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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.
Sensor
Chip
Archimedes
Resonant Mass Measurement
Measuring Particle Mass in Fluid
-150 -100 -50 0 50 100 150
-400
-300
-200
-100
0
Fre
quen
cy S
hift (m
Hz)
Time (msec)
200
1. 3.
2.
1.
2.
3.
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
Buoyant Mass Measurement
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 Generation Sequencing
• Semiconductor Cleaning
• Food Scenting
5 m
Generic example of ability of RMM to differentiate between
bubbles and lipid droplets
Effect of Loading Pressure on Bubbles
5psi
10psi
20psi
30psi
35psi
Bubbl
es
As PLoad increases,
number of bubbles
decreases
0.265 0.269 0.272
0.24
0.357
0.15
0.2
0.25
0.3
0.35
0.4
0 10 20 30 40Me
an
Siz
e (
um
)
Loading Pressure (psi)
294 267
247
35 22
0.15
50.15
100.15
150.15
200.15
250.15
300.15
350.15
0 10 20 30 40
Nu
mb
er
of B
ub
ble
s
Loading Pressure (psi)
As PLoad increases,
mean bubble size
increases
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
5psi
10psi
20psi
30psi
35psi
Bubbles prepared by using agitation method
Sample contains bubbles + excess lipid
Samples used for each Archimedes measurement aliquoted from same
vial
Loading Pressures (psi): 35, 30, 20, 10, 5
Total number particles (lipid + bubble) counted per experiment: 500
Lipids
As PLoad increases, number of lipid
particles increases
206 233 253
465 478
0
100
200
300
400
500
600
0 10 20 30 40
Nu
mb
er
of L
ipid
P
art
icle
s
Loading Pressure (psi)
› Shake Time Bubb
les
Lipid
s
20 seconds
45 seconds
90 seconds
20 seconds
45 seconds
90 seconds
Mean size clearly increases with shake time –may be
due to coalescence
Lots of lipid particles at 20sec shake time
161
10 1
0
20
40
60
80
100
120
140
160
180
0 20 40 60 80 100
Num
be
r o
f lip
ids
Shake Time (sec)
188
286
395
150
200
250
300
350
400
450
0 20 40 60 80 100
Siz
e (
nm
)
Shake Time (sec)
Effect of Gas Pressure
Bubb
les 6 psi
11 psi
16 psi
Lipid
s 6 psi
11 psi
16 psi
Change in mean bubble size does not
seem significant
Not much difference in 6 and 11 psi
samples, but 16 psi has many more lipids.
Suspect that higher pressure is preventing
bubbles from forming, hence more lipids
Shelf Life – USA bubble samples shipped
March 24th, 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
= the particle velocity
= laser wavelength
q = scattering angle
› Frequency shifts determined
by Fourier transformation and
phase analysis light scattering
f = 2 sin(q/2)/
› Measured electrophoretic mobility converted into zeta
potential using Henry’s equation
Typical ultrafine bubble size distribution measured by NTA
Con
cen
trati
on
(10
6 b
ub
ble
s/m
L)
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
