10138455 FYP Final Draft

Immiscible Liquid-Liquid Interface in High Throughput
Microfluidics, and Development of Vapour within a
Microfluidic System
Seán M. Cunningham
ID Number: 10138455
Supervisor: Dr. Tara Dalton Ph.D.
Final Year Project report submitted to the University of Limerick:
21st
of March 2014
Report submitted in partial fulfilment of the requirements for a Bachelors of
Engineering in Biomedical Engineering

- 2 -
University of Limerick
Department: Mechanical, Aeronautical and Biomedical Engineering
Title: Immiscible Liquid-Liquid Interface in High Throughput
Microfluidics, and Development of Vapour within a Microfluidic
System
Degree Award: Bachelors of Engineering in Biomedical Engineering
Author: Seán M. Cunningham
ID Number:10138455
Supervisor: Dr. Tara Dalton Ph.D.
Date of Submission: 21st
of March 2014

i
Declaration of originality
I declare that this is my work and that all contributions from other persons have been
appropriately identified and acknowledged
Signed: ______________________________
Seán M. Cunningham
Date: ________________________________

ii
Abstract
The rate at which microfluidics has developed has been hindered by the
development of vapour within the channels of the microfluidic system. This
development of vapour reduces the efficiency of the microfluidic system. The vapour
is developed within the system and is not drawn in from outside the system.
In this report, the hypothesis is that the effect of spacing of droplets, within a
train of 3 droplets has an effect on the development of vapour. This is done using
statistical analysis of a large sample size of droplets. This report discusses the effects
the forces within the system have on the development of vapour. This is done with
the use of observation that have been noted during the experimental trail, and with
the use of non-dimensional characterisation of the fluid flow. With the aid of the
statistical results and observations, it is possible to discuss trends in the conditions
required for the development of vapour within the channels of the system.
Concluded from this report is that the formation of vapour does not solely rely on the
spacing of the droplets within the train. Properties of the fluid such as high surface
tension, and the design of the manifold also affect the development of vapour within
the channels of the microfluidic system.

iii
Acknowledgements
I would like to thank my family especially my parents for their support both moral
and financial throughout my time at University of Limerick as an undergraduate and
without which I would not have been able to complete this work.
I would also like to thank my supervisor Dr. Tara Dalton, for her valued assistance
throughout the course of this project and for her willingness to share her knowledge
of the subject area
Dr. Eric Dalton, Chris Hayes and Conor McCarthy for their assistance in aiding me
in the instrumentation and building of the test rig required for this project.

iv
Table of Contents
Abstract.......................................................................................................................................... ii
Acknowledgements....................................................................................................................... iii
Index of Figures ..............................................................................................................................v
Index of Tables................................................................................................................................v
Nomenclature................................................................................................................................ vi
1. Introduction.............................................................................................................................1
2. Objectives ...............................................................................................................................3
3. Background.............................................................................................................................4
3.1 Addition of surfactants..........................................................................................................6
3.2 Bookending...........................................................................................................................9
4. Theory...................................................................................................................................11
5. Method..................................................................................................................................14
5.1 Procedure ............................................................................................................................14
5.1.1Testing Sequence..........................................................................................................16
5.2 Apparatus......................................................................................................................17
5.3 Material Used......................................................................................................................21
6. Results and Discussion .........................................................................................................22
6.1 Results.................................................................................................................................22
6.1.1 Percentage Occurrence.................................................................................................22
6.1.2 Error Analysis..............................................................................................................22
6.1.3 Non-Dimensional Analysis of flow. ............................................................................23
6.2 Discussion...........................................................................................................................25
6.2.1 Occurrence of cavitation..............................................................................................25
6.2.2 Temperature affects viscosity ......................................................................................31
6.2.3 Effects of vapour on the system...................................................................................34
6.2.4 Analysing experimental data........................................................................................35
6.2.5 Surfactants and bookending.........................................................................................40
6.3 Summary of Discussion ......................................................................................................43
7. Conclusion............................................................................................................................45
8. Recommendations.................................................................................................................46
References.....................................................................................................................................47
Appendix.........................................................................................................................................a
Appendix A.....................................................................................................................................a
Appendix B ..................................................................................................................................... f
Appendix C .....................................................................................................................................g

v
Index of Figures
FIGURE 1: DEVELOPMENT OF VAPOUR IN MICROFLUIDICS CHANNEL.......................................................1
FIGURE 2: SURFACTANTS STRUCTURE.....................................................................................................6
FIGURE 3: VARYING THE SURFACTANT COMPARED TO % FAILURE .........................................................8
FIGURE 4: BOOKENDING PLACEMENT .....................................................................................................9
FIGURE 5: OPERATION CURVE FOR SYRINGE PUMP................................................................................11
FIGURE 6: DIPPING HEIGHTS .................................................................................................................14
FIGURE 7: DIPPING TIMINGS AND HEIGHTS FOR 5 SECONDS SPACING ....................................................15
FIGURE 9: BUILT TEST RIG ...................................................................................................................17
FIGURE 8: APPARATUS SET UP...............................................................................................................17
FIGURE 10: SCHEMATIC OF CIRCUIT LAYOUT........................................................................................18
FIGURE 11: DIAGRAM OF CONTROL.......................................................................................................19
FIGURE 12: MEAN OCCURRENCE OVER THE SAMPLE SIZE TESTED, WITH BEST FIT.................................22
FIGURE 13: STANDARD ERROR OF THE SAMPLE MEAN.........................................................................23
FIGURE 14: DEVELOPMENT OF VAPOUR IN THE SYSTEM. .......................................................................25
FIGURE 15: DROPLET ACCUMULATION ON THE OUTLET IN MANIFOLD ..................................................26
FIGURE 16: EFFECT DROPLET ACCUMULATION ON FLOW RATE .............................................................26
FIGURE 17: ACCELERATION OF FLUID WITH CAPACITANCE EFFECT.......................................................27
FIGURE 18: TRAINS OF 3 DROPLETS.......................................................................................................27
FIGURE 19: TENSILE FORCES APPLIED DURING DEVELOPING FLOW THAT CAUSES CAVITATION.............29
FIGURE 20: PERCENTAGE OCCURRENCE OF VAPOUR FOR 5S, 15S, AND 20S SPACING ............................37
FIGURE 21: DROPLET ACCUMULATES TO THE OUTLET OF LINE 1 DUE TO HIGH SURFACE TENSION. .......40
FIGURE 22: DROPLET DETACHES FROM THE OUTLET OF LINE 1 DUE TO LOW SURFACE TENSION...........41
Index of Tables
TABLE 1: RESULTS OF SURFACTANT TESTING .........................................................................................7
TABLE 2: STANDARD ERROR OF THE SAMPLE MEAN..............................................................................23
TABLE 3: CHARACTERISTICS OF PD5 OIL .............................................................................................23
TABLE 4: NON- DIMENSIONAL NUMBERS ..............................................................................................24
TABLE 5: CHANGE IN VISCOSITY WITH TEMPERATURE..........................................................................31
TABLE 6: VARIANCE OF THE EXPERIMENTAL DATA...............................................................................35
TABLE 7: RESULTS OF VAPOUR FORMATION AT 10 SECOND SPACING ....................................................36
TABLE 8: DENSITY AND SURFACE TENSION...........................................................................................41

vi
Nomenclature
Symbol Name Units
a Acceleration
g Acceleration due to gravity
Bo Bond Number Dimensionless
Ca Capillary number Dimensionless
l Characteristic length m
ρ Density of carrier fluid
Density of the droplet
F Force N
m mass kg
P Momentum N.s
π Pi Dimensionless
r Radius m
Re Reynolds number Dimensionless
n Sample size Dimensionless
Standard deviation Dimensionless
Standard error of the sample mean Dimensionless
γ Surface tension N/m
v Velocity of fluid m/s
µ Viscosity Pa.s
Q Volume Flow Rate
We Weber number Dimensionless

