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EXPERIMENT 1

USE OF MICROPIPETTOR AND


SPECTROPHOTOMETER
Y. A. Azucena, R. M. Baladad, S. C. Baquiran, T. J. Bautista 4BIO5
Department of Biological Sciences, College of Science, University of Santo Tomas, Espana
Avenue, Manila

Keywords:
accuracy, precision, micropipettor,
spectrophotometer

Summary

In the activity, the micropipettor and the


spectrophotometer were used to be able to test the
accuracy and precision of students in using such
devices. Bromphenol blue and distilled water were
used in the experiment as testing agents. In the first
procedure a specified amount of distilled water
combined with different amounts of bromphenol
blue
were
subjected
for
test
using
a
spectrophotometer set at 540nm basing on
absorbance.
In the second procedure specified
amounts of distilled water were subjected for test
for ease of recognition and are used for
Introduction
different sizes of micropipettors. With
respect to volume transferred, white tips
The micropipette or micropipettor is a
typically transfer between 0.5-10 L, yellow
precision pump fitted with a disposable tip,
tips are for 10-100 L, and blue tips from
designed to accurately transfer volumes in
100-1000 L. In deciding which micropipette
the microliter range. Micropipettors work by
to use for volumes that can be measured by
air displacement. The operator depresses a
two types, it is practical that one use the
plunger that moves an internal piston to one
micropipettor with smaller volume capacity
of two different positions. The first stop is
to attain accuracy in measurement.
used to fill the micropipette tip, and the
A spectrophotometer is an instrument
second stop is used to dispense the
that
measures the concentration of solutes
contents of the tip.
in
solution
by measuring the amount of the
The three types of micropipettors are
light that is absorbed by the solution in a
distinguished according to the volume
cuvette. This experiment involves the use of
ranges that they each can transfer, each
a
UV/VIS
spectrophotometer
which
recognizable by a number at the top of the
operates
in
the
ultraviolet
and
visible
plunger, and by color. The large volume
regions of the electromagnetic spectrum.
micropipettor is blue and can assist in
The spectrophotometer measures light
moving liquids 100-1000 L, the mid-range
intensity as a function of wavelength by
micropipettor is yellow and has a capacity of
diffracting the light beam into a spectrum of
10-100 L, and the small volume
wavelengths, detecting the intensities with a
micropipettor is red and capable of moving
charge-coupled device, and displaying the
0.5-10 L of liquid. Different sizes of
results as a graph on the detector and then
pipettes tips are often likewise color-coded
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the display device. As the concentration of


the absorbing substance increases, less
light is transmitted and more light is
absorbed by the solution.
A UV/Vis spectrophotometer utilizes two
light sources. The first is a tungsten halogen
lamp used as the source for visible light.
This lamp consists of a tungsten filament
enclosed in a glass envelope, with a
wavelength range of about 330 to 900 nm.
They are generally useful for measuring
moderately dilute solutions in which the
change in color intensity varies significantly
with changes in concentration. The second
lamp is usually a deuterium arc lamp that
can measure from 190-380 nm. The
electrical excitation of deuterium at low
pressure produces a continuous UV
spectrum. The mechanism for this involves
formation of an excited molecular species,
which breaks up to give two atomic species

and an ultraviolet photon. The GENESYS


10 UV/Vis spectrophotometer, however,
utilizes a xenon flash lamp which provides a
high energy light source with a short warm
up time and long lamp life. They measure in
both the UV and Visible regions of the
electromagnetic spectrum from 190 - 1100
nm. Xenon lamps typically provide 80
flashes per second giving them their
distinctive bird-like noise.
A blank is a solution that is identical to the
sample solution but does not contain the
solute that absorbs light. This measurement
removes any absorption of light and is
therefore used to set the absorbance to
zero.

Materials and Methodology


The materials and equipments used for the
first procedure of testing accuracy and
precision in using a micropipettor were
Bromphenol blue, microcentrifuge tubes,
micropipettors,
micropipettor
tips,
GENESYS 10 uv-vis spcetrophotometer,
vortex mixer, semi-micro cuvettes and
distilled water. For the second procedure,
the materials and equipments used were
micropipettors, micropipettor tips, analytical
balance, distilled water, and parafilm or
aluminium foil.
Testing accuracy and precision in using
a micropipettor
Testing the accuracy of operators
The spectrophotometer was warmed up and
was set at 540nm. 1 mL of distilled water
was placed in each of five microcentrifuge
tubes. Following the instructions on how to
use the micropipettors, successive amounts
of bromphenol blue were added. The
volumes of the bromphenol blue added
were 0.5, 1.0, 1.5, 2.0 and 2.5 L
respectively. Each tube was then vortexed

until the dye was in solution. The


spectrophotometer was zeroed with distilled
water. The contents of the microcentrifuge
tubes were transferred to the cuvettes. After
which the absorbance of the dye solutions
were read and were recorded. Finally, the
results were graphed.
Testing precision of operators
1 mL of distilled water was placed each of
five microcentrifuge tubes. With the
micropipettors, 2.5 L of bromphenol blue
was added by each member in each
microcentrifuge tube. The tube was ten
vortexed until the dye was in solution. The
spectrophotometer was zeroed with distilled
water. The contents of the microcentrifuge
tubes were transferred to the cuvettes. After
which the absorbance of the dye solutions
were read and were recorded. The standard
deviation and the coefficient of variation
were calculated, where:
SD = ([Nx^2 (x)^2 ]/(N-(N-1) ) )
CV = SD/Mean

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Testing accuracy and precision in using


a micropipettor
Testing accuracy of operators
Parafilm or aluminium foil was placed in a
an analytical balance. Tare was pressed
after it was weighed. Using a micropipettor,
50 L of distilled water was added into the
parafilm. The weight was recorded up to the
fourth decimal place. Tare was pressed
again and the procedure was repeated until
all the members of the group finished
performing the procedure. The average
weight and standard deviation were
computed. Finally, the entire procedure was
repeated using 100 L and 500 L distilled
water.

