Faculty of Engineering and Information Technology

Chemical Engineering Department

Chemical Technology Lab. I (10626478)

UV-Vis Spectrophotometer experiment

(Reaction Experiment #5, Performed on: November 27, 2023)

Prepared by:

Ameer Abu Hayat

Areen Al-shaer
Nadeen Hawash
Sara Rabaya

Submitted to:

Dr. Majd Eshtaya

Eng. Maha Fuqha

January 6, 2024
Table of Contents

Abstract......................................................................................................................................2
Introduction................................................................................................................................3
Theory........................................................................................................................................3
Methodology and experimental method.....................................................................................3
Results and sample of calculation..............................................................................................3
Discussion..................................................................................................................................3
Conclusion..................................................................................................................................3
References..................................................................................................................................3
Appendices.................................................................................................................................3

List of Figures

Figure 1: UV-Vis Spectrophotometer........................................................................................5

Figure 2: Cuvette........................................................................................................................5
Figure 3: Calibration curve for red dye solution........................................................................7
Figure 4: Calibration curve for yellow dye solution..................................................................7
Figure 5: Calibration curve for green dye solution....................................................................8

List of Tables

Table 1: Results for red dye solution.........................................................................................7

Table 2: Results for red dye solution.........................................................................................7
Table 3: Results for green dye solution......................................................................................8

1
Abstract

The objectives are to understand the functionality of the UV-Vis spectrophotometer and how
it works, be able to construct a calibration curve for a certain solution, and apply the Beer-
Lambert equation to estimate the unknown concentration of a given solution (i.e., dyes).

The UV-Vis Spectrophotometer should be turned on and left to warm up for 10 minutes. Any
leftover cuvettes should be removed. The device should be calibrated using a blank, with
water used for dye solutions. The targeted solution, Methylene Blue, should be scanned
between 200-800 nm to determine the maximum absorbing wavelength. Repeat steps for
other dye solutions. Methylene Blue solutions with different concentrations (0 to 35µM)
should be prepared and observed at λ max. The absorbance of unknown solution concentrations
should be found, and their concentrations determined using the Beer-Lambert equation.

2
Introduction

The goals are to be able to build a calibration curve for a particular solution, comprehend the
operation of the UV-Vis spectrophotometer, and use the Beer-Lambert equation to estimate
the unknown concentration of a given solution (i.e., dyes).
UV-Vis spectrophotometry is a fundamental technique used in analytical chemistry to
measure the absorption of ultraviolet (UV) and visible (Vis) light by molecules in a solution.
This powerful method relies on the interaction between electromagnetic radiation and matter,
specifically the ability of molecules to absorb light at characteristic wavelengths.
In this experiment, our aim is to explore the principles of UV-Vis spectrophotometry by
analyzing the absorption spectra of different chemical compounds. By measuring the
absorbance of solutions at various wavelengths, we can identify the wavelengths at which
these compounds absorb light and quantify their concentrations. This data will provide
insights into the nature of the molecules present in the solutions and their behavior
concerning light absorption.
Furthermore, this experiment will demonstrate the application of UV-Vis spectroscopy in
quantitative analysis, allowing us to construct calibration curves and determine the
concentrations of unknown samples based on their absorbance values. Understanding the
principles and applications of UV-Vis spectrophotometry is crucial in fields.
Through this experimental exploration, we aim to deepen our understanding of molecular
absorption and the practical utility of UV-Vis spectrophotometry in characterizing chemical
substances.

https://www.studocu.com/my/document/monash-university-malaysia/biochemistry/
experiment-1-uv-vis-spectrophotometer-practical-lab-report-answer/14746951
https://www.jove.com/v/10204/ultraviolet-visible-uv-vis-spectroscopy-principle-and-uses

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Theory

Beer's Law, also known as the Beer-Lambert Law, is an equation that relates the attenuation
of light to the properties of a material. The law states that the concentration of a chemical is
directly proportional to the absorbance of a solution. The relationship is most often used in
UV-visible absorption spectroscopy. Note that Beer's Law is not valid at high solution
concentrations.