1
1. Introduction
In high throughput microfluidics, droplets are placed in train within a carrier
fluid. The carrier fluid used is oil based and the droplets are aqueous. These two
immiscible fluids have a high interfacial tension and do not mix. The leading
concern with developing high throughput microfluidic devices is the development of
vapour/gas bubbles within the channels of the system. These bubbles within the
system disrupt the flow and the efficiency of the microfluidic system is reduced.
Below Figure 1, show a
schematic of the creation and
development of the vapour
within the micro-channel. As the
droplet moves down along the
line the vapour increases until it
reaches equilibrium. The size of
the vapour varies hugely but the
volume of vapour does not affect
the outcome as any vapour in the line will result in a reduction in the flow rate. This
reduction in the flow rate effects the velocity of the droplets as it travels down the
line. As the velocity is affected the sequencing of the system becomes out of sync.
This results in errors in the collected data.
The development of vapour is unexplained and is very intermittent. Vapour in
the channel reduces the flow rate in the channel, resulting in a reduction in flow rate
over all of the lines. This is because all lines are connected via a common manifold.
This reduction in flow rate reduces the velocity of the flow. This reduction in
velocity disrupts the sequencing of the process, especially as the droplets move over
the thermo-cycle. If the velocity is reduced, it causes the time spent in the denaturing
process in the thermo-cycle to be extended. This will result in the DNA being
exposed to elevated temperatures for an extend period of time and which will cause
over heating of the DNA. This ultimately will destroy the DNA. This in turn affects
the detection methods of the system. Also droplet recognition is affected by the
presence of vapour, which will result in a loss of data.
Creation of Vapour
Development of Vapour
Figure 1: Development of vapour in microfluidics channel

2
There are many hypotheses that already have been addressed with regard to the
creation of vapour within the closed microfluidic system. These are, boiling,
influence of charge, thermal effect, decreasing pressure drop and potential degassing,
permeability effects and static on tubing and droplet throughput. All of the above
have resulted in the creation of vapour within the closed system, and therefore have
been unsuccessful. These previous experiments will help to develop a hypothesis to
base future research on, which will be carried out over the course of this project.
This will be done with the development of an experimental rig. This experimental rig
will be able imitate the volume flow rates and upper temperatures which are
experienced in the thermo-cycling process. As the development of vapour is very
intermittent and infrequent this will require a large volume of statistical results. The
gathered data, from the proposed hypothesis will be compared with previous bodies
of work which have been carried out is Stokes Institute on this topic.
The problem occurs in the lines in the system, when the immiscible fluid train
passes over the initial 95˚C heated plate where an irregular occurrence of vapour
development in the micro-channel occurs. The phenomenon is sporadic and
infrequent in its occurrence, but is frequent enough for a reduction of efficiency in
the microfluidic system. As the droplets move along in train, cavitation occurs on the
surfaces on the leading or trailing droplet in the train. This cavitation causes a rapid
drop in pressure and allows for the expansion of a vapour bubble to develop within
the system. The vapour develops adjacent to the aqueous droplet.

3
2. Objectives
1. Develop a hypothesis for this study, by using previous experimental data
conducted about this problem.
2. To develop an experimental rig, based on the proposed hypothesis.
3. Build proposed test rig and produce relevant data.
4. Apply proposed hypothesis to experimental rig.
5. To gather statistical data on the occurrence of vapour/gas formation.
6. Develop a greater understanding of the forces that affect the formation of
vapour.
7. To reduce or eliminate the occurrence of vapour/gas formation within the
lines of the system. By applying the greater understanding of forces that
effect vapour formation.
8. Discuss results and observation of the occurrence of vapour formation in
relation to the proposed hypothesis.
9. The gathering of the data to compare with previous pieces of work, around
this topic and document the finding in a final year project.

4
3. Background
As part of the high throughput microfluidics, the trains of droplets must pass
over a thermo-cycle. Thermo-cycling is the process in which the train of droplets
passes over heated plates varying from 60˚C to 95˚C. This is done to amplify a
desired section of DNA as part of the genotyping process, this is done by using PCR
(Polymerase chain reaction), which relies on TaqMan chemistry. Thermo-cycling
allows for the hydrogen bonds to break in the DNA strands. This happens at the
higher operating temperature (95˚C), this is known as denaturing. At the lower
operating temperature (60˚C), allows for primers to hybridise to the different strands
on the separate strand of the DNA. This is carried out 40 times. This amplifies the
result to 2^40. This amplified DNA is then able to be detected using optic
processing. The use of optics will not be discussed in the report as it is outside of the
scope of the project.
PCR is a method to synthesize new strands of DNA that is complementary to
the template of the sample DNA. First DNA is heated to 95˚C, this breaks the
hydrogen bonds between the two stands and they separate, and this is called
denaturing. Primers are then used to attach to the target DNA. The primers anneal to
the DNA and allow the TAQ polymerase to attach to the nucleotides. This then fills
out the rest of the DNA strand. By filling out the strand it creates a complete copy of
the initial sample DNA. This then makes it possible to amplify a specific section of
DNA. PCR uses specific primers that are particular to the section of DNA wished to
be amplified. As this process is repeated it has an exponential amplification.
The problem occurs when the immiscible fluid train passes over the initial
95˚C heated plate where an irregular phenomenon occurs. The phenomenon is
sporadic and infrequent in its occurrence, but is frequent enough for reduction of
efficiency of the PCR system. As the droplets move along in train, cavitation occurs
on the surfaces on the leading droplet in the train. This cavitation causes a rapid drop
in pressure and allows for the expansion of a vapour bubble to develop within the
system. The vapour develops adjacent to the aqueous droplet. It tends to form on the
leading droplet in the train.

5
Vapour developing in the lines has been a persistent problem and many
hypotheses have been drawn and explored recently within Stokes Institute. Many
have been investigated but few have contributed considerably to understanding the
phenomenon. The hypotheses that have been explored are the following:
1. Decreasing pressure drop and potential degassing: Experiments were carried
out by varying the ID of the tubing used. This is to see the effects that
pressure has on the system. The aqueous liquid was degased to investigate
the effect that dissolved gases have on the occurrence of vapour being present
in the lines. Degassing the liquid did have an effect on the occurrence as it
was seen to reduce the presence of vapour. It did not eliminate the occurrence
completely. (Deschamps & Delerue, 2012) Also explored under this
hypothesis, were a positive displacement pressure gradient and also a gravity
fed pressure gradient. These had no effect on the development of vapour
within the line of the system and the frequency of vapour was still
maintained.
2. Droplet throughput: This hypothesis was investigated by varying the number
of droplets in a train. Form this it was concluded that there is a higher
tendency for vapour to develop on the first in a train of droplets. This did not
eliminate the occurrence of vapour nor did it reduce the frequency of the
event. (Deschamps & Delerue, 2012)
4. Thermal effect: This hypothesis was explored by varying the temperature of
the heated plates. The experimental data for this hypothesis showed that at
lower temperatures, the occurrence of vapour was reduced and at elevated
temperatures, above 70˚C vapour did form within the lines. Elevated
temperatures of 95˚C are required for the PCR process. From this
experimental data, it has been observed that the conditions must be very
specific in order for vapour to develop within the system. (Deschamps &
Delerue, 2012)
5. Permeability effects and static on tubing: Different materials have been used
for the tubing. Vapour still develops within the lines. This has ruled out the
cause being the PTFE tubing. (Deschamps & Delerue, 2012)

6
3.1 Addition of surfactants
Surfactants are widely used in microfluidics. Surfactants act by stabilizing
droplet interfaces. (Dalton & Dalton, 2013) This is done as a surfactant bolting the
droplet and carrier interfaces together. This interface is known as the liquid-liquid
interface. The surfactant does this by having two components to its structure. The
surfactant is comprised of a hydrophilic head and a hydrophobic tail. The
hydrophilic head binds to the aqueous droplet. The hydrophobic tail binds to the oil
based carrier fluid. With the combination of both of these components it allows for
the droplet interface to be stabilized. This is shown below in Figure 2. By bolting the
interface together it reduces the possibility for cavitation to occur and this then
reduces the possibility for vapour to develop within the system. The surfactants used
also have a lower surface tension. This will greatly increase the Bond number of the
system (Hager, 2012)
Research has been carried out in Stokes Institute with the use of surfactants.
These tests were carried out with two different droplet types within the carrier oil.
The two droplets used were a water droplet and Triton - X100. This experiment will
be the model followed for further experiments. As the variables such as temperature,
droplet size, flow rate, train size, and apparatus set up will be the same for future
experiments.
Droplet
interface
Hydrophilic
head
Hydrophobic
tail
Figure 2: Surfactants structure

7
Table 1: Results of surfactant testing
Boiling,
pt (ºC)
Density,
, (g/cm3)
Viscosity,
, (mPa.s)
Surface
tension, ,
(mN/m)
Number of
Gas event
Carrier oil – PD5 268 0.92 4 24.2 n/a
H2O 100 1 1 72.8 3
Triton - X100 233 1.03 240 33.0 0
(Dalton & Dalton, 2013)
Referring to the experimental data shown above in Table 1. It can be seen
that 3 vapour events occurred when the water was present in the droplet. This
compared to zero vapour events when Triton - X100 is present in the droplet. The
surface tension can be seen to be an effect on the occurrence in cavitation. This is
due to the fact the difference in surface tension between the PD5 Oil and water is Δ
53.6mN/m. This is considerably higher than the difference in surface tension
between the Triton - X100 and PD5 oil of Δ 9.7mN/m. The difference in interfacial
tension is considered to be a major contributing factor cause of cavitation.
It was speculated that the interfacial tension between the water and oil was a
trigger for the development of gas bubbles; to test this, the interfacial tension of the
interface was modified with the introduction of surfactant to the oil. The surfactant
chosen were an equal mixture of Span 80 and triton x100, these were mixed with
Silicone oil at percentage ratio of 0, 0.001 and 0.01%; and the presents of vapour
was monitored; these experiments were repeated 15 time (30 times for the 0.01%
mixture); (Dalton & Dalton, 2012)

8
Figure 3: Varying the surfactant compared to % failure
Figure 3 shows the power law fit, in relation to the occurrence of vapour and
the presence of surfactants as a percentage weight of the carrier oil. This shows that
there is a direct relationship between the presence of surfactants and the occurrence
of vapour.
The presence of surfactants is not an ideal solution. This is due to the fact that
surfactants must be present with the biological material in the droplet. This will
cause problems during the detection stage, as the results may be askew due to the
presence of surfactants.

