MAHSA University

Department of Biomedical Sciences

Bachelor of Biomedical Sciences. (HONS.)
BMC 112 Laboratory Science & Instrumentation 1

Name: AISSATA DIAWARA

Student ID: BBSH 22106335

Course: Bachelor In

Biomedical Science

Practical Title: Use and calibration of pipettes for volume transfer and usage of
analytical balance.

Module Leader: Dr Wong

PRACTICAL: 2

INTRODUCTION

Pipettes are a type of scientific instrument used to measure and transfer extremely small
quantities of liquid. Any difference in amounts dispensed can have an impact on an
experiment's outcomes, hence accuracy and precision in pipette measuring are essential.
Every few months, the pipette calibration needs to be checked to ensure accuracy. The
calibration procedure aids in determining whether or not the apparatus is dispensing the
correct quantities so that it can be corrected if it is not.

1) GLASS PIPETTES
Pipetting of volumes greater than 1.0 ml is usually performed with glass pipettes, which
may be of the “volumetric” or “graduated” type. These require pipette filler firmly
attached to the end of the pipette to draw liquid into the glass tube (Remember: NEVER
PIPETTE BY MOUTH!!)
 Some different types of pipette
fillers

Different fillers cater for pipettes of different diameter, but the two should form an
effective seal to prevent loss of liquid. There are also different types of filler, using
rubber bulbs with valves, ratchet mechanisms or motors to fill and empty the pipette
safely (see diagram above). If necessary, these will be demonstrated for you. The outside
of glass pipettes may be wiped with tissue to prevent the transfer of liquid clinging to the
glass, and making the volume inaccurate. However, when doing this, avoid drawing
liquid from the pipette tip by wicking, i.e., capillary action.

Ejection of liquid from glass pipettes usually relies on gravity, but the last few drops are
either ejected completely (“blow-out” pipettes) or allowed to drain out when the pipette
tip is touched to the side of the receiving vessel.

2) ADJUSTABLE MICROPIPETTES

Micropipettes are called a lot of different names, most of which are based on the
companies which manufacture. For example, you might hear them called “Gilsons”, as a
large number of these devices used in laboratories are made by this company. Regardless
of the manufacturer, micropipettes operate on the same principle: a plunger is depressed
by the thumb and as it is released, liquid is drawn into a disposable plastic tip. When the
plunger is pressed again, the liquid is dispensed.

The tips are an important part of the micropipette and allow the same device to be used
for different samples (so long as you change your tip between samples) without washing.
They come in a number of different sizes and colours, depending on the micropipette
they are used with, and the volume to be dispensed.

The most commonly used tips are:

Large Blue – 200-1000µL Small Yellow – 2-200µL Small White - <10µL

They are loaded into tip boxes which are often sterilized to prevent contamination. For
this reason tip boxes should be kept closed if they are not in use. Tips are loaded onto the
end of the micropipette by pushing the end of the device into the tip and giving two sharp
taps. Once used, tips are ejected into a sharps disposal bin using the tip eject button.
Never touch the tip with your fingers, as this poses a contamination risk.

Accuracy is the term used to describe how near a value is to a true value, whereas
precision is the term used to indicate how reproducible the values are. If consistent bias
exists in an experiment, the data may be precise but inaccurate. These characteristics are
determined by calculating the mean and the error in a set of data. The standard deviation
measures the spread of the data about the mean value. It is useful in comparing sets of
data which may have the same mean but a different range. For example, the following
two data sets have the same mean value: 15, 15, 15, 14, 16 and 2, 7, 14, 22, 30. However,
the second is clearly more spread out. If a data set has a low standard deviation, the
values are not spread out too much. The smaller the standard deviation, the greater the
precision, i.e. the water is being dispensed more reproducibly.

A simple method for checking the accuracy of pipettes is to repeatedly weigh a volume of
liquid of known density. Pure distilled water has a density of 1g ml -1, so the weight in
grams should give the volume in milliliters directly. By repeating the pipetting/weighing
exercise many times, the data can be analyzed by descriptive statistics.

MATERIALS
Graduated glass pipettes (1ml),
Pipette bulb / filler,

Micropipette (200 ul & 1000 ul)

Micro-tips (yellow & blue),

Beakers (20 ml),

Top-pan balance

Distilled water
METHODS
A. Students are instructed and demonstrated how to use and care for pipettes (glass
pipettes and micropipettes) of different range.
B. Students are alarmed with the various errors that occurred while transferring the
liquid using the pipettes.
C. Selection of proper micropipette tips.
D. Students will be familiarised with forward and reverse pipetting technique.

