The document is a detailed overview of single beam spectrophotometers, covering their history, construction, operation, and applications. Developed in 1940, these instruments significantly improved the speed and accuracy of chemical analysis, with various models evolving over time to enhance performance. Applications range from scientific research to forensic analysis, though they have limitations like time consumption and lack of instrumental variation compensation.

1. Single Beam
Spectrophotometer

2. Township Campus,Lahore
Submitted to: Dr.Mehr-un-Nisa
Submitted by: Muhammad Kashif
(mcf 1700986)
M.Kamran Nazir
(mcf1701055)
Nabeel Ahmed
(mcf1701195)
Sohaib Arshad
(mcf1701178)
Abdul-Qadeer
(1701258)
Programme: M.Sc Chemistry(E)
Semester I
Subject: Advance analytical
Chemistry
Course code: CHEM3114

3. Contents:
.History and Introduction
.Constuction and Instrumentation
.Working
.Applications & Advantages
.References
The History of Spectrophotometer
A spectrophotometer measures either the amount of light reflected from a sample
object or the amount of light that is absorbed by the sample object.
It is a device to measure the brightness of various portions of a spectra.
Spectrophotometry is the study of the spectra, this study is built upon the belief that each
chemical element has its own spectrum.
The spectrophotometer was invented in 1940, by Arnold J. Beckman and his colleagues at
National Technologies Laboratories, the company Beckman had started in 1935. They were
led by project leader Howard H. Cary. The spectrophotometer was the company's greatest
discovery.
Before 1940, the chemical analysis process was a long venture taking weeks to complete
with only 25 percent accuracy according to the MIT's "Inventor of the Week" archive. In
1940, when the Beckman DU Spectrophotometer was introduced, it simplified the process
greatly, requiring only a few minutes for analysis. According to the same source, this test
offered
99.99 percent accuracy on the analysis . This instrument set the standard in chemical
analysis[1]
.

4. In the beginning there were performance issues with the spectrophotometer. These
problems led to changes in design. The model B spectrophotometer used a quartz prism
instead of a glass prism this improved the UV capabilities of the device. Model C soon
followed with changes that raised wavelength resolution in the UV and three subsequent
Model C spectrophotometers were made. In 1941 the Model D, also known as the Model
DU, was produced with a hydrogen lamp and other improvements. This design remained
essentially unchanged from 1941 to 1976 when it was discontinued[2]
.
In 1981 Cecil Instruments produced a spectrophotometer that was microprocessor
controlled. This automated the device and improved the speed. This spectrophotometer was
more reliable than others made in this era. From 1984 to 1985, development was made in
double beam versions of the instrument which developed into the Series 4000 model. With
the 1990s came the addition of external software that provided PC control and onscreen
displays of the spectra. Today,the development of the spectrophotometer continues and its
applications range from science and medicine to crime scene investigation and law
enforcement.
The DR/4000 Spectrophotometer is a powerful scanning spectrophotometer that gives you
advanced capabilities and performance. It has ultraviolet and visible spectrum analysis
capabilities with a range of 190 to 1100 nm, customer calibration, kinetics, time course and
multi wavelength readings capabilities
Introdution
This is called as single beam spectrophotometer because all the measuresments are
based on the varying intensity of a single beam of light in single optical path. The primary
advantage is that it has fewer component and is less complicated than alternative design.
Hence, it is easier to construct and less expensive.With a single path and a minimum

5. number of optical components single-beam system typically provide very high light
throughput.
The primary limitation is that it provides no means to compensate for instrumental
variations during an analysis such as change in source intensity. The resulting signal
variability can limit the proformance capabilities of a single beam system.
UV-VIS 230 UV-Visible Spectrophotometer is the one of the best single beam
spectrophotometer still availble in the market and it ha high efficiency. The UV 230 is an
intelligent ultra-violet/visible spectrophotometer that is well suited to the qualitative and
quantitative analyses required by today's laboratories. With its wide wavelength range and
narrow bandwidth design, the instrument provides extraordinary flexibility and high
resolution at an exceptional price. It features a standard printer port for hard copy. The UV-
Vis Spectrophotometer also features power interruption protection, easy operation[3]
.
The UV/Vis Spectrophotometer operates over a wavelength range of 190 - 1100 nm
with a 5nm bandwidth. The spectrophotometer features a standard printer port and a
standard RS- 232 serial port. The UV/VIS 230 has the capacity to analyze up to four
cells.
.Instrumentation and Construction:
Spectronic 20 is designed for use in the visible region of the spectrum. It consisits of
following parts:

