BOE 201

BIOLOGICAL INSTRUMENTATION

2021/2022 SEMESTER 1

PROJECT-BASED LAB: BUILD YOUR OWN

SPECTROPHOTOMETRY

Topic: Principles and techniques of basic spectrophotometer and atomic

absorption spectrophotometer

Name: Lee Chi Yien

Matric Number： 151877

Lecturer: Dr Yazmin Bustami

Date of submission: 12/1/2022

 A) Introduction

Spectrophotometer is an instrument in which used to measure the absorbance of

light intensity by a sample after light is transmitted through it. It is useful in the field of
spectrophotometry to measure the light absorbance of a chemical substances after light
had passing through it. The principle of spectrophotometry is the components in the
analyzed sample will absorb or transmit the light over a certain range of wavelength.
(Tom, 2021) According to Beer-Lambert law, the absorbance of molecules in solution
is directly proportional to the concentration of sample.

The electromagnetic spectrum consists of a continuum of waves with different

wavelength and energy. Energy and Wavelength is inversely related. When the
electrons in an molecules absorb energy from the electromagnetic radiation, they will
be excited to higher energy level. This phenomenon called absorption. Whilst the
electron in the atom jump from its excited state to the ground state, electromagnetic
radiation is released, this called emission. (Boyer)The picture below shown is several
regions of electromagnetic spectrum.

Figure 1 Electromagnetic spectrum

In this practical, we are going to focus on the basic spectrometer, Ultraviolet-Visible

(UV-VIS) absorption spectrometry and atomic absorption spectrophotometer (AAS).

Ultraviolet-Visible (UV-VIS) absorption spectrometry. This instrument is used to

measure the absorption of ultraviolet or visible light with discrete wavelength by the
molecules or atoms in the sample tested. Thus, the predicted the Below shown are the
specific wavelength range of ultraviolet light and visible light.

• Ultraviolet (UV), λ=180-340nm

• Visible, λ=340-800nm

This instrument consists of 5 components, light source, monochromator, sample

chamber, detector and a recorder. The working principle of a light spectrophotometer
is a beam of light from the light source (tungsten lamp or deuterium lamp) is directed
towards a monochromator. Monochromator consists of two slits and prism. The light
pass through the slits and prism, producing a beam of light with single wavelength. The
light is then transmitted to the sample. The molecules in the sample will then absorb
the energy of the light. Finally, the detector detect the effect of photon converting into
electrical current, and the results will be shown on the recorder. (Tom, 2021) (Visser,
2021) (Boyer)

Atomic absorption spectrometer (AAS) is an instrument used to determine the

concentration of specific metal atoms or ions by measuring the absorption of light by
the atoms or ions in the sample. The light used is commonly in the visible and ultraviolet
region of electromagnetic spectrum. The principle of these spectrometer is to measure
the amount of light absorption by the free atoms in their ground state. (Visser, 2021)
The difference between the basic spectrophotometer with atomic absorption
spectrometer is that AAS uses an atomizer by flame to atomize the sample. The working
mechanism of this spectrometer is the sample (e.g salt solution) is placed into the plane
and being flamed. The solvent is vaporized, and the solute molecules is converted into
gas molecules while further heating, eventually the gas molecules dissociates into
atoms. Some of the atoms absorb energy and promoted to high energy level, and they
are said to be in excited state. When the atoms come back from the excited state to
ground state, they will emit energy, this called photon emission. Atomic absorption
spectrometer will detect the radiation emitted from the atoms, and thus determined the
concentration of the sample. (Visser, 2021)

Nowadays, spectrophotometers are widely applied in many fields, for instance,

DNA and RNA analysis, pharmaceutical analysis, bacteria culture and so on. On the
contrary, the atomic absorption spectrometer is used in metal tracing analysis, food and
beverage, chemical sectors and so on. (Visser, 2021) (Boyer)

B) Objective
1. To learn the techniques of analyzing the wavelength of the light emitted by
using a paper spectrophotometer.
2. To master the principle of basis spectrophotometer and atomic absorption
spectrophotometer.
3. To understand the law and calculation involved in the analyzing of spectrum
results.
C) Materials and apparatus
1) Paper template
2) Scissors
3) Tape
4) Paper board (black colour)
5) Unused compact disc (CD)
6) Camera detector (i.e. webcam, laptop)
7) Light source (i.e. torchlight, portable light)

