Particle separator utilizing

ultrasonic standing waves in

microfluidic channels

Andreas Sandin
Experimental Biophysics
Lund University
May 30, 2006

CONTENTS
1
2
3
4
5
6
7

Abstract .................................................................................................................................. 2
Purpose of this project............................................................................................................ 3
Basic theory............................................................................................................................ 3
Particle separator designs ....................................................................................................... 4
Band formation....................................................................................................................... 6
Experimental setup................................................................................................................. 6
Measurements: Separation of blood and fat lipids in cream .................................................. 6
7.1 Results and conclusions .................................................................................................. 7
8 Lateral movement of a particle in the flow ............................................................................ 9
9 Measurements: Separation of plastic particles of different size........................................... 10
9.1 Results and conclusions ................................................................................................ 10
10 Acknowledgements ............................................................................................................ 13
11 References .......................................................................................................................... 13

1 Abstract
Separation science is a wide and well investigated area. Techniques such as many kind of
filters, centrifugation or dielectrophoresis has been developed during the years. Many of these
applications are used in the medical area which demands high performance and polite devices.
Blood washing is an important application where it is desirable to separate red blood cells
(erythrocytes) from the carrier medium, the blood plasma. This could be the case when the
plasma contains high levels of drugs, inflammatory or coagulation.
Blood washing is also necessary during very complicated surgeries, such as cardic surgery
where the blood loss is evident. This is a better alternative than transfusion of new blood to
the patient in such matter to eliminate transfusion transmitted disease and immunologic
reactions to allogenic blood. Another thing to advocate blood washing is that fresh blood is
today an article in short supply.
Currently, centrifuge-based methods are utilized for blood washing. But this technique suffers
from two major drawbacks. First it can not remove lipids sufficiently enough which will leak
out in the blood from surrounding fat tissue. Lipid micro emboli can affect different organs
such as the brain and kidney by obstruct the capillary system in the body[1]. Secondly the
centrifuges expose the red blood cells to strong deformation forces which lead to the cells to
break up and cause a hemoglobin leakage in the plasma.
The department of electrical measurement at Lund institute of technology has developed a
new technique which is free from these problems.
This technique uses an ultrasonic standing wave in a micro fluidic channel to separate
different particles from each other.
Particles that are exposed to an ultrasonic standing wave field are affected by an acoustic
radiation force and will be collected depending on its acoustic properties.

2 Purpose of this project

The aim of this project was first to simulate a blood wash of bovine blood contaminated with
fat globules from cream by using an ultrasonic wave in a micro fluidic channel. This was
made to get a view of the application and to get a clear understanding in the basic theory and
idea of this technique. In the second part the separation efficiency of particles of different
sizes by changing the amplitude of the wave, was investigated.

3 Basic theory
All waves can be reflected. If a wave is reflected back onto itself it will interfere with itself.
This creates an interference pattern both constructive and destructive which will occur as
antinodes and nodes respectively. A sound wave is a longitudinal wave. It is called
longitudinal because the molecules in the medium are moving back and forth in the same
direction as the wave. The molecules are vibrating sinusoidal and it is useful to see them as
they are connected by springs with a displacement from equilibrium of the individual air
molecules. At a node the molecular displacement has its maximum value and at the anti node
it has its minimum value.

Fig 1. A model of the molecules in which the wave is traveling through.

An acoustic standing wave can mathematically be described as:
2t
2x
(1)
y = A cos
+
+ sin
2
2
T

Where (y) describes the displacement to a certain position (x) at the time (t). A is the
amplitude of the wave and is the wavelength. T is the period time and the phase shift
between two traveling waves is expressed with . The cosine part describes the wave in space
and the sinus part describes the time dependence. If we have the case of a standing wave the
phase shift is fixed, by looking at the cosine part it is easy to see that the distance between two
consecutive nodes is /2. Formula 1 can then be rewritten in terms of pressure:
2t
2x
p = p 0 sin

cos
T

(2)

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A standing sound wave can create an acoustic force on particles in the x direction. This force
has been theoretically expressed by Yosioka and Kawasima[2]:

p 2V
4x
Fr = 0 c w ( , ) sin

2
5 2 w c

= c
2 c + w w

(3)
(4)

w = Compressibility of medium

c = Compressibility of the particles

w = Density of medium

c = Density of the particles

Vc = Volume of the particle

The direction of the force is dependent on the sign of the -factor. To separate two types of
particles they need to have opposite signs of the -factor. One particle type will end up in the
pressure node and the other type in the pressure anti node. This effect can be used to separate
different particles in a continuously and laminar flow. A flow is laminar if there is no
turbulence in the liquid. Particles will keep their lateral position even after leaving the
acoustic force field.
For example if the particles are erythrocytes with 0.3 and lipid particles with -0.3, the
erythrocytes will be dragged to the centre node while the lipid particles are forced out to the
anti nodes. However, can not be zero thus there will be no net force on the particle.