6. 1.Tungsten lamp
Tungsten lamp is a major source of providing light.It is linked with the photo tube which is
used to measure intensity of light.The light is emitted from this lamp and is entered in the
entrance slit.
2.Diffraction Gratting
Light rays pass through objective lens and strike with the diffraction grating.Diffraction
grating is used as a barrier and it is linked with the wavelength cam which is used to adjust its
position.
3.Light control:
Light control is basically a metal rod which is used to adjust the wavelength of rays.It is
linked with the an occluder.An occluder is situated on the upper side of light control.
4.Sample
It consists of solute and solvent but its major part consists of solute.Zero reading shows
solvent and final reading is the reading of the solute.Zero reading on the right side is used for
the solvent and reading of solute is obtained on the digital reader.
5.Photo detector
Photo detector is used to convert electromagnetic waves into electrical waves.They are also
present in our eyes.They pass electrical rays towards the amplifier..Amplifer enhances the
power of the photo rays.
6.Digital reader:
It is used to show the final reading of electrical rays (light rays).And final reading is obtained
from right to left side of the reader[4]
.
Range of spectronic 20 spectrophotometer:
The range of the above mentioned spectrophotometer is from 340 nm to 950 nm and
accuracy of wavelength is + 2.5 nm (Picture of instrument is given on next page).

7. Wavelength
CamLight
Control
SampleMeasuring
Phototube
Filter
Occluder
Diffraction
Grating
LampReference
Phototube
Objective
Lens
Entrance Slit
Field
Lens
Light Gate
Diagram: Internal instumentation of spectronic 20
Diagram: General construction of single beam spectrphotometer

9. SPECTRONIC 20 Spectrophotometer
The sequence for sample measurement is:
a. Select wavelength using Wavelength Control.
b. With sample compartment empty and cover closed, adjust Zero Control
so that the meter reads zero (on the left side of the dial).
c. Insert reference blank into the sample compartment and setAbsorbance Control to zero
(onthe right side of the dial).
d. Insert unknown sample into the sample compartment and read
measurement from meter in percent transmittance or absorbance[5]
.

10. General Working :
A lamp provides the source of light. The beam of light strikes the
collimator or lens.The lens then makes these radiations parallel and fall them
on the prism or sometimes diffraction grating(In optics, a diffraction grating
is an optical component which splits and diffracts light into several beams
travelling in different directions.).The prism or grating then separates the
light into its component wavelengths. The grating is rotated so that only a
specific wavelength of light reaches the exit slit.(The entrance slit,prism and
exit slit collectively form the monochromator).Then the light interacts with
the sample placed in the transparent cuvette.Here some of the amount of light
is absorbed by the sample solution placed in the cuvette.Then the light that is
not absorbed by the sample strikes with detector where its transmittance is
measured.Transmittance refers to the amount of light that passes through the
sample. Absorbance is a measurement of light that is absorbed by the sample.
The detector senses the light being transmitted through the sample and
converts this information into a digital value as shown in figure[6]
.
Prism splittinglight Wavelengthof differentcolours of visible light

11. .Why some of light is absorbed by the sample solution?
Some amount of light that is coming from the exit slit is absorbed by the
sample solution.The reason is that the electrons of the molecues present in the
sample solution absorb some specific amount of energy from the photons of
the light which have specific wavelength and go into an excited state i.e
into higher energy level.In this way,light energy is converted into heat energy[7].
In this way electrons are promoted to a higher energy molecular orbital after
absorbing that monochromatic light.
Explanation according to Beer-Lambert’s Law:
Spectrophotometers work on the principle of Beer-Lambert’s law.With the
help of this law,one calculate the Transmittance of light.And so,absorbance
can also be calcultulated.
Transmittance:
The ratio of intensities of the transmitted and incident light gives
transmittance. In other words,
Transmittance is the amount of light energy that is transmitted through the solution
without absorbing.It is measured in percantage T(i.e % transmittance)[8]
.
Transmittance = IT/I0 × 100

12. Where IT = Intensity of transmitted light
through the sample
And I0 = Intensity of the incident light (i.e blank cuvette having
pure water )
A 100 % value of T represents a transparent substance present in the
cuvette while zero value of T represents that object is totally opaque meaning
that it has absorbed all wavelength of light and has not transmitted any
wavelength of light.
Absorbance:
Absorbance is the amount of energy of specific wavelength in
terms of photons that is absorbed by the electrons of molecules present in the
sample.It is given as:
Absorbance : -log10T
We can also write it as : -log10 IT/I0
Here T resents the transmittance of a substance[4].
Beer-Lambert’s Law is given as:
Absorbance ∝ b .c
A= ε.b.c
Where ‘ε’ is propotionality constant known as molar absoptivity
coefficient.We can also use here ‘a’ which means same thing.“b” is the path
length and “c” is the concentration of the sample.
This equation states that the quantity of light absorbed by a substance dissolved in
a fully transmitting solvent is directly proportional to the concentration of the
substance (i.e ‘c’) and the path length (i.e b ) of the light through the solution.
It means that if there is more path length (i.e ‘b’) of the cuvette in which sample
is placed,more absorbance of light will take place and as a result less transmission

