SUTD: 10.

006 Chemistry and Biology 1: The Natural World: 1D Designette

2015

Design and Fabrication of Microfluidic Device for Lab-on-a-Chip Chemistry

Microfluidics, the manipulation of fluid streams in micro-scale dimensions, is of growing
technological interest because of its diverse applications, including medical diagnostics and
environmental monitoring. Microfluidic devices are becoming increasingly important because
(i) they require only small reagent volumes (nano-to-picoliters) and (ii) their small channel
widths facilitate efficient analytical experiments. For example, microfluidic channels have
been used to deliver samples for DNA manipulation and protein analysis; such samples
usually come only in small volumes due to their high cost. Furthermore, microfluidic
networks built on lab-on-chip devices offer high-throughput, miniaturized and mobile platform
for experiments.
A microfluidic channel provides a useful platform to perform chemical and biological
reactions in confined environment. In this 3-session laboratory exercise, we are going to
learn:
(1) how chemistry knowledge is used to design materials with desirable physical and
chemical properties
(2) how different materials are used in a design sequence to prototype a device for certain
function: in this case, a microfluidic device with Y-shaped channel for interfacing two
fluids
(3) how chemical and biological reactions are visualized in a microfluidic device and how the
results are compared with those in bulk solutions.
Background
Lets have a brief introduction of Polymer Chemistry before going into the details of the
laboratory process. A rational design and synthesis procedure could help us tailor the
structure and architecture of a polymer that would impact its various physical (e.g., flexibility)
and chemical (e.g., surface adhesion) properties.
Polymers are macromolecules formed by linking large numbers of much smaller
molecules called monomers. These chemicals have a large molecular weight and many
repeating units or chemical structures. Polymerization refers to the reactions by which the
monomers are combined. Curing is another common terminology used to describe
polymerization or the hardening process. Two types of classifications can be used to
group the polymers. One classification is based on polymer structure: condensation and
addition. The other classification is based on polymerization mechanism: step and chain
polymerizations.

Generally,

most

condensation

polymers

are

produced

by

step

polymerizations and most addition polymers are produced by chain polymerizations. The
most important difference in chain and step polymerizations is in the identities of the species
that can react with each other. As the name suggests, step polymerizations proceed by the

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stepwise reaction between the functional groups of reactants as in condensation reactions,

COOH with NH2 and COOH with OH for example. In chain polymerization, an initiator is
used to produce an initiator species R* with a reactive center which may be a free radical,
cation, or anion. Chain polymerization occurs by the propagation of the reactive center by
the successive additions of large numbers of monomer molecules in a chain reaction.
(George Odian, Principles of Polymerization, Wiley-Interscience, 2004)
The process of polymerization can be controlled by manipulating reaction conditions,
including temperature, choice of compounds involved in building the polymer, and the ratio
of the compounds involved.
parameters

are

polymerization

important
rates,

These reaction
in

controlling

polymer

molecular

weight, and architecture such as branching

and crosslinking. Polymers can have a wide
range of properties that are dependent on their
molecular structure and architecture (see
Figure 1). Depending on the properties,
polymers have extensive uses ranging from Figure 1. Increased cross-linking in the right-hand
"wash and wear" clothing to rubber tires and polymer sample compared to the left-hand sample
even

protective

enamels

and

paints. (cross-links shown in red) results in a stiffer polymer.

Innovations in polymer chemistry constantly (http://mrsec.wisc.edu/Edetc/LEGO/PDFfiles/bookcha

bring

both

improved

and

entirely

new p3.PDF)

technological applications for polymers. We

will now discuss on three polymers that we will use for this laboratory course. You will be
learning how structure and architecture of a polymer can influence its physical and chemical
properties
(1) polydimethylsiloxane (PDMS)
PDMS is one of the most common materials used to create
microstructures in microfluidic chips. It can be molded into different shapes
during polymerization. It can serve as an elastic stamp that can transfer
patterns onto glass, silicon or other surfaces. The chemical formula for
PDMS is CH3[Si(CH3)2O]nSi(CH3)3, where n is the number of repeating monomer [SiO(CH 3)2]
units. The long SiO bond linkage in PDMS offers flexibility to the polymer backbone,
whereby PDMS can behave as a very flexible rubber. The CH3 group makes the polymer
surface hydrophobic(water-fearing).
(2) Norland Optical Adhesive 81 (NOA 81) (Thiolene based polymer)
NOA 81 is a polymer containing thiolene. Thiolene has two moieties: Thiol (-SH) and ene