E. Accuracy & precision determination:

1. Using pipette filler, fill a 1 ml graduated glass pipette to the 1 ml (1000µl) mark
with water. Carefully wipe the pipette and, placing the pipette tip lightly against
the wall of the weighed container, allow the contents to drain into the container.
Note the weight of the solution dispensed (to three decimal places). Repeat this
10 times, zeroing the balance between additions, and tabulate the results.
2. Using a P1000 adjustable pipette dispense 1000µl (1ml) into your container and
note the weight of the solution dispersed. Again, repeat this 10 times, zeroing the
balance between additions, and tabulate the results.
3. Continue to investigate the accuracy of your pipetting by dispensing 10 aliquots
of each the following volumes from the pipettes indicated. In all cases, record the
weight of the aliquots dispensed (to three decimal places).
a) 200 l (0.2 ml) from a 200 adjustable pipette.
b) Successive 200 l aliquots from a 1 ml graduated pipette.
4. Tabulate the weight data under headings for ease of interpretation.
5. Determine the mean (average) weight for each pipette/volume combination.
6. Record the difference between each value and the mean calculated by subtracting
the mean individually from each of the numbers (ignore any negative signs).
7. Determine the standard deviation for each set by using the below formula:
RESULTS
Accuracy & precision determination
The data can be grouped as follows:
(A) 1000 µl (P1000 pipette)

X (x - x̄) (x - x̄)2

1.02 -0.02 0.04

1.01 -0.01 0.01

0.98 0.02 0.04

0.99 -0.01 0.01

1.00 0 0

0.99 -0.01 0.01

0.99 -0.01 0.01

1.00 0 0

0.99 0.01 0.01

1.03 0.03 0.09

∑X =10 ∑(x - x̄)2 = 0.31

Mean:

∑ x 10
X̄ = = =1
n 10

X̄ = 1
Standard Deviation =
√ ∑ (x − x̄ ) 2
(n −1)

=
√ 0.31
9
= √ 0.034 = 0.18

Standard Deviation = 0.18

(B) 1000 µl (1 ml graduated pipette)

X (x - x̄) (x - x̄)2

1.00 -0.005 0.04

1.00 -0.005 0.01

1.01 0.01 0.04

1.01 0.01 0.01

1.00 -0.005 0

1.00 -0.005 0.01

1.01 0.01 0.01

1.01 0.01 0

1.01 0.01 0.01

1.00 -0.005 0.09

∑X =10.05 ∑(x - x̄)2 = 0.000625

Mean:

∑ x 10.05
X̄ = = =1
n 10

X̄ =
1.005
Standard Deviation =
√ ∑ (x − x̄ ) 2
(n −1)

=
√ 0.000625
9
= √ 0.000069 = 0.0083

Standard Deviation = 0.0083

(C) 200 µl (P200 pipette)

X (x - x̄) (x - x̄)2

0.240 0.026 0.000676

0.220 0.006 0.000036

0.200 0.014 0.000196

0.200 0.014 0.000196

0.210 0.004 0.000016

0.220 0.006 0.000036

0.220 0.006 0.000036

0.220 0.006 0.000036

0.200 0.014 0.000196

0.210 -0.004 0.000016

∑X =2.14 ∑(x - x̄)2 = 0.00144

Mean:

∑ x 2,14
X̄ = = = 0.214
n 10

X̄ =
0.214

Standard Deviation =
√ ∑ (x − x̄ ) 2
(n −1)

=
√ 0.00144
9
= √ 0.00016 = 0.0126

Standard Deviation = 0.0126

(D) 200 µl (1 ml graduated pipette)

X (x - x̄) (x - x̄)2

0.200 0.026 0.000676

0.210 0.006 0.000036

0.220 0.014 0.000196

0.210 0.014 0.000196

0.190 0.004 0.000016

0.200 0.006 0.000036

0.190 0.006 0.000036

0.190 0.006 0.000036

0.210 0.014 0.000196

0.220 -0.004 0.000016

∑X =1.04 ∑(x - x̄)2 = 0.00124

Mean:

∑ x 1.04
X̄ = = = 0.104
n 10

X̄ = 0.104

Standard Deviation =
√ ∑ (x − x̄ ) 2
(n −1)

=
√ 0.00124
9
= √ 0.000137 = 0.0117

Standard Deviation = 0.0117

DISCUSSION

Use the forward approach during standard pipetting:

This method is frequently used for pipetting and mixing a sample or reagent
into another liquid. It is advised for aqueous solutions, such as buffers, diluted
acids, or alkalis
1. press the operational button all the way to the start.

2. Dip the tip into the solution until it is 1 cm deep, then gradually release the
operating button. Withdraw the tip from the liquid, wiping the edge of the
reservoir with it to drain any extra liquid.

3. Gently press the operational button to the second stop to dispense the liquid
receiving vessel. The tip will be emptied as a result. By sliding it along the
vessel's wall, remove the tip from the container.
The operational button ought to be set to the ready state at this time. Use the reverse
method for solutions with high viscosity or a propensity to foam:

This method is suggested for precisely pipetting small volumes and is frequently used
with air displacement pipettes. The possibility of sample splash, foaming, or bubble
formation is eliminated by reverse pipetting.