13. will be obtained.Because more distance has to be covered by the photons of light
while passing through the solution and there will be more interaction between the
photons and absorbing substances.Hence they will be absorbed more (i.e their
energy will be taken up and absorbed by the electrons of the absorbing
molecules).And less transmittance will be obtained.
Similarly If there is more concentration ( i.e c) of absorbing materials in the
sample of cuvette,the result will be the same(meaning more absorbance and less
transmittance)[9]
.
Units:
In uv spectroscopy,the concentration c of the sample solution is measured in mol L-1
and the Path length b of the light path in cm. Thus, given that absorbance is unitless,
the units of molar absorptivity ε are L mol-1 cm-1.[10]
c = mol L-1
b = cm
ε = L mol-1 cm-1
EXAMPLE:
Guanosine has a maximum absorbance of 275 nm.ϵ275= 8400M−1
cm−1
.ϵ
275=8400M−1
cm−1
and the path length is 1 cm. Using a spectrophotometer, we
find that A275=0.70. What will be the concentration of guanosine[11]
?
SOLUTION:
Since,
Absorbance = A = 0.70
Molar absoptivity =ϵ =8400
M-1
cm-1
)
Path length = b = l=1cm
Concentration =c= ?
To solve this problem, we use Beer's Law.

14. A =ϵlc
0.70 = (8400) (1 cm)
Next, divide both side by [(8400 ) (1cm)],we get the answer
c = 8.33x10-5 mol/L
Graph:
We can plot a graph between concentration and absorbance.Drawing
Concentration in mol L-1
on X-axis and absorbance on Y-axis.A straight line is
obtained which indicates that as concentration of a sample is increased,absorbance
also increases in the the same ratio.Similarly graph between path length b and
absorbance c can be plotted[12].
Graph 1:Graph between concentration c (in molL-1
) and absorbance
A(no units) indicating directly proportional relation.

15. Graph 2: Graph between pathlength b (or c) in cm absorbance A.
> Applications of Single Beam
Spectrophotometer
Before the spectrophotometer was developed scientists had no reliable and rapid method to
determine the chemical makeup of a substance. Spectrophotometer radically changed all of
that. Some applications of spectrophotometer are given as
1. It is used extensively in astronomy
2. It is also used in archeology
3. It is used to determine the deviation of light by a glass prism
4. It is used to determine refractive index
5. It is used to study the chemical bonding and to determine
concentrationof substances(nucleic acid for example which do not
absorb visible light)
6. Spectrophotometers are used to identify the unknown compounds
7. It is used to study atmosphere of distant planets
8. They are also used in chemistry to find out which chemicals are
present in chemical samples
9. Spectrophotometers can also be used for colour determination within
range of 380-700nm
10. It is also used in forensic sciences for investigation purposes
11. It is also used in microbiology to study of micro-oragnisms likebacteria
12. It is also used in pharmaceutical , food and paint industries
13. It is used in the diagnosis of diseases which is detected by the samples taken
from the body are analysize in three areas Chemistry , Hematology and
microbiology

16. 14. It is used to explain absorbance vs time graph in kinetics
15. It is used in the single point calibration for equilibrium constant
determination
16. It is also used in quantitative analysis of organic compounds
17. It is also used for study the progress of a chemical reaction
Advantages and disadvantages :-
There are following advantages of single beam spectrophotometer
 It has simple construction
 It has less complicated system
 It is easy to use
 It is not very much expensive
 It has fewer parts which can be easily handled
 It has high light throughput
 It measures accurately
There are following disadvantages of single beam
spectrophotometer
 It consumes much time as compare to other designs
 We cannot make variations during experiment
References:
1.The Journal of Biological Chemistry A Classic Instrument
2. Beckman DU Spectrophotometer and Its Inventor, Arnold
Inventor of the Week Archive: Arnold Beckman
3. Cecil Instruments: History of Cecil Instruments
4. Analytical Chemistry by D.A Skoog,D.M West F.J Holler and S.R Crouch,,8th
edition
5. https://web.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy
6.https://www.lsteam.org/projects/videos/how-does-spectrophotometer-
work

17. 7.https://www.andor.com/learning-academy/what-is-light-an-overview-
of-the-properties-of-light
8. https://cellbiologyolm.stevegallik.org/node/7
9.https://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/
Kinetics/Reaction_Rates/Experimental_Determination_of_Kinetcs/Spect
rophoto metry
10.https://life.nthu.edu.tw/~labcjw/BioPhyChem/Spectroscopy/beerslaw.
html
11.https://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistr
y/Spectroscopy/Electronic_Spectroscopy/Electronic_Spectroscopy_Basi
cs/The_Beer-Lambert_Law
12.https://public.iorodeo.com/docs/colorimeter/lab_2.html