Indonesia International Institute for Life Science

General Chemistry Laboratory

Laboratory Protocol Developer and Supervisor(s) Information

Protocol Developer: Katherine, Leonny Y. Hartiadi, Pietradewi Hartrianti, Ivanna Williantarra, Ulung
K, Alya Ghina Aqila Arham, Fadillah Putri Patria

Email: katherine.k@i3l.ac.id, leonny.hartiadi@i3l.ac.id, pietradewi.hartrianti@i3l.ac.id,

khoe.kusumo@i3l.ac.id, alya.arham@i3l.ac.id, fadillah.patria@i3l.ac.id

Supervisor(s) Email
Pietra Dewi Hartrianti pietradewi.hartrianti@i3l.ac.id
Agnes Anania Triavika S. agnes.sahamastuti@i3l.ac.id

Safety Notice
Everyone working in i3L laboratories is potentially exposed to chemicals. Some of these chemicals
are potentially very harmful. Please review the “Code of Good Laboratory Practice” and the following
to ensure that all work can be conducted without significant risk.

● Do not embark on a new unfamiliar procedure until you have been fully trained.
● While working in i3L laboratories, laboratory coats must be worn and always fastened.
● Remove your laboratory coat and any other protective equipment and wash your hands before
leaving the laboratory.
● Only authorized staff, students and visitors are allowed in the laboratories. Students are not allowed
in the laboratories except at scheduled class times without prior permission. Undergraduate work
must be always supervised by a member of academic staff.

Indonesia International Institute for Life Science – General Chemistry Laboratory

Fundamental of Laboratory Practice I consists of the following session:

Session Topic
1 Introduction to Laboratory Practice and Safety
2 Laboratory Measurement and Mathematics
3 Micropipette, Solution Preparation and Serial Dilution
4 Inorganic Nomenclature: Binary Compound and Oxidation Number
5 Stoichiometry of Chemical Reaction
6 The Law of Chemical Equilibrium and Le Chatelier’s Principle
7 Review of session 1 to 7
8 Mid exam
9 pH and Buffer Solutions
10 Acid Base Titration
11 Paper Chromatography – Separation of Color Pigments
12 Density and Specific Gravity
13 Calorimetry
14 Solubility and Boiling point
15 Review of session 9 to 14
16 Final Exam

Indonesia International Institute for Life Science – General Chemistry Laboratory

 Micropipette, Solution Preparation and Serial Dilution

Session 3
Aim Identify critical steps in using micropipette, solution preparation and simple dilution

Overview
Following last session (laboratory math), the students are going to perform solution preparation using
micropipette. In laboratories, the technique of preparing correct amount of concentration is one of the
most important skill aside from laboratory math. Solution preparation and dilution require accurate
measurement of the samples or reagents, the use of the right equipment along with the correct technique.

Micropipette is commonly used to transfer accurate amount of solutions (usually in microliters). Such
accuracy however, is highly dependent on the user as correct technique of pipetting is needed to produce
reproducible experiments. The main objective of this practical session is to practice accurate pipetting
technique along with the introduction of serial dilution method.

Based on the mechanism of action, there are two types of micropipette: air-displacement and positive
displacement. In majority of cases, air displacement is the type that is commonly used. Air-displacement
micropipette works like a syringe with an air cushion between the piston and the sample. When the
plunger / operating button is pressed to the 1st stop, the piston will expel the same volume of air as
indicated on the volume setting.

