Mateusz Zatoński Gr.17
Influance of concentration of a solution on its absorbance.
Introduction:
As a Medical student I am an inquisitive person and seek answers to how scientists get the
knowledge about which we then learn during lectures. By performing experiments like this I
get to have a deeper understanding of what is the human body composed of.
Research question:
To what extent does the increase in concentration (0.25%, 0.5%, 1%, 2%) of methylene blue
solutions have an influence on the absorbance of the solution [±0.001Au].
Background information:
Spectrophotometry is a quantitative measurement technique that allows scientists to investigate
the optical properties of materials over a wide wavelength range, from the ultraviolet to the
visible and infrared spectral regions. It involves measuring the ratio of two radiometric
quantities as a function of wavelength.
Beer’s law, also called Lambert-Beer law or Beer-Lambert law, in spectroscopy, a relation
concerning the absorption of radiant energy by an absorbing medium. Formulated by German
mathematician and chemist August Beer in 1852, it states that the absorptive capacity of a
dissolved substance is directly proportional to its concentration in a solution. The relationship
can be expressed as A = εlc where A is absorbance, ε is the molar extinction coefficient (which
depends on the nature of the chemical and the wavelength of the light used), l is the length of
the path light must travel in the solution in centimetres, and c is the concentration of a given
solution.
Hypothesis:
With the increase in concentration (0.25%, 0.5%, 1%, 2%) the absorbance of the solution
[±0.001Au ] will also increase with the highest absorbance occurring at the highest
concentration and the lowest absorbance occurring at the lowest concentration.
Variables:
Independent variable
● The concentration of the solution (0.25%, 0.5%, 1%, 2%)
Dependent variable
● absorbance of the solution [±0.001Au ]
Table 1. Controlled variables
Controlled Variables
How will they be controlled?
Why will they be controlled?
All tested samples of solution are In order to restrict any unwanted
The volume of the 1ml and are measured using a fluctuations in the results caused by the
tested sample
mechanical pipette[± 0.02ml]
inconsistency of the volume of solution
samples used all will be precisely 1ml
The same cuvette
All tested samples of solution
will
be
tested
in
the
spectrophotometer [± 0.001μm]
using the same calibration and
the solutions will be measured
starting with
the
lowest
concentration sample, thus
avoiding the need to wash the
cuvette every time.
With eliminating the amount of different
cuvvets used in the experiment posible
inaccuracies will be eliminated thus the
measurements can be more reliably
compeared to echother.
Equipment:
●
●
●
●
●
●
●
●
●
●
●
●
Spectrophotometer [± 0.001μm]
mechanical pipette[± 0.02ml]
spectrophotometer cuvette
Deionized water (for measurement )
Deionized water (for cleaning)
4% methylene blue solution 1ml
Paper towels
Lab coat
4 test tubes
Marker
Pen and paper
beaker
Methodology:
1.
2.
3.
4.
5.
Prepare and label with a marker 4 test tubes (1, 2, 3, 4).
Add 1 ml of distilled water to each test tube using a mechanical pipette[± 0.02ml]
Introduce 1 ml of the 4% methylene blue solution into the first test tube.
Mix the solution several times using the mechanical pipette.
Transfer 1 ml of the newly made solution from test tube 1 to test tube 2 using a
mechanical pipette[± 0.02ml]
6. Repeat the consecutive dilutions three times.
7. Set the spectrophotometer to the wavelength to 520nm.
8. calibrate the apparatus using a blank (cuvette with distilled water).
9. Start the measurement from the lowest concentration (sample no. 4), thus avoiding the
need to wash the cuvette every time.
10. Record the absorbance result after each reading by writing it down on paper.
11. Pour the contents of the cuvettes into the prepared beaker and rinse with distilled water.
Safety:
All activities performed in the lab require focus and caution, while in the laboratory students
must wear lab coats, During the experiment, it is vital to keep the workplace clean and in order.
And after the experiment is done dispose of all the liquids into the drain.
Data presentation and analysis:
Raw data:
Table 2. Absorbance levels of methylyn blue solutions of different concentrstions
Concentration Absorbance [±0.001Au ]
2%
0.395
1%
0.155
0.50%
0.071
0.25%
0.030
Results and analysis:
Graph 1. Absorbance levels of methylyn blue solutions of different concentrstions
As one can observe both from the graph and from the equation of the best fit line there is a
strong positive correlation between the concentration of the methylene blue solution and the
level ofabsorbance of the solution [±0.001Au]. with the highest absorbance of 0.395Au
occurring at the highest concentration of 2% and the lowest absorbance 0.030Au occurring at
the lowest concentration of 0.25%.
Conclusions:
To what extent does the increase in concentration (0.25%, 0.5%, 1%, 2%) of methylene blue
solutions have an influence on the absorbance of the solution [±0.001Au].
The experiment depicts that there is a strong positive correlation between the concentration of
methylenel blue solution and concentration increase (0.25%, 0.5%, 1%, 2%). Which proves the
hypothesis corect and is further backed by the fact that absorbance is often used in gauging the
purity and concentration of molecules (for example proteins) in a solution, which may be
essential in comparative experiments of different protein.
Expansion;
- More types of solutions, for example, Phenol red, methyl orange.
- More concentrations
- More trials
Bibliography:
1. Cooksey,
Catherine
et
al.
"Spectrophotometry".
NIST,
2022,
https://www.nist.gov/programs-projects/spectrophotometry.
2. 2. Wójcik, Dr n. biol. Aneta et al. Skrypt Do Ćwiczeń Z Biochemii. ŚLĄSKI
UNIWERSYTET MEDYCZNY W KATOWICACH.
3. 3. Rafferty, John P.. "Beer’s law". Encyclopedia Britannica, 20 Nov. 2019,
https://www.britannica.com/science/Beers-law. Accessed 24 March 2022.