9
3.2 Bookending
Bookending was successful, as the vapour/gas bubble tends to form adjacent
to the first or second droplets. Two droplets with less interfacial tension (di-
propylene glycol DPG) are placed at the front and back of the droplet train. This is
illustrated in Figure 4 below. This is done because it was noted from observation that
the higher probability of vapour/gas bubbles forming, is in the first two and last two
droplets. (T.M. Dalton. 2012)
Figure 4: Bookending placement
Bookending has shown positive results for a limited number of droplets in a
train. Vapour occurrence has been reduced by the use of bookending, but irregular
results have been recorded when applied to a droplet train size of more than 10
droplets. It has been recorded that above a droplet train size of 10, the development
of vapour in the system becomes irregular and sporadic. This means that bookending
is not a viable solution for large droplet train sizes.
Below can be seen the properties of the fluid used in bookending
experiments. The surface tension of H20 is much greater than DPG, this results in
H20 droplets having a much lower Bond number.
Table 2: Properties of PD5 oil, H20, and DPG
Boiling,
pt (ºC)
Density,
, (g/cm3)
Viscosity,
, (mPa.s)
Surface
tension, ,
(mN/m)
Number of
Gas event
Carrier oil – PD5 268 0.92 4 24.2 n/a
H2O 100 1 1 72.8 3
DPG 233 1.03 150 33.9 0
Lower interfacial
tension Aqueous Droplet

10
From the results of the bookending testing, this raises a theory that droplets
within the train have an effect upon other droplets in the same train. Hypothesis is
that the spacing of droplets has an effect on the occurrence of cavitation and
therefore vapour/gas bubbles being present in the train of droplets. This will be done
to compile statistical results at different spacing between droplets, to see if droplet
spacing has an effect on cavitation and vapour/gas.
3.3 Conclusion of previous experiments
Surfactants and bookending showed positive results as vapour/gas was not
created. Adding surfactants is the equivalent to fastening the two immiscible fluids
together reducing the possibility of cavitation. Cavitation is the creation of vacuum,
which then allows the vapour/gas to form. This was successful as it does reduce
cavitation. Not ideal as there must be a surfactant present in the biological material.
Bookending was successful, as the vapour/gas bubble tends to form adjacent
to the first or second droplets. Two droplets with less interfacial tension are placed at
the front and back of the droplet train. This is done because it was noted from
observation that the higher probability of vapour/gas bubbles forming, is in the first
two and last two droplets.
From the finding with regards to bookending, this raises a theory that
droplets within the train have an effect upon other droplets in the same train.
3.4 Hypothesis of the study
Hypothesis is that the spacing of droplets with in the train of droplets, has an
effect on the occurrence of cavitation and therefore vapour/gas bubbles being present
in the system. This will be done to compile statistical results at different spacing
between droplets, to see if droplet spacing has an effect on cavitation and
vapour/gas.

11
4. Theory
Syringe pump: The flow rate is maintained by the pump, irrespective of the systems
pressure drop. (Newport, 2014).
Figure 5: Operation curve for syringe pump
This means that if the numbers of tubes is reduced the volume flow rate is not
reduced and the volume flow rate is then disbursed over the remaining tubes. This
causes an increase in the volume flow rate in the remaining available lines. This is
illustrated above in Figure 5.
Approximate Radius:
(1)
Velocity of carrier fluid (Engineers Edge 2000)
(2)

12
Capillary number: A dimensionless group used in analysis of fluid flow that
characterizes the ratio of viscous forces to surface or interfacial tension forces.
(Saylor & Bounds, 2012)
(3)
Reynolds number: A dimensionless group used in analysis of fluid flow that
characterizes the ratio of viscous forces to inertial forces (The Engineering Tool
Box, 2005)
(4)
Webber Number: A dimensionless group used in analysis of fluid flow that
characterizes the ratio of inertial forces to surface or interfacial tension forces.
(Saylor & Bounds, 2012)
(5)
Bond Number: A dimensionless group used in analysis of fluid flow that
characterizes the ratio of gravitational forces to surface or interfacial tension forces
(Hager, 2012)
(6)
Ohms law: This defines the relationship between pressure drop resistance, and
volume flow rate (Millikan & Bishop, 1917)

13
(7)
Newton’s second law: states that the net force acting upon an object is equal to the
rate at which its momentum changes with time. (Feynman, 2005)
(8)
Momentum: (Feynman, 2005)
(9)
Statistic error: Standard Error of the Sample Mean: (Harper, 2005)
√
(10)

14
5. Method
5.1 Procedure
Testing is carried out by varying the spacing of the droplets. This is done by
programming a timing sequence between two different heights in the FESTO™
Configurator Tool. The heights used were 88mm and 84mm, but will be varied
depending on the well size and volume of fluids used. Clearance of 2mm either side
of the liquid interface for dipping. This ensures that the dipping head is only
extracting one fluid at a time. Figure 6 below shows the heights that are used for the
dipping sequence. In the Up position the system is extracting the carrier fluid
(Silicon Oil PD5) and in the Down position the system is extracting the aqueous
droplet solution. Dist. A is 2mm.
Figure 6: Dipping heights
Dist. A
Dist. A
Up Down
Silicone Oil
Water

15
At the given flow rate of 15µl/min, in order to create the droplets, a pick up
time of 0.12s is required within the water. This remains constant for the entire
experiment as the flow rate does not change. This is to ensure uniform droplets
throughout all of the experiments. These parameters are inputs for the FESTO™
Configurator tool. This cycle should be run for 10 trains of droplet. Where there are
3 droplets in each train. To alter the spacing of the droplets the timings for the
pickup in the silicon oil is varied for 5sec, 10sec, 15sec and 20sec. This dipping
sequence can be seen below in Figure 7. This graph show the dipping sequence
between two height, 88mm and 84mm can be seen. The liquid-liquid interface is at
86mm.
Figure 7: Dipping timings and heights for 5 seconds spacing
The heated plates are controlled by a LabView™ Program. Refer to
Appendix B. All four of the heated plates are heated to 95C and are maintained at
this temperature by the PID controller. The PID controller used is a virtual
instrument that is created in LabView™
The Harvard syringe pump is set to refill 0.360ml/min across all of the
24lines. This then allows the manifold to distribute the flow rate over these 24 lines
to create a flow rate of15µl/min, that is required in each line. Ensure that all
connections to and from the manifold are airtight. This is due to the syringe pump
Liquid-Liquid Interface
of well

16
being a negative displacement pump, and any air in the system will cause a reduction
in flow rate. This is as the syringe and all lines must be entirely full of silicon oil to
maintain this flow rate. If an air bubble is present it will act as a capacitor in the
pump, which will create an error in the flow rate.
The Harvard syringe pump is allowed to pump 2ml of fluid before the
dipping sequences start; this is to ensure that the flow is fully developed. When fully
developed is flow is established begin the dipping sequence on the FESTO™
Configuration Tool.
Using IC Capture on a personal computer that is connected to an Imaging
Source CCD camera that has been mounted above the second heated plate using a
table clamp camera mount. Imaging Source CCD camera is connected via a USB 2.0
connection. IC Capture allows for video to be captured which will be used to gather
results. Ensure that the camera is focused in the lines on the second heated plate as it
may be difficult to distinguish between vapour and aqueous droplet on analysing the
results.
To begin testing allow for droplets to appear on the first heated plate and
begin recording the trains as they pass the camera. Testing times vary as the spacing
between droplets gets greater. After each test is complete, stop recording and infuse
the syringe pump. Repeat the testing 15 times. 15 tests are chosen as it gives a good
statistical size as there will be 10,800 droplets monitored after 15 tests. This is
because the occurrence of vapour is inconsistent and irregular so a large sample size
is required to gather accurate statistical results with a low error.
5.1.1Testing Sequence
The experiments are carried out by varying the timing between each droplet in
the train of droplets; this is done by varying the dipping times between each droplet
in the train. The first experiment carried out was a spacing of 10 seconds; this is
chosen as it is a bench mark. This experiment was comprised of 5 runs, with 15 tests
in each run. This lead to a total number of 54,000 droplet sample size. The following
experiments for 5 seconds, 15 seconds and 20 seconds all consist of 2 runs with 15
tests in each test. This gives a sample size of 21,600 droplets in each experiment. A
constant time of 30 seconds is kept between each train of droplets.

