DLS, Sample

Preparation,
Applications, and
Limitations
A comprehensive guide to understanding particle
characterization using light scattering technology
What is Dynamic Light Scattering (DLS)?
Dynamic Light Scattering is a powerful, non-destructive
optical technique used to measure particle size in
suspensions and solutions. It has become an essential
analytical tool across various scientific disciplines.

• Measures particles from 1 nanometer to several microns

• Provides real-time analysis with minimal sample preparation
• Critical for quality control in pharmaceutical and
nanotechnology industries
Historical Background of DLS
The journey of DLS technology spans several decades of
scientific advancement:

• Initial development in the 1960s by pioneering physicists

studying molecular diffusion
• Significant theoretical advancements throughout the 1970s
• First commercial DLS instruments appeared in the 1980s
• Digital correlators revolutionized the technology in the 1990s
• Modern systems feature user-friendly interfaces and
sophisticated algorithms
Basic Concept of DLS
At its core, DLS analyzes the random motion of particles in solution:

• Particles undergo constant Brownian motion in liquid media

• A laser beam illuminates the sample
• Scattered light intensity fluctuates as particles move
• These fluctuations are measured over microseconds
• Analysis of the fluctuation patterns reveals particle size
The Physics Behind DLS
Laser Illumination
Coherent monochromatic light source directed at sample
solution

Particle Scattering
Suspended particles scatter incoming light in all directions

Interference Patterns
Scattered light waves create constructive and destructive
interference

Intensity Detection
Photodetector captures time-dependent intensity fluctuations
Role of Brownian Motion
Brownian motion is the fundamental physical
phenomenon that makes DLS possible:

• Particles suspended in liquid undergo random,

constant movement
• Motion results from collisions with surrounding
solvent molecules
• Smaller particles move faster and cover greater
distances
• Larger particles move more slowly due to increased
drag
• This size-dependent velocity difference is the key to
DLS analysis
Light Scattering Principles
Scattering Regimes in DLS

• Rayleigh Scattering: For particles much smaller than the

wavelength of light (d < λ/10)
• Mie Scattering: For particles comparable to or larger
than the wavelength
• Scattering intensity is proportional to d6 for small particles
• Refractive index difference between particles and medium
affects scatter strength
• Larger particles produce more directional forward scattering
Measurement Process Overview
Sample Preparation
Properly diluted and filtered sample is loaded into a clean cuvette

Laser Illumination
Sample is illuminated with a focused laser beam at specific wavelength

Scattering Detection
Photodetector positioned at fixed angle (typically 90° or 173°) measures scattered light

Signal Processing
Digital correlator analyzes intensity fluctuations to generate correlation function

Data Analysis
Software applies algorithms to extract particle size distribution from correlation data
Correlation Function in DLS
The correlation function is the mathematical heart of DLS
analysis:

• Represents how quickly intensity fluctuations lose their

relationship over time
• Faster-moving (smaller) particles cause more rapid
correlation decay
• Slower-moving (larger) particles maintain correlation longer
• Decay rate directly relates to the diffusion coefficient
• Complex algorithms extract size distribution from
correlation data
Calculation of Hydrodynamic Diameter
The Stokes-Einstein Equation

Where:

• D = Diffusion coefficient (from correlation

function)
• kB = Boltzmann constant
• T = Absolute temperature
• η = Viscosity of the medium
• Rh = Hydrodynamic radius
Key Metrics Reported
by DLS
1 Hydrodynamic Diameter (Z-average)
The intensity-weighted mean diameter of the particle
population, including any solvent layer moving with the
particle
2 Polydispersity Index (PDI)
A dimensionless measure of the breadth of the size
distribution, ranging from 0 (perfectly uniform) to 1 (highly
polydisperse)
3 Size Distribution Profiles
Presented as intensity, volume, or number distributions to
show the relative contribution of different size populations
Typical DLS Instrumentation
Core Components of a DLS System

• Laser Source: Typically He-Ne (633 nm) or solid-state

(532 nm)
• Optical System: Focuses laser and collects scattered light
• Sample Chamber: Temperature-controlled environment
for cuvette
• Detector: Avalanche photodiode or photomultiplier tube
• Digital Correlator: High-speed processor for real-time
correlation
• Computer System: For data analysis and user interface
Sensitivity and Size Range
DLS offers impressive versatility in particle size
analysis:

• Detects particles from sub-nanometer to

several microns
• Optimal performance range: 1 nm to 1 μm
• Lower limit depends on particle contrast and
instrument sensitivity
• Upper limit restricted by sedimentation of large
particles
• Highly sensitive to large particles or aggregates
Summary: DLS Measurement Principle
Laser Illumination
Brownian Motion
Monochromatic light source
Particles move randomly in illuminates sample
suspension, with movement
speed inversely related to size Light Scattering
Moving particles cause
fluctuations in scattered light
intensity
Size Calculation
Correlation Analysis
Diffusion coefficient converted
to hydrodynamic diameter Time-dependent intensity
fluctuations analyzed
mathematically
Importance of Sample Preparation in DL
Sample preparation is the foundation of reliable DLS
measurements:

• Directly impacts data quality, accuracy, and

reproducibility
• Poor preparation leads to artifacts and misleading
results
• Cannot be compensated for by instrument adjustments
• Requires understanding of both the technique and
sample properties
• Often the most overlooked aspect of DLS analysis
Essential Requirements
for DLS Samples
Dominant Brownian Motion
Particles must be suspended and freely moving in liquid
medium without sedimentation or floating

Stable Dispersion
Sample must remain uniformly dispersed throughout
measurement without aggregation or phase separation

Appropriate Size Distribution

Relatively narrow size distribution for optimal analysis;
multimodal distributions are challenging
Sample Cleanliness
Contaminants can severely compromise DLS measurements:

• Dust particles (typically micron-sized) overwhelm signal

from smaller particles
• Even trace amounts of large contaminants skew results
dramatically
• Filtration through 0.2-0.45 μm membrane filters often
necessary
• Use dust-free sample preparation environment when
possible
• Clean cuvettes with filtered solvents and lint-free wipes
Selecting Dispersing Media
1 Compatibility Factors 2 Common Dispersing Media
• Chemical compatibility with sample material • Ultrapure water for hydrophilic materials
• Stability of dispersion in the medium • Buffered solutions for biological samples
• Appropriate viscosity (known and stable) • Organic solvents for hydrophobic materials
• Suitable refractive index (different from • Surfactant solutions for stability enhancement
particles)
Sample Concentration Guidelines
Finding the optimal concentration balance is critical:

• Too concentrated: Multiple scattering effects distort results

• Too dilute: Insufficient signal-to-noise ratio for reliable
measurement
• Optimal concentration depends on particle size, material,
and optical properties
• Visual appearance should be slightly turbid but still
transparent
• May require trial measurements at different concentrations
Manufacturer Recommendations by Size
Particle Size Range Minimum Concentration Maximum Concentration

< 10 nm 0.5 mg/mL Varies by material

10-100 nm 0.1 mg/mL 50 mg/mL

100 nm - 1 μm 0.01 mg/mL 10 mg/mL

> 1 μm 0.1 mg/mL 10 mg/mL

Visual Check: Turbidity Indicator
Using Visual Appearance as a Guide

• Ideal: Slightly milky but still transparent

• Too dilute: Completely clear, indistinguishable
from solvent
• Too concentrated: Opaque or deeply colored
• Rule of thumb: You should be able to see
through the sample
• For colored samples, dilute until partially
transparent
Achieving Monodispersity
Sonication
Uses ultrasonic waves to break up aggregates and disperse
particles uniformly throughout the medium

Filtration
Removes large particles and contaminants while narrowing
the size distribution

Centrifugation
Separates particles by size/density to isolate specific
fractions or remove aggregates
Steps in Preparing a DLS Sample
Initial Dissolution
Dissolve or disperse sample in appropriate filtered solvent

Homogenization
Apply gentle mixing or sonication to achieve uniform dispersion

Filtration
Pass through membrane filter (0.2-0.45 μm) to remove dust and large contaminants

Dilution
Adjust to target concentration within manufacturer's recommended range

Equilibration
Allow sample to equilibrate to measurement temperature (typically 10-15 minutes)
Avoiding Aggregation
Particle aggregation is a common challenge that
compromises DLS results:

• Handle samples gently to prevent shear-induced

aggregation
• Avoid excessive vortexing that can introduce air
bubbles
• Use surfactants or stabilizers when appropriate
• Adjust pH to optimize surface charge and stability
• Consider steric stabilization for sensitive samples
• Monitor sample stability over time with repeat
measurements
Minimizing Bubbles
Air bubbles interfere with DLS measurements and must be
eliminated:

• Bubbles scatter light intensely and move unpredictably

• Ultrasonic degassing can remove dissolved gases
• Use gentle pipetting techniques to avoid introducing air
• Allow samples to stand briefly before measurement
• Centrifuge at low speed if bubbles persist
• Inspect sample visually before measurement
Measuring Multiple Concentrations
Benefits of Concentration Series Analysis

• Identifies concentration-dependent effects

• Reveals potential particle interactions
• Confirms measurement reliability
• Determines optimal concentration range
• Provides insight into sample stability

Prepare a series of dilutions from the same stock solution and measure each under identical conditions. Plot size vs. concentration to
Storage and Handling
Precautions
1 Temperature Considerations
Store samples at appropriate temperature to prevent degradation
or aggregation. Allow samples to equilibrate to measurement
temperature before analysis.