To calculate the concentration C:

Lambert-Beer Law:

A=Ɛ∗I∗C

Where:

A: The absorptivity of the solution.

ε: The molar absorptivity, L /mol.cm.
l: The cuvette path length, cm.
C: The concentration of the solution, mol/L

4
Methodology and experimental method

Apparatus:

 GENESYS 10S UV-VIS Thermo Science Spectrophotometer (Figure 1)

Figure 1: UV-Vis Spectrophotometer

 1 cm width Quartz/Glass or plastic cuvette

Figure 2: Cuvette

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Procedure

1) The UV-Vis Spectrophotometer is to be turned on and left for 10 minutes to warm up.
2) Any previous leftover cuvettes are to be removed.
3) The device is to be calibrated by using an appropriate reference (blank), with water being
used in the case of dye solutions.
4) The targeted solution (Methylene Blue) is to be scanned between 200-800 nm to determine
the maximum absorbing wavelength (λmax).
5) Step 4 is to be repeated for other dye solutions (green, yellow, red dyes).
6) Methylene Blue solutions with different concentrations (0 to 35µM) are to be prepared,
and their absorbance at λmax is to be observed.
7) The absorbance of unknown solution concentrations is to be found, the Beer-Lambert
equation is to be applied, and their concentrations are to be determined.

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Results and sample of calculation

Table 1: Results for red dye solution

Red dye
Sample # Concentration (ppm) Absorbance 508 nm
1 0 0
2 20 0.268
3 40 0.599
4 60 0.984
5 80 1.394

1.6
1.4
Absorbance (A)

1.2 f(x) = 0.01752 x − 0.0518000000000001

1 R² = 0.993276984609809
0.8
0.6
0.4
0.2
0
0 10 20 30 40 50 60 70 80 90

Concentration (ppm)

Figure 3: Calibration curve for red dye solution

Table 2: Results for red dye solution

Yellow dye
Sample # Concentration (ppm) Absorbance 426 nm
1 0 0
2 1 0.077
3 2 0.041
4 3 0.029

0.09
0.08
Absorbance (A)

0.07
0.06
0.05
0.04 f(x) = 0.0051 x + 0.0291
0.03 R² = 0.0426568265682658
0.02
0.01
0
0 0.5 1 1.5 2 2.5 3 3.5

Concentration (ppm)

Figure 4: Calibration curve for yellow dye solution

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 Table 3: Results for green dye solution

green dye
Sample # Concentration (ppm) Absorbance434 nm
1 0 0
2 1 0.232
3 2 0.31
4 3 0.58
5 4 0.779

0.9
0.8
Absorbance (A)

0.7 f(x) = 0.1906 x − 0.00100000000000006

0.6 R² = 0.980774547198092
0.5
0.4
0.3
0.2
0.1
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Concentration (ppm)

Figure 5: Calibration curve for green dye solution

Unknown:

Red dye
Sample # Concentration (ppm) Absorbance 508 nm
1 0 0
2 20 0.268
3 x 0.552
4 40 0.599
5 y 0.761
6 60 0.984
7 80 1.394

0.599−0.552 40−x
=
0.599−0.268 40−20

X = 37.16 ppm

0.984−0.761 60− y
=
0.984−0.599 60−40

Y = 48.415 ppm

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Discussion

1. Comment on the results

The purpose of this experiment was to provide laboratory students practice using the
spectrophotometer. The regular light scattering and absorption patterns of chemical structures
are exploited by this apparatus. In more detail, it finds substances that block light at specific
wavelengths and generates a number that represents the absorption. Each substance absorbs
and scatters this light differently at particular wavelengths. Spectrophotometry can be used to
detect unknown chemicals and calculate the concentration of a material.
The relationship of four dye (Red, Green and Yellow) and founded max wavelength happen
when dye is red(508nm). Also, when concentration unknown take absorbance value
and with curve drown before by Appling Lambert-Beer can found concentration. It's
very important to know when the connection decreases, the light passes quickly and
affects the absorbance. The absorbance for the same material constant and here when
drowning graph by using low absorbance is constant. When concentration of dye
decrease absorbance decrease.