17
5.2 Apparatus
Below is Figure 9, in this image is the complete test rig used to carry out
experimentation.
Figure 8: Built Test Rig
Experimental Apparatus
Above is Figure 8, which shows the layout of the apparatus. (Dalton & Dalton, 2013)
The rig consists of 24 lines, of 400µm ID tubing made of PTFE (Teflon). 24 lines
have been chosen as it gives a large population size of droplets and therefor increase
the likelihood of observing vapour developing in the lines. The length of the tubing
is arbitrary to the experimental outcome but one meter is used as it fits along all the
heaters.
Figure 9: Apparatus set up

18
5.2.1Thermal Control
The 24 lines pass over four 10 ohm heated plates which are controlled by a PID
controller, created on LabView™. The Labview™ program allows for the creation
of four individual PID controllers. LabView™ is a virtual instrument engineering
workbench. LABView™ is run on a PC with a Data Acquisition Card (DAQ) and
with DAQ assistant. A DAQ assistant is a driver that allows the LabView™ code to
read in put channel on the DAQ card.
PID (Proportional, Integral, Derivatives) this describes how the error is
treated before being summated to the system. This is a looped system as the
temperature of the heaters is monitored and corrected to maintain a constant
temperature. Four thermocouples are used to monitor the temperature on the heaters;
the thermocouples relay the temperature reading into a thermocouple reader and are
then accessed by the LabView™ program. A 25 volt power supply is used to power
the heaters; this is the voltage that the PID controller controls. The use of individual
PID controllers, in addition to 5 Volt solid states relays (SSR). This allows for tight
control of the temperature of the heated plates through the DAQ card and
LabView™ programs. The solid state relays act as a switch which is controlled by
the PID controller. This allows for large voltages to be switched off or on to maintain
the temperature. The circuit design is illustrated below in Figure 10.
Figure 10: Schematic of circuit layout.

19
The PID controller corrects the error that is present as the heater tries to
maintain a fixed temperature, 95˚C. The PID uses 3 operations to maintain the
temperature on the heaters. These 3 components are: proportional component,
Integral component, and derivative component. These 3 components handle the error
differently and can also be controlled individually of each other. A component can
be neglected by assuming it is zero. This can be done to develop a simpler
controller.
LabView™ 9.0 is used along with a National Instruments DAQ MX data
acquisition card, along with a DAQ assistant driver. This was a major stumbling
point as the DAQ assistant drivers. The drivers supplied with in the service package
was not sufficient, so an online install was required from the National Instrument
website was required in order to remedy this (National Instruments, 2013). Below in
Figure 11 can be seen the closed loop system used to control the heaters.
Figure 11: Diagram of control
The FESTO™ dipping stage used to control the creation of droplets. The
FESTO™ stage moves between two different heights in the well, as discussed in
section 5.1. This allows for the immiscible fluids to be picked up separately. The
timing set in each of the fluids determines the volume of liquid that is to be picked
up and therefore creates the droplet trains. The FESTO™ stage is powered by two
power supplies. A 48Volt and 24Volt power supplies are used to do this. This is

20
because the dipping stage is made up of two components, the driver and the stage.
The stage is programmed and controlled through the driver. This driver is
programmed through a software package, FESTO configuration tool. This software
allows the control of the timing required for the stage, in order to develop droplets
within the carrier fluid. The timings required are reliant on a continuous flow rate,
which is created using a negative displacement Harvard syringe pump.
5.2.2 Pumping system
A Harvard syringe pump is used to create a negative pressure gradient. This
negative pressure gradient is used to carry the fluids through the lines of the system.
This pumping set up is known as a negative displacement pump. This negative
displacement pump allows for a constant flow rate to be created. This in turn allows
for the FESTO™ stage to create droplets within the carrier fluid. In order to connect
the syringe pump up to the 24 lines a manifold is required to do this. The manifold
design allows for an even distribution of the pressure gradient, created by the syringe
pump. This is important as all lines should maintain the same flow rate. This is to
insure that all of the droplets, across all lines are of uniform size. The operational
flow rate in the lines is 15µl/min. This is to mimic the genotyping (PCR) flow rates
used.
All fluids will be degassed where possible; this will remove any dissolved
gases within the carrier fluid or the aqueous droplet fluid. For this deionized water is
used. The tubing used is PTFE (Teflon). PTFE is chosen as it is ridged and will not
deform under the negative pressure applied to the system by the negative
displacement pump. All linkages and connection are sealed using cyanoacrylate
(superglue) and heat shrinks which will ensures an airtight seal. This is done as a
negative displacement pump is used. If there is a breach in the system atmospheric
air will be drawn into the system, the cyanoacrylate and heat shrink ensure air tight
seals on the connections. Glass syringes are used as glass will not deform under the
pressures experienced by the pumping of the fluid. This type if syringe will ensure
that all seals and connections will stay airtight as there is not any deformation.

21
5.2.3 Visual Detection
The visual detection system is an Imaging Source CCD camera and zoom
lens able to record at 15 FPS. This is recorded using a software package IC Capture
as an .AVI file. The camera is mounted above the second heating plate and will
record any vapour that will be created. All files are then recorded and stored on a
personal computer. The focusing of the camera must be fine, as it is difficult to
distinguish between aqueous droplets and vapour bubbles in the system.
5.3 Material Used
The tubing used in all 24 lines is 400µm ID PTFE. This tubing is impermeable to
silicon oil and water. It has ridged walls which will not deform under the given
negative pressure. Silicon oil is used as the carrier fluid as it is the carrier fluid of
choice for the PCR system

22
6. Results and Discussion
6.1 Results
6.1.1 Percentage Occurrence
Experiment 1: Spacing 5s
Figure 12: Mean occurrence over the sample size tested, with best fit.
All results recorded are shown above in Figure 12. Experiment 2, resulted in
the lowest occurrence of vapour. This experiment was the first experiment carried
out. The spacing for this experiment was 10 second between each droplet in the train.
Experiment 1, resulted in the highest occurrence of vapour in the system.
This experiment was the third experiment carried out. The spacing for this
experiment was 5 seconds between each droplet in the train.
6.1.2 Error Analysis
Standard error of the mean is calculated to justify the sample size. This error
is present as statistically it is impossible to gather data for 100% of the population
size. This error can be negligible if a sample size is justified.
y = -4E-07x + 5E-06
0.00000%
0.00010%
0.00020%
0.00030%
0.00040%
0.00050%
0.00060%
0.00070%
1 2 3 4
%Occurence
Experiments No
% Occurrence

23
Figure 13: Standard Error of the Sample Mean
These standard errors of the sample mean, this is a standard deviation that provides a
measure of the potential error in estimating the population figure from the sample
figure. This is shown in Figure 13. All of the errors are of the same order of
magnitude. Therefore it is considered to be a negligible error. This is characterised
below in Table 2. (Harper, 1991)
Table 3: Standard error of the sample mean
% ERROR Mean %Error/Mean
Experiment 1
(5Sec)
4.42635E-09 0.0006% 0.074019%
Experiment 2
(10Sec)
6.50538E-09 0.0002% 0.392837%
Experiment 3
(15Sec)
4.42635E-09 0.0004% 0.106917%
Experiment 4
(20Sec)
8.8527E-09 0.0005% 0.192450%
6.1.3 Non-Dimensional Analysis of flow.
Table 4: Characteristics of PD5 Oil
PD5 oil characteristics
0.0000000%
0.0000001%
0.0000002%
0.0000003%
0.0000004%
0.0000005%
0.0000006%
0.0000007%
0.0000008%
0.0000009%
0.0000010%
1 2 3 4
standarderrorofmean
Experiments No.
Standard Error of the Sample Mean

24
Density (ρ) kg/m^3 920
Viscosity (μ) Pa.s @ 21C 0.004
interfacial tension (γ) N/m 0.042
Volume flow rate (Q) µl/mn 15
ID of tubing (D) µm 400
Velocity in Line (m/s) 0.0020
Temp (Deg C) 95
Viscosity (Pa.s) @95C 0.0030634
Table 5: Non- dimensional numbers
Non-Dimensional numbers
Reynolds number 0.183121
Bond number 0.00299
Webber number 3.47E-05
Bond/Webber 86.12523
Capillary number 0.00019
From calculating the non-dimensional number it is possible to see what
forces dominate in the system.
Low Reynolds number: This show that the viscous forces dominate over the inertial
forces. The Reynolds number determines that the flow regime is laminar. As the
Reynolds number is much less than one, this means that the flow is in the Stokes
flow regime, as it is highly laminar.
Low Bond number: This shows that the surface tension forces dominate the
gravitational or body forces. The system is greatly dominated by surface tension as
the Bond number is much less than one. This is relevant to the design of the
manifold as will be discussed later.
Low Webber number: This shows that the inertial forces are negligible in
comparison to the surface tension forces. This also confirms that the dominating
forces in the system are the surface tension forces.
Low Capillary number: This shows that the viscos forces are dominated by the
surface tension forces, this means that the system in entirely dominated by the
surface tension forces.

