Pinched Flow Fractionation: Continuous Size

Separation of Particles

Jaime Latas, Ikboljon Adakhamjonov, Madan Parajuli, Ehtashamul Rahat

Master Science in Smart Systems Integration

Heriot-Watt University
Edinburgh, Scotland
2016

Abstract – Micro-Nanofluidics has been already become
popular in especially medicine, biology, food industry and
chemical fields to analyse and diagnose samples. Particle
separation has always been great importance in the
development phase of them. Currently, several methods of
particle separation are available. This paper reviews the
pinched flow fractionation particle separation method which
was first demonstrated by Yamada et al [1]. This method
contributed further development of continues size separation
of particles utilizing a laminar flow profile in a microchannel
having a pinched segment and aligning to one sidewall in the
pinched segment by another liquid flow without particles. This
method is considered passive separation technique where the
mixture of fluid and particles coming out of the pinched
segment is separated by the spreading streamlines according to
their sizes [2]. In the laboratory experiment, we showed the
two different size polymer beads separation with our design,
discussions, explanations and results.
Keywords: Particle separation, passive sorting, pinched flow
fractionation, continuous polymer beads separation
I. INTRODUCTION
Accurate size separation and size measurement of
various particles, including polymer beads, ceramics,
cells, and pharmaceutical emulsions, are some of the
most important technologies in the fields of industrial
production, environmental assessment, and chemical or
biological research[2]. By employing the unique
characteristics of microscale flow phenomena, various
techniques have been established for fast and accurate
separation and sorting of microparticles in a continuous
manner. A few examples of such fluidic-only separation
methods include the following: size-exclusion, entropic
trapping[3], deterministic lateral displacement[4] and
pinched flow fractionation[2]. In this work, we used
pinched flow fractionation method to develop a
microfluidic system for accurately separating particles
based on their size.
Pinched Flow Fractionation (PFF) is a continuous size-
dependent fluidic separation technique relying on
laminar flow in micrometer or nanometer-size channels.
One powerful aspect of PFF in terms of fluidic LOC
components is that it works without any applied external
fields. Furthermore, since PFF is a continuous process it
is applicable both in regards to separation on one hand,
and in diagnosis combined with other up- or down-
stream continuous processes on the other hand[1]. In
PFF, particles with different sizes in a fluid stream are
pushed against one wall of a narrow channel, the pinch,
by the flow of an injected diluent. Due to their alignment
along that wall, the center of mass of the particles follow
different streamlines. When the particles enter a wider
channel, they separate as the streamlines spread[5].
The principle of PFF is illustrated in Figure 1. Two inlet
channels, the sample inlet and the buffer inlet, merge
together in one narrow channel, the pinch segment,
which ends out in a much broader channel, the
broadening segment. A sample containing particles is
introduced via the sample inlet channel into the pinch
segment. The sample particles are aligned (pinched)
against the wall in the pinch segment, regardless of size,
using the fluid flow from the buffer inlet channel. A
particle is pinched when the width of the sample fluid is
smaller than the radius of the particle. As the particles
move into the broadening segment, the distance from the
channel wall to the center-of-mass of the particles is
amplified, and the particles are separated according to
their size. The distance from the channel wall to the
center of the particles in the broadening segment is
denoted y’. The two main requirements for PFF to work
are laminar flow and a difference in size of the particles
to be separated. The particles are assumed to follow a
