The New Zetasizer Ultra - Anamet · The New Zetasizer Pro and Ultra 17 31 May 2018 The new...

31/05/2018

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© Malvern Panalytical 2018

The New Zetasizer UltraAdvance With Confidence

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Dr Mike Kaszuba

Technical Support Manager

E-mail: [email protected]


Zetasizer Ultra

• In addition to the features covered for the Zetasizer Pro, the Zetasizer Ultra has a number of other unique features

31 May 2018Title of the presentation2

Additional Features

mailto:[email protected]

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The New Zetasizer Pro and Ultra

31 May 20183

The new instruments at a glance

Zetasizer Pro Zetasizer Ultra (additional features) Back and forward detection angles Back, side and forward detection angles

ZS XPLORER software Multi Angle Dynamic Light Scattering (MADLS )®

Adaptive Correlation Particle Concentration measurementIntelligent Data Quality Advice Low volume disposable capillary sizing cell

Constant Current zeta potential mode Extended upper size limit (low volume disposable capillary sizing cell

Optical filter wheel


Zetasizer Ultra

Advance with Confidence

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Multi Angle Dynamic Light Scattering MADLS Video


MADLS®

• Polydisperse samples above the Raleigh region show particle size angular dependence resulting in different answers dependent on the measurement angle used

• Requires particle refractive index and absorption values

MADLS combines DLS measurements from three angles To produce a single, angular independent, size distribution with higher resolution

See the whole sample

Laser

Side

ForwardsBack

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MADLS

• MADLS ® gives higher resolution• Enables ~2:1 resolution compared to 3:1

typical of single-angle measurements

• Provides a more complete view of your sample – one answer rather than two or three different answers

• MADLS will reduce the need to duplicate analyses with other techniques such as TEM/SEM – saving time and expense

See the whole sample

MADLSSingle Angle

“The Zetasizer Ultra has dramatically increased our sample throughput, enabling us to accelerate our particle development cycle from 12 months to around a month.”

Pavel Abdulkin, Head of Particle Works

Blacktrace Holdings Ltd.


MADLS

• Important that samples are prepared at the correct concentration for all angles • Free of multiple scattering or number fluctuation issues

• MADLS operates best at sizes below 500nm

• A sample concentration calculator has been developed and will be incorporated directly into ZS XPLORER software

Green = concentration goodAmber = number fluctuations probableBlue = multiple scattering probable

Ensure Correct Sample Concentration For All Angles

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Particle Concentration

Simple, calibration-free particle concentration measurements



• Extension of MADLS measurement - calibration free -works best below 500nm

• Unique capability that complements NTA as it can reach into the sub-50nm region, which is more of a challenge for NTA

• Works for all homogenous (not mixture) sample types over a relatively broad concentration range (size-dependent)

• Gives total particle concentration and the particle concentration for each mode present in the sample

Easy, calibration-free concentration measurements

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• Measuring a material with known Rayleigh ratio (typically toluene) allows the system to be characterised at all scattering angles and attenuators so the sample scattering can be normalized

• Characterization data is stored on instrument and done at final build so all Zetasizer Ultra instruments will have a characterization data set to use straight from the box• Users may run additional characterizations from the instrument page if required

• It is important that accurate size results are obtained as the particle concentration relies on accurate size data – poorly reproducible size data leads to poor concentration data!

• Particle concentration is like a MADLS measurement but the sample buffer scattering needs to be known

How does it work ?



• Applicable to a wide range of sample types• Low density materials (e.g. liposomes and polymer

particles) • Higher density materials (e.g. silica, metallic nanoparticles

such as gold)

• Particle concentration measurements require the following:• Particle/molecule optical properties (refractive index and

absorption)• Dispersant refractive index and viscosity• Dispersant scattering intensity (measurable)

Sample types and requirements

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• Dynamic concentration range for some typical material types – no need or less need to dilute Easy, Calibration Free Concentration Measurements

1.00E+071.00E+081.00E+091.00E+101.00E+111.00E+121.00E+131.00E+141.00E+151.00E+161.00E+171.00E+181.00E+191.00E+20

1 10 100 1000

Par

ticle

con

cent

ratio

n [#

/mL]

Diameter [nm]