25
6.2 Discussion
6.2.1 Occurrence of cavitation
The camera used; records at a frame rate of 15 frames per second. Therefore it is
possible to put a time scale on the formation of the vapour. This is illustrated below
in Figure 14. The development of vapour is a rapid process and happens in under a
second. This also strengthens the argument that the development of vapour requires
very specific conditions in order to develop as it occurs so rapidly.
Figure 14: development of vapour in the system.
Above Figure 14 can be seen. This figure shows the development of vapour in
the system. The vapour begins on the surface of the droplet and then expands as the
droplet travels down the line. The way in which the vapour develops, strengthens the
argument of cavitation on the surface of the droplet. This rapid drop in pressure, due
to the creation of a vacuum, allows the vapour to develop on the surface of the
Frame 1
0.06 sec
Frame 3
0.18 sec
Frame 5
0.30 sec
Frame 12
0.72 sec
Frame 14
0.93 sec
Frame 7
0.42 sec
A B C
D E F
Cavitation Occurs
1mm 1mm 1mm
1mm 1mm 1mm

26
droplet. This vacuum is created by a tensile force applied to the liquid-liquid
interface.
An observation that has been noted within the
manifold of the system. Droplets that have passed
through the system are collected in the manifold. Due
to the droplets high surface tension it remains
attached to the outlet of the line, and acts like a valve,
by increasing resistance in the line.
This is also confirmed by the Bond number, when
calculated is much less than one (0.00299). This
shows that the system is highly dominated by the
surface tension. More than one droplet is required to
reduce the flow rate significantly. This can be seen in
Figure 15. This increases the resistance will reduce the volume flow rate in the lines
where the droplet remains attached. This follows Ohms law when applied to fluid
dynamics (Millikan & Bishop, 1917). This decrease in volume flow rate in the block
line will then distributed over the remaining unblocked lines. This follows the
discussed theory in section 4; Figure 5. This then causes a decrease in velocity of the
lines that are blocked. When the droplet has accumulated at the outlet and can no
longer maintain its attachment to the outlet. It drops off and the line becomes
unblocked. This unblocking causes a rapid increase in the volume flow rate to that
line. This rapid increase causes the
trains in the line to accelerate. This is
illustrated in Figure 16 below; this will
force the flow in the lines to re-
develop.
The droplet therefore acts as a
valve on the end of the outlet of the
line. The droplet detaches due to the surface tension to mass ratio. This is because as
more droplets accumulate at the outlet
Figure 15: Droplet accumulation on
outlet in manifold
Figure 15: Droplet accumulation on
the outlet in manifold
Droplets accumulating on
the outlets of the lines
Droplet detaches
Figure 16: Effect droplet accumulation on flow rate

27
Direction of Flow
of the line the volume of droplets increase, the droplet detaches with the surface
tension forces can no longer over comes the gravitational forces as the volume of the
droplet accumulation increases.
Figure 17: Acceleration of fluid with capacitance effect
Above can be seen Figure 17, a schematic of how the capacitance effect
travels down the line and how the mass of the proceeding droplets effects the
creation of vapour downstream. As the droplet that is attached to the outlet of the
manifold detaches, it acts as a valve. A train consists of 3 droplets.
This opening of the valve returns the volume flow rate to the line. This
increase in volume flow rate, results in an increase in velocity, which applies
acceleration to the line. This therefore means the fluid flow in the line must re-
develop. As the acceleration of the fluid travels down the line, it has a capacitance
effect as the flow become fully developed again, beginning from upstream to
downstream. This can be seen above in Figure 18. Train “A” is the first to see the
Cavitation
Higher Velocity
Mass
Interface
Direction of Flow
Figure 18: Trains of 3 droplets

28
applied acceleration. From Newton’s second law. (Feynman, 2010)
(8)
This means now that the flow at Train “A” is now fully-developed flow. As the
train “A” is now fully developed, therefore the acceleration of train “A” is zero and
travels at a constant velocity. This therefore means that train “A” has momentum
(Feynman, 2005)
(9)
If the interface between train “A” and “B” can withstand the tensile force applied by
the acceleration of train “A”, train “B” will experience the acceleration as it
develops. This tensile force is only experienced by the liquid-liquid interface as train
“B” is developing. Once train B is fully developed the acceleration of train “B” is
equal to zero. This means that train “A” and “B” have momentum as they both are
moving at the same constant velocity. This allows the addition of train “A” and “B”
masses.
As train “B” becomes fully developed, the liquid-liquid interface between
trains “B” and “C” experiences this applied acceleration as the flow around train “C”
is developing. The mass of both train “A” and train “B” are experience. This
increases the force, as the mass is increased. (Mass of train “A” and “B”)
If the liquid-liquid interface between train “B” and train “C” can withstand
the force applied by train “A” and “B” as it develops. This means that train “C” can
now become fully developed as well. This therefore increases the force as the mass
of “A”, “B”, and “C” can be summated as all three trains are now fully developed.
Therefore the liquid-liquid interface behind train “C” experiences the peak
force as the mass of train “A”, “B”, and “C”, are all summated and are fully
developed. The peak force is applied to the liquid-liquid interface of the leading
droplet. This is where cavitation is at its highest to occurrence. This is because the
mass at a higher velocity has increased i.e. more momentum upstream

29
Figure 19: Tensile forces applied during developing flow that causes cavitation
Above in Figure 19 can be seen. In this figure the tensile forces are applied to the
liquid-liquid interface. Force 1 is applied to the droplet is at a lower velocity. Force 1
is also applied due to a higher viscosity caused by the carrier fluid being at a lower
temperature. This will be discussed further in later sections. Force 2 is applied as the
upstream flow becomes fully developed at the new higher velocity. Force 2 is only
applied to the surface of the droplet as it developing. Force 2 is applied to the liquid-
liquid interface is due to the momentum change in the fluid due to the acceleration as
the flow that the droplet is it is developing. This increase in velocity is due to the
droplet detaching from the outlet of the line that acts like a valve. Therefore
cavitation occurs when force 2 exceeds the liquid-liquid interfacial force. This tensile
force is due to the mass of trains “A”,”B”, and “C” multiplied by the increase in
velocity.
The momentum of the fluid is given by. (Feynman, 2010)
(9)
This means that the fully developed flow has momentum. This allows for the
mass of the trains can be summated, thereby increasing the tensile force experienced
as the flow at the droplet is developing.

30
As the numbers of trains that become fully developed increases, so does the force
applied, this is because the number of trains increases the mass thus increasing the
tensile force experienced in developing flow: (Feynman, 2010)
(8)
This tensile force is only experienced as the train is developing. Once the train is
developed the acceleration of the train is zero and the train of droplets now have a
momentum. This is allows for the summation of the masses of the fully developed
trains.
Derivation of Theory
From Newton’s second law
(8)
As acceleration is rate of change in velocity:
(8.1)
(8.2)
Therefore the force is the rate of change in velocity:
(8.3)
(Feynman, 2010)

31
6.2.2 Temperature affects viscosity
The effect temperature has on the system. As the temperature is increased it
will reduce the viscosity of the PD5 oil.
Table 6: Change in viscosity with temperature
Temp (Deg ˚C) 95 21
Viscosity (Pa.s) 0.0030634 0.0043362
As the viscosity decrease with an increase in temperature, which increases the
rate the fluid can deform. This allow for the tensile force to be applied faster to the
liquid-liquid interface. This increases the peak force that the liquid-liquid interface
experiences. The increase in peak force is because the fluid can become fully
developed faster. Resulting in an increase in the force experienced at the surface of
the droplet. This allows cavitation to occur. Cavitation also occurs faster the higher
the temperature, this will result in a more rapid pressure drop.
The tensile force is applied to the liquid-liquid interface is due to the momentum
change in the fluid due to the acceleration as the flow the droplet is it is developing.
The reduction in viscosity has an effect on the rate of momentum change as the fluid
is developing. This then allows it to be possible to show the difference in peak forces
experienced at the liquid-liquid interface at two different temperatures.
By increasing the temperature from 21˚C to 95˚C reduces the viscosity by 29%.
This is a considerable decrease. As the momentum is converted into a force by
dividing by the time at which the force acts over, and if the viscosity is reduced by
29% this will increase the tensile force that will be applied to the liquid-liquid
interface which causes cavitation.
By using the frame rate from the camera. As the camera is recording at 15fps,
refer to Figure 14. By looking at A-B in this figure it is clear that there is no
cavitation at A, but two frames later in B, cavitation occurs. Therefore it is possible
to get a time scale to convert the momentum into a force. The time in which
cavitation occur is 0.12 sec. Therefore it is possible to find the force applied to the
liquid-liquid interface by knowing the volume of the liquid proceeding cavitation
location and by knowing the volume flow rate increase as the droplet valve is
opened. This opening in the valve results in an acceleration of the fluid in that line.