D) Methods:
i) Basic spectrophotometer:
Video Link: https://youtu.be/yHnxYANuwmQ

Figure 1 With Light

Figure 2 Without Light

ii) Atomic absorption spectrophotometer (AAS)
1. The sample is screwed on tightly.
2. The power switch of atomic absorption spectrophotometer is turned on.
3. The acetylene gas tank is turned and following with the air flow.
4. The desired level of air flow and fuel are adjusted by turning the coarse
adjustment knobs and fine adjustment knobs.
5. The control is turned to AIR and the flame is lighted up by using a lighter
when the FUEL knob is turned on.
6. A blue flame should be observed, if yellow flame is produced the oxidant
level should be decreased.
7. The sample tubing is placed in distilled water and flushed for 30 seconds.
8. Focus on leaking condition. If no leaking occurs, the instrument is zeroed,
whereas when leaking occurred, flame is turned off and re-screw the
sample slot, then reignite the flame again.
9. The sample tubing is placed in the sample and the readings are taken when
the readings are stabilized.
10. The sample tubing is placed back into the distilled water to rinse for 1
minute after all samples are measured.
11. Turn off the FUEL knob.
12. Turn off the AIR knob followed by turning off the air flow.
13. The acetylene gas tank is turned off.
14. The power switch is turned off.
15. The sample slot is unscrewed to let dry
 E) Results and Discussions:
a) Basic spectrophotometer
1) State your observation (intensity, wavelength, etc)

Based on the result of absence of light source, the spectrum is black. The light intensity
is around 8% to 12%. This probably due to leaking of light into the paper
spectrophotometer. There is no observable wavelength of light can be deduced.

Based on the result when light source is applied, the light spectrum composed of red
light, green light and blue light. The wavelength of the light falls in the region of the
visible light region which is between 350 nm to 750 nm. The red light has the highest
intensity of approximately 61%. Then followed by green light approximately of 58%
and the blue light with intensity of around 45%. Based on the result obtained, the red
light is around the range of wavelength 300nm to 550nm. Green light has the range of
wavelength of around 400nm to 700nm. Last, for the blue light with range of
wavelength around 500nm to 800nm.
2) Discuss your observation:
i. State the principle.

The principle of the technique of spectrophotometer is to measure the

concentration of components in a sample by measuring the amount of light
absorbed by chemical substance in a sample (Absorbance) with the measure
of the intensity of light that is not absorbed by the chemical substances
(transmittance).

ii. Identify the functions for each component involved in this

experiment.

Torchlight, act as a light source of spectrophotometer. The compact disc

act as the monochromator which produce a monochromatic light with single
wavelength. The slit on the paper spectrophotometer allow the light to pass
through. Lastly, the webcam in which function as a detector of the
wavelength of light and it is then be analyzed by the spectral analyzer
software to measure the wavelength and the light intensity of light.

iii. Troubleshooting/ problems encountered for this experiment.

One of the problems encountered during the experiment is leaking of

light into the paper spectrophotometer due to not complete sealing. Leaking
of light will affect the precision of the results. The light leak into instrument
through the incomplete seal part, and thus the folding part of paper
spectrophotometer need to fold and seal again using cellophane tape to make
sure no leaking of light occur. Next, the light intensity from the light source
may affect the spectrum result. This is because over exposure of light to the
detector will influence the reading of spectrum. Hence, the light intensity of
light source should adjust to optimum level. Furthermore, the results
obtained was inaccurate because the calibration steps is not taken.
3) Questions:
i. Explain the relationship between absorbance (A) and transmittance
(T).

In spectrophotometry, absorbance is to measure the amount of light

being absorbed by the components in a sample when the light is allowed
to pass through the sample. On the contrary, transmittance is to measure
the light that not being absorbed by the components in a sample and
being transmitted when the light beam is allowed to pass through the
sample. Below shows the expression for transmittance and absorbance:

a) Transmittance:
𝑰
T (%) =𝑰𝟎 x 100%

T= Transmittance
I= Transmitted intensity of light
Io= Incident intensity of light

b) Absorbance:
A=-log10 T

A= Absorbance
T= Transmittance

Based on the expression, there is a logarithm relationship between the

transmittance and absorbance. Zero absorbance is corresponding to 100%
of transmittance.
ii. Calculate the concentration of bovine serum albumin (BSA) if 40%
of light beam transmitted through a 2 cm of cuvette (assume the
molar absorption coefficient, e is 43,824 M-1 cm-1).
A= Elc
A= absorbance
E= absorption coefficient
L= path length of light through the sample (cm)
C= concentration of absorbing material in the sample
Solution:
40% transmittance=0.398 absorbance
A= Elc
43824
0.398=1𝑀 𝑥 1𝑐𝑚 x 2cm x c
1𝑀 𝑥 1𝑐𝑚 1
c =0.398 x x 2𝑐𝑚
43824

c= 4.541X 10-6M
The concentration of bovine serum albumin (BSA) is 4.541M

iii. Calculate the %T, if the concentration of BSA in a 2 cm of cuvette

is 0.025 mg/mL

Step 1: Calculate the concentration of BSA.