1. Press the control button all the way to the second stop.
2. Dip the tip down 1 cm, then gradually depress the operational button. The tip
will fill after this action. Withdraw the tip from the liquid and wipe it against the
reservoir's edge to wipe away extra liquid.
3. Press the working button slowly and steadily all the way to the first stop to
dispense the liquid into the receiving vessel. Keep pressing the button in this manner.
4.There will be some liquid in the tip that should not be dispensed. Slide the tip along the
vessel's wall to remove it.
5.The operational button should be released to the ready position.
1. List out three errors that could be involved in pipetting.
1. Not Accounting for the Viscosity of a Sample
 2. Dispensing Liquids Too Quickly3
3. Contamination By “Double Dipping”4
4. Cleaning Irregularly—or Not at All5
5. Taking a Break Only at the End of a Project

1. List and explain the functions of alphabets marked on rubber bulb pipette-filler.

Types of Pipette and Its Funsctions

The pipette is one of the most used scientific instruments and is used to transfer various
substances and fluids. Following is an explanation of the many pipette kinds according
to function:

1. Drop Pipette

This drop pipette is employed to extract liquids with a minimal degree of accuracy. This
means that you must trust your judgement and not be able to be certain how many
millilitres of fluid you drained into the pipette.
drop pipette for the medicinal syrup

The material used to make this pipette is often borosilicate glass, which is much stronger
than regular glass. The rubber pipette used to extract the liquids is located in the tip.

2. Measuring Pipette

The only difference between the measuring pipette and the drop pipette is that the
precision level of the measuring pipette is a little bit higher.

The filler or rubber bulb, a type of sucking rubber that must be pushed to take the fluid,
must be used in conjunction with the measuring pipette.

The pressurised filler was then released gradually until it sucked the fluids, and the
measuring pipette was dipped into the fluid that was intended to be collected.
The only difference between the measuring pipette and the drop pipette is that the
precision level of the measuring pipette is a little bit higher.

The filler or rubber bulb, a type of sucking rubber that must be pushed to take the fluid,
must be used in conjunction with the measuring pipette.

The pressurised filler was then released gradually until it sucked the fluids, and the
measuring pipette was dipped into the fluid that was intended to be collected.

3. Volumetric Pipette

The glass or plastic volumetric pipette has a one to one hundred mililiter capacity.
A volumetric pipette is one that is specifically made for taking liquid measurements and
is calibrated inside using the volume. Therefore, the pipette marked with a 10 ml capacity
can only be used to dispense 10 ml of fluid.

The volumetric pipette differs from the measuring pipette in that the tube is located in the
middle of the pipette's shape. However, the usage process is identical to that of the
measuring pipette.

Make sure the pipette's interior is clean before using it. Avoid allowing any bubbles to
form when ingesting the liquids.

Suck the liquids 2-3 cm over the line so that we may modify after removing the rubber
bulb with our thumbs. We may use our fingertips to adjust the fluid as it gradually
descends.

The low basin should be adjusted such that it is parallel to the line. After that, transfer the
liquids to the container.

When using the volumetric pipette to transfer the fluids, there are various things to avoid:

• there will be evaporation. The amount of fluids will increase if this occurs. Don't forget
to wet the volumetric pipette before usage or it could not be accurate.

• Pay close attention to the room temperature. The volumetric pipette will indicate the
ideal measurement temperature, which is often around 20°C. The volume of the fluids
can expand and contract as a function of temperature.

• Avoid wiping the pipette tip with material having a high capilarity since it will absorb
the liquid. As a result, the measurement will be off.

• Avoid sucking the fluid at such an oblique angle because doing so will cause the fluid to
leak from the pipette's tip.
4. Burette Pipette

The burette pipette is an unique pipette used in the titration procedure to determine the
degree of reactant contamination.

Several parts of this pipete include:

• Stopwatch • 

 Measuring instrument that is used with a funnel to pour liquids inside

Pipettes can be straightened using a ring stand or clamp.

Use the distilled water to clean it before using. After that, add the liquids and keep an eye

on the input volume.

Place the erlenmeyer flask with the reactant fluids underneath the burette, and slowly

rotate the fluids inside using the faucet.

To react with the second fluids inside the Erlenmeyer tube, pay attention to the fluid

volume.

5. Micro Pipette
The pipette can be used for the same purpose as a volumetric pipette, but it has more
micrometer-level precision.
The plunger button, which is used to draw fluids into the pipette, is one of its component
pieces.
• Scale Volume for fluid volume adjustments.
• Function Wheel for selecting options from a separate menu, such as mixing, pumping,
and sucking.

CONCLUSION
The aforementioned considerations guarantee that you buy a micropipette that will
serve you well for a long time and enhance your lab's liquid handling procedures.
High precision liquid handling equipment is designed and developed by Microlit for
renowned laboratories worldwide. It provides a cutting-edge and cutting-edge
micropipette range that enhances an exceptional user experience in real-world
laboratory settings. Visit our website to view the full selection of our products.

REFERENCES
de Groot, Martin. "Calibrating a micropipette." Int. J. Metrol 25.1 (2018): 19-25.
de Groot, M. (2018). Calibrating a micropipette. Int. J. Metrol, 25(1), 19-25.
de Groot, Martin. "Calibrating a micropipette." Int. J. Metrol 25, no. 1 (2018): 19-25.
de Groot, M., 2018. Calibrating a micropipette. Int. J. Metrol, 25(1), pp.19-25.
de Groot M. Calibrating a micropipette. Int. J. Metrol. 2018;25(1):19-25