MAHSA University

Department of Biomedical Sciences

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

PRACTICAL: 2

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

AIM

1) To provide students with the opportunity to familiarise themselves with different

types of pipette and pipette filler, and to practice pipetting procedures
2) Use of micropipette and analytical balance to determine imprecision and
inaccuracy of micropipettes
3) To illustrate the importance of accuracy, precision, bias and error in practical
procedures involving measurement.

INTRODUCTION

The purpose of this laboratory experiment is to exercise pipette calibration and gain
familiarity on the proper usage of laboratory pipettes. Pipettes are useful during
laboratory experiments due to an important feature in that they offer an accurate and
precise method of transferring desired volumes of liquid. There are three types of pipettes
most commonly found in a laboratory setting which are volumetric, measuring, and
mechanical micropipettes. In order to effectively take advantage of instruments that offer
accurate and precise measurements, proper usage must be enforced as well as frequent
calibration.
Following proper standard operating procedures on the usage of pipettes will greatly
increase the accuracy and precision of each measurement. The relative error of the user
will decrease as well as a reduced standard deviation will be observed. Certain measures
must be enforced such as keeping devices clean and dry prior to each use. Improper use
of laboratory equipment will result in a higher standard deviation. Frequent calibration of
laboratory equipment enables frequent inspection on the functionality of the instrument at
question. During a pipette calibration procedure, the mechanical parts that enable the
instrument to perform properly are examined. This ensures that the quantities that are
being measured are accurate to maintain the integrity of the data being collected. When
the mechanical parts of a measuring instrument have been compromised, it is likely that
the accuracy of the instrument will deviate away from the accepted value at a higher
probability.
To conduct the pipette calibration, aliquots of water are repeatedly pipetted and weighted.
A mean and standard deviation is calculated which are utilized to calculate the coefficient
of variation (CV). The coefficient of variation takes the standard deviation and divides it
by the mean and multiplied by 100. This describes the amount of variability compared to
the mean which gives insight on the precision and accuracy of the pipette being
calibrated. A CV is acceptable if it is less than or equal to 3%. A CV greater than 3% is
not acceptable and cannot be used in collecting parametric data. A CV greater than 3%
indicates that the pipette exhibits mechanical failure that interferes with the precision and
accuracy of the measuring device. To achieve parametric data that best suits the actual
measurement calibration, must be conducted frequently.

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
 1. Place small plastic weighing boat on analytical balance.

 2. Depress pipette plunger until resistance is expressed.

 3. Place the tip of the pipette into beaker containing water.

 4. Slowly release plunger until fully extended to collect aliquot of water.

 5. Remove excess water on pipette tip.

 6. Deliver the water to the weighing boat by fully depressing the plunger on the micropipette.

 7. Record the weight of the water delivered on the weighing boat on a pipette calibration
form.

 8. Using the same pipette tip repeat procedure of weighing water aliquots 5 times for 3 trials.

 9. Calculate the CV following the formula

 a. CV= Standard Deviation in Volume/Average Volume x 100

A. 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:

Standard Deviation, σ=

RESULTS

1. Accuracy & precision determination

The data can be grouped as follows:
(A) 1000 µl (P1000 pipette) 1000 µl (1 ml graduated pipette)
(B) 200 µl (P200 pipette) 200 µl (1 ml graduated pipette)

DISCUSSION
1. Describe the protocols of both forward & reverse pipetting (micropipette) (use
diagram).
2. List out three errors that could be involved in pipetting.
3. List and explain the functions of alphabets marked on rubber bulb pipette-filler.

CONCLUSION
REFERENCES

Learning Outcome

By the end of this practical, students know the following:

1) Identify the range of pipettes

2) Able to use (measure accurately and transfer completely) and care of the pipette

3) Understand the methods of forward and reverse pipetting in micropipette

4) Understands and appreciate the use of micropipette and top-pan / analytical balance
to determine imprecision and inaccuracy of micropipettes