Figure 2. (a)The wave profile in a micro fluidic channel with two different particles
positioned, one in the pressure node and the other one in the pressure anti node. (b) Top view
of the continues separation [3].
It is important to have a sufficiently strong force to separate many particles in a short time.
According to formula 3 the force is increased with increased pressure amplitude. This works
well but only to a certain critical limit. The increased power dissipation will heat up the
surrounding medium to a critical temperature and even create gas bubbles. There is also a risk
for acoustic streaming to occur if the pressure amplitude is high.

4 Particle separator designs

The micro fluidic channel was made in Silicon. Silicon is preferable because of its good
acoustic properties. The fabrication process is well known and by using photolithography and

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anisotropic wet etching, perfectly vertical walls can be obtained with a hydrophilic surface.
The channel was 375 m wide and 125 m deep.

Figure 3. Cross section of the micro fluidic channel with an ultrasonic standing wave in
fundamental resonance mode orthogonal to the flow direction [4].
The ultrasonic waves were generated by a standard 2 Mhz resonant piezoelectric crystal, PZT
(PZ26 Ferroperm Piezoceramics AS, Kvistgard, Denmark). This PZT must be attached to the
channel without preventing the waves to propagate into the channel. By using an ultrasonic
gel which is used in mammography investigation this problem can be minimized. To enclose
the channel a boron-silicate glass lid was bonded to the silicon wafer. The device had two
inlets and two outlets but only one inlet was used so one of them was plugged with piece of
metal to avoid leakage.
a)

b)

Fig. 4 a) Top view of the separator chip. b) Bottom view of the separation chip.

5 Band formation
To create a stable standing wave in the micro fluidic channel it is important to choose
frequency which fits well to the dimension of the cross section of the channel.
The choice of ultrasonic frequency is given by:
c
f =
(5)

Where c is the speed of sound in the liquid.

In these experiments the fundamental resonance mode (/2) was used. This will create a
single band formation in the middle of the channel. It is also possible to operate at even higher
harmonics with the possibility to generate multiple bands.

Fig. 5 Different modes of standing pressure waves in a micro fluidic channel[5].

6 Experimental setup
The outlets and the inlet of the chip were connected with Teflon tubes with an inner diameter
of 0.8 mm. The two outlet tubes were used to collect the sample after the separation and the
length was varied depending on how much of the sample that was needed for further
investigations. To create a stable flow in the channel, an under-pressure was created by two
syringe pumps (SP260P, World Precision Instruments Inc, Sarasota, FL, USA) connected to
the outlet tubes. The PZT was supplied via a high frequency power amplifier (Model 75A250,
Amplifier Research, Southerton, PA, USA) and the frequency was set by a function generator
(Model HP 3325B, Hewlett-Packard Inc, Palo Alto, CA, USA). The signal amplitude was
controlled by a digital oscilloscope (Model HP 54503A). Finally the separator was placed
under a bright field microscope connected to a CCD-camera to study the process in real time.

7 Measurements: Separation of blood and fat lipids in cream

By separating bovine blood and cream in an ultrasonic particle separator, a realistic
simulation of blood washing can be made.
The cream that was used was coffee cream with a fat content of 12 %.
The frequency was 2 MHz which was the resonance frequency of the PZT, to get a standing
wave of the first order in the channel. The signal voltage was varied from 0 to 20 Vpp and the
flow rate was 50 l/min. To measure the separated samples in the outlet tubes a centrifuge
(Haematokrit 2010, Hettich Zentrifugen, D-78532 Tuttlingen, Germany) was used. The

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sample was put in to a thin glass pipe with the top of the pipe directed to the centre of the
centrifuge.
A centrifuge separates the particles by density. The erythrocytes in the blood have a larger
density than the fat in the milk so they were collected in the bottom of the pipe. In the middle,
water and other particles in the milk and the blood was collected and at the top the lipid
vesicles with the lowest density. Each sample was centrifuged for two minutes at 13000 rpm
after which of the participation of blood and fat could easy been read out in the pipe.
Assume that:
A = relative particle fraction collected from the centre outlet
B= relative particle fraction collected from the side outlets
Then the separation efficiency of the blood was determined as the ratio of the percentage
fraction blood from the centre outlet to the percentage total fraction blood in the fluid at all
three outlets:
A
Separation efficiency = 100
(6)
A+ B
This was made in the same way for the cream but with the B as numerator in formula 6.