In the first part of the experiment a


spectrophotometer was utilized to correlate
absorbance with the amount dye.

Testing precision of operators


Parafilm or aluminium foil was placed in a
analytical balance. Tare was pressed after it
was weighed. Using a micropipettor, 50 L
of distilled water was added into the
parafilm. The weight was recorded up to the
fourth decimal place. Tare was pressed
again and the procedure was repeated until
all the members of the group finished
performing the procedure. The average
weight, percent error (% error), standard
deviation (S.D.) and standard error of the
mean (S.E.) were calculated, where:
% error=(standard value-experimental
value)/(standard value)100
SE= SD/(N)
Results & Discussion
The following formula were used to
calculate for the standard deviation and
coefficient of variation

[ N x 2 ( x )2]
SD=
N (N 1)

CV =

SD
Mean
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Table 1.2 Accuracy of operator


Bromphenol
blue volume
(L)
0.5
1.0
1.5
2.0
2.5

Standard
Deviation

Absorbance (540nm)

Trial 1
Trial 2
Trial 3
Average
0.251
0.251
0.251
0.251
0.281
0.281
0.281
0.281
0.342
0.342
0.342
0.342
Absorbance versus Volume of BPB
0.356Average0.356
0.356
0.356
0.413
0.413
0.413
0.413

0
0
0
0
0

Coefficient
Variation
0
0
0
0
0

Average absorbance (540 (nm)

0.5 1 1.5 2 2.5 3


Bromphenol blue volume (L)

Table 1.3 Testing precision of operators


Absorbance

Student
1
2
3
4

Trial 1
0.489
0.520
0.457
0.554

Trial 2
0.488
0.520
0.457
0.554

The results show the amount of dye in the


solution is directly proportional to the
amount of light absorbed. The increasing
amount of Bromphenol blue in the solution
resulted to a higher absorbance reading.

Trial 3
0.489
0.520
0.457
0.555

Average
0.489
0.520
0.457
0.554

Standard
Deviation

Coefficient
Variation

5.7710-4
0
0
5.7710-4

1.1810-3
0
0
1.0410-3

In the second part of the experiment,


micropipettors were used to transfer the
water sample and weigh them in numerous
trials. The micropipettors were used to test
their accuracy and precision.

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Table 1.4 Weight of different water samples by 1 member of the group


Volume
of water
(L)
50
100
500

Trial 1
0.491
0.0999
0.5011

Student

Weight
(g)

0.0495

2
3
4

Standard
Deviation

Weight of sample (g)

0.0495
0.0484
0.0546

Trial 2
0.495
0.0992
0.4980

Trial 3
0.489
0.1008
0.4979

Trial 4
0.497
0.1004
0.4976

Trial 5
0.496
0.0997
0.4997

Average
0.0494
0.1
0.4989

Table 1.5 Weight of different water samples by members of the group


50L
100L
Weight
Weight
Deviation
%Error
Deviation
%Error
(g)
(g)
310-4
310-4
1.410-3
4.810-3

0.60%

0.1005

-810

0.60%

0.1003

-610

2.81%

0.1003

-610

9.64%

0.1005

-810

%Error

-3

0.34%

0.5002

-4

0.60%

0.4983

-4
210

0.04%

-4

0.60%

0.4988

-4
-310

0.06%

-4

080%

0.4997

0.1004

0.4993

Standard
Deviation

2.810-3

1.210-4

8.610-4

References
Gaines, P. (2014). Accuracy, Precision,
Mean and Standard Deviation. Retrieved
from

Deviation

0.80%

0.0505

The values obtained were close to each


other because of the proper transfer of
water using the appropriate micropipettor.
The minor difference of the different values
may be due to the adherence of water on

500L

-4

Average

Accuracy is defined as the closeness of the


measured value to the true value while
precision is defined as the closeness of one
measured value to other measured values.
The lower %error means that the value
obtained is more accurate. The lower
standard deviation and coefficient variation
means that the value obtained is more
precise.

3.4410-4
6.2010-4
1.49810-3

-1.710

-1.210

-3

0.24%

the pipette tip or because the same pipette


tip with water residue was used.
Conclusion
The
results
obtained
from
using
micropipettors in weighing the water sample
produced accurate and precise values. In
the utilization of a spectrophotometer, an
increase in the absorbance of a substance
results to a decrease in the transmittance.
Moreover, the increase in the concentration
of the BPB cuases an in increase in the
absorbance.

http://www.inorganicventures.com/accuracyprecision-mean-and-standard-deviation.

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Reusch, W. (2013). Visible and ultraviolet


spectroscopy. Retrieved 23 September
2015,
from
http://www2.chemistry.msu.edu/faculty/reus
ch/virttxtjml/spectrpy/uv-vis/spectrum.htm

Campbell, M. (2002). How to Use a


Micropipettor. Retrieved 23 September
2015,
from
http://www.bio.davidson.edu/molecular/Prot
ocols/pipette.html

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