Air displacement micropipette comes in many volume capacities (from 0.2 µl to 10 mL) and is
commonly described by adding “P” to the number of the maximum volume of the micropipette (i.e. A
micropipette with the volume range of 200 – 1000 is commonly described as P1000). To aid the transfer
of the liquid, disposable plastic tips are attached to the bottom shaft of the micropipette. There are
different sizes of the tips and must be matched with the pipette volumes. (Note: NEVER use a
micropipette without the tip)

Anatomy of an Air-Displacement Micropipette

Plunger / Push Button – It has 3 positions: Resting, 1ST Stop, 2nd Stop
Volume Adjustment Knob - Some micropipettes have a separate knob to
adjust the volume, in other models the plunger button serves as a volume
knob as well
Tip Ejector Button – to discard used tips
Volume Indicator /Display – Some micropipettes employ a 3 digits
volume display (Thus, 1000 µl in a P1000 is shown as 100, while 20 µl in a
P20 is shown as 200, with the last 0 represents tenths of microliters); In
other type of micropipettes, the volumes are represented clearly (1000 for
1000 µl and 20.0 for 20 µl)

Indonesia International Institute for Life Science – General Chemistry Laboratory

 8 Steps to Achieve Accurate Micro pipetting [Forward Pipetting]
1) Set the volume knob.
2) Fit the appropriate tip Gently press down the shaft with rotation motion (“Press and twist”). Do
NOT stab the tip (“jacking” the tip).
3) Depress the plunger into the 1st stop position.
4) As the pipette is held vertically (90 o), immerse the tip slightly below the surface of the liquid
(around 2-4 mm).
5) Aspirate (draw) the liquid, by slowly release the plunger to the resting position. Wait for one second.
6) As the tip is positioned at an angle (10-45o) against the inside wall of the receiving container,
depress the plunger smoothly to the first stop position to dispense the liquid.
6) Wait for one second and then depress the plunger to the second stop to discharge the residual liquid.
This step is usually called “blow out” or purge.
7) While holding the plunger at the 2nd stop position, slowly remove the tip from the receiving
container by sliding it along the inner wall.
8) Release the plunger to the rest position.

NOTES
● Pre-wet the pipette tips (before step 5) 3-5 times to improve the accuracy of pipetting.
● At step 6 – the angle is created by shifting the container, while the micropipette should be held in
vertical manner.
● ALWAYS use a micropipette tip
● ALWAYS pipette in a slow, smooth action
● ALWAYS immerse the tip slightly below the surface, to prevent droplets buildup on the outer wall.
● ALWAYS discard the pipette tips when pipetting different samples/chemicals or different
concentrations of the same samples/chemicals.
● ALWAYS return the knob to the highest volume, to reduce tension of the spring.
● If possible, NEVER use the lowest capacity of the micropipette if different range of micropipette is
available (i.e. use P200 for 100 µl instead of P1000)
● NEVER hold the micropipette horizontally when the tip is connected to the micropipette (especially
if there’s liquid inside)
● NEVER move the plunger too fast to prevent chemicals moving into the micropipette shaft.

Indonesia International Institute for Life Science – General Chemistry Laboratory

Serial Dilutions
If the target concentration is much smaller than the initial concentration, it is very hard to directly dilute
the solution without compromising the accuracy. Furthermore, if the initial volume of sample / reagents
are relatively small, it’s technically impossible to accurately transfer a very minute amount of solution
and expect a homogenized final solution. Therefore, the dilution process is usually carried out in a
stepwise manner.

A serial dilution is a step wise dilution series in which the concentration decreases by the same quantity
(same Dilution Factor) in each successive step. Dilution factor of 1/2, 1/5, and 1/10 are commonly used.
The main disadvantage of serial dilution is that an error in the initial steps will be carried over to the
subsequent dilutions and will result in a multiplicative error.

For a 0.01 mM glucose solution to be made from a 1 M stock, the initial stock can be diluted 5 times
with the dilution factor of 1/10: 1) 5 empty tubes are initially filled with 0.9 mL of water. 2) 0.1 mL of
the 1M solution is then added to the first tube, resulting in a 0.1 M solution (after careful mixing) 3)
With a new pipette tip, 0.1 mL of the 0.1 M solution is then added to the second tube, resulting in a 0.01
M solution. 4) Repeat the process until a final tube of 0.01 mM glucose solution is created.