2. What is the principle of ultraviolet-visible (UV-Vis) spectroscopy and how does it

work?

UV-Visible Spectroscopy is a technique that uses the absorption of ultraviolet or visible light
by chemical compounds to create distinct spectra. This process involves the interaction
between light and matter, where matter undergoes excitation and de-excitation, resulting in
the formation of a spectrum. When matter absorbs ultraviolet radiation, its electrons undergo
excitation, transitioning from a ground state with low energy to an excited state with high
energy, always equal to the amount of radiation absorbed. (1)

Regardless of the exact design used in a UV-vis spectrophotometer, the spectroscopy

procedure typically goes as follows:

1. A blank sample is analyzed in a spectrophotometer to calibrate the device for accurate

reading, containing only the solvent for the actual sample solution preparation.

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2. The prepared solution is then placed in the cuvette, which is typically made from fused
quartz in UV-vis spectrophotometers. The reason for this is that quartz has a high absorbance
cutoff of >380nm and won’t absorb the UV wavelengths, unlike glass or plastic.

3. The analysis procedure begins with the sample being placed, and a light source sends a
beam through the wavelength selector and the sample. In UV-vis spectroscopy, the light
source is typically a single xenon lamp capable of both UV and visible light wavelengths.
Some UV-vis spectrophotometers use a dual light source solution.

4. The photodetector absorbs light from a sample, interpreting it and displaying it in an

absorption spectrum graph. This graph maps the amount of light absorbed by the sample at
specific wavelengths, providing a comprehensive analysis report. (2)

3. What is the Beer-Lambert Law?

Beer's Law may be written simply as:

A = ε b c = log10 (I0/I)
Where: A is absorbance (no units), ε is the molar absorptivity with units of L mol-1 cm-1, b is
the path length of the sample, usually expressed in cm, c is the concentration of the
compound in solution, expressed in mol L-1, I0: incident light, I: transmitted light (3)

4. What is the Absorbance Spectrum?

Absorbance spectroscopy is a spectrometer technique that measures the intensity of light

absorbed by a sample based on wavelength. This information can reveal the electronic
structure of an atom or molecule, sample concentration, phase changes, or composition
changes. The technique outputs the intensity of detected light within the visible light region,
allowing for the calculation of absorbance at different wavelengths. UV-Vis absorbance
spectroscopy is particularly useful for characterizing materials with energy transitions
between 1-4 eV, such as chemical dyes, chromophores, conjugated organic materials, solar
cells, and OLEDs. If a significant absorbance peak is present, it can also measure the
concentration of a specific material.

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Useful ways which you can use absorbance spectroscopy:

 To determine the glass transition temperature of polymer films.

 To track reactions in your solution using universal indicators.
 To test electrochromic materials (which change colour depending on the electrical
stimulus).
 To measuring the size of certain nanoparticles.
 Determine Solution Concentration and Optical Density. (3)

5. How can you create a calibration curve for a specific solution?

To construct the calibration curve, use a computer program such as Excel to plot the data as
signal vs. concentration. Use the standard deviation of the repeated measurements for each
data point to make error bars. Remove portions of the curve that are non-linear, then perform
a linear regression and determine the best-fit line. (4)

Conclusion

the greater the absorbance the greater concentration .The absorption was noticeably affected
by the color change, and it looked that the red and green dye had absorbed more light than the
yellow ones.

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References

1. Principle of UV-Visible Spectroscopy. Byjus. [Online] October 19, 2023.

https://n9.cl/4v2k99.
2. What Is UV-Vis Spectroscopy. tec5 USA. [Online] October 19, 2023. https://n9.cl/twg01.
3. Helmenstine, Ph.D., Anne Marie. Beer's Law Definition and Equation. ThoughtCo.
[Online] February 11, 2020. [Cited: October 19, 2023.] https://n9.cl/z5iyl.
4. [Online] https://chemistry.as.virginia.edu/.

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Appendices

Raw data sheet

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