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 95C 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

Experiment 2: Spacing 10s

Experiment 3: Spacing 15s

Experiment 4: Spacing 20s

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

% O

ccu

ren

ce

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

stan

dar

d e

rro

r o

f m

ean

Experiments No.

Standard Error of the Sample Mean

24

Density (ρ) kg/m^3 920

Viscosity (μ) Pa.s @ 21C 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) @95C 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

0.0000650538238691624%

Total 0.000598

%

Variance 0.00000000004232%

Average 0.000598%

0.00138% = 1 occurrence in 720 droplets

Std.Err

0.000000442635206378713%

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%

0.001380%

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%

Std. dev 0.000151%

Total 0.0001

7%

Variance

0.000000000228528%

Average 0.000166%

0.00138% = 1 occurrence in 720 droplets

Std.Err

0.000000650538238691624%

0.00276%= 2 occurrence in 720 droplets

Total droplets = 54,000 droplets

24 lines Temp 95˚C Q=0.36 ml/mn (15µl/mn per line) 3 droplet train 10 seconds between each droplet 30 seconds between each train

Dip 0.12s

c

Experiment 3 (15 seconds spacing)

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

0.0000650538238691624%

Total 0.000414

%

Variance 0.00000000004232%

Average 0.000414%

0.00138% = 1 occurrence in 720 droplets

Std.Err

0.000000442635206378713%

0.00276%= 2 occurrence in 720 droplets

Total droplets = 10,800 droplets

24 lines Temp 95˚C Q=0.36 ml/mn (15µl/mn per line) 3 droplet train 15 seconds between each droplet 30 seconds between each train

Dip 0.12s

d

Experiment 4 (20 seconds spacing)

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%

Variance 0.00000000016928%

Average 0.00046%

0.00138% = 1 occurrence in 720 droplets

Std.Err

0.000000885270412757426%

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 20 seconds between each droplet 30 seconds between each train Dip 0.12s

e

Experiment 3(5Sec) Experiment 1

(10Sec) Experiment 5(15Sec) Experiment 2

(20Sec)

0.0

00

59

8%

0.0

00

16

56

%

0.0

00

41

4%

0.0

00

46

0%

% Occurence

0.00000%

0.00010%

0.00020%

0.00030%

0.00040%

0.00050%

0.00060%

0.00070%

1 2 3 4

% O

ccu

ren

ce

Experiments

Mean with Standard Error

f

Appendix B

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 Word Count: 11952

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