32
By taking an example of a 30 second spacing of PD5 oil and calculating the
momentum. This is calculated by assuming that there is even distribution of the
volume flow rate from the syringe pump over the 24 line. This will result in a flow
rate of 15 µl/min and applying it over the 30 seconds for which it is dipped in PD5
oil, gives a volume of 7.5 µl and then convert to m^3. Then by multiplying the
volume by the density to determine the mass. By multiplying the mass by the
velocity of the flow, which is 0.0020 m/s. This gives a momentum figure of
1.64809E-12 N.s.
As the viscosity is reduced by 29% at 95˚C and the time taken for cavitation took
0.12 s (Refer to Figure 13 between A-B) therefore it must take 29% longer at 21˚C
which is 0.1548 s.
To convert the momentum in to a force, the momentum must be divided by the
time which the force is applied.
This means as the temperature is increased the viscosity of the PD5 oil is reduced,
this allows the fluid to deform faster, which results in a peak force increase of 29%.
Resulting to the liquid-liquid interface experiencing a higher tensile force at higher
temperature.
This is just an example of how the reduction in viscosity can affect the tensile
force applied to the liquid-liquid interface. This doesn’t mean that cavitation occurs
between these two forces. This just shows the effect that temperature has on the
force applied. Elevated temperature reduces the viscosity and this increase the tensile
force applied to the liquid-liquid interface. This is because at elevated temperatures it
allows for the fluid to deform faster.

33
All of the recorded cavitation even occurred on the first heater. This is
because PD5 oil had heated up to 95˚C which reduces the viscosity. Also the first
heater is far enough downstream where, and the maximum amount of volume is up
stream of the cavitation location. This maximum volume upstream of the cavitation
location, maximise the momentum force experienced by the liquid-liquid interface.
This is the location where all effects are optimised.
Figure 20: Location of where cavitation occurs.
Above in Figure 20 shows the location where vapour develops in the system.
Vapour develops on the first heater but is then present in the lines for the remainder
of the experiment.
The line affected, will have a reduced flow rate. This will affect the all of the
other lines as all lines have a common manifold. As the resistance in the affected line
increases, the volume flow rate is reduced. This reduction in volume flow rate will
then be disbursed over the other lines, resulting in a slightly increased volume flow
rate.

34
6.2.3 Effects of vapour on the system
After cavitation occurs, the droplet does decelerate as the vapour forms. This
deceleration is caused by the increase in resistance in the line, due to the vapour
being present. This results in a reduction in the volume flow rate. This follows Ohms
law directly when applied to fluid dynamics (Millikan & Bishop, 1917). A reduction
in the volume flow rate will reduce the velocity in the line.
This reduction in volume flow rate manifests itself into the other lines within the
system. This is due to the fact that all lines have a common manifold. As a syringe
pump maintains flow rate despite the pressure drop along the lines. (See section 4
Figure 5) This is due to Ohms law again (Millikan & Bishop, 1917). The pressure
gradient is maintained by the syringe pump as the resistance increases, due to the
presence of vapour. The volume flow rate decreases in the line where vapour is
present, as the pressure gradient is maintained. This will therefore affect the velocity
of all of the lines.
This results in a reduction in velocity in the lines with vapour and an increase in
the velocity with the lines with no vapour present. This variation in velocity will
disrupt the three stages in PCR. Denaturing, annealing and, extending processes, this
is because the velocity of the lines must be tightly controlled during these stages.
This will become difficult if the velocity of the lines is not uniform. This is most
important for the extension stage of PCR, as a tight control in the time the droplets
spend in this stage determines how well the DNA amplifies (MIT Open Course
Ware, 2012). Therefore any reduction or increase in the velocity will cause a varied
amount of amplification. As the amplification follows a power law the effects of
change in velocity could be greatly increase or decrease the amplification. As the
time spent in the extension stage must be tightly controlled. This depends how early
or late the increase or decrease in velocity occurs during the thermo-cycling process.

35
6.2.4 Analysing experimental data
By using a large sample size the statistical results obtained from
experimentation have been with an acceptable range of error. This has been shown
by calculating the standard error of the mean (Refer to Table 2). Table 2 therefore
shows that the sample sizes of the experiments are justified. By calculating the
Standard Error of the Mean, in relation to the mean occurrence for each of the four
experiments. All four standard error of the sample mean are below 0.40%, this is a
negligible error and the sample size is justified. This is an acceptable error to have.
In order to show that the results are independent of sample size the variance
of the results were calculated and shown to be of the same order. This shows that the
results are independent of the sample size.
Table 7: Variance of the experimental data
Spacing between droplets Variance from the mean
5 second 0.00000000483761%
10 second 0.000000003067628%
15 second 0.00000000423322%
20 second 0.00000000437793%
(Harper, 1991)
This also shows that the sample size is irrelevant. This as all results lie within the
same order of magnitude. As the variance is so small it’s is considered

36
The occurrence of vapour reduces considerably as the spacing is increased to
10 seconds. This reduces the occurrence of vapour to 0.000166% this is the lowest
occurrence of vapour that was. This is 9 occurrences in 54,000 droplet sample size.
Table 8: results of vapour formation at 10 second spacing
Experiment 1 ( 10 second spacing)
Run 1 Run 2 Run 3 Run 4 Run 5
Test 1 0.000% 0.000% 0.000% 0.000% 0.001380%
Test 2 0.000% 0.000% 0.000% 0.000% 0.000%
Test 3 0.000% 0.000% 0.000% 0.00276% 0.000%
Test 4 0.000% 0.000% 0.000% 0.000% 0.000%
Test 5 0.000% 0.000% 0.000% 0.000% 0.000%
Test 6 0.000% 0.001380% 0.000% 0.000% 0.001380%
Test 7 0.000% 0.000% 0.000% 0.000% 0.000%
Test 8 0.000% 0.000% 0.000% 0.000% 0.000%
Test 9 0.000% 0.000% 0.000% 0.000% 0.00276%
Test 10 0.000% 0.000% 0.000% 0.000% 0.000%
Test 11 0.000% 0.000% 0.000% 0.000% 0.000%
Test 12 0.000% 0.000% 0.000% 0.000% 0.000%
Test 13 0.000% 0.000% 0.000% 0.000% 0.000%
Test 14 0.000% 0.000% 0.001380% 0.000% 0.000%
Test 15 0.000% 0.000% 0.000% 0.001380% 0.000%
% over
15 0.0000000% 0.0000920% 0.0000920% 0.0002760% 0.0003680%
Total 0.00017%
Table 7, shows the results from the first experiment, done at 10 seconds
spacing. This was the first experiment carried out. This set of results yielded the
lowest occurrences of vapour events. As this was the first experiment carried out,
this means that there was no droplets present in the manifold to act as a valve on the
outlet of the line. It can be seen that the occurrence in vapour increases as the
number of runs increase. As the number of runs increase this increases the number of
droplets being present in the manifold, this will increase the likelihood that droplets
will accumulate at the outlet of the line. This increase in accumulation will increase
the likelihood of the droplet to act as a valve on the outlet of the line. As this
happens it can be seen that the number of vapour events over the 15 lines does
increase (highlighted in the red box). This experiment yielded the lowest result as it
was the first experiment carried out. The lower results can be attributed to no
droplets being present in the manifold accumulating at the outlet. This therefore