streamline corresponding to their center position when
they are pinched to the channel wall of the pinching
segment. In the case of non-spherical particles, it is the
smallest dimension which defines the separation. This is
experimentally demonstrated with red blood cells in [6].
Figure 1: (a) The principle of
pinched flow fractionation (PFF).
Two fluidic inlet channels, the
sample inlet and the buffer inlet,
merge together in one narrow
channel, the pinch segment, which
ends out in a much broader channel,
the broadening segment. Fluid from
the buffer inlet is used to focus the
fluid and align the particles from the
bead inlet against the channel wall
in the pinch segment. When the
particles enter the broadening
segment, different sized particles will
follow different streamlines in the
fluid and separate accordingly.
(b+c) Cross-sectional view of the
pinch situation for the smallest
spheres[1].
Important design parameters are illustrated in Figure 1.
These are the width of the pinch segment, , the width
of the broadening segment, , and the channel height,
. Especially, and are important parameters
regarding separation. is more relevant in clogging
issues[1].
In this laboratory work, particle sizes of 58um and
165um of diameter are used for separation. Design chip
was fabricated in PMMA with 3 layers and subtractive
laser manufacturing technology was used. In our design,
we made the broader segment as cone shaped and it
contains six outer channels with the same width at the
end of the broader segment and another wider channel at
the bottom of these six channels to make the buffer go
faster. The main idea is that when the particles move into
the cone shaped broadening segment, they will separate
and follow different path and finally come into any of
the small channels at the end and being collected.
II. DEVELOPMENT OF THE LABORTORY
The lab was divided in various steps, first of all the
designing step, where the chip was designed using
AUTOCAD, following a fabrication step, where the
actual chips were fabricated using a laser and finally the
chips were tested.
After the test process, several weaknesses were detected
in the first chip created, therefore it was necessary to
improve the design of the structure, leading to a loop
process of design-fabrication-test in order to try getting
the best results.
Figure 2: Characteristics of the input channels and
the mixer channel.
A. Chip Designing step
To model a good chip, it is needed to know both
requirements and our design is suitable for it; therefore,
it is necessary to understand the behavior of the
particles.
The purpose of the chip was to divide to different sized
particles. The particles are mixed in a laminar flow,
which means that depending on the size of the particles
they will follow the same line inside the flow. In this
laboratory work, a Pinched Flow Fractionator (PFF) was
implemented.
PFF make the different sized particles to follow a
constant path along the entire structure, consequently it
is possible to divide the different sized particles in a
same flow.
The PFF has four main structures, two initial flow
channels, one flow with the particles mixed, and the
other with a clean flow, the channel where those two
flows are going to be mixed, the cavity where the
particles are going to be divided in different paths and to
finish the structure, different channels, where it is
possible to find particles with the same size. The basic
design of the PFF can be seen in Fig 2 and Fig.3.
In this laboratory, particles sizes of 58um and 165um of
diameter were supposed to be separated; therefore, the
minimum channel size was four times bigger than the
biggest particle radius.
The designed chip was fabricated in PMMA, due to the
excellent optical properties, transparency, stability,
strength, bio-compatibility, it can be used in medical
applications. Before moving the material to the laser an
extra thin layer of paste was clayed over the middle layer
faces so that after the laser cut, all the layers could be
mounted together to finish the final chip design shown in

