Experiment 1: An introduction to Biology Laboratory

Objectives:
To expose and familiarize the use of pH meter, micropipette, spectrophotometer and weighing
scale basic techniques and some of their applications in scientific research.

Introduction:
The study of cell and Molecular Biology involves a vast number of methods and techniques as it
studies the structural unit of the living system. Laboratory instrument are devised to achieve
accuracy and precision as it deals with up to microscopic level. The micropipettor is one of the
most commonly used instruments in science laboratories. Pipettes are used to accurately measure
and dispense relatively small volumes of liquid. A small volume micropipettor has a range of
0.5- 10 mL, mid-range micropipettor can handle 10- 100 mL of liquid and a large volume
micropipettor can measure up to 100- 1000 mL. It is precision instrument and should be handled
with utmost care.
While for pH meter is an instrument used to measure acidity or alkalinity of a solution
also known as pH. pH is the unit of measure that describes the degree of acidity or alkaline
between
scale 0 to 14. The quantitative information provided by the pH value expresses the degree of the
activity of an acid or base in terms of hydrogen ion activity. The pH value of a substance is
directly related to the ratio of the hydrogen ion [H+] and the hydroxyl ion [OH-] concentrations.
If the H+ concentration is greater than OH-, the material is acidic (pH value is less than 7) while
if the OH- concentration is greater than H+, the material is basic (pH value greater than 7). If
equal amounts of H+ and OH- ions are present, the material is neutral, with a pH of 7. Acids and
bases have free hydrogen and hydroxyl ions, respectively. The relationship between hydrogen
ions and hydroxyl ions in a given solution is constant for a given set of conditions, either one can
be determined by knowing the other. Compare to pH papers which change color with varying pH
levels. These papers have limitations on their accuracy and as such, their use is limited.
(Compton, 2012)

Every chemical compound absorbs, transmits, or reflects light (electromagnetic

radiation) over a certain range of wavelength. Spectrophotometry is a measurement of how
much a chemical substance absorbs or transmits. Spectrophotometry is widely used for
quantitative analysis in various areas chemical engineering, clinical applications and industrial
applications. A spectrophotometer is an instrument that measures the amount of photons (the
intensity

of

light)

absorbed

after

it

passes

through

sample

solution.

With

the

spectrophotometer, the amount of a known chemical substance (concentrations) can also

be determined by measuring the intensity of light detected. (Atkins, 2006)

Figure 1: Basic structure of Spectrophotometers.

Lastly, Jove (2015) did mention that the analytical balance is the common piece of
equipment in the scientific laboratory. These highly precise instruments can measure down to ten
thousandths or even hundred thousandths of a gram. The triple beam balance is a type of scale
that uses counterweights on a three rider beams to counterbalance the sample in the pan. On the
other hand, modern analytical balances use a complex system of electronic sensors to accurately
mass a substance. When weighing, a weigh boat or weigh paper is used to hold the substance
being weighed and protect the weighing pan. Before weighing a substance, analytical balances
are tarred to subtract the weigh boat or weigh paper and re-zero the scale. Tarring refers to
setting the scale back to read zero and allows for a substance to be accurately weighed. Aside
from weighing chemical substances, analytical balances are used to animals or insects, and
centrifuge tubes in ultracentrifugation experiments.

Methodology
Part 2:
Part A: Visual Estimation of pH
0.1 M solutions (100mls) of the K2HPO4 and KH2PO4 was prepared and set up accordingly
followed the Table A-1.2A

5 drops of Bromothymol Blue was mix in the tube of 1, 3, 5, 7, 9, and 11.

The changes of color was observed and recorded.

Section A:

Used pH 7 buffer to standardize the pH meter and rinsed the electrodes

also measured the pH using standard pH buffer 4.01.
The pH of the six solution of 2, 4, 6, 8, 10, and 12 tubes (as prepared in
Part A) was measured. The electrodes was keep rinsing in between the
solution.
For the same solution, pH indicator paper was used to measure the pH as
rapid method for the small volumes or radioactive solution.
The measured value from part A and B was observed and recorded and
correlate the value from part B to the expected pH value from the table A1.2A.

