted into zeta potential to compare materials under different experimental conditions.

  The fundamental physical principle is electrophoresis. A dispersion is applied in a cell with two electrodes.

  An electric field is applied across the electrodes, causing any charged particles or molecules to migrate toward the oppositely charged electrode. The speed at which any charged particles or molecules move is the electrophoretic mobility, which is related to the zeta potential of the particles or molecules.