Figure 3: Characteristics of the

cavity and the channels.
Figure 4: Three layers of PMMA
The equation 1 is used to find the position of the particle
center at the outlet.
In this case, the subtractive manufacturing technology
R/D=y/H Equation 1 used was based on laser technology. The laser
technology emits a concentrate beam that heats the
The parameters for the design can be found in table 1. surface of the material and evaporates it, creating the
Table Styles desired design.
The laser used is “Epilog Mini Helix 40 Watts” a
Pinched flow fractionator parameters (mm) commercial CO2 laser with a 457x305mm cutting area
Y R D H and can cut up to 102mm on the cutting table, the laser
26.36 29 4x82,5 300 spot size is approximately 80um and the minimum
75 82,5 4x82,5 300 resolution is 200 um.
Once the chip is finished, two syringe’s needles were
glued to the input holes of the first layer of the chip in
order to test the design.
Table 1

Due to the fabrication process, different limitations in

the size of the chip had to be considered. A minimum
channel size of 300um, the total chip size and the
distance from the holes and the limits of the chip were
considered. Also, it was necessary to build three layers
to contain the fluid inside the chip.
AUTOCAD was the software used in this laboratory to
design the PFF.

Figure 5: Final chip with three

B. Chip Fabrication step
layers and two syringe needels.
In this lab, apart from learning how to rapid prototype C. Testing the chip
and test a microfluidics chip, subtractive manufacturing
processes were also shown.
Testing is one of the most important steps when
developing new microfluidics chips. During the
laboratory, the results obtained during the tests were used
to improve the chip design. The set up for the test
involved several machines and materials.
Two automatic constant flow machines with syringes,
shown in Figure 6, were used during our tests to
introduce polymer beads contained solution to the mixer
contained viscous water solution.
Figure 6: Constant flow machine
and syringe.
A portable microscope was used to obtain images and
videos of the particles inside the chips. In this case, we
used a “Dinolite Camera”, see Figure 7.
Figure 7: Dino-Lite camera.
III. RESULTS
Figure 8:Particles with different
size
Figure 8 shows the two different size particles; one is
blue colored with size 58um and another one is yellow
colored with size 165um. The purpose of experiment is
to separate these two particles in two different channel.
Figure 9 shows the connecting point of the two inlet
channels. The blue one is the buffer line and the white
one contains the experimental particle.
Figure 9: Connecting point of two
inlet channels
Figure 10 shows the particles flowing into the pinched
segment and it can be seen from the figure that the
particles are aligned (pinched) against the upper wall in
the pinch segment. Figure 11 shows the unexpected
buffer showed up into the micro channels during the
experimental. These bubbles affected the flow path of
the small particles until their removal.
Figure 2.3: Particles flowing into the
pinched section
Figure 11: Bubbles inside the
microchannel
Figure 12 shows the entry point to the broadening
segment. It can be seen from the figure that when both
sized particles enter into the broadening segment from
the pinched segment, most of the small sized blue
particle goes up from the connecting point because of
the amplification of the distance from the channel wall
to the center-of-mass of the particles. The bigger yellow
colored particle just followed the path below the smaller
particle path.
Figure 12: Entry point to the
broadening segment
Figure 13 shows the paste remaining in the channel after
the liquids flowing for some time. It blocked the flow of
the particle, eventually Figure 14 shows the final
collecting channel where it can be visible that most of
the small particles followed the upper channel designed
for collecting particles and the bigger yellow colored
particles followed the another channel.
Figure 13: Paste remaining in the
channel
Figure 14: Collecting particles in
different channels
IV. DISCUSSION
A. Effect of size of the chips
The first chip with greater width and length of flow
did not work as expected. The greater flow length
causes the diffusion of the particles in the channels.
The diffusion distance is proportional to the square
root of time of flow. Time of flow depends upon the
velocity of the liquid and the length of the channel.
The objective of the experiment to separate the
particles was not achieved using the first chip. The
second chip was designed with shorter width and
length of flow. This reduced the diffusion distance
but the separation of particles was partially
achieved. Experiments with two different size of
microfluid chips helped to conclude that there are
other parameters associated with the separation of
particles.
B. Effect of fabrication process
The PFF technique assumes that the particles do not
perturb the fluid flow and interact with the channel
walls [1] but it was observed that some particles
were stuck into the channel. There appear to be two
reasons for this problem. Firstly, the remains of the
paste in the channel, which was used to attach the
microfluid chips was trapping the particles.
Secondly, the laser cutter used to fabricate
microchips was not efficient to fabricate the sharp
channels of microchips. The rough channels and the
paste were affecting the particle separation by
obstructing the flow of liquid. Moreover, another
challenge was the injection of fluid to the
microchannel. In particular, there was observed
some issues regarding blockage of needles and
solution leakage from the junctions of needles.
C. Effect of bubbles formation
The PFF method utilizes only the laminar flow
profile inside the microchannel [2] but the bubbles
formed during the experiment were obstructing the
laminar flow of liquid. The bubbles were formed
due to the leakage in the connection of pipes to the
channel and air in the pipes. The fluid was flowing
the path around the bubbles which created the
problem in particle separation. The bubbles were
removed with gravity effect which helped to
observe the laminar flow of liquid with particle
separation. The effect of bubbles formation and the
reasons of bubbles formation should be effectively
considered during the experimental setup. The
elimination of bubbles in the channels is necessary
to achieve the desired behaviour of the fluid.
D. The effect of flow rate
The increment of flow rate decreases the flow time
of the fluid. Flow time is related to the diffusion
