UV/VISIBLE SPECTROSCOPY:

DEMONSTRATING THE USE OF BEERS LAW IN UV/VISIBLE

SPECTROSCOPY
By
Lungelo Mkhwanazi (21103379)
Abstract
The purpose of this experiment was to demonstrate the use of Beer’s law in UV /visible
spectroscopy. To perform the experiment a ……… Dual beam UV/ visible spectrophotometer
operating at a fixed wave length of 392nm and a pair of 1cm quartz absorption cells was used.
A 500mL stock solution OF 0.35M nickel nitrate hexahydrate at 0.35M concentration this
solution was referred to as the stock solution. The stock solution was used to prepare five
100mL Ni(NO3)2∙6H2O Standard solutions of different concentrations, the lastly 100mL sample
X was prepared using 45Ml of the stock solution mixed with de-ionized water. The
spectrophotometer was used to measure the absorbance of the standard solution Samples.
The graphical interpretation of the concentration and absorbance was used to determine the
concetration of sample x
INTRODUCTION

Ultraviolet-Visible (UV-Vis) spectroscopy is concerned with measurement of the interaction of

outer shell electrons with electromagnetic radiation in the range 190 nm to 700 nm. Electrons of
organic compounds with a high degree of conjugation, transition metal ions and charge transfer
complexes are active in this part of the spectral region. Compounds with the ability to interact
with or absorb UV-Vis radiation are given the general name chromophores .This experiment
was concerned with chemistry and analytical science and introduces the concepts of qualitative
and quantitative analysis through use of UV-Vis spectroscopy.

Qualitative Analysis is defined as analysis in which substances are identified or classified on the
basis of their chemical or physical properties, such as chemical reactivity, solubility, and in this
experiment, UV Vis absorption characteristics. Quantitative Analysis, on the other hand, refers
to analysis in which the amount or concentration of an analyte is determined. This experiment
will demonstrate that Quantitative Analysis can also be performed by using UV Vis
spectroscopy.

UV- Vis spectroscopy involves measurement of the fraction of incident electromagnetic radiation
that is either absorbed or transmitted by a sample. The measurement can be performed at a
single wavelength or across a broad range of wavelengths when a spectrum. is produced.
Compounds can then be characterized qualitatively and identified by comparing the absorption
spectrum produced with the spectra of known compounds.

The basis of UV- Vis spectroscopy is shown in Figure below:

Figure 1: Basis of a uv vis measurement

This process can be described by The Beer Lambert Law which relates the incident light intensity (Io) at
the front of a cell of length, L, (in centimetres) of a single absorbing species of concentration, c, to that
which is transmitted by the sample [i.e., the intensity of light (I) which emerges from the cell].  is known
as the molar extinction coefficient and is a measure of the probability of absorption of a photon1 of light
by a compound.
The Beer Lambert law is expressed formally in equation:

log 10 (Io/I) =- log 10 (% T/100%) =  c L = A Equation 1.

Another simplified equation of Beer–Lambert law relates the absorption of light by a solution to
the properties of the solution according to the following equation: 

A = εbc Equation 2

where ε is the molar absorptivity of the absorbing species, b is the path length, and c is the
concentration of the absorbing species. As expressed in the equation above this means that the
absorption of light by a solution is directly proportional to the concentration of the solution. The
experiment looked at investigating and proving the correlation between absorbance and
concentration of a solution stated by beers Lambert law. This study is important because several
industries use uv spectrophotometers in quality analysis and instruments like this help processes
function more efficiently with accurate well proven study’s that prove that the results the
machine produces are accurate.
Materials and methods

Material used

 ……….. Dual beam UV/visible spectrophotometer with a fixed wavelength of 392nm

 One pair of matched 1cm quartz absorbtion cells
 500ml Volumetric flask
 100 ml volumetric flask’s
 Pippete
 Ni(NO3)2∙6H2O salt
 De-ionized water

