GBE 111

BIOLOGY I LABORATORY

EXPERIMENT 5
(Spectrophotometric Analysis)

Submitted by: Türkiz Sundu

Course Instructor: Assist. Prof. Dr. Hüseyin Çimen

Submitted to:
T.A. İlayda Set
T.A. Buğra Onat
T.A. Seda Karakaş
T.A. Neslihan Karakaya

Experiment Date: 26.10.22

Submission Date: 30.10.22

Yeditepe University
Genetics and Bioengineering Department
Istanbul
1. AIM
The aim of the experiment is determined the absorbed amount of light of the different
concentrations of solutions, determination of the concentration of the unknown solutions
by using of Beer and Lambert law and the determination of maximum wavelength for
KMnO4. Preparation of calibration curve and absorption curve plots.

2. THEORY
Spectroscopy can be defined as study of the interactions between light and matter or
examination of matter by used of electromagnetic radiation. Each matter has different
features to reflect, absorb, reflect the electromagnetic radiation in ways that are specific to
them and this differentiation can be defines as what kind of covalent bonds are existed in
matter and every matter has a different maximum wavelength rate such as DNA’s 260 λ
and protein’s maximum wavelength is 280 λ. These properties are used by spectroscopy to
determine detect and characterize composition of the sample [1]. In addition,
spectrophotometry is a technique which is used for examine the optical properties with the
wavelengths by used of absorbed or transmitted ultraviolet and visible light spectrums
reflection or transmission properties and for the genetic and bioengineers there are some
applications which is used by spectrophotometry [5]. These applications are protein
estimation, DNA/RNA estimation, protein estimation, enzymatic assay, measuring the
concentration, ELISA, determination numbers of bacteria in a sample. Protein
estimation/assay is accurate determination of the concentration (UV/VIS) of protein by
used of spectrophotometer. Determination of DNA/RNA purified concentration is made
by used of UV spectroscopy, it is made by nucleic acids placed into UV
spectrophotometer. Enzymatic assays are defined as measure the enzymatic activity, by
used of enzymatic spectrophotometric assay technique course of the enzymatic reactions
can be determined by a monitor a change in how much light the assay solution absorbs
[3,4].

Spectrophotometer is an instrument which is used for analysing the absorption or

transmission of the sample by used of electromagnetic radiation, UV, visible spectrum in
order to measure the concentration of the solutions or determination of the wavelength
maximums for the organic compounds. Spectrometers can be separated into different
groups according to their light sources such as single beam photometer, double beam
spectrophotometer, UV-spectrophotometer, visible spectrophotometer, fluorescence
spectrophotometer. In order to measurements are made with spectrophotometer there are
some requirements such as cuvette and blank. To explain, cuvette is an apparatus used for
placing the sample into the spectrophotometer. In addition, blank is needed when the
spectrometer is used, as a blank everything can be used except significance analyte, after
the blank’s and the other concentrations wavelengths are determined, calculations and the
graphs can be prepared. The basic principle of spectrometer can be explained by steps.
Firstly, the sample is placed into cuvette and the cuvette is placed into the
spectrophotometer, the light beams are sent into sample, then the light is absorbed and
emitted to the detector. The detector is determined the accurate wavelengths and the
results are shown on computer monitor, by the obtained wavelengths specimen’s
concentrations can determined or the substances can be identified [2].

Beer and Lambert law can be defined as every matter absorb light and emitted, emitted
lights caused to objects appears coloured and the absorbance relationship between
transmission was formulated by beer and lambert law. Formula equation is A = E x b x c.
A is defined as absorbance, E is defined as molar absorptivity, b is defined as length of
path, c is defined as molar concentration of observing compound. By using this equation
concentration can be determined [2].

Spectrophotometer is used for determination of samples wavelengths.

Figure1. Typical spectrophotometer device with the components [2].

3. METERIALS
1. Cuvette
2. Distilled water
3. Falcon tube
4. Micropipette and tips
5. Spectrophotometer
6. Ethanol
7. 4x10-5 M KMnO4 solution

4. METHODS
1. Cuvettes were cleaned with ethanol.
2. Cuvettes were numbered from 1 to 5 besides unknown and blank cuvettes were
labelled.
3. From the KMnO4 falcon tube 400 µl and from the distilled water falcon tube 600 µl
were transferred to the first cuvette.
4. From the KMnO4 falcon tube 500 µl and from the distilled water falcon tube 500 µl
were transferred to the second cuvette.
5. From the KMnO4 falcon tube 600 µl and from the distilled water falcon tube 400 µl
were transferred to the third cuvette.
6. From the KMnO4 falcon tube 700 µl and from the distilled water falcon tube 300 µl
were transferred to the fourth cuvette.
7. From the KMnO4 falcon tube 800 µl and from the distilled water falcon tube 200 µl
were transferred to the fifth cuvette.
8. Blank was filled 1 mL with distilled water.
9. The unknown cuvette was filled randomly concentration from distilled water and
KMnO4 solution.
10. KMnO4 solution was used selection of wavelength for analysis, absorbances
measurements was made with 20 nm spaced intervals over the 400–600 nm
wavelength range and the peak value was as a 520nm determined.
11. Absorbed amount of light was determined for the blank cuvette with
spectrophotometer.
8. Absorbed amount of light was determined for the first cuvette with spectrophotometer.
9. Absorbed amount of light was determined for the second cuvette with
spectrophotometer.
10. Absorbed amount of light was determined for the third cuvette with
spectrophotometer.
11. Absorbed amount of light was determined for the fourth cuvette with
spectrophotometer.
12. Absorbed amount of light was determined for the fifth cuvette with spectrophotometer.
13. Concentration-absorbance and maximum absorbance plots were made from data.
14. Unknown solution’s concentration was determined by data from plot.

