The outline and latest status of fine bubble measurement techniques Dr Stephen Ward-Smith Malvern Instruments Ltd
Techniques used for characterising fine and ultrafine bubbles › Particle tracking analysis (aka Nanoparticle tracking analysis, NTA, PTA) › Resonance Mass Measurement › Dynamic Light Scattering (aka Photon Correlation Spectroscopy) › Laser Diffraction › Zeta potential › Others (electrozone sensing, ultrasonics, static multiple light scattering, image analysis) › All are sizing techniques, bar Zeta Potential which is a measure of particle charge
Brief Summary Technique Size Range Laser Diffraction <100nm to >2mm Dynamic Light Scattering <1nm to >1 micron NTA <30nm to >1 micron Archimedes <35 nm to > 2micron Image Analysis <1um to >3 mm › Particle size ranges will depend on the sample and the sensor used.
Resonant Mass Measurement Archimedes Sensor Chip
Measuring Particle Mass in Fluid 0 Frequency Shift (mHz) 1. 3. -100 -200 1. -300 2. 3. -400 2. -150 -100 -50 0 50 100 150 200 Time (msec)
Buoyant Mass Measurement The buoyant mass of a particle is always measured relative to its surrounding fluid. A particle of dust will therefore have a negative buoyant mass in water, and a bubble will have a positive buoyant mass in water
Resonant Mass Measurement › Populations of particles with negative and positive buoyant can be detected using RMM. › The limit of detection for RMM with ultrafine bubbles is around about 100nm based on the sensitivity of the instrument and the mass differential of the fluid and 100nm bubbles. › RMM is unique in its ability between dust particles and ultra fine bubbles of the same size in the same sample. › However as proven with NTA some ultrafine bubbles will be generated beyond the limit of detection for RMM. › Efforts are being made to push the limit of detection to smaller sizes with RMM.
What are Microbubbles? • Gas: Air, Perfluorocarbon, Sulfur Hexafluoride, etc… – High Molecular Weight Gas • Shell: Polymer, Lipid, Albumin, etc… • Size: Typically < 8 μm for Contrast Agent Applications • Microbubble Contrast Agent – Molecular Imaging – Blood Perfusion-Based Imaging – Gene Therapy • DNA Fragmentation for Next 5 m Generation Sequencing • Semiconductor Cleaning • Food Scenting
Generic example of ability of RMM to differentiate between bubbles and lipid droplets
Effect of Loading Pressure on Bubbles 350.15 294 267 300.15 247 Number of Bubbles 250.15 As P Load increases, 5psi 200.15 number of bubbles Bubbl 10psi 150.15 decreases 100.15 es 20psi 35 22 50.15 30psi 0.15 0 10 20 30 40 35psi Loading Pressure (psi) 0.4 0.357 0.35 Mean Size (um) As P Load increases, 0.3 0.272 0.265 0.269 mean bubble size 0.24 0.25 increases 0.2 0.15 0 10 20 30 40 Loading Pressure (psi)
Update: Measuring bubbles with RMM › Bubbles measured successfully during 2 customer demos in 2015 › During both demos used standard operating conditions Pload 35psi. Able to demonstrate measurement of lipids and bubbles. › Concern that 35psi loading pressure may cause bubbles to collapse › US customer provided us with samples to study loading pressures Bubbles prepared by using agitation method Sample contains bubbles + excess lipid 5psi Samples used for each Archimedes measurement aliquoted from same Lipids 10psi vial 20psi Loading Pressures (psi): 35, 30, 20, 10, 5 Total number particles (lipid + bubble) counted per experiment: 500 30psi 35psi 600 Number of Lipid 465 478 500 Particles 400 253 300 206 233 200 100 0 0 10 20 30 40 Loading Pressure (psi) As P Load increases, number of lipid particles increases
› Shake Time 450 395 400 Bubb Size (nm) 350 les 20 seconds 286 300 45 seconds 250 188 90 seconds 200 150 0 20 40 60 80 100 Shake Time (sec) Mean size clearly increases with shake time – may be due to coalescence Lots of lipid particles at 20sec shake time Lipid 20 seconds 161 180 s Number of lipids 160 45 seconds 140 90 seconds 120 100 80 60 40 10 20 1 0 0 20 40 60 80 100 Shake Time (sec)
Effect of Gas Pressure Bubb Change in mean bubble size does not les seem significant 6 psi Not much difference in 6 and 11 psi 11 psi samples, but 16 psi has many more lipids. 16 psi Suspect that higher pressure is preventing bubbles from forming, hence more lipids Lipid s 6 psi 11 psi 16 psi
Shelf Life – USA bubble samples shipped March 24 th , 2016 5 days 25 days 70 days *These are the bubbles sent in March *Excellent shelf life
Zeta potential of bulk ultrafine bubbles: effects of salt, pH and surfactant
Zeta Potential • Zeta potential measurement results in an absolute value reported in [mV] and serves as a predictor of suspension stability. High Zeta Potential Low or Zero Zeta Potential Unstable suspension Stable suspension
Electrophoretic Light Scattering (ELS) Measured parameter is the frequency shift of the scattered light. The frequency shift is proportional to the electrophoretic mobility, which is a function of the particle surface potential. Hence ELS gives us information regarding the charge on the particle.
Measuring Zeta Potential › Electrophoresis = movement of a charged particle relative to the liquid it is suspended in under the influence of an applied electric field Particles velocity dependent on: Zeta potential Field strength Dielectric constant of medium Viscosity of the medium
Laser Doppler Electrophoresis › Scattered light is frequency (Doppler) shifted › Frequency shift f = 2 sin( q /2)/ = the particle velocity = laser wavelength q = scattering angle › Frequency shifts determined by Fourier transformation and phase analysis light scattering › Measured electrophoretic mobility converted into zeta potential using Henry’s equation
Typical ultrafine bubble size distribution measured by NTA Concentration (10 6 bubbles/mL)
Ultrafine bubbles generated in a salt solution are less stable than those generated in distilled water
Adding salt to a suspension of ultrafine bubbles reduces their stability
Generating ultrafine bubbles in a low pH medium reduces their stability isoelectric line
Lowering the pH of a suspension of ultrafine bubbles reduces their stability
Adding surfactant to a suspension of ultrafine bubbles increases their stability
Further work › Do DLS / Zeta in series with each other to see the effect Zeta potential has on size. Would expect bubbles in systems with zeta potential in the -30 mV to + 30 mV area to grow larger over time compared to those outside this area. › Need to do some daily monitoring experiments.
Acknowledgements › Archimedes team at Malvern Instruments › US customer for supplying bubble / lipid samples › Mostafa Barigou and group at University of Birmingham
Thank you for your attention -Any Questions? Steve Ward – Smith - stephen.ward-smith@malvern.com 28
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