distance. The experiment was performed at different
flow rates of fluid with particles and buffer. The
pinching effect and particle separation were more
clearly observed at the increased flow rate of the
fluid. The effect of bubbles in the channels was
more severe with the low flow rate of the fluid. The
observed behaviour of fluid with different flow rate
infers that flow rate is the important parameter that
should be considered during the experiment.
E. Effect of dyed buffer
To study the behaviour of buffer in the pinched flow
fractionation process, the buffer was dyed with blue
colour. The pinching effect was clearly observed
during this process. The difference in flow rate of
fluid with particles and buffer was visible. The ratio
of flow rate of fluid with particles to buffer was
maintained above 5 which helped in the separation
of particles. The desired behaviour of the buffer and
the particles with respect to the flow rate was
observed.
F. Collection of separated particles
Particles with two different diameters were used in
the experiment. Particles with small diameter were
expected to flow through the first outlet while
particles with bigger diameter were expected to
flow through the second outlet. It was visible that
the density of smaller particle was more in the first
channel and less in the second channel while the
density of bigger particle was more in the second
channel. The aim of the experiment was to separate
the particle and collect in the different channel
which was partially achieved.
V. CONCLUSION
We have reviewed the continuous polymer beads/particle
separation method which particle size determines the
position in the design. Some critical direct parameters
are width of the pitch, width of the expansion segment
area, the distance from channel joining section to
expansion area and radius of the particle. Furthermore,
the laser beam tolerance, surface roughness, the space in
between the layers, geometry of microchannel and the
flow rate can affect the particle separation. This method
can be applied for virus separation, analysing the sizes
and sorting them to diagnise in various fields. Better
results can be achieved modifying the design and
implementing other factors that could improve particle
sorting, however the principle is the same.
VI. INDIVIDUAL STATEMENT
A. Jaime Latas
1) Contribution to the project
During the laboratory, I participated in all the parts of
the design, fabrication and test processes.
I designed the second version of the microfluidics chip
with some improvements, like smaller overall chip size.
I didn’t take part in the fabrication process, I only
watched the process of applying the paste layer to the
PMMA and how the laser cut the design.
In the test process, I was in charge of the microscope
over the chip in order to get the best pictures of the chip
and the particles and the overall setup of the test
environment.
Finally, I was in charge of writing the “Development of
the Laboratory” and “Testing of the chip” sections.
2) Three Key points learnt in this project
The first thing I learnt while doing this laboratory was
the understanding of the complete process to fabricate a
microfluidics chip. It is a loop of generating new designs
from the feedback received in the testing step.
The second thing is how a pinched flow fractionator chip
works and how the particles inside a laminar fluid
behaves, during the testing process we were able to
observe for real what we have studied in the lectures.
And finally, we had to face up with two of the main
problems of a microfluidic chip, the low rate of flow that
a chip can achieve and the problems of interconnections
of the chip with the external flow.
3) Further development
As we didn’t have more time to polish the chip, the first
thing I would do is finishing the chip in order to
precisely separate the particles from the fluid.
I would start by using another laser, in order to obtain
sharper features in the channels and trying to maintain a
small amount of roughness in those channels. By doing
this we would have avoided the particles to get stuck
inside the chip.
Also, I would find another way to paste the layers
because from the test we could see that the unwanted
paste was trapping the particles, if that were not possible
I would try to pattern the design on the paste film and
then put the film in the PMM layers.
Another improvement to the chip would be to reduce the
size of the output channel to make sure that only the
certain size particles go through the certain channel.
As the chip was meant to separate particles, a good
addition would be small reservoirs where the particles
could be seen well.
Thinking about a new way to introduce the liquid to the
microfluidics chip would be to improve the control of
the flow rate of the liquid with the particles contained
solution and viscous mixer solution.
B. Ikboljon Adakhamjonov
1) Contribution to the project
I took part all laboratory sessions. I drew the first design
in AutoCAD based on my colleagues’ calculations and
modified the second design which lead us to the third
design. I actively took part in laser cutting process.
In the test process, I mostly took my part controlling
flow rate of solutions.
In the writing part, I was responsible for writing abstract,
conclusion and most importantly for merging and
collecting my colleagues’ reports and merging them
together in one document with coherence and writing
style.
2) Three key points learnt in this project
- I improved my AutoCAD design skills
- I built a complete image of process steps, setups,
microfluidic systems in my mind.
- How the particle separation is dependent on the
flow rate of solutions. The best result was
 obtained when the flow rate was 6-7 times faster
in buffer.
- When I redesigned the third model with thinner
middle layer, the bubble formation was less than
before. So, I concluded that 2 times as big as
large particle is optimum space for our case to
avoid bubble formation.
3) Further Development
- In the future work, I would recommend to
carefully design the first design based on
literatures and limitation factors that might arise
from laser cutting machine and surface
roughness of the PMMA.
- To design the drainage as a fourth layer to
collect the solution in order to avoid solution
come from the third layer’s holes.
A. Madan Parajuli
1) Contribution to the project
2) Three key points learnt in this project
3) Further Development
B. Ehtashamul Rahat
1) Contribution to the project
2) Three key points learnt in this project
3) Further Development
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