2 Light Sensitivity
Protect photosensitive samples from light exposure. Use amber
vials or aluminum foil wrapping for storage of light-sensitive
materials.

3 Contamination Prevention
Use only ultra-clean cuvettes, pipettes, and containers. Work in a
clean environment to minimize dust introduction. Close sample
containers promptly after use.
Sample Loading Tips
Best Practices for Cuvette Loading

• Fill to appropriate volume (typically 0.8-1 mL)

• Avoid under-filling or over-filling
• Prevent liquid on outside of cuvette
• Eliminate fingerprints with lint-free wipe
• Handle cuvette by top edges only
• Check for bubbles and tap gently to remove
• Position cuvette correctly in holder (note optical windows)
Calibration and Blank Runs
Proper calibration ensures accurate and reliable
measurements:

• Run blank measurements with filtered dispersant

medium
• Verify count rate is low and stable for the blank
• Use standard reference materials to validate system
performance
• Common standards: polystyrene latex beads, silica
nanoparticles
• Run standards periodically to track instrument
performance
• Document calibration procedures and results
Practical Example: Protein Nanoparticle
Sample Preparation Protocol

1. Prepare protein in appropriate buffer (pH 7.4

PBS)

2. Filter buffer through 0.22 μm membrane

3. Prepare concentration series (0.1-10 mg/mL)
4. Centrifuge at 10,000g for 5 minutes to remove
aggregates
5. Collect supernatant and transfer to pre-rinsed
cuvette

6. Equilibrate at 25°C for 10 minutes before

measurement
Practical Example: Polymeric Micelles
Critical Considerations for Polymeric Systems
• Ensure concentration exceeds critical micelle concentration (CMC)
• Use gentle dissolution methods to prevent polymer degradation
• Allow sufficient time for micelle formation and equilibration
• Filter through appropriate pore size to retain micelles while removing dust
• Consider temperature effects on micelle stability and size
• Monitor stability over time with repeated measurements
Troubleshooting Common
Issues
High Count Rate / Noise
• Likely causes: Dust contamination, concentration too high
• Solution: Filter sample, dilute, clean cuvette

Poor Data Quality

• Likely causes: Low concentration, laser attenuation
• Solution: Increase concentration, check instrument settings

Size Inconsistency
• Likely causes: Aggregation, sample instability
• Solution: Modify solvent, add stabilizers, adjust pH
Recap: Preparation Best Practices
Cleanliness
Concentration
Ensure dust-free samples,
Balance between signal
cuvettes, and solvents through
strength and multiple
filtration and proper handling
scattering effects

Consistency
Dispersion
Standardize preparation
Achieve stable, homogeneous
protocols for reproducible
suspension without
results
aggregation
DLS Applications Overview

Pharmaceuticals & Nanomaterial Research Environmental Science

Biologics
Size analysis of engineered Monitoring colloidal particles in
Characterization of drug delivery nanoparticles, quality control natural waters, studying pollutant
systems, protein aggregation during synthesis behavior
studies, formulation stability
Nanoparticle Size Characterization
DLS is essential for engineered nanoparticle development:

• Rapid size analysis of metal nanoparticles (gold, silver)

• Quality control of silica nanoparticle synthesis
• Monitoring size during surface modification steps
• Ensuring batch-to-batch reproducibility
• Studying stability in different media
• Real-time monitoring of nanoparticle growth
Biopharmaceutical Formulations
Critical Applications in Biologics Development

• Detection and quantification of protein aggregation

• Monitoring stability of therapeutic proteins during storage
• Quality control of monoclonal antibody formulations
• Screening of buffer conditions for optimal stability
• Assessment of thermal and mechanical stress effects
• Evaluation of freeze-thaw cycle impact on size distribution
Polymer and Micelle Analysis
Applications in Polymer Science