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(C=C; carbon-carbon double bond). The details of the chemical composition of the product
are not disclosed by the manufacturer. For details of the physical properties, we can refer to
the information published by the manufacturer.
(http://www.norlandprod.com/literature/81tds.pdf)

(A)

(B)

Figure 2: Possible functional groups present in NOA 81.

(A) 2-Propenamide, n,n-methylenebis; (B) A thiol group
As mentioned earlier, the polymerization process can be controlled through manipulation
of reaction conditions (e.g., temperature). For some polymers such as adhesives or coating
materials, the polymerization (i.e., curing, or hardening) process can be induced and
controlled by UV light. These UV curable polymers offers several unique advantages
compared to thermally cured polymers; lower energy consumption, wider range of
formulation with varying viscosities for convenient processing and application on different
surfaces. One of the prime areas of the application for UV curable polymers is to provide
clear coatings on wide range of substratesfrom metals and wood to floors and paper. Each
of these substrates demands a different set of properties from the UV curable coating. For
instance, coatings on metallic substrates must have good adhesion and be able to resist
deformation induced by stresses experienced during use.
All UV curable systems consist of four basic components for successful applications:
photoinitiator, oligomer, monomer, and additive. Light emitted from a suitable UV source
causes the photoinitiator to fragment into reactive species. These fragments subsequently
initiate a rapid polymerization process with monomers and oligomers in the systems to form
a crosslinked, durable polymer. The oligomers provide the photocured polymer with its basic
physical properties. Monomers such as acrylates are used in UV curable systems to provide
final film properties and viscosity control of the polymer. Monomers are also important in
determining the speed of curing, crosslink density and final properties.

Additives are

included as dispersants for pigment dispersion, color pigments, and stabilizers for both
thermal and UV protection.
(3) Polyvinyl chloride (PVC)
This is the material used in 3D printer and you can find detailed information about this
polymer from the textbook, Oxtoby, page 1105-1110.

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Overview of Experiments
Now we know how the chemical structures result in polymers with different physical and
chemical properties. Over the next three to classes, we will conduct the experiment in the
following three steps:
1) Creating a mold of a Y-shaped channel with NOA 81(Day 1)
2) Molding with PDMS (Day 2)
3) Assembling channel and testing for leakage; conducting experiments in the
microfluidic device (Day 3)
First, we will use these building blocks in different steps of a molding process to
prototype a microfluidic device. The schematic illustration of Y-shaped channel is shown in
Figure 3. We provide three types of channels with different dimensions of width (W) and
height (H). Each group is provided with one of the three types of channels with W=H=300
m, W=H=500 m or W=H=800m. Think how the dimension of the channels may influence
the flow of fluids.

Figure 3. Schematics of the channel dimensions for the microfluidic device.

At the beginning of your first lab session, each group will be given a PDMS master mold
prepared by your instructor in advance. You will use this PDMS master mold to make your
own microfluidic device. The overview of fabrication process of this PDMS master mold is
shown in Figure 4. The initial mold has been prepared using a 3D rapid prototyping printer.
The PDMS stamp provided to you was created by casting PDMS on the initial mold followed
by curing and subsequent releasing of the PDMS. Notice that the feature of the original mold
is complementary to the feature of the PDMS stamp (i.e. if one is convex, the other is
concave because they are created by molding and transferring the pattern.)

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Figure 4. Schematics of fabrication of the PDMS master mold.