Method

 A 500 ml of 0.35M nickel nitrate hexahydrate stock solution was prepared by mixing
50.89g of Ni(NO3)2∙6H2O salt with de-ionized water in a 500ml volumetric flask.
 A 100 mL of 0.035 M, 0.070 M, 0.140 M, 0.175 M and 0.260 M Ni(NO3)2 ∙
6H2O standard solutions were prepared from the 0.35 M stock solution.
 45 mL of the 0.35 M nickel nitrate hexahydrate stock solution was transfered into a 100
mL volumetric flask and made up to mark with de-ionized water. This sample was than
labelled as Sample X.
 The instrument parameters were set according to the operating manual
 To zero the spectrophotometer de-ionized water was measured.
 All the standard samples were measured for abasorbance
 Sample X absorbance was measured.
Results and discussion

Sample prep calculations

Mass of Nickel nitrate enhydroxide salt required to prepare 500 ml stock solution.

m
c=
Mr∗V

m=C × M × V

¿ ( 0.35 ) ( 290.79 ) ( 0.5 )

¿ 50.89 g∋( NO3 ) 6 H 2 O ¿

Sample calculation of the volume of stock solution used to prepare the standard solutions:

0.035
C i V i=C f V f ∴ V 1= ×100=10 ml
0.35

∴ 10 ml of stock solution was used to produced 100 ml of 0.035 M ¿(NO 3 )6 H 2 O¿ .

Table of stock solution used to prep the standard solutions at the required concentrations

stock solution
volume calculated Concentration of a 100ml
Sample (mL) standard solution (M)
1 10 0.035
2 20 0.07
3 40 0.14
4 50 0.175
5 75 0.26
Results
Table of absorbance and concentrations measured

Concentration of a 100ml
Sample ID standard solution (M) Absorbance
STD1 0.035 0.178
STD2 0.07 0.353
STD3 0.14 0.701
STD4 0.175 0.89
STD5 0.26 1.335

Sample X had a measured absorbance of 0.800.

Graphical representation of results

Concentration vs Absorbance graph

1.6
1.4 1.335

1.2
1 0.89
Absorbance

0.8 0.701

0.6
0.353
0.4
0.178
0.2
0
0 0.05 0.1 0.15 0.2 0.25 0.3
concentration of nickel nitrate hexahydrate

Using the calibration curve the concentration of ¿( NO 3 )6 H 2 O¿ in sample X from calibration

curve was 0.157 M.
4.4 Equation for the relationship between absorbance and concentration.

x=nickel concentration
y=nickel absorbance
y=5.381 x−0.00 8
4.5 Concentration of nickel in the sample using the equation
0.847+0.008
x= =0.159 M
5.381
4.6 Calculation of nickel (ii) in sample X using beer’s Law

Caverage¿ 0.136M
Absaverage¿ 0.64424

A=εbc
0.64424
ε= =¿4.74L.mol-1.cm-1
0.136 ×1 cm

0.847
c= =0.159 M
5.331 ×1 cm

Discussion
By Using the results from the experiment to plot a absorbance vs concentration graph the
concentration of sample X was extrapolated and found to be 0.157 M. The results proved that
absorbance of a solution was directly proportional to the concentration of the solution , this is
evident in the linear correlation of concentration and absorbance where the absorbance of the
solution increased with the concentration of the solution.

Conclusion
Beers law states that absorbance is proportional to the path length through the sample and the
concentration of the absorbing species , from the study performed it was found that this is true
as the result from the study replicated this law by means of a linear graph. From the results we
can also deduce that there was a high potential for error during the preparation of the samples
and the errors would have been visible when the sample absorbance was being measured and
the absorbance vs concentration point did not form a straight line but in the study this was not
the case as it was possible to extract the sample X concentration graphically. In industries the
spectrophotometer has the ability to give accurate results fast this helps processes function
faster and better with trust worthy quality results.
References
Layout of tables and figures