Part 3:

Spectophotometer is
warmed up about 20
minutes and set up at
the range 540 nm.

10 mls of distillled
water was pipette in
eight tubes

Bromophenol blue
(1.25% w/v): O.5, 1,
2, 4, 10, 20, 50, and
100 l was added in
the tubes by using the
micropipettors.

The reading value

obtained from the
spectrophotometer
was recorded

The
spectrophotometer
was set to zero with
distilled water and the
dye solution had been
transferred from least
concentrated to most
concentrate into the
same cuvette.

Part 4:

Basic weighing function: Turned on the balanced scale.

Container was placed on top of the balance, balanced weights to zero then them
sample was placed in the container.

Weight of the sample was recorded.

Result
Part 2:

Figure 2: pH indicators results using Bromothymol Blue.

Figure 2.1: pH indicator color range.

Table 1: Comparison of pH results using two different method.

Solution (tube)

pH meter

pH paper

5.45

6.24

6.65

7.08

10

7.53

12

8.21

Part 3:
Table 2: Wavelength results for different concentration solution.
Solution

Bromophenol blue

Absorbance value

1
2
3
4

concentration(l)
0.5
1
2
4

(nm)
0.006
0.010
0.082
0.012
6

5
6
7
8

10
20
50
100

0.028
0.064
0.153
0.287

Absorbance (abs) versus concentration of bromophenol blue (w/v)

R = 0.95
Absorbance
Linear (Absorbance)

Figure 3: Absorbance (540 nm) versus concentration of Bromophenol blue (w/v)

Part 4:
Table 3: Weight of micropipette results.
Amount micropipette (l)
200
500
700
1000

Weight (g)
0.2012
0.5010
0.6947
1.0081

Discussion

pH is the unit to describe the degree of acidity or alkaline (0 to 14). The objective of this
experiment was to expose on basic skills the use of pH meter and also pH indicator. Based from
the result obtained in figure 1 shows different range of pH color from tube 1, 3, 5, 7, 9, and 11 by
mixing each tube with the Bromothymol blue. As obtained, the comparison between figure 2
and figure 2.1 shows that the tubes 1 and 3 solution was yellow color means the value of pH is 5,
while for the tubes 5 and 7 solutions shows the pH value was 7 and for the tube 9 and 11 turned
to blue color indicates the pH value was 9. Bromothymol blue solution commonly used as pH
indicator. Bromothymol blue changes color over a pH range from 6.0 (yellow) to 7.6 (blue).
Tube 9 and 11 turned in blue color because the levels of acid in solution with Bromothymol blue
indicator was low, for the tube 5 and 7 its turned to the mid pH range between acidic and base
while the level of acid increases, the solution will gradually take on a yellow as shown in figure
2.
Based from the results obtain in table 1 shows two different method use to indicate the pH by
using the pH meter and also pH indicator paper. From table 1, the value recorded from tube
solution 2, 4, 6, 8, 10, and 12 shows slightly different from the table A-1.2A. The value was near
to the expected pH value for each tubes solution for an example tubes 2, the pH recorded was
5.45 near to the expected value 5.59 while for the solution 4, the pH meter value was equally
same to the expected value. This is because the pH value of a substance is directly related to the
ratio of the hydrogen ion [H+] and the hydroxyl ion [OH-] concentrations. If the H+
concentration is greater than OH-, the material is acidic (pH value is less than 7) while if the OHconcentration is greater than H+, the material is basic (pH value greater than 7). If equal amounts
of H+ and OH- ions are present, the material is neutral, with a pH of 7. However, based from the
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pH paper indicates that the value obtained was more different to the expected value at table A1.2A. This is because the pH paper or Litmus paper is often used to differentiate an acid from a
base. When pH is less than 4.5, the paper turns red. This indicates that the substance is an acid. If
pH is greater than 8.3, the paper turns blue, indicating that the substance is a base. In neutral
conditions, pH paper is a purple color. Based from these 3 methods of pH measurement, the
more accurate method to measure pH is by using pH meter because the value of pH meter
because the value obtained was almost same and nearly to pH expected.
From experiment part 3 tackles on the familiarization of two commonly used laboratory
equipment spectrophotometer. A specific amount of Bromophenol blue was dropped and mixed
with each of the eight test tubes. Based from the table 2 by using the spectrophotometer, the
absorbance for 0.5L of Bromophenol blue was 0.006nm, for 1.0L Bromphenol blue it was
0.010nm, 0.082nm was recorded for 2.0L of Bromophenol blue, for 4.0L Bromophenol blue
was 0.012nm, for 10.0 L Bromophenol blue show 0.028nm, for the 20.0 L the results show
0.064nm, while for the 50 L show the result obtained 0.153nm and lastly for the 100 L the
result was 0.287nm. The results show that there is a direct relationship between the amount of
dye in the solution and the amount of light absorbed by the solution. As the amount of dye
increases, the amount of light absorbed also increases. For the 100 L, the solution was more
concentrated than others it will have a very high absorbance because there are lots of molecules
to interact with the light rather than 0.5 L the value was expected as the concentration was low,
so that the interaction between the light and molecular will be less. When photons encounter the
molecules in the sample of the solution, the molecules may absorb some of them, reducing the
number of photons in the beam of light and decreasing the intensity of the detected signal. This
means the higher the concentration of the solution the higher the absorbance of the wavelength
will be.
From the absorbance, the concentration of the sample solution can be determined which states
that there is a linear relationship between the absorbance and concentration of a sample.
Based on figure 3 shows the plotted graph of the amount of the absorbance versus Bromophenol
blue shows the linearity of the absorbance which shows the higher the concentration of the
higher the absorbance. However, there was an error occur at solution 3 which the absorbance