With the PDMS stamp given to you, each group achieves the following goals over the
three sessions.
Lab Session 1. Fabrication of daughter mold with NOA (Week 6, Cohort Day 1)
1.1 Fabricating of the daughter mold (material: NOA81) using the PDMS stamp provided
1.2 Curing the daughter mold
Lab Session 2. Casting PDMS on the mold (Week 6, Cohort Day 2)
2.1 Weighing and mixing PDMS and initiator of polymerization
2.2 Casting PDMS on the daughter mold created
2.3 Degasing bubbles from PDMS
2.4 Curing PDMS
Lab Session 3. (Week 6, Cohort Day 3)
(A) Preparation of microfluidic device
3.1 Releasing the PDMS channel from the daughter mold
3.2 Creating inlets and outlets on the PDMS channel
3.3 Assembling the PDMS channel with tubing
3.4 Sealing the PDMS channel with a glass slide
(B) Chemical and biological reactions in the channel
Two scenarios are used to study how the microfluidic device can be used to perform
chemical and biological reactions on a small-scale.
4.1 An acid-base reaction in the channel
4.2 Colorimetric detection of protein by BCA Protein Assay.

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Lab Session 1 Manual (Week 5)

A. Materials and equipment:
PDMS Master Mold (made by instructors beforehand), plastic dropper, glass slides, Petri
dish, UV light source, oven, desiccator, camera, and Scotch tape.
B. Chemicals:
Norland Optical Adhesive 81, ethanol
C. Procedure:
Part A: Preparation of NOA 81 Mold
1. Clean a glass slide with water, then ethanol, and leave it
to dry in air, Label your groups slide with (a) cohort
number (b) group number.
2.

Clean both sides of the PDMS Master Mold with Scotch

tape to remove any dust particles on the surface. Then,
place the PDMS stamp in contact with the glass surface
with the Y-channel facing down. Apply pressure by
placing your finger on the top of the mold to drive the air
bubbles out.

3.

(This step needs good ventilation)

Place NOA 81 to cover the two inlets at the end of the
branched Y channel.

4.

Using a plastic dropper, apply negative pressure (i.e.

suction) from the inlet that is not covered with NOA 81.
Slightly press the head of the dropper, place the
opening of the dropper on the inlet, and release the
pressing of the head. Confirm that the channel starts to
be filled with NOA 81 (apparent from the change of the
reflection of the channel). Once you fill the entire
channel, wipe all remaining NOA 81 with tissue paper.

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2015

Expose the PDMS filled with NOA 81 under the UV

lamp. This process consists of three exposures; each
duration of the exposure is ~10 min.
(i) First expose the UV light from the side of the
PDMS stamp for 10 minutes.
(ii) Then expose it for another 10 minutes from the
side of the glass slide by flipping the PDMS and
glass upside down.
(iii) After two exposures, remove the PDMS stamp
and you will see the channel structure made of NOA
81. Finally, expose the NOA 81 with the UV light
from the top (without PDMS) for another 10 min
(iv) Take a picture of the Y-shaped channel for
yourrecord
The UV lamp should be positioned an inch or so above
the substrate. (Caution: Please dont put your hands
under the UV lamp while its turned on)

6.

7.

Put your device in a petri dish, and give it to the

instructors. The instructors will collect all devices, and
place it in the oven preset at 65 C for overnight curing.
Clean up and have your instructor sign on your lab
handout before you leave

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Lab Session 2 Manual

A. Materials and equipment:
Petri dish, disposable cups, stirring stick, balance, vacuum, oven, desiccator, camera
B. Chemicals:
Sylgard 184 elastomer kit (poly(dimethylsiloxane))
1.

Find the NOA 81 mold made in a Petri dish. (You may

want to take a picture of your cured NOA 81 mold). You
place PDMS in the Petri dish, and cure on the top of the
mold to obtain the PDMS channel complementary to the
mold.
a. Weigh the PDMS pre-polymer (30 g) in a
disposable cup, add in curing agent (3.0 g), and
fully mix (~5 min) by stirring with the ice-cream
stick.
b. Place the PDMS mixture in the Petri dish
containing the mold
c. Put a lid on the Petri dish, and place it in the
desiccator under the vacuum for 15-20 minutes.
Make sure that most of the bubbles are removed
from PDMS or staying at the surface of the
PDMS.
Tip: Steps b and c are crucial to obtaining a
transparent PDMS mold for clear visualization in
Lab Session 3.
d. Hand in the sample with degassed PDMS to the
instructor. They will be placed in the oven at
65C overnight for curing

2.

Label your groups petri dish with (1) cohort number, (2)
your group number.

3.