• Characterization of polymer nanoparticles

• Determination of micelle formation and CMC
• Size analysis of dendrimers and star polymers
• Monitoring polymer degradation kinetics
• Studying stimuli-responsive polymer behavior
• Investigation of polymer-polymer interactions
Liposome and Vesicle Characterization
DLS is invaluable for lipid-based drug delivery systems:

• Rapid size determination of liposomal formulations

• Quality control during manufacturing
• Stability assessment during storage
• Effect of drug loading on vesicle size
• Impact of surface modifications (PEGylation)
• Batch release testing for pharmaceutical applications
Extracellular Vesicle Analysis
Applications in EV Research

• Size profiling of exosomes (30-150 nm)

• Characterization of microvesicles (100-1000 nm)
• Quality control of EV isolation protocols
• Monitoring EV purity and concentration
• Studying EV stability in biological fluids
• Assessment of storage condition effects
Environmental & Colloidal Studies
Environmental Applications

• Monitoring colloidal particles in natural waters

• Studying pollutant transport mechanisms
• Analysis of nanoparticle behavior in ecosystems
• Water treatment process optimization
• Food and beverage colloidal stability
• Cosmetic formulation development
Industrial Quality Control
1 Manufacturing Process Control
DLS provides rapid feedback during production of nanoparticles,
emulsions, and suspensions, allowing real-time adjustments to
maintain consistent size specifications.

2 Batch Release Testing

Size and distribution data serve as critical quality attributes for
batch approval, ensuring consistency across production runs and
compliance with regulatory requirements.

3 Stability Monitoring
Regular DLS testing during storage provides early detection of
aggregation, settling, or other instability issues before they become
visible or affect product performance.
High-Throughput Screening
Modern DLS systems enable rapid analysis of multiple samples:

• Automated plate-based systems for 96 or 384-well formats

• Temperature ramp studies for stability assessment
• Formulation screening across multiple conditions
• Accelerated stability testing protocols
• Minimal sample volume requirements (20-50 μL)
• Integrated software for data analysis and comparison
Real-World Example: Vaccine Development
DLS in mRNA Vaccine Platforms

During the COVID-19 pandemic, DLS played a crucial role in

the rapid development and quality control of lipid
nanoparticle (LNP) mRNA vaccines:

• Characterization of LNP size (~80-100 nm)

• Optimization of formulation parameters
• Stability assessment during storage
• Batch release testing for manufacturing
• Evaluation of freeze-thaw resilience
Real-World Example: Water Treatment
Colloidal Monitoring in Water Systems

• Detection of nanopollutants in water sources

• Optimization of coagulation/flocculation processes
• Monitoring membrane fouling potential
• Assessment of filtration efficiency
• Characterization of natural organic matter
• Quality control of drinking water treatment
Research Example: Quantum Dots
DLS in Quantum Dot Development

DLS provides critical size information for quantum

dot synthesis and applications:

• Monitoring growth during synthesis

• Quality control of size-dependent optical
properties
• Assessment of surface functionalization
• Stability testing in various media
• Batch consistency verification
Comparison: DLS vs. Electron
Microscopy
Dynamic Light Scattering
• Rapid analysis (minutes)
• Statistical average of millions of particles
• Measures in native liquid state
• Non-destructive technique
• Limited resolution for multimodal distributions
• No shape information

Electron Microscopy
• Time-intensive sample preparation and imaging
• Direct visualization of individual particles
• Requires vacuum conditions
• Sample may be altered during preparation
• Excellent resolution of size and shape
• Limited statistical sampling
Advantages of DLS in Routine Analysis

Rapid Measurement
Complete analysis in minutes, enabling high sample throughput and real-time process monitoring

Minimal Sample Requirements

Small volume needs (as little as 20 μL) and non-destructive nature preserve valuable materials

Automation Capability
Modern systems offer walk-away operation with temperature control and multiple sample handling

High Sensitivity
Capable of detecting particles across a wide size range, from sub-nanometer to several microns
Limitations of DLS: Overview
Despite its utility, DLS has important limitations to consider:

• Assumes spherical particle shape in calculations

• Limited resolution for polydisperse samples
• Biased toward larger particles in mixtures
• Cannot distinguish between particle types
• Requires transparent or translucent samples
• May be affected by sample-specific artifacts
Sensitivity to Large Particles &
Agglomerates
The Disproportionate Impact of Large Particles

• Scattering intensity scales with d6 for Rayleigh scatterers

• A single 1 μm particle scatters as much light as a million
100 nm particles
• Even trace amounts of large particles or dust severely
skew results
• Intensity-weighted distributions particularly vulnerable
• Can lead to significant overestimation of average size
• Difficult to distinguish between sample aggregates and
contaminants
Multiple Scattering Complications
Consequences of High Concentration

