Name: Anastasija Anastasovska

Student number:113
Lab group: a2
Date: 8.11.2022

2nd laboratory exercise:

Spectrometry

Introduction
The aim of this laboratory was to:
  Learn to determine the absorption spectrum of an unknown concentration of KMnO4
solution,determine the λmax and construct an absorption spectrum curve.
 Construct a standard curve with the known concentration of KMnO4 solutions and determine the
unknown concentration of KMnO4 using the previously constructed standard curve.
In order to conduct this lab, we used a machine called the spectrometer. The spectrometer measures the
transmittance and absorption of the sample. In the first experiment we used the spectrometer to determine
the λmax (wavelength where the solution has its maximum absorbance) of the unknown concentration of
KMnO4 solution. We did this by testing the absorption spectrum of the x mM KMnO4 solution for a
wavelength range of 440-600 nm. The results that we used to create the absorption spectrum curve
showed us the radiation observed by the solution at different wavelengths.
In the second experiment we constructed a standard curve for the known concentration of the KMnO4 in
order to find out the concentration of the unknown concentration of KMnO4 solution. For this we used
λmax from the first experiment. By doing this we determined whether our substance follows the Lambert-
Beer law which states that the concentration of the sample and path length is directly proportional to the
absorbance of the light.

log Io/Ι = k  C  ℓ

Methodology – experiment 1
Step 1: we used 2 cuvettes. We filled one with 5ml distilled water and another with 5 ml of unknown
concentration of KMnO4 (x mM KMnO4). We used the distilled water as a blank.
Step 2: we set the spectrometer at the length of 440 nm, and we put the blank inside the spectrometer in
order to adjust the absorbance to 0.
Step 3: we put our second cuvette with the solution in the spectrometer and wrote down the results which
the spectrometer showed us for the absorbance.
Step 4: we repeated the previous 2 steps with a wavelength range from 440 nm-600 nm.
Step 5: we constructed the absorbance spectrum and got our result for λmax

Results- experiment 1

Wavelength-λ(nm) Absorption- OD
440 0.165
460 0.149
480 0.223
500 0.396
520 0.501
540 0.416
560 0.280
580 0.142
600 0.054
Table 1- Absorption spectrum of aqueous x mM KMnO4
From table 1 we concluded that λmax is at 520 nm, because it has the highest absorbance of optical density.

Absorption spectrum of x mM KMnO4

0.6

0.5

0.4

0.3 OD
OD

0.2

0.1

0
440 460 480 500 520 540 560 580 600 620
wavelenght (nm)

This is how the absorption graph looked once I constructed in on Excel.

The x axis is the wavelength λ(nm) and the y axis is the absorption OD.
From the graph we can see that as the wavelength increases the OD increases to a certain point (reaching
its maximum) and then decreases.

Methodology- experiment 2
Step 1: we prepared 6 different test tubes each filled with different volumes of H2O and 1 mM KMnO4.
Step 2: using the spectrometer we observed the absorbance of the different solutions at the wavelength of
λmax which we determined already from the previous experiment. We used the first tube as a blank
(because it only contained distilled water)
Step 3: we calculated the known concentration of KMnO4 after dilution, and then constructed the
standard curve for KMnO4 on Excel.
Step 4: we used the standard curve in order to calculate the unknown concentration of KMnO4 from the
first experiment.
Step 5: we calculated the εmM of KMnO4 at λmax.

Results- experiment 2
Using the law of dilution, we calculated the concentration of test tubes 2-6. (We didn’t calculate the
concentration of test tube number 1 because we used it as a blank)
C1 x V1 = C2 X V2
Test tube 2: 1mM x 0,6= C2 x 6
C2=0.1 mM KMnO4
 Test tube 3: 1mM x 0.9= C2 x 6
C2=0.15 mM KMnO4
Test tube 4: 1mM x 1.2= C2 x 6
C2=0.2 mM KMnO4
Test tube 5: 1mM x 1.5 = C2 x 6
C2=0.25 mM KMnO4
Test tube 6: 1mM x 1.8= C2 x 6
C2=0.3 mM KMnO4

Test tube Concentration KMnO4 mM OD

1 0 0
2 0.1 0.163
3 0.15 0.307
4 0.2 0.343
5 0.25 0.5
6 0.3 0.573
Table 2 Absorbance of known concentrations of KMnO4 at λmax

standard curve
0.7

0.6
f(x) = 1.946 x − 0.01
0.5 R² = 0.986337495721026
0.4
OD

0.3

0.2

0.1

0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35
mM concentration of KMnO4