Clean up and have your instructor sign on your lab

handout before you leave.

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Lab Session 3

2015

Manual (Week 6)

Part A
A. Hazards
Sharps Ensure safe use and disposal of razor blades and needles. Keep its cap in safe
place when the needle is in use.
B. Materials and equipment:
27 -gauge needle, glass slide, polyethylene tubing PE 20 (inner diameter: 0.28mm, outer
diameter 0.61mm), paper binders (x2), transparent Scotch tape, biopsy punch, tweezers (x1
pair), knife, (x1), syringe (1mL, x1), camera.
C. Chemicals:
Deionized (DI) water, ethanol
D. Procedure:
1.

Cut around the edge of the Petri dish using a razor

blade and use a tweezers to peel out the PDMS stamp.
Peel off the glass slide from the PDMS stamp and cut
out the PDMS stamp around the edge of the glass slide.
Alternatively you can cut around the edge of the glass
slide (~1mm inside of the edge).
You will obtain a rectangular stamp of PDMS.

2.

The PDMS channel needs to have inlets and outlets.

Using the biopsy punch create holes for inlets and
outlet.
- Put the PDMS stamp on a flat surface (channel
side up). You can use the back of the lid of the
Petri dish.
- Put the biopsy punch in the position of the inlets
and outlets, and apply the pressure and
penetrate all the way down to the surface.
- Take the punch out, and remove the plug with a
pair of tweezers
- The plug should come out automatically when
you make the hole with the biopsy punch. If not,
then penetrate the hole again using the punch to
clear the plug.

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Do the same for all three inlets/outlets.

3.

Assemble the microfluidic device. Clean the surface of

the PDMS (channel side) using the Scotch tape to
remove all dust particles. Place the PDMS channel
against the glass slide and press down until the two
surfaces are in contact and make a good seal.

4.

Insert three ~4 cm, ~4 cm and ~4 cm lengths of tubing

into the inlets and outlet. It is suggested to cut the tubing
end at 45o before inserting into the PDMS. The slanted
tip makes it easier to insert.
Insert syringe needle into the end of the outlet tubing.
Caution: use tweezers to guide the end of the outlet
tubing; be careful not to get fingers punctured.
Assemble a syringe with the needle in the outlet.

5. To ensure that the sealing is sufficient, fill 1~2 mL of deionized water in the syringe.
Push the syringe carefully, and allow the water to flow into the Y-shaped channel to
make sure that there is no leakage and blocks in the channel
6. In case leaks are found, try different ways of troubleshooting (e.g. clips, tapes, 5min Epoxy) [Supplementary Information]. If necessary, remove the PDMS channel
from the glass slide, and clean the surface of the glass with water and ethanol, and
the surface of the PDMS with Scotch tape.
7. Label your groups slide with (1) cohort number, (2) your group number. Take a
photo of your assembled microfluidic device.

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Part B
We have fabricated the microfluidic channel master and fabricated a device with a Yshaped channel. Now we will learn how to use microfluidic devices for chemical reactions.
Specifically, the following two scenarios are explored: (1) an acid-base reaction in the
channel (2) Colorimetric detection of protein (Bovine Serum Albumin, BSA) by BCA Protein
Assay.
A. Hazards
Sharps: Ensure safe use and disposal of razor blades and needles. After use, make sure to
dispose them in the sharp waste.
Chemicals: HCl (aq) and NaOH (aq) are corrosive and can cause burns at any area of
contact. Wear lab-coat, goggles and gloves.
B. Materials and equipment:
Syringe (1mL x1), 1.5mL vials (x5), pH paper, camera, 27-gauge Needle
C. Chemicals:
0.05 M HCl solution with phenolphthalein (C 20H14O4) indicator, 0.1 M NaOH solution for acidbase reaction. BCA working reagent (green in color) and protein sample (BSA; 1 mg/mL) in
PBS (Phosphate(0.1M), NaCl (0.15 M), pH 7.2) buffer.
1. Acid-base reaction in the channel
Note: For better visualization and photo-taking, you can flip the device upside down, so that
the glass slide is facing up. In this case, make sure you hold the glass slide horizontally (do
not tilt it sidewaysthink about why).
a. Insert one of the inlet tubings into vial 1, which has an acid solution (HCl, 0.05 M) with
phenolphthalein (C20H14O4) indicator. Note the color of the solution.
b. Insert the other tubing into vial 2 with the basic solution (NaOH, 0.1 M). Note the color of
the solution.
c. Connect the syringe and needle to the outlet tubing with tweezers (Proceed with
caution!).