Materials and Equipment

CHEMICAL USED EQUIPMENT USED

Sucrose 10 mL graduated cylinder
37% (w/w) Hydrochloric Acid 50 ml beaker
Tris Base 10 ml graduated pipette
Glycine 25 ml volumetric flask
SDS Stirring rod
1 M CuSO4.5H2O Dropper
Glycerol Balance
Micropipette
10 ml volumetric flask
Graduated cylinder
PCR tube
Microcentrifuge tube

Indonesia International Institute for Life Science – General Chemistry Laboratory

Techniques
You have to read this before you come to the laboratory.
1. Prepare standard solution (Appendix 2)
2. Preparing sample for spectrophotometric measurement (Appendix 2)

Procedure
A. Micropipette Exercise
A1. Accuracy of Repetitive Pipetting 1
1) Transfer 6 mL of water using 1 mL micropipette into a 10 mL graduated cylinder.
2) Observe the amount of water in graduated cylinder and write down the difference between the
estimated result and the observed measurement.

A2. Accuracy of Repetitive Pipetting 2

1) Using a 10 µl micropipette, aspirate 2 µl CuSO4 Solution and transfer it into a PCR tube
2) Into the same tube, transfer more CuSO4 Solution in the following manner: 4 µl, 6 µl, 8 µl, 10 µl.
3) Using a 200 µl micropipette, aspirate 30 µl of the solution from the PCR tube to a clean PCR tube.
Observe whether there is an excess solution remaining in the tube / pipette tips.

A3. Reverse Pipetting for Transfer Viscous Solution

Reverse pipetting technique is often used for the pipetting the following sample conditions: viscous
liquids (i.e. glycerol), volatile solvents (i.e. chloroform), very small volumes (i.e. < 1.0 µL), and when
the solution has a tendency to foam. During the aspiration, there’s an extra amount of air (corresponds
to the amount of purged air) to compensates for the extra liquid which remains inside the tip during
dispensing. No prior pre-wetting is required.
1) Press the plunger / push button to the 2nd stop.
2) Immerse the tip +/- 3-4 mm below the surface of the glycerol.
3) Aspirate 500 µL of glycerol by slowly release the plunger. Wait for 1-2 seconds to ensure all liquid
has moved up to the tip.
4) At an angle against a clean microcentrifuge tube, depress the plunger smoothly to the 1st stop to
transfer the glycerol to the new container.
5) Depress the plunger to the 2nd stop to a waste container to do a complete purge.
Note: If the pipette tip needs to be reused for subsequent pipetting, maintain the plunger at 1st stop and
aspirate the liquid (restart from step 2)

B. Solution preparation
1. Make 10 mL of 3% (w/w) Sucrose Solution in water using 50 mL Beaker!
2. Prepare 25 mL of 1 M Hydrochloric Acid from 37% (w/w) Hydrochloric Acid! Please note that
37% HCl is very strong acid and is corrosive. Therefore, you have to do it inside fume hood and
cover your hands using gloves. Take 37% HCl using only glassware (do not dip plastic tips into
strong acid!). For dilution of strong acid, ALWAYS add acid into water! Put 80% of the amount of
water needed into volumetric flask, slowly add strong acid through the flask wall (do not carelessly
drop it directly into the solution). Let the solution cool down a bit and add remaining water until
the volumetric marking.
3. Prepare 10 mL 1x working stock of SDS PAGE Running Buffer from 10x SDS Page Running
Buffer (1L) (30 grams of Tris Base; 144 grams of Glycine; 10 grams of SDS).