At high concentrations, light can scatter from multiple

particles before reaching the detector:

• Shorter apparent path length of scattered light

• Faster correlation decay than expected
• Underestimation of particle size
• Non-linear relationship between concentration and
count rate
• Complex mathematics required for correction
Polydispersity and Interpretation Challeng
Resolving Power Limitations
• Cannot reliably resolve peaks differing by less than 3:1
in size
• Poor resolution of multimodal distributions
• Algorithm assumptions favor log-normal distributions
• High PDI values (>0.3) indicate potential interpretation
issues
• May report "average" size that doesn't represent any
actual population
• Different weighting schemes (intensity, volume,
number) yield different results
Limited Structural Information

Spherical Assumption Rod-Shaped Particles Complex Structures

DLS calculations assume Elongated particles report as Proteins and other complex
particles are perfect spheres, larger than their actual width due macromolecules yield only an
leading to inaccuracies for non- to rotational diffusion effects apparent hydrodynamic size, not
spherical structures actual dimensions
Sample Refractive Index Constraints
Optical Property Considerations

• Sufficient refractive index difference needed between

particles and medium
• Low contrast materials may not scatter enough light
• Refractive index errors lead to inaccurate size calculations
• Absorption by colored samples can reduce signal quality
• Multiple scattering in high-contrast systems complicates
analysis
• Complex calculations required for core-shell or coated
particles
Low Concentration Limitations
Signal-to-Noise Challenges

• Minimum concentration threshold required for reliable

detection
• Smaller particles require higher concentrations
• Low scattering intensity leads to poor statistics
• Background scattering becomes proportionally significant
• Longer measurement times may compensate but risk
sample changes
• Special low-volume cuvettes can help concentrate limited
samples
Sample Preparation-Related Pitfalls
Dust Contamination Improper Dispersion Solvent Incompatibility
• Ambient dust particles • Insufficient sonication or mixing • Poor particle-solvent
typically 1-10 μm • Leads to artificial aggregates interactions
• Dramatically skews intensity • • Can cause aggregation or
Results in overestimated
distribution particle size sedimentation
• Requires filtration and clean • May require surfactants or
handling stabilizers
Temperature Sensitivity of Diffusion
Critical Role of Temperature Control

• Diffusion coefficient directly affected by temperature

• 1°C change alters diffusion by approximately 2-3%
• Temperature gradients cause convection currents
• Sample must equilibrate to measurement temperature
• Viscosity changes with temperature affect calculations
• Some samples exhibit temperature-dependent
aggregation
Alternative Techniques: When is DLS Not Enough?
Nanoparticle Tracking Analysis (NTA)
Tracks individual particles to provide concentration and better resolution of mixed populations

Transmission Electron Microscopy (TEM)

Provides direct visualization of size, shape, and morphology at nanometer resolution

Analytical Ultracentrifugation (AUC)

Separates particles based on size and density for improved resolution of complex mixtures
How to Address DLS
Limitations
1 Complementary Techniques
Combine DLS with orthogonal methods like electron microscopy,
field-flow fractionation, or size-exclusion chromatography to build
a more complete picture of particle characteristics.

2 Advanced Data Analysis

Utilize regularization algorithms, multi-angle DLS, or depolarized
scattering to extract more information from complex samples and
overcome basic limitations.

3 Sample Optimization
Carefully adjust sample preparation parameters like
concentration, filtration, and dispersion techniques to minimize
artifacts and maximize data quality.
Best Practices for Reliable DLS Measurement
Validation Controls
Standardized Protocols Regularly measure standard
Develop and follow detailed SOPs reference materials to verify
for sample preparation and instrument performance
measurement 1
Multiple Replicates
Perform repeated measurements
to ensure statistical reliability

Operator Training
Thorough Documentation
Ensure thorough understanding of
Record all sample details,
both theory and practical aspects
preparation steps, and
measurement parameters
Summary and Key Takeaways
Dynamic Light Scattering is a powerful analytical technique when
properly applied:

• Provides rapid, non-destructive particle size analysis in solution

• Sample preparation critically impacts measurement quality
• Optimal for monodisperse samples in the 1 nm to 1 μm range
• Applications span pharmaceuticals, nanomaterials, and
environmental science
• Limitations include sensitivity to large particles and poor
resolution of mixtures
• Best results achieved by understanding principles, limitations,
and best practices
• Often most valuable when combined with complementary
techniques