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d. Pull the syringe steadily, and allow the

solutions from vial 1 and 2 flow into
the Y-shaped channel simultaneously.
Observe the color change in the
channel as the flow continues.
Check whether there is any
difference between the region near
the inlet and that toward the outlet.
Consider the reasons for the color
change and the difference
between regions if there is any.

Figure 6. Acid-base reaction. Images were

captured with a standard digital camera
through the eyepiece of a compound
microscope.

e. Stop pulling the syringe and disconnect it from the outlet tubing. Observe what happens to
the solution remaining in the channel. Consider the reasons for this change.
f. Take a piece of pH paper, dispense a few drops of solution from the syringe to the pH
paper. Record the value of pH.
g. Rinse and clean the syringe, syringe needle and vials with DI water.

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2. Colorimetric Detection of Protein by the BCA assay

a) Insert one of the inlet tubings in the vial with BCA Working Reagent (WR).
b) Insert the other inlet tubing in the vial with 1mg/ml of the BSA (protein) solution in PBS
buffer.
c) Reconnect the syringe and needle to the outlet tubing with tweezers (Proceed with
caution!).
d) Pull the syringe steadily, and allow the solutions from vial 1 and 2 flow into the Y-shaped
channel simultaneously.
e) After drawing in both the fluids in the channel and some in the syringe, stop pulling the
syringe, and disconnect it from the outlet tubing.
f) Let the solutions sit in the channels, for a few minutes, ~4-5 minutes.
g) Observe if the color of the solution in the channel and in the syringe changes with time.
Record the time taken for the color to change in the channel and the time taken to change in
the syringe.
h) Check if theres any difference between the color intensity in the channels and in the
syringe.
i) If color doesnt change after 5 minutes, put your microfluidic device on the hot plate, and
set it to 35C. Check if the color intensity changes. If yes, then consider the reasons for this
change. (This step is optional)
3. Clean up and have your instructor sign on your lab handout before you leave.

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Supplementary Information:
Note on 5-min Epoxy
If every monomer forming a polymer has only two reactive sites, then only chains or
rings can be made. However, if some or all of the monomers in a polymer have three or
more reactive sites, then they can be cross-linked to form sheets or networks. One important
example of cross-linking is 5-min Epoxy, which is formed from two different chemicals (i.e.
copolymer), termed as the "resin" and the "hardener". The resin contains an epoxide group
(

) at either end. This can be seen from the structure of a commercially available

resin EPON 862 in Figure 7. The hardener consists of polyamine monomers, for example,
triethylenetetramine (TETA) whose structure is shown in Figure 7. When these compounds
are mixed together, the amine groups react with the epoxide groups to form a covalent bond.
Each -NH group can further react with an epoxide group, which results in a polymer that is
heavily cross-linked, and is thus rigid and strong.

Figure 7: Reaction schematic using

EPON 862 and TETA. Source: Polymer, Vol 48, pg 21742178, 2007

Note on the BCA Reaction Assay

The BCA Protein Assay is a formulation based on bicinchoninic acid (BCA) for the
colorimetric detection and the quantitation of total protein. This method combines the
reduction of Cu2+ to Cu+1 by protein in an alkaline medium with the highly sensitive and
selective colorimetric detection of the cuprous ion (Cu +) using a unique reagent containing
the bicinchoninic acid. This assay primarily relies on two reactions.

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a) Firstly, the peptide bonds (electron-rich) as well as some specific amino acid residues in
the protein sample reduce Cu 2+ ions present in the BCA reagent to Cu+. The amount of Cu2+
reduced is proportional to the amount of protein present in the solution.
Protein + Cu2+ (aq) Cu+(aq)
b) Next, two molecules of bicinchoninic acid (BCA) bonds (dative bond) with each Cu+ ion ,
forming a purple-colored complex that strongly absorbs light at a wavelength of 562nm. The
color intensity increases with increase in the protein concentration.