7.1 Results and conclusions

The result showed that the separation process was successful for both the mixtures with an
efficiency of at least 83 %.
Bovine blood has almost the same properties of human blood and the consistency is quite
inert. So was the cream and to avoid clogging it was necessary dilute the sample. They were
diluted with common NaCl solution because that is a natural medium in the body and will not
affect the red blood cells.
Both of the mixtures were tested without any acoustic wave and the side- and centre outlet
tubes then had the same fraction blood and cream. This shows clearly that erythrocytes and
lipids behave in the same way in a laminar flow and that the acoustic radiation force is the
only thing that will give rise to separation.
The voltage can be seen as proportional to the amplitude of the standing wave.
The 25 Volume % sample needed a voltage of 17 Vpp to get the best and most stable flow
while the more diluted sample only needed 10 Vpp. This shows that a higher concentration of
particles needs a stronger acoustic force to separate them. High concentrations will disturb the
standing wave because the speed of sound is different in the particles than in the liquid
medium which will affect the resonance frequency according to formula 5.
If the Voltage was increased to even more than 17 Vpp, the flow became unstable and the
separated blood in the middle started to show fluctuations. This was due to that the PZT
quickly became hot because of the increased power dissipation. The gel which attaches the
PZT to the channel became dry and sticky and it lost its acoustic properties.

(a)

(b)

Figure 6. (a) A mixture of 25% blood and 25% cream flowing through the chip without
ultrasonic wave. (b) The same mixture with the ultrasonic wave turned on. The flow rate was
50 l/min and a Voltage of 17 Vpp.
(a)

(b)

Figure 7. (a) A mixture of 12.5% blood and 12.5% cream flowing through the chip without
ultrasonic wave. (b) The same mixture with the ultrasonic wave turned on. The flow rate was
50 l/min and a Voltage of 10 Vpp.
Content of
the volume
before
separation

frequency

amplitude

Flow rate

Content of
the volume
after
separation, in
the centre
outlet

Content of
the volume
after
separation,
in the side
outlets

Blood
25 %
50
21 %
2%
2 Mhz
17 Vpp
l/min 3 %
Cream
25 %
20 %
Blood
12,5 %
50
12 %
0,5 %
2 MHz
10 Vpp
l/min 1 %
Cream
12,5 %
5%
Table 1. Separation efficiency for two different diluted mixture of blood and cream.

Separation
efficiency

91 %
87 %
96 %
83 %

8 Lateral movement of a particle in the flow

The primary force acting on a particle in a laminar flow is, according to equation 2, the
acoustic radiation force, Fr. It is also a viscous drag force, Fd which is directed in the opposite
direction. These forces acts perpendicular to the direction of the flow. Assume that the
channel flow is along the y-axis and the position over the width of the channel is denoted x.
The drag force is described by [5]:
Fd = 3dx

(7)

Where d is the particle diameter, x is the velocity perpendicular to the flow and is the
dynamic viscosity of the fluid.
This is the same force which prevents a body from falling faster than a certain maximum
velocity when falling out from an air plane. Or when falling into a river, the viscous drag
force will accelerate the body to make it follow the stream.
The resulting movement of the particle along the x-axis is then governed by this equation:
p 02V p w
( , ) sin 4x 3dx
mx = Fr + Fd =
(8)
2

The Volume of a spherical particle is given by

4 3 d 3
r =
3
6
Expressing the mass of the particle in terms of the volume times the density, p, the equation
becomes:
2 d 3

p 0
w
3
d
6
( , ) sin 4x 3dx
x
(9)
p =

6
2

With some rearrangement, the acceleration of the particle is given by the following equation.
p 2
x = 0 w
2
p

( , ) sin 4x 18 x
2

pd

p 02 w
,
C1 =
2 p

C2 =

x +

C1
4x
x + C 2 sin
=0
2
d

(10)

18

(11)

p
(12)

This differential equation describes a typically expression for a damped free vibration model
[6] where C1/d2 is the damping factor. The damping factor shows that larger particles are
damped less then smaller particles.