Indonesia International Institute for Life Science – General Chemistry Laboratory

C. Serial Dilution
Using serial dilutions, design a standard curve covering 0.4 ppm until 4000 ppm of CuSO4.5H2O using
a 1 M CuSO4.5H2O solution in exponential manner.
1) Make a 6-points of standard curve (6 different concentrations) using microcentrifuge tubes. (1 tube
= maximum 1 mL). One of them should be the blank.
2) Transfer 150 µl of each concentration into a 96-well plate. Make a duplicate for each concentration.
3) Measure the absorbance of CuSO4 solution at 645 nm wavelength (it will be done by lab assistant).
4) A calculation of the correlation coefficient (r2) would be done by the lab assistant. A linear
correlation will result in standard curve with r2 close to 1.0.
1 ppm = 1 mg/L; M.W. CuSO4.5H2O = 249.677 g/mol

Data observation
A. Micropipette exercise
1. Compare the volume obtained by repetitive pipetting and find the % error (for the A1).
2. Is there any excess liquid observed in A2?
3. What are problems you face when you try to pipette viscous liquid?

B. Solution preparation
1. What is the actual weight of sucrose that you add?
2. What are problems you face when you try to dilute strong acid?
3. What is the actual weight of Tris base, glycine and SDS that you add?

C. Serial dilution
1. What are the concentrations of CuSO4.5H2O solution?
2. What is the r2 of your standard curve?

Data analysis and Discussion

A. Micropipette exercise
Analyze whether it is acceptable to use repetitive pipetting based on the % error that you get! What is
the common final volume result when you use repetitive pipetting? Why reverse pipetting is useful for
viscous liquid?

B. Solution preparation
What is the limit of % weighing error? Were the weighed materials within those limits? Why you
need to be careful when you dilute strong acid or base?

C. Serial dilution
What is the use of serial dilution? What its advantage and disadvantage? What is r2 indicate of?

Prelab activities (answer in separate paper)

1. Student A prepare a 10 mL 5 M NaCl solution at 20°C. Student B prepare the same solution at
80°C. Will the final weight of the solution prepared by A be the same as B? Why?
2. Prepare a calculation algorithm to convert:
a. solution with known M to % w/v and vice versa
b. solution with known M to % w/w and vice versa
3. How many mL of 5 M solution is needed to prepare 10 mL of 1 M solution? How much solvent
should be added?

Indonesia International Institute for Life Science – General Chemistry Laboratory

4. Find the MSDS for the chemicals used in this session. Which compounds are flammable, toxic by
inhalation, toxic by ingestion, or other routes of exposure? How do you handle them? What kind
of PPE is needed when handling the chemicals? How should they be disposed?

References
1. Jespersen, N.D., J. E. Brady, & A. Hyslop. 2012. Chemistry: The Molecular Nature of Matter 6th
Edition. Wiley Pub.: USA

Approved by Acknowledged by

Sanjaya Mulya Waani apt. Audrey Crystalia, S. Farm, M.Sc.

Head of Laboratory Head of Department, Pharmacy

Indonesia International Institute for Life Science – General Chemistry Laboratory

Indonesia International Institute for Life Science – General Chemistry Laboratory
 Appendix 1: Laboratory Equipment

Figure 1. Common equipment found in lab

(Source: Garcia, et al. 2016. Experimental Organic Chemistry)

Indonesia International Institute for Life Science – General Chemistry Laboratory

 Figure 1. Common equipment found in lab
(Source: Garcia, et al. 2016. Experimental Organic Chemistry)

Indonesia International Institute for Life Science – General Chemistry Laboratory

 Figure 1. Common equipment found in lab
(Source: Garcia, et al. 2016. Experimental Organic Chemistry)

Indonesia International Institute for Life Science – General Chemistry Laboratory

 Appendix 2: Laboratory Techniques

1. Using Graduated Cylinders

A graduated cylinder is a cylinder marked with different scale increments. It comes in many
different size and different scale increments. Before you begin to measure, you need to first determine
the scale increment. To do this, subtract the values of any two adjacent labeled graduations and divide
by the number of intervals between them.