Molecular weight of BSA is 66430.3 g/mol
1mg/mL= 1g/L
0.025 𝑔 1 𝑚𝑜𝑙
c= 𝑥
1𝐿 66430.3 𝑔

c=3.76334x10-7 mol/L / 3.76334x10-7 M

Step 2: Calculate Absorbance(A) of BSA

A= Elc

43824
A=1𝑀 𝑥 1𝑐𝑚 𝑋 2𝑐𝑚 𝑥 3.7633x10-7 M

A=0.032985
 Step 3: Calculate the %T of BSA

A= -log10 T

𝑰
T = 𝑰𝟎

𝑰
T (%) =𝑰𝟎 x 100%

𝑻
0.032985= - log10 𝟏𝟎𝟎%

T=10-0.032985 X 100%

T= 92.68%

iv. Compare between ii and iii.

Based on the calculation, the absorbance in (ii) (0.398) is higher
as compared to absorbance in (iii) (0.032985). This is because the
concentration of BSA in (ii) (4.541X 10-6M), is higher than in (iii)
which is 3.76334x10-7 M.
According to Beer-Lamber law, the concentration of a sample is
directly proportional to the total amount of light absorbance. The
greater the concentration of a sample, the greater amount of light is
absorbed by the components in the sample. (Boyer)
b) Atomic absorption spectrophotometer

1. Based on your Reading:

a. Draw the instrumentation setup

(Boyer)

b. Explain the principle involved

Atomic absorption spectrophotometer (AAS) applied the principle in which the

atomized gaseous atoms which is in its ground state can absorb electromagnetic
radiation of a specific wavelength. (Visser, 2021) The gaseous molecules is being first
being thermal dissociated in the flame producing metal atoms in ground state. Then,
the metal atoms will absorb the electromagnetic radiation of specific wavelength and
provided energy to excite the atom but some does not. Thus, some radiation are
transmitted and be detected by the detector. The amount of absorption of radiation is
then used to determine the concentration of the sample. For this, Beer Lambert Law is
applied and it is stated that the amount of light absorbed is directly proportional to the
number atoms excited from the ground state in the flame. (Visser, 2021)

2. Questions:

a) What is the main application of Atomic Absorption Spectrophotometer (AAS)?

Atomic absorption spectrophotometer (AAS) is being widely used to trace

presence of metal in a sample. It is also being used in the field of pharmaceutical
industry, clinical usage for biological fluid analysis, environment studies and food
industry. (Visser, 2021)

b) Compare Atomic Absorption Spectrophotometer (AAS), Atomic Emission

Spectrophotometer (AES) and UV-Vis spectrophotometer.

Atomic absorption spectrophotometer (AAS) measure the amount of light

absorbed by the gaseous atoms, whereas the atomic emission spectrophotometer
measure the amount of light emitted by the excited atoms. Both atomic absorption and
emission spectrophotometer is used to determine the concentration of specific metals
or element in a sample whereas the UV-Vis spectrophotometer used to determine the
concentration of a sample depends on the light absorbance. Atomic absorption and
emission spectrophotometer involves metallic atoms, and the valence electron is being
excited. On the contrary, the UV-visible light involves molecules, and the electron in
the molecular orbitals is being excited. In Atomic absorption spectrophotometer,
monochromatic light source provide energy to excite the gaseous atoms whereas flame
is used in Atomic emission spectrophotometer to excite the electrons of gaseous atoms.
To compare the components in AAS and UV-Vis spectrophotometer, UV-visible
spectrophotometer use a lamp as a light source and use a monochromator to generate a
beam of light with specific wavelength while the AAS use a metal cathode lamp to
generate electromagnetic radiation with few wavelengths and monochromator to
produce a specific wavelength.
References
Boyer, R. (n.d.). Biochemistry Laboratory Modern Theory and Techniques. (S.
Edition, Ed.) Pearson Education.

Tom, J. (2021, June 30). UV-Vis Spectroscopy: Principle, Strengths and Limitations
and Application. Retrieved from January 11, 2022
https://www.technologynetworks.com/analysis/articles/uv-vis-spectroscopy-
principle-strengths-and-limitations-and-applications-349865

Visser, D. (2021, December 16). Atomic Absorption Spectroscopy, Principles and

Applications. Retrieved January 11, 2022, from
https://www.technologynetworks.com/analysis/articles/atomic-absorption-
spectroscopy-principles-and-applications-356829