obtained was 0.082, this error might cause from wrongly micropipette the amount of
Bromophenol blue and maybe because of the cuvette.
Lastly for the part 4 experiment shows that the value recorded in table 3 shows four different
amount of solution by using micropipette. The purpose of this experiment was to familiarize
students with the operation of the analytical balance. Because all analytical direclt based on the
measurement of mass. The volume of distilled water was measure by using micropipette with
volume 200L, 500L, 700L and 1000L. Based on table 3, when the volume of distilled water
increase, the weight of distilled water also will be increase. This is due to the density of water
increase.

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Conclusion
The objective of this experiment is to expose students with pH meter, micropipette,
spectrophotometer and weighing scale basic technique. This experiment was achieved the
objective. For pH measurement experiment the uses of 3 steps to measure pH by using drops of
Bromothymol blue into the buffer solution, pH meter and lastly pH paper it can be concluded
that pH meter is more accurate to measure pH of the solution compared to the two others
method. In spectrophotometer experiment the result shown in figure3, the absorbance of solution
increase when the concentration of Bromophenol blue solution increases. The results show that
there is a direct relationship between the amount of dye in the solution and the amount of light
absorbed by the solution. As the amount of dye increases, the amount of light absorbed also
increases. For the 100 L, the solution was more concentrated than others it will have a very
high absorbance because there are lots of molecules to interact with the light rather than 0.5 L
the value was expected as the concentration was low, so that the interaction between the light and
molecular will be less. Lastly, for part weighing balanced, when the volume of distilled water
increase, the weight of distilled water also increase this is because the density of distilled water
also increase. As recommendation proper measuring and using the micropipette was important to
get the accurate result and the cuvette use for the spectrophotometer must be in proper condition,
wipe the fingerprints at the clear parts.

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Question
Part 1:
1) -Do not allow the tip of the micropipette to touch the receiving liquid. Some of the liquid
may be drawn back up into the micropipette, contaminating the sample for the remainder
of the micropipette sequence.
-Never hold the micropipette sideways when liquid is inside the tip.
-Never flame the tip of the micropipettor.
2) Care :
-Disassemble and clean the pipette
-Remove the tip ejector
Separate the lower half of the pipette from the top half
-Decontaminate each component as it is removed from the pipette
-Remove the seal and o-ring assemblies
-Inspect visually for worn and cracked parts
Maintainance:
-Treat micropipettes very gently as they are precision instruments.
-Keep upright when in use to prevent liquids running inside the shaft of the pipette.
-Do not leave pipettes lying on the workbench where they can be knocked off and
damaged.
-Check all tips are securely fitted to pipette.