37
means that the spacing of the droplets is not the main effect on the development of
vapour in a line.
After the first experiment (10 seconds spacing) the manifold had droplets
present in it. This could be a factor in why the results at 5 seconds, 15 seconds and
20 seconds vary very little. As there were droplets already present in the manifold to
act as a valve of the outlet of the line. This is the main effect on the development of
vapour. This can be seen in Table 7. With reference to run 1, no vapour is created as
there is no droplets present in the manifold. The only liquid present in the manifold
is PD5 oil, which is the carrier fluid that is being pumped. As the number of tests
increased, so does the volume of droplets in the manifold. This also shows an
increase in the vapour occurrences recorded. (Ref to table 7)
During the experimental trials, it was noted that vapour tended to develop in
certain lines more regularly. As droplets may more prone to accumulate at the outlets
of certain lines than others. This may be due to uneven surface finish of the outlets of
these lines. This would increase the likelihood of droplets accumulating on the outlet
of the line.
Figure 20: Percentage occurrence of vapour for 5s, 15s, and 20s spacing
The trend as the spacing is varied between droplets (Ref Figure 20). It can be
seen that at 5 second spacing has the highest occurrence of vapour developing. This

38
shows that the tight spacing of droplets within the train have an effect on the
occurrence of vapour formation. This exhibits the highest occurrence of vapour of
0.00060%. These figures show an occurrence of 13 vapour occurrence in a sample
size of 21,600 droplets.
Figure 20 shows the percentage occurrence of vapour for 5 second, 15 second
and 20 second spacing. For this the 10 second spacing experiment results are
excluded as number of droplets present in the manifold was so low. The trend line in
the figure shows that there is a decrease in the occurrence of vapour as the spacing is
increased. This could be explained as the spacing is decreasing the rate at which the
droplets enter the manifold is increased. This means that at a shorter spacing of 5
seconds the likelihood of two droplets accumulating at the outlet of the line is higher.
This is why a slightly elevated occurrence of vapour at shorter spacing. This means
at a longer spacing, the longer the droplet must stay attached to the outlet, in order
for the next droplets to come accumulate at the outlet and act as a valve.

39
The effect of the droplet valve was evident over the entire set of test results
gathered. In all of the tests carried out there was no presence of vapour in any of the
first tests carried out (refer to Appendix A). There is no vapour present for any of the
first tests in run one, across all of four line spacing. This is relevant as for the first
tests there would not be any droplets in the manifold to accumulate at the outlet of
the lines. Therefore there is no droplet valve present.

40
6.2.5 Surfactants and bookending
6.2.5.1 Surfactants
The spacing of droplets has a minor effect on the occurrence of vapour development
in the line. The major cause for vapour development is the accumulation of droplets
on the manifold outlet.
Figure 21: Droplet accumulates to the outlet of line 1 due to high surface tension.
Shown above in figure 21 is a schematic showing an accumulation of droplets on
the outlet of the line. These droplets adhere to the outlet due to the high surface
tension of the liquid. The droplet also adheres to the surface of the outlet due to the
low Bond number (Hager, 2012). This because the surface tension forces (F1) is
dominating the gravitational forces (F2). This means that if the droplet is to detach, it
means that a high volume of accumulated droplets are required to overcome the
surface tension forces.
Droplet Valve

41
In previous experimental data gathered with the use of surfactants in the droplet.
These results with surfactants shows that there if a reduction in the occurrence of
vapour (refer to Table 1, in section 3).
Figure 22: Droplet detaches from the outlet of Line 1 due to low surface tension.
This is due to the fact that Triton-X100 has a much lower surface tension, and
therefore less volume of an accumulation is required for the gravitational forces (F2)
to overcome the surface tension forces (F1). This means that a droplet of Triton-X100
will not stay attached to the outlet of the line for long enough to cause the flow rate
to be reduced in the line. As the droplets of Triton-X100 do not accumulate on the
outlet of the line, there is no droplet valve present to disrupt the flow rate in the line.
Triton-X100 has similar density as water,
Table 9: Density and surface tension
Density, ,
(g/cm3)
Surface tension, ,
(mN/m)
PD5 0.92 24.2
H20 1 72.8
Triton-X100 1.03 33.0
Table 8, compares the surface tension values of H20 and Triton-X100. As H20 has
a much higher surface tension value than Triton-X100, this means that the Bond number for
the H20 system will have a much lower Bond number (Hager, 2012). This means in a
100per cent H20 droplet will require more volume to detach from the outlet of the line

42
6.2.5.2 Bookending
Droplets with lower interfacial tension are place on the front and back of the train
of droplet. This means the numbers of droplets with a high interfacial tension are in
cased between droplets with a lower interfacial tension. Droplets with lower
interfacial tension contain di-propylene glycol (DPG). As these droplets, with lower
interfacial tension must exit the line first. As these droplets exit first and have a
lower surface tension force the droplet will not adhere to the surface of the outlet. As
these droplets exit first it may be possible that a di-propylene glycol (DPG) residue
is left on the outlet. This DPG residue may cause the high surface tension droplets to
detach and not accumulate at the outlet.
Bookending results show that for a larger train size of droplets, the creation of
vapour become sporadic and irregular. These results may be due to the DPG residue
left by the leading droplets, with lower interfacial tension on the outlet to be
removed and wear off. This would then cause droplets of higher surface tension to
accumulate on the surface of the outlet. It would then be possible the accumulation
of droplets to create a droplet valve at the outlet. This then would lead to an increase
in the presence of vapour in the lines.

43
6.3 Summary of Discussion
 Low Bond number in the system means that the surface tension forces
dominate over the gravitational forces. The Bond number is very much less
than one (0.00299) (Hager, 2012).
 Due to the water droplets having a high surface tension, and that the system
is dominated by a very low Bond Number, this means that within the
manifold, on the outlets, it allows for the adherent of the droplets on the
surface of the outlet of the line.
 This adherent has been observed in the manifold over the course of the
experimental trial.
 As the droplets adhere to the surface of the outlet, an accumulation effect
occurs. This accumulation effect decreases the volume flow rate in that line.
Creates a droplet valve on the outlet of the line.
 As the body forces of the accumulation of the droplets exceed the tensile
forces, this is due to the volume of the accumulation increasing. The droplet
detaches opening the droplet valve.
 The droplet detaches and the volume flow rate is restored to the line causing
the fluid in the line to accelerate. This caused the line to redevelop with a
capacitance effect from the proximal end of the line to the distal end.
Proximal in relation to the syringe pump.
 As the flow redevelops, a tensile force is applied to the fluid as it develops.
This tensile force increases as the mass of the redeveloped fluid increased
from the proximal end to the distal end.
 This tensile force is peak at the distal end, as the most amount of mass is
developed upstream. This is why cavitation occurs over the first heated plate.
 This tensile force acts on the liquid-liquid interface, causing cavitation to
occur.
 Increasing the temperature reduces the fluids viscosity, resulting the fluid
deforming at a faster rate. This increases the force experienced at the liquid-
liquid interface, causing cavitation to occur at the liquid-liquid interface.
 The combination of both the tensile force and reduction of viscosity
contribute to cavitation of the liquid-liquid interface.

44
 From the results obtain, when no droplets are in the manifold no vapour is
created. As the number of droplet in the manifold increases so too does the
vapour events.
 The spacing of the droplets does affect the vapour events. This is due to the
rate at which the droplets enter the manifold affects the vapour events.
Resulting in more droplets enter the manifold in a shorter space of time,
contributing to a quicker accumulation of droplets at the outlet.
 When surfactants are present in the droplets the surface tension is reduced,
increasing the Bond number and the droplets are unable to accumulate at the
outlet within the manifold.

45
7. Conclusion
1. Hypothesis was developed using previous experimental data. This was done as
by carrying out experiments, where the distance between the droplets is varied.
2. Experimental rig was developed and was based on the specific requirements
needed to explore the hypothesis.
3. Experimental rig was built, calibrated and relevant data obtained.
4. Relevant data was obtained as the experimental trials were designed on the
previous experimental trials carried out. This was done so the results would be
comparable. The hypothesis was applied to gather relevant data.
5. Statistical data was gathered on the occurrence of vapour within the lines of the
system. A large sample size was used to develop the statistical results. All results
are available in Appendix A
6. A greater understanding of the forces within the system was developed. This was
done by using non-dimensional number to characterise the forces within the
system.
7. This greater understanding was used along with observations while carrying out
the experiments.
8. Results of experimental trials and observations discussed, within the context of
the formation of vapour and the proposed hypothesis.
9. Statistical data gathered and compared to previous data gathered on this topic.
Report produced with findings of the FYP.

46
8. Recommendations
 Change geometry of outlet cross-sectional area (CSA), reducing the CSA,
this will increase the stress applied by the body force and also reduce the
stress applied by the surface tension forces at the outlet. The CSA must be
reduced as to increase the stress applied by the body forces of the droplet.
This will result in no accumulation of droplets at the outlet
 Possible removal of the use of the manifold in the system. Hence reducing
the chance of droplet accumulation. This will also allow for better control in
the velocity of each line.
 Coating the outlets with a hydrophobic coating. Droplets will not adhere to
the outlets if hydrophobic coating is present.
 Extending the outlet to the bottom of the manifold. This will reduce the
accumulation of the droplets at the outlet surface.