Concentration range for gold, protein and latex

max PS latex min PS latex max goldmin gold min protein max protein

• A study with National Physical Laboratory (NPL, UK) shows data has good agreement with nominal values

• Error is in the region of ~30 % which is excellent, considering NPLs work shows classical technologies such as SAXS, ICP-MS, UV-Vis all give approx. ~20% error and take significant time and operator skill


Low Volume Disposable Sizing Cell

High quality data with minimal sample usage

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• The cell holder allows 1 mm x 1 mm high quality glass capillaries to be used

• Minimum measurable volume is 3 µL

• Capillaries are disposable but can be cleaned and reused

• Cell is used with the side detection angle of the Zetasizer Ultra

• Can be filled using gel-loading tip or simply capillary action to draw-up the sample

• Enables repeatable analysis at ultra-low sample volumes – high quality data at reduced cost

Low volume with high quality data



• Hindered convection/sedimentation in the capillary cell allows for larger particles to be measured without the need to density-match

• PS latex measured to 10 µm diameter

• Useful for anyone with larger material present in their sample and for top-down processes (e.g. milling)

Extended Size Range

Erro

r %

Nominal Particle Size (nm)

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The New Zetasizer Pro and Ultra

31 May 201817

The new instruments at a glance

Zetasizer Pro Zetasizer Ultra (additional features) Back and forward detection angles Back, side and forward detection angles

ZS XPLORER software Multi Angle Dynamic Light Scattering (MADLS )®

Adaptive Correlation Particle Concentration measurementIntelligent Data Quality Advice Low volume disposable capillary sizing cell

Constant Current zeta potential mode Extended upper size limit (low volume disposable capillary sizing cell

Optical filter wheel


Zetasizer Ultra

Applications

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Surfactant Micelles

• Micelles are 3D aggregated structures dispersed in a liquid colloid• For micelles to be formed, the unimers have to display an amphiphilic character, with a

large solubility difference between the hydrophilic and hydrophobic segments• Continuous addition of surfactant induces a saturation of molecules on the surface and

this is point where unimers start aggregating to form micelles i.e. critical micelle concentration (cmc)

Introduction

C < CMCC < CMC C > CMCC > CMC


Micelle CharacterizationZetasizer Ultra

Unfiltered resultsUnfiltered results Steady-state resultsSteady-state results

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Micelle CharacterizationCritical Micellization Temperature (CMT)

• With the new Zetasizer Ultra, there are several ways the user can study the critical micellization temperature of surfactants

• An established method to achieve this is to monitor the derived count rate with increasing temperature

• Owing to the relationship between particle’s diameter and intensity of scattered light, any increase in the particle size will be readily noticed in the count rate



• When both unimers and micelles are present in a solution, DLS may not be able to resolve the two populations

• MADLS gives higher resolution size results, particularly suitable for multimodal samples

MADLSMADLS 3-angle measurement3-angle measurement

Back

Forward

Side

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Micelle CharacterizationMADLS


• Particle concentration results show that with increasing temperatures the particle concentration decreases

Micelle CharacterizationParticle Concentration

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• From the initial and final particle concentration values (before and after micellization, respectively), it is possible to calculate an approximate aggregation number (N):

푵 = 푰풏풊풕풊풂풍푷풂풓풕풊풄풍풆푪풐풏풄풆풏풕풓풂풕풊풐풏푭풊풏풂풍푷풂풓풕풊풄풍풆푪풐풏풄풆풏풕풓풂풕풊풐풏

• The N obtained of ~ 64 unimers per micelle, is in line with those reported in the literature using SLS (N = 68)

• Zhou, Z. K. & Chu, B. (1998) Light-Scattering Study on the Association Behavior of Triblock Polymers of Ethylene-Oxide and Propylene-Oxide in Aqueous-Solution. J. Colloid Interface Sci. 126, 171–180



Gold Nanoparticles

• Particle Works (brand of Blacktrace Holdings Ltd) produce gold nanoparticles which must be highly monodisperse and have high batch-to-batch consistency, • CV values as low as 5% for the size distribution and 2.5% for the batch consistency

• Currently, Particle Works use TEM as primary technique for characterizing their samples for QC

• TEM is time consuming and expensive requiring an experienced user to perform the analysis

