Report

UV Visible Spectrophotometry

Lab partners: Amil Ahamdov, Muhammad Ismayilov, Sarkhan Valiyev, Nurali

Nuralizade, Aykhan Abdurrahmanli, Emil Asadullayev
Group: 3
Date performed:01/01/2023
Date submitted:03/01/2023

French-Azerbaijani University
 Table of Contents
Purpose 3

Introduction 4

Experimental Procedure 5

Results and Discussion 6

Conclusion 7

Bibliography 7
 Introduction
Electronic Spectroscopy spectroscopic is a method used to determine, characterize, and
quantify a broad range of molecular compounds. Spectrophotometry is a branch of electronic
spectroscopy concerned with the quantitative measurement of the reflection or transmission
properties of a material as a function of wavelength[1]. UV-Vis spectrophotometer is an
instrument used to measure light absorbance across the ultraviolet and visible ranges of the
electromagnetic spectrum. The aim of this practical work is to determine molar extinction
coefficient of fuchsin solution with different concentrations using UV-Vis spectrophotometer,
and estimate concentration of unknown sample by using previously obtained results.

For this experiment, five different solutions with concentrations of 1μM, 2μM, 4μM, 6μM,12μM are
prepared. Each has its own unique abundance values, as concentrations increase, abundance values also
increase due to direct proportionality between them.
 Principles
The Beer–Lambert law states that the absorbance of a solution is directly proportional to the
concentration of the absorbing species in the solution and the path length[2]. Therefore a
UV-Visible spectrophotometer can be used to measure the amount of light transmitted through a
sample by ratioing the intensity of the incident light to the intensity of the transmitted light(See
Pic. 1). To perform these calculations, it is required to determine the rate of absorbance changes
with change of concentration. The rate is determined from a calibration curve.

Picture 1

Operating principle of UV-Vis Spectrophotometer

UV-Vis spectrophotometers use UV/visible wavelength

range to light a sample and subsequently measure the intensity of absorbed, transmitted, or
reflected light after passing through a sample.

The main components of a spectrophotometer are:

– A light source that generates a broadband of electromagnetic radiation
across the UV-visible spectrum
– A wavelength selector separates the broadband radiation into wavelengths
– A sample area, where the light passes through or reflects off a sample
– One or more detectors to measure the intensity of the reflected or
transmitted radiation.
Samples for UV/Vis spectrophotometry are often liquids. Samples are typically placed in a
transparent cell, known as a cuvette. Most cuvettes are made of quartz glass or fused silica.
Glass and plastic cuvettes are also common, although glass and most plastics absorb in the
UV, which limits their usefulness to visible wavelengths[3]. After being placed in the
spectrophotometer, the molecules of a sample are lighted by a light source. Absorption of
UV-vis light excites molecules that are in ground-states to their excited-states[4].

A detector is used to convert the light that has passed through the sample, into an electronic
signal which is then sent to a computer. Generally, detectors are based on photoelectric
coatings or semiconductors. The most common detector used in UV‑Vis spectroscopy is
photomultiplier tube (PMT)[5][6].

Procedure
1) Firstly, five solutions with 1μM, 2μM, 4μM, 6μM,12μM were prepared by adding
4.17ml, 8.34 ml, 16.68ml, 25ml, 50ml fuchsin in each solution accordingly. Then ethanol
is poured until each of them has 50 ml volume.

2) When the solutions are ready, they are placed in a UV visible spectrophotometer by
using quartz cuvette for measuring absorbance of each of them. Before placing solutions,
cuvette filled with distilled water as a reference sample. Then Least concentrated
solution is put first in cuvette, otherwise high concentrated solution will have effect on
lower concentrated solution and give wrong absorbance values which will lead higher
errors

3) After obtaining absorbance values of mixtures at 542 nm wavelength, the dependence

curve is plotted between Absorbance and Concentration to determine molar extinction
coefficient which is 68723 using Excel.

4) After that using this molar extinction coefficient value concentration of unknown sample
is measured as 2.13μM.
 Results and Discussion

Sample N Concentration(μM) Abundance molar extinction

coefficient(L/mol*cm)

1 1 0.064 68723

2 2 0.129 68723

3 4 0.219 68723

4 6 0.422 68723

5 12 0.809 68723

So final equation obtained from chart given above is:

f(C) = 68723*C*l ± 0.015

For given five solutions extinction molar coefficient is: ε=68723 L/mol*cm
Absorbance value of unknown sample measured as 0.144 from which concentration
value can be obtained which is 2.13μM

Error = (68723/50000)*100% = 137%

 Conclusion
Electronic Spectroscopy spectroscopic is a method used to determine, characterize, and
quantify a broad range of molecular compounds. UV-Visible spectrophotometer is used in this
experiment to observe absorbance values of compounds in different wavelengths. In our case
545 nm equivalent absorbance values are needed.
In general, molar extinction coefficients can be estimated for solutions over a wide range of
concentrations from 1μM to 12μM and by using obtained molar extinction coefficient value
concentration of unknown samples can be calculated. Reliability of molar extinction
coefficient value is influenced by the number of solutions. As the number of solutions
increases, estimated molar extinction coefficient also become more accurate. Results from the
error shows that the experimental value of molar extinction coefficient is 37% higher than the
theoretical value which is due to mistakes while creating solutions. So in theory concentration
of unknown sample have to be approximately 37% higher than expected.

Bibliography
1. Allen, DW; Cooksey, C; Tsai, BK (Nov 13, 2009). "Spectrophotometry". NIST. Retrieved Dec 23,
2018.
2. Metha, Akul (22 April 2012). "Derivation of Beer–Lambert Law".
3. Skoog, Douglas A.; Holler, F. James; Crouch, Stanley R. (2007). Principles of Instrumental Analysis
(6th ed.). Belmont, CA: Thomson Brooks/Cole. pp. 169–173
4. Ninfa AJ, Ballou DP (2004). Fundamental laboratory approaches for biochemistry and
biotechnology. Hoboken: Wiley. p. 66.
5. Link: https://micro.magnet.fsu.edu/primer/digitalimaging/concepts/photomultipliers.html
6. Marcello P. , Maurizio A.,Vitorino T.(Nov 14, 2018) “UV-Vis spectroscopy”.