47
References
T.M. Dalton,a and E. D. Dalton,a.. (2013). Cavitation in microfluidic plug flow. The
Royal Society of Chemistry 2013. 1 (1), 1-3.
T.M. Dalton,a. (2012). On the liquid-liquid interface in high throughput
microfluidics. Stokes Institute. 1 (1), 1-15.
[14] Romain Deschamps and Benjamin Delerue. (2013). Microfluidics Project.
ICAM Technical Report. 2 (1), All.
Engineers Edge. (2000). Volumetric Flow Rate- Fluid flow. Available:
http://www.engineersedge.com/fluid_flow/volumeetric_flow_rate.htm. Last accessed
19 Mar 2014
The Engineering Tool Box. (2005). Reynolds Number. Available:
http://www.engineeringtoolbox.com/reynolds-number-d_237.html. Last accessed 19
Mar 2014.
Feynman, Richard P.; Leighton, Robert B.; Sands, Matthew (2005). The Feynman
lectures on physics, Volume 1: Mainly Mechanics, Radiation, and Heat (Definitive
ed.). San Francisco, Calif.: Pearson Addison-Wesley. ISBN 978-0805390469.
Feynman, Richard P.; Leighton; Sands, Matthew (2010). The Feynman lectures on
physics. Vol. I: Mainly mechanics, radiation and heat (New millennium ed.). New
York: BasicBooks.
Hager, Willi H. (2012). "Wilfrid Noel Bond and the Bond number". Journal of
Hydraulic Research 50 (1): 3–9.
MIT Open Course Ware. (2012). Polymerase Chain Reaction (PCR) | MIT 7.01SC
Fundamentals of Biology. Available:
http://www.youtube.com/watch?v=OK7_ReXhVaQ. Last accessed 18th Mar 2014.
Robert A. Millikan and E. S. Bishop (1917). Elements of Electricity. American
Technical Society. p. 54.
National Instruments. (2013). Install NI LabVIEW and NI-DAQmx Driver.
Available:
http://www.ni.com/gettingstarted/installsoftware/dataacquisition.htm#Installing NI-
DAQmx. Last accessed 18th Mar 2014.
Dr David Newport. (2014). Syringe Pumps in Microfluidics. ME6008. 1 (Lec 3), 15-
22.

48
Rosen MJ and Kunjappu JT (2012). Surfactants and Interfacial Phenomena (4th
ed.). Hoboken, New Jersey: John Wiley & Sons. p. 1
John. R. Saylor and Garrett D. Bounds. (2012). Experimental Study of the Role of
the Weber and Capillary. TRANSPORT PHENOMENA AND FLUID
MECHANICS. 10.1002 (1), p2-3.
[15] WM Harper (1991). Statistics. City of London Polytechnic: Financial Times
Prentice Hall. 300.

a
Appendix
Appendix A
Experiment 1 (5 seconds spacing)
Run 1 Run 2
Test 1 0.000% 0.000%
Test 2 0.000% 0.000%
Test 3
0.001380
%
0.001380
%
Test 4 0.000% 0.000%
Test 5
0.001380
%
0.001380
%
Test 6
0.001380
% 0.000%
Test 7 0.000%
0.001380
%
Test 8 0.000%
0.001380
%
Test 9
0.001380
% 0.000%
Test 10 0.000% 0.000%
Test 11
0.001380
%
0.001380
%
Test 12 0.000% 0.000%
Test 13
0.001380
% 0.000%
Test 14
0.001380
%
0.001380
%
Test 15 0.000% 0.000%
% over
15
0.000644
%
0.000552
%
Std.
dev
0.000065053823869162
4%
Total
0.000598
%
Varian
ce 0.00000000004232%
Averag
e 0.000598%
0.00138% = 1 occurrence in 720
droplets Std.Err
0.000000442635206378
713%
0.00276%= 2 occurrence in 720
droplets
Total droplets = 21,600 droplets
24 lines
Temp 95˚C
Q=0.36 ml/mn (15µl/mn per
line)
3 droplet train
5 seconds between each
droplet
30 seconds between each train
Dip 0.12s

b
Experiment 2 ( 10 second spacing)
Run 1 Run 2 Run 3 Run 4
Run 5
Test
1 0.000% 0.000% 0.000% 0.000%
0.0013
80%
Test
2 0.000% 0.000% 0.000% 0.000% 0.000%
Test
3 0.000% 0.000% 0.000%
0.0027
6% 0.000%
Test
4 0.000% 0.000% 0.000% 0.000% 0.000%
Test
5 0.000% 0.000% 0.000% 0.000% 0.000%
Test
6 0.000%
0.0013
80% 0.000% 0.000%
0.0013
80%
Test
7 0.000% 0.000% 0.000% 0.000% 0.000%
Test
8 0.000% 0.000% 0.000% 0.000% 0.000%
Test
9 0.000% 0.000% 0.000% 0.000%
0.0027
6%
Test
10 0.000% 0.000% 0.000% 0.000% 0.000%
Test
11 0.000% 0.000% 0.000% 0.000% 0.000%
Test
12 0.000% 0.000% 0.000% 0.000% 0.000%
Test
13 0.000% 0.000% 0.000% 0.000% 0.000%
Test
14 0.000% 0.000%
0.0013
80% 0.000% 0.000%
Test
15 0.000% 0.000% 0.000%
0.0013
80% 0.000%
%
over
15
0.0000
000%
0.0000
920%
0.0000
920%
0.0002
760%
0.0003
680%
Std.
dev 0.000151%
Total
0.0001
7%
Varia
nce
0.000000000228
528%
Aver
age 0.000166%
droplets
Std.E
rr
0.000000650538
238691624%
droplets
24 lines
Temp 95˚C
line)
3 droplet train
droplet
Dip 0.12s

c
Run 1 Run 2
Test 1 0.000% 0.000%
Test 2 0.000%
0.001380
%
Test 3 0.000% 0.000%
Test 4
0.001380
% 0.000%
Test 5 0.000%
0.001380
%
Test 6 0.000%
0.001380
%
Test 7 0.000% 0.000%
Test 8 0.000%
0.001380
%
Test 9 0.000% 0.000%
Test 10 0.000% 0.000%
Test 11 0.000% 0.000%
Test 12 0.000% 0.000%
Test 13
0.001380
% 0.000%
Test 14 0.000% 0.000%
Test 15 0.00276%
0.001380
%
% over
15
0.000368
%
0.000460
%
Std.
dev
0.000065053823869162
4%
Total
0.000414
%
Varian
ce 0.00000000004232%
Averag
e 0.000414%
droplets Std.Err
0.000000442635206378
713%
droplets
24 lines
Temp 95˚C
Q=0.36 ml/mn (15µl/mn per line)
3 droplet train
15 seconds between each droplet
Dip 0.12s

d
Run 1 Run 2
Test 1 0.000% 0.000%
Test 2
0.001380
% 0.000%
Test 3 0.000%
0.001380
%
Test 4
0.001380
% 0.000%
Test 5 0.000% 0.000%
Test 6
0.001380
%
0.001380
%
Test 7 0.000% 0.000%
Test 8
0.001380
% 0.000%
Test 9 0.000% 0.000%
Test 10 0.000% 0.000%
Test 11*
0.001380
% 0.000%
Test 12
0.001380
%
0.001380
%
Test 13 0.000% 0.000%
Test 14 0.000% 0.000%
Test 15 0.000%
0.001380
%
% over
15
0.000552
%
0.000368
%
Std.
dev
0.000130107647738325
%
Total 0.00046%
Varian
ce 0.00000000016928%
Averag
e 0.00046%
droplets Std.Err
0.000000885270412757
426%
droplets
24 lines
Temp 95˚C
line)
3 droplet train
droplet
Dip 0.12s

e
Experiment 3
(5Sec) Experiment 1
(10Sec) Experiment 5
(15Sec) Experiment 2
(20Sec)
0.000598%
0.0001656%
0.000414%
0.000460%
% Occurence
0.00000%
0.00010%
0.00020%
0.00030%
0.00040%
0.00050%
0.00060%
0.00070%
1 2 3 4
%Occurence
Experiments
Mean with Standard Error

g
Appendix C
Turnitin Originality Report
Seán Cunningham 10138455 by Seán Cunningham
From FYP Submission (FYP1314)
 Processed on 20-Mar-2014 12:35 PM GMT
 ID: 407877502
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