5. RESULTS
In experiment 5 mL 4 x 10 -5 molar KMnO4 was used, in order to solution was prepared
3.1606x10-7 gram KMnO4 was used. The calculation was made this formula; (Mw=158.03
g/mol)
Molarity of KMnO4 solution (n/L) = mole of KMnO4 (n) / volume of KMnO4 (L)
Mole of KMnO4 (n) = mole mass of KMnO4 (g) / used mass of KMnO4 (g)
4x10-5 M = 2x10-5 mole / 0.5 L
2x10-5 mole = 158.03 g / 3.1606x10-7 g

This graph is used for to determination of the wavelength maximum for KMnO4.

Absorbition Curve for KMnO4

0.25
0.2
Absorbition

0.15
0.1
0.05
0
350 400 450 500 550 600

Wavelength

Graph1. Absorption curve plot for KMnO4.

This graph is used for to solve E x b result by slope of the plot and determination of
unknown solution concentration can be calculated.
 KMnO4 Calibration Curve
0.07
0.06

Absorbition
0.05 f(x) = 0.096 x − 0.018
0.04 R² = 0.937347436940602
0.03
0.02
0.01
0
0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85

Concentration

Graph2. KMnO4 calibration curve plot

Plot of the KMnO4 concentration-absorbance curve was used to calculate E (molar
absorptivity) x b (length of path) then the results was determined by slope of the plot and
slope of the plot was determined 0.096.

Preparation of the calibration curve is used for the determination of the unknown solution
concentration, unknown solution concentration was determined 0.302, however the actual
value was 0.3, by used of formula percentage error was calculated 0.66%.

% error = (| experimental value - accepted value | / accepted value) x 100

6. DISCUSSION
Blank can be defined as just a sample of the solvent and blank consist except of analyte.
To explain, the spectrophotometer can zero out these background readings and only
provide values for the chemical of interest, the solution to be tested should be devoid of
the one compound we are looking for so ingredients of the blank can be changed by which
chemicals are used to prepare the concentrations. For example, in KMnO 4 experiment as a
blank water was used because KMnO4 was diluted with water, then the wavelength value
of the blank was determined and after the all concentrations wavelengths were measured,
blank was used to calculations of the concentrations absorptions by subtracting the blank
value from the wavelength.

After the absorptions were determined max absorbances plot and calibration curve was
prepared. To analyse calibration curve, the results were shown linear on prepared plot. On
plot a standard curve is seen, standard curve can be likened to ruler, so it can be provided
to find accurate determination of the concentration. On calibration curve plot, the results
are deviated for the 0.6 and 0.7 concentrations from the trendline. However, these
concentrations are still close enough to the trendline which means that accuracy of the is
high. To analyse, the absorption curve maximum value for the wavelength is 520 λ and
this wavelength value is shown on 0.203 absorption value. Until reach the value of 520 λ,
the graph is increasing and after the maximum value the graph is decreasing, which is
shown on graph.1. The maximum value determination was need to measure the
concentrations absorption value. R2 can be defined as how closely the trendline approaches
the real data in the context of analysis is known as fit, so the accuracy rate can be shown
with R2 and on the calibration curve which is shown on graph.2. the value of R 2 is 0.9373,
so it is shown the plot was prepared close to fittest one and the equation of the plot was
given after the data are placed and the equation is y=0.096x - 0.018 so by this equation
slope of the plot is shown well the slope is 0.096, E (molar absorptivity) x b (length of
path) result is given by the slope of the plot.

After the calibration curve plot was prepared, the equation is given by the graph, by using
the equation, unknown solutions concentration was determined. Actual concentration of
the unknown solution was 0.3, however by using the equation for the unknown solution
concentration was calculated 0.302. After the calculation percentage error of the unknown
solutions concentration was calculated and it is resulted 0.66%. Percentage error was
related to equation and the prepared graph. The graph was close to the fittest one. That is
why percentage error was resulted 0.66%.

To sum up, after the concentrations and blanks wavelengths are determined, calibration
curve and the absorption curve was prepared, then the resulted plots are interpreted.
Unknown solutions concentration was determined by the equation and then the percentage
error was calculated. Then, by used of slope of the calibration curve plot, E x b was
calculated.
7. REFRENCES
1. Ball, D. W. (2001). The basics of spectroscopy (Vol. 49). Spie press.
2. Morris, R. (2015). Spectrophotometry. Current Protocols Essential Laboratory
Techniques, 11(1), 2-1.
3. Stuart, B. H. (2006). Infrared spectroscopy of biological applications. Encyclopedia of
Analytical Chemistry: Applications, Theory and Instrumentation.
4. Nilapwar, S. M., Nardelli, M., Westerhoff, H. V., & Verma, M. (2011). Absorption
spectroscopy. In Methods in enzymology (Vol. 500, pp. 59-75). Academic Press.
5. Larsson, J., & Malmqvist, E. Absorption spectroscopy.