Figure 8: Complex formed between Cu+1 and BCA reagent:

Source: www.lifetechnologies.com

The amount of protein present in a solution can be quantified by measuring the absorption
spectra

and

comparing

with

protein

solutions

with

known

concentrations.

The

macromolecular structure of protein, the number of peptide bonds and the presence of four
particular amino acids (cysteine, cystine, tryptophane and tyrosine) are reported to be
responsible for the color formation caused by more than the mere sum of individual colorproducing functional groups.
References:
1) This experimental design is adapted from the paper published in Journal of Chemical
Education: Chemistry in Microfluidic Channels, by Matthew C. Chia, Christina M. Sweeney,
and Teri W. Odom (Vol. 88, pp. 461, 2011). Figure 2 adapted from a book chapter Norland
Optical Adhesive 65 as Holographic Material by J.C. Ibarra et al. It is only for internal use,
and please do not distribute outside of this course.
2) Instruction manual for Pierce BCA Protein Assay Kit.
3) Smith, P. K., et al. (1985). Measurement of protein using bicinchoninic acid. Anal Biochem
150: 76-85
4) Wiechelman, K., et al. (1988). Investigation of the bicinchoninic acid protein assay:
Identification of the groups responsible for color formation. Anal Biochem 175: 231-7

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Lab Report Format and Requirements

Before and during each Lab Session
Please complete the lab handouts for each lab session (attached at the end of this handout)
and then have your handout signed by one of the instructors before you leave.

Lab Report
You are required to write and submit an individual lab report (maximum of 10 pages, font
size 12). The due date is 29th June, 2015 sharp at noon. The lab report must be typed
using Microsoft Word or an equivalent word processing software. The report needs the
following information:
(0) Your full name, group number, group members names.
(I) Objectives (Please write down the objectives for each lab session. Dont write it in
phrases (e.g. To make a microfluidic device) but write in complete sentences. (5 pts)
(II) Methods (procedures) (20 pts)
(III) Observations during the procedures (20 pts)
(IV) Results and Discussion (20 pts)
Note: Sections (III) and (IV) should not be compiled as one section in your lab report. In
Section III, you write down your observations, whereas in section (IV), you need to write
down your interpretations based on your observations/findings from the three lab
sessions.
(V) Summary Conclusions, what you learned from the experiment, what worked and
what did not, what would you do differently? (5 pts)
(VI) References (if any)
(VII) Answers to the following post-lab questions
(a) If we do not have 3D printer, how would you create the Y-shaped block that the
PDMS master mold is made of? (2 pts)
(b) (i) Calculate the volume of a drop of blood with diameter of 2 mm (assume it is
spherical).(2 pts)
(ii) Now imagine you are adding this drop of blood in the microfluidic channels
with the dimensions specified in Figure 2. Calculate the volumes of the drop in
the channel.(2 pt)

Assume that the blood spreads across the entire channel and estimate the
length of the channel (state how long they are).

(iii) How do the volumes will differ if (1) w = h = 500 m and (2) w = h= 800 m?

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(2 pts)
(iv) Compare the volume of the channel and the volume of the blood drop. (2 pt)
(c) What are the advantages of UV curable process? (2 pts)
(d) Describe the observations during the HCl (aq) - NaOH (aq) reaction (step 2d-2f)
and explain the reason. (2pts) (You can also attach your photo to explain your
answer)
(e) For the colorimetric assay, did you observe any difference in the rate of color
change between the channel and the syringe? If yes, then suggest a possible
reason for your observation. (2 pts)

Grading
These three sessions of labs contributes to 100 points out of the total 1000 points of
chemistry (refer to the Syllabus found on e-dimension). The lab will be evaluated based on
the following:

Lab handouts (notes taken during lab): All the lab handouts must be
submitted with the lab report. (10 pts)

Lab reports including the post lab questions (86 pts))

Conduct during lab classes (e.g. punctuality, observing safety rules,

housekeeping) (4 pts)

A note on plagiarism
Please make sure to write the report in your own words. Do not copy directly from
your lab manual. If making use of web-based resources, please cite the references
accordingly.

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