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Part 2:
1)

A pH meter provides a value as to how acidic or alkaline a liquid is. The basic principle
of the pH meter is to measure the concentration of hydrogen ions. Acids dissolve in water
forming positively charged hydrogen ions (H+). The greater this concentration of
hydrogen ions, the stronger the acid is. Similarly alkali or bases dissolve in water forming
negatively charged hydrogen ions (OH-). The stronger a base is the higher the
concentration of negatively charged hydrogen ions there are. A pH value of 7 indicates a
neutral solution. Pure water should have a pH value of 7. Now pH values less than 7
indicate an acidic solution while a pH value greater than 7 will indicate an alkaline
solution. A solution with pH value of 1 is highly acidic and a solution of pH value of 14 is
highly alkaline. A pH meter will be made up of a probe, which itself is made up of two
electrodes. This probe passes electrical signals to a meter which displays the reading in
pH units. The glass probe has two electrodes because one is a glass sensor electrode and

the other is a reference electrode.

2) Calculation:
NaOH 20% w/v
Volume 200 ml
20
g
200 ml=40
100
mol
Tris 1.0 M (MW 121.1 g/mol)
Volume 1000 ml
1.0 mol
g
1000 ml
121.1
=121.1 g
1000 ml
mol
EDTA 0.2 M (MW 372.2 g/mol)
Volume 100 ml

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100 ml

0.2 mol
g
372.2
1000 ml
mol

7.44 g

3) Calculation:
Tris 10 mM (MW 121.1 g/mol)
Volume 100 ml
10 mM
g
100 ml
121.1
=0.12 g
1000 ml
mol
EDTA 1 mM (MW 372.2 g/mol)
Volume 100 ml
1 mM
g
100 ml 1000 ml 372.2 mol =0.037 g
4) The buffer capacity depends on the concentration of acid or base in a buffer. For a good
buffer, the acid concentration to be about the same as the base concentration. Thus, the
closer the acid and base concentrations are to each other the more they approach 1 which
results in a log (1) which equals 0. So good buffers mean their pH will be around pKa.
This also means a better buffer capacity.
5) Buffer capacity defined as maximum amount of either strong acid or strong base that can
be added before a significant change in the pH will occur depend on how high the
concentration of the acids and bases are in the buffer. This is because if the
concentrations of acid/base are extremely large, than it will take much more of an added
acid/base to change much of the buffer's acid/base. Buffer capacity can be also defined as
quantity of strong acid or base that must be added to change the pH of one liter of
solution by one pH unit.

Part 3:

14

1) A spectrophotometer consists of two instruments, namely a spectrometer for producing

light of any selected color (wavelength), and a photometer for measuring the intensity of
light. The instruments are arrange so that liquid in a cuvette can be placed between the
spectrometer beam and the photometer. The amount of light passing through the tube is
measured by the photometer. The photometer delivers a voltage signal to a display
device, normally a galvanometer. The signal changes as the amount of light absorbed by
the liquid changes. If development of color is linked to the concentration of a substance
in solution then that concentration can be measured by determining the extent of
absorption of light at the appropriate wavelength.
2) Spectrophotometers are instruments that are commonly used for measuring the
transmittance or reflectance of solutions or transparent. This instrument is basically used
in field biochemistry because UV visible spectrophotometers are used in the quantitative
determination of solutions of transition metal ions and highly conjugated organic
compounds.
3) There is a logarithmic dependence between the transmission of light through a substance
and the concentration of the substance, and also between the transmission and the length
of material that the light travels through. The percent transmittance multiplied by 100, is
usually converted absorbance.

References:

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Campbell, M.K., Farell, S.O, Biochemistry. 6th ed. Philippines: Cengage Learning AsiaPte. Ltd.
(2009)
Atkins, Peter and Julio D.P. Physical Chemistry for the Life Sciences. New York: Oxford
University Press, 2006
Chang, Raymond. Physical Chemistry for the Biosciences. USA: University Science Books,
2005.
Gore, Michael. Spectrophotometry & Spectrofluorimetry. New York: Oxford University Press,
2000.

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