• Particle Works were therefore keen to explore whether the Zetasizer Ultra could be used to reduce the amount of TEM analysis required

Particle Works

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Gold Nanoparticles

• 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

Particle Works

Sample Name Target Size (nm)Au-2-016-7 10

Au-2-016-13 15Au-2-17-6 20 20Au-2-037-4 50 50Au-2-041-3 50 50


Gold Nanoparticles

• MALDS results showed size populations much larger than the expected 10nm• No 10nm peak appears in the distribution• Sample failed to meet specification

• TEM image also shows that there are multiple large particles but also shows the expected 10nm particles

• 10nm particles being masked in the MADLS measurement by the larger particles

Au-2-016-13 Target Size 10nm

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Gold Nanoparticles

• MADLS suggests that sample is within specification • Narrow size distribution measured

• TEM measurements disagree • Majority of the populations fall within the expected range, • However, there is a tail of particles below target size

• MADLS measurements unable to pick up smaller sizes• Scattering of the sample more skewed towards the larger sizes masking smaller particles



Gold Nanoparticles

• MADLS gives monomodal, narrow distribution• Sample meets specification and confirmed by TEM • Sample commercially viable


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Gold Nanoparticles

• TEM shows uniform size distribution that falls within specifications • Rings around many of the particles• Unclear if these are real

• MADLS gives narrow distribution within specifications• If rings were around particles when dispersed,

DLS distribution would be much wider and larger in size

• Likely that some of the surfactant in the dispersion has dried around particles causing these visible rings in TEM

• Sample is commercially viable



Gold Nanoparticles

• MADLS shows unexpected small peak below 10nm• DDLS used to determine whether peak is due to translational diffusion of particles

• i.e. confirm whether another population present in the sample• Vertically-polarized backscatter measurement similar to backscatter measurement• Horizontally-polarized measurement has a much higher relative intensity for the smaller size

peak than the higher size peak• Small size peak is due to rotational diffusion of non-spherical particles


All polarizations Vertical Horizontal

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Gold Nanoparticles

• 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 out of specification samples have been identified using the Zetasizer Ultra alone


Conclusions


Liposomes

• Liposomes used were Formumax HSPC/CHOL liposomes prepared and diluted in GibcoPBS at pH 7.2

• Zetasizer Ultra and NanoSight NS300 were used to measure the liposomes at 25°C• Samples were measured 5 times on the Zetasizer Ultra using the particle concentration

measurement• Using the NanoSight NS300, 5 videos of 60 seconds each were recorded while using

the syringe pump and a 405nm laser

Experimental

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Liposomes

• Size results across the dilution range measured by NTA and MADLS show a consistent mean size value of around 90nm

• From 1:10 to 1:1000000 dilution, the mean size value does not significantly change


Particle Size Zetasizer Ultra and NS300


Liposomes

• Size distributions for the 1:10,000 dilution measured with MADLS and NTA

• NTA is able to pick up on a small shoulder of liposomes that are slightly larger than the main population

• These are too few and too close in size to be detected with MADLS


Particle Size Zetasizer Ultra and NS300

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Liposomes

• Good agreement between Zetasizer Ultra and NS300 for the 10,000x and 100,000x dilutions • Both techniques are measuring in a suitable

concentration range• Less agreement between the two techniques at

the 1,000,000x dilution• Zetasizer measuring about twice as many

particles as Nanosight• At this concentration, scattering intensity is

very low in the Zetasizer at side scatter• Difficult to autocorrelate

• Result expected to be less accurate than at the higher concentrations


Particle Concentration on Zetasizer Ultra and NS300


Liposomes

• Zetasizer Ultra’s MADLS based concentration measurement demonstrated to be able to measure both the size and concentration of liposomes with good agreement with NTA in the concentration range where they overlap

• Zetasizer Ultra shows wider concentration range at which it can measure the sample• NTA able to detect a small shoulder of larger liposomes• Both techniques are complementary

• MADLS provides rapid size and concentration results for a wide range of concentrations with minimal sample preparation

• NTA able to work at lower sample concentrations and provide more size distribution detail

Conclusions

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www.malvernpanalytical.com

Thank you for your attention.Any questions?

[email protected]

http://www.malvernpanalytical.com

mailto:[email protected]

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