A 50 mL graduated cylinder. The value difference of two adjacent label is 50 –

40 = 10. There are 10 intervals between the label. So each graduation is 10 mL
divided by 10 intervals = 1 mL for each interval.

To determine the volume of liquid in the cylinder,

hold it up to the eye level and view the height of the
liquid in the cylinder with your eyes directly level with
the liquid. The liquid in the cylinder will most likely
curve downward. This phenomenon is called meniscus.
It is important that when you read the liquid level, you
position should be on eye – level with the meniscus. Do
not read from above or below eye level. The line you
are reading should be the center of the meniscus. For
water, this is the bottom of the meniscus. For mercury (found in thermometer), the reading is taken from
the top of the meniscus since the mercury is curved upward.

Handling graduated cylinders

- Pouring liquids
Graduated cylinders are tall and thin, so they easily tip over, break and spill out their contents. So
when pouring liquid, hold the top of cylinder with one hand while pouring the liquid with other
hand. Never crouched down so your head is on the lower position than the cylinders.
- Cleaning and drying
If you need to use the cylinder directly after washing, take a piece of paper towel a few inches
longer than the graduated cylinder. Then fold the paper towel lengthwise, then until the folded paper
towel has a circumference that will allow it to fit inside the cylinder. Insert the folded paper towel,
and thrust it up and down a few times to absorb the liquid from the inside surface of the cylinder.
Else put the cylinder upside down on a hanger rack, or put it horizontally to dry.

Indonesia International Institute for Life Science – General Chemistry Laboratory

2. How to use pipette filler

Source : Learning the basics – how to work with volumetric instruments. Brand GMBH. Retrieved
from www.brand.de.

3. How to read marking on pipette

To deliver (TD) – the graduation line indicates the volume delivered by the pipette
To contain (TC) – the graduation line indicates the volume contained in the pipette
TC calibrated pipette contain error caused by water holdback by the adhesion force with the wall
of the pipette. The residual liquid left in the tip must be discharged by blowing out. TD calibrated
pipette has already considered the error. Therefore, the residual liquid left in the tip must not be
discharged into the vessel, such as by blowing out.

4. Using measuring pipette

There are two types of measuring pipettes: Mohr and serological pipettes.
Mohr pipettes Serological pipette
The graduation mark always end before tip The graduation mark continue to the tip

These pipettes require a controlled delivery of
the solution between the upper fill mark and
the intended lower mark. The mark between
the last graduated line and the tip are not
calibrated. Therefore, should not be used for
measurement.
Partial measurements using the last few
milliliters of the pipette in or near the
delivery tip are inaccurate (and thus
discouraged) due to changes in the
diameter of the pipette. The opening in
the tip is large, allowing liquid to flow
faster.

5. Using volumetric pipette

Volumetric pipette is used to deliver only one volume. Should be used when accuracy and
reproducibility are crucial, because these can achieve accuracy to four significant figures.
On a volumetric pipette, the specifications indicate:
how much liquid will be transferred if the liquid is drawn up to the calibration line on the neck

Indonesia International Institute for Life Science – General Chemistry Laboratory

• the temperature at which the calibration was made

1. How to prepare a standard solution with a volumetric flask:

a. Insert the precisely weighed amount or measured volume of substance.
b. Rinse the weighing cup or volumetric pipette with water. The rinsing solution is then poured
into the volumetric flask.
c. Fill the flask with distilled water to about half. Swirl the flask to dissolve and mix the contents
thoroughly.
d. Top up the flask with distilled water to just below the ring mark.
e. Top up the remaining volume, using a wash bottle or pipette, until the meniscus is exactly at
the ring mark. Important: meniscus must be read at eye level. The wall of the flask must not be
wetted above the mark.
f. Close the flask and shake upside down to mix contents.
Source : Learning the basics – how to work with volumetric instruments. Brand GMBH. Retrieved
from www.brand.de.

Indonesia International Institute for Life Science – General Chemistry Laboratory