This is how the standard curve looked like after I constructed it in excel.
The x axis is the values of mM concentration of KMnO4, and the y axis is the values of OD.
R² = 0.9863 represents how close our values were in comparison to the standard curve line.
In order to find the unknown concentration of KMnO4 we used the standard curve and the equation y =
1.946x - 0.01. This equation was automatically made by excel if we want to calculate the concentration
value for other OD values. It comes from the equation y=mx+c
 y = 1.946x - 0.01
where y is the absorbance value and x is concentration that we need to calculate. In order to do this we
use the absorption value from table 1 (absorption value of λmax) 0.501.
y +0.01 0.501+ 0.01
x= → x= → 0.26 mM
1.946 1.946
The concentration of the unknown x KMnO4 is 0.26 mM
We calculated the εmM of KMnO4 at λmax using the Lambert-Beer law.
OD=0.501
C=0.26
I= 1cm
λmax = 520nm

OD
OD=K x C x I → εmM =
CxI
0.501
→ =1.92 M-1cm-1
0.26 x 1 cm

Discussion- experiment 1 & 2

Conducting the first experiment was not a problem, the instructions were easy to follow, the data was
collected efficiently, and the construction of the absorption curve was made with no difficulty. However,
I faced some difficulty during the second experiment. The problem occurred during the dilution of the
concentration of 1mM of KMnO4 with water. When I measured how much solution of KMnO4 to put in
the test tubes I made a mistake of using a pipette that I haven’t used before and added 1 ml less of
KMnO4 in each test tube. We can see this error also in the construction of the standard curve. If we
connected all the dots on the graph, it wouldn’t be a straight line. R² = 0.9863 this value represents how
close we were to having values that would make the line straight (1 being the exact value without any
mistakes), which means that we had an error of 0.0137 for each point on the standard curve.
From the second experiment we can conclude that by connecting all points in a straight line the substance
follows the Lambert-Beer law meaning the absorbance of this substance was proportional to its
concentration.
Answers to questions

absorption spectrum of x/2 mM KMnO4 solution

0.3

0.25

0.2

0.15
OD

0.1

0.05

0
420 440 460 480 500 520 540 560 580 600 620
wave length (nm)
1.
What we know now from Beer-Lamberts law is that the absorption is prportional to the concentration of
the solution, so that means when the concentration is half compared to the original conentration the
absorption will aslo be half. This graph comfirms this hypothesis (the absorbance is half of what it was in
the first graph.)
2. Because the substancens don’t react with one another the concentration of the substances remains the
same in the solution. This means that all the wavelenghts of each substance can be absorbed. The
maximum will be at 382 nm and it will be twice as high ( because both substance A and B absorb at the
same wavelenght)
3. when we mix the same volume of 2 different substances with equal concentration and by using the law
of dilution we can conclude that the volume of the solution will increase twice the volume of the
substances individually and the concentration will decrease by half. Beer lamberts law tells us that the
concentration and the absorbance are proportional, meaning if the concentration decreases by half the
absorbance will toothe absorbance for each wavelenght will decrease by half. The only exeption is the
wavelenght of 382 nm, because both substances absorb at the same wavelenght the absorbance at 382 nm
will remain unchanged.
C1 x V1= C2 x V2
1 x 10 = C2 x 20
½=C2
4. If we want to measure absorbance in the UV range we use quartz cuvettes, this is because plastic and
glass absorb UV light and can cause interference. We usually use plastic cuvvetes for radiation
photometry of 400-700 nm.
5. PH can affect the absorption spectrum of an indicator. The absorption spectrum is changed when the
mollecular structure of the solute is changed. As we know from before changes in pH can change the
molecular structure of a substance. As the pH of a indicatro gets higher so does the absorbance.
Conclusion
Spectrometry helps us to better understand molecules and how they can behave under different
circumstances. The first experiment taught us how to construct an absorption spectrum and from that
information how to determine the maximum absorption. In the second experiment we learned how to
construct a standard reference curve and from that determine the unknown concentration of the first
substance. With this new information we know how to find the exact values of concentration, construct an
absorption curve and standard curve for different substances.