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9 Measurements: Separation of plastic particles of different size

In this part of the experiments the separation efficiency of different large particles at different
applied voltage was measured. The major aim was to see if the efficiency can be related to the
size of the particles in the same way as the theoretical measurements.
The measurements were made at a Voltage 0f 0 Vpp, 2 Vpp, 4 Vpp, 6 Vpp and 8 Vpp. At even
higher the flow became unstable.
The particles had the size of 1, 3, 5 and 8 m in diameter. They were made of Polystyrene
mixed in distillated water. Furthermore the separation efficiency was determined in the same
way as in experiment 1 according to formula 6. Afterward the separation the medium was
collected and analyzed using a multisizer ( model 3, Beckman Coulter) to determine the
concentration of the particles. 150 ml of all the samples were collected by using two 0.15 m
long Teflon tubes connected to the syringe pumps. The flow rate was 50 l/min so all the
separations lasted for at least 3 minutes, after the flow had been stabilized. Between the
separations the system was rinsed with NaCl for cleaning and to remove air bubbles.

9.1 Results and conclusions

The Voltage is proportional to p0 and in theory the acoustic force is changed by the square of
the amplitude of the wave. This appearance can roughly be seen for the 3 micron and 5
micron particles (fig. 8).
According to figure 8 the separation efficiency is increasing with increased Voltage apart
from the 1 micron particles. Its efficiency is even higher without the any acoustic wave. This
can be explained by the fact that small particles have a propensity to be collected in the
middle of the channel in a laminar flow. This is because the flow profile is parabolic with a
slower flow in the periphery. Why they are not affected by increasing amplitude is probably
due to that the acoustic radiation force is not sufficient to attract them.
The results indicate that 1 m particles are simply too small to separate with this chip.
A limitation of the particle separation has been reported by Augustsson [5] where the critical
particle was estimated to approximately 1,8 m.
1 m
3 m
5 m
8 m
58.29
46.80
47.11
45.28
56.35
60.95
65.90
72.37
51.63
65.04
76.57
94.93
52.76
62.59
72.46
85.70
55.80
91.00
97.96
95.95
Table 2. Separation efficiency in percentage for different diameter of the
particles at different applied voltage.
0Vpp
2Vpp
4Vpp
6Vpp
8Vpp

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Fig. 8 Separation efficiency of different size of the particles as a function of the Voltage
p0. Constant frequency of 2 Mhz and a flow rate of 50 l/min
The best efficiency was found at a voltage of 8V (figure 9a) for particles of 5 and 8 m.
Equation 12 describes the lateral movement of the particles which shows that large particles
are less damped and therefore they can move easier to the centre of the channel. At an applied
voltage of 2V, 4V and 6V the efficiency shows a linear relationship for the particle size. One
theory has been estimated by simulations which support this linear relationship[5].
1
(13)
y
d
where y is defined to be the distance along the y-axis that a liquid element travels when
moving 80% of the distance from the channel wall to the center of the channel.
This y can approximately been related to the separation efficiency by a converted
relationship because with a high efficiency only a short distance in the flow direction (y-axis)
is needed for the particles to be separated. This approximation which gives this linear relation:
1
1

d efficiency
d efficiency

(14)

However, this particle separation technique demonstrated in this project offers not only the
ability to wash cells, but also to sort particles by there size which can be useful for instance in
biomedical and chemical applications. For example it could be used to manipulate and control
different reaction by moving certain particles over bands in the channel for a given period of
time.

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a)

b)

c)

d)

e)

Fig 9. Graphs over separation efficiency depending the on the size of the particles at a) 0V,
b) 2V, c) 4V, d) 6V and e) 8V

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10 Acknowledgements
I wish to express my gratitude to doctoral student Per Augustsson for all his kind help and
guidance during my work. His engagement and expertise in this area of research made it
possible for me to complete this project in an enjoyable way. I also want to thank the Electric
Department of Measurement at Lund Institute of Technology for letting me use all the
necessary equipment.

11 References
[1] F Petersson, A. Nilsson, H. Jnsson and T. Laurell, Carrier Medium Exchange through
Ultrasonic Particle Switching in Microfluidic Channels, Anal. Chem, 77, 1216-1221 (2005).
[2] K. Yosioka and Y. Kawasima, Acoustic radiation pressure on a compressible sphere,
Acustica, 5, 167-173 (1955).
[3] F Petersson, A. Nilsson, C. Holm, H. Jnsson and T. Laurell, Separation of lipids from
blood utilizing ultrasonic standing waves in microfluidic channels, Analyst, 129, 938-943,
(2004)
[4] A. Nilsson et al. Acoustic control of suspended particles in micro fluidic chips, Lab Chip,
4, 131-135 (2004).
[5] P Augustsson, Ultrasonic Control of Particles in Microfluidic Channels, Masters thesis,
Department of Measurement at Lund Institute of Technology, 27/01 (2006).
[6] J.L. Meriam, L.G. Kraige, Engineering Mechanics, Dynamics, Fourth Ediditon (1998).

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