This task will explore various chemical and biological processes, and one of the main physical research methods will be spectroscopy.
Spectroscopy can vary in terms of the technique used, and its capabilities range from determining the structure of substances to the movement of galaxies. As a rule, a spectrum refers to the dependence of a physical quantity (energy, radiation intensity, absorption, etc.) on the wavelength of light.
A real beam of light is always a composite object. It consists of several elementary beams of different colors—waves with different wavelengths. A real beam can be separated into elementary beams using a prism or a diffraction grating.
For example, we can use a prism to obtain a rainbow from sunlight. If we measure the intensity of different colors in the rainbow with a sensor and plot a graph of this intensity against wavelength, we get something similar to Fig. 1(upper left).
The resulting graph is called the spectrum of solar emission radiation in the visible range.
An incandescent lamp, a fluorescent lamp, and LEDs can be used as radiation sources. Using a prism or a diffraction grating, we can obtain rainbow-like images and measure the intensity for different colors in them (see the other graphs in Fig. 1). The graphs show the intensity with which the light source emits in a given wavelength range.
Light interacts with the objects around us, and the nature of this interaction determines the visible color of the object. In this task, we will study the absorption of light by various solutions. For example, a solution of malachite green absorbs blue light (wavelength 450 nm) and red light (wavelength 600 nm), but does not absorb green light (wavelength 500 nm). Therefore, white light passing through the solution becomes green (Fig. 2).
Quantitatively, this absorption process is described by the ratio of the intensity of incident light $I_0$ to the intensity of transmitted light $I$:
$$A= \log_{10} \frac{I_0}{I}.$$
Now let's imagine that we shine a beam of fixed color (laser) into a solution of malachite green. The green laser will pass through the solution with virtually no absorption ($I_\mathrm{gr} \approx I_{0,\mathrm{gr}}$), while the blue laser will be strongly absorbed ($I_\mathrm{bl} \ll I_{0,\mathrm{bl}}$).
Therefore, to describe the absorption process, we need to specify the wavelength $\lambda$ we are working with:
$$A(\lambda) = \log_{10} \frac{I_0(\lambda)}{I(\lambda)}.$$
The dependence $A(\lambda)$ is called the absorption spectrum. Figure 2 shows the absorption spectrum of malachite green.
The absorption spectrum $A(\lambda)$ is described by the Beer-Lambert law and depends on the substance of which the object is composed, the concentration of this substance $n$, and the thickness of the sample $b$:
$$A(\lambda) = c b \cdot \varepsilon(\lambda),$$
where $\varepsilon(\lambda)$ is a coefficient unique to each substance.
If the sample consists of substances $A$ and $B$, their absorptions are added together:
$$A(\lambda) = b\left(c_A\,\varepsilon_A + c_B\,\varepsilon_B\right).$$
If any substance that absorbs green light is added to a solution of malachite green, the solution will become very dark, as it will absorb almost all visible light passing through it.
To analyze the chemical processes occurring in this task, you will need to measure the absorption spectra of copper sulfate solutions and $\rm pH$ indicators in solutions of varying acidity. You will also obtain absorption spectra of photosynthetic pigments from two algae and use them to try to explain the efficiency of photosynthesis when microorganisms are illuminated with different colors.
In this part of the task, you will perform electrolysis of an aqueous solution of $\rm{CuSO_4}$ and calculate with the charge that passed through the solution during electrolysis using various methods.
During the electrolysis process, you will observe:
By analyzing the results of these observations, you will calculate the charge that contributed to a particular effect and compare it with the total charge that flowed through the solution, measured by the direct method $Q = I\cdot t$, where $I$ is the current flowing through the solution and $t$ is the time of electrolysis.
Balance the equations for the reactions occurring in the solution at the anode
and cathode:
Assume that no other reactions occur at the anode and cathode.
Write down and balance the overall equation for the electrolysis of an aqueous solution of $\rm{CuSO_4}$.
Prepare $V_0=150$ mL of copper sulfate with a molar concentration of $c_0=0.400~\mathrm{M}$. You are given copper sulfate powder ($\rm CuSO_4 \cdot 5 H_2 O$). How many grams $m_{bs}$ of powder are needed to prepare the specified solution? It can be assumed that the volume of the resultant copper sulfate solution is the same as the volume of the added water.
We will call the resulting solution “solution A2.” Pour $5$ mL of solution A2 into Answer tube A2.
In accordance with instruction G2, perform electrolysis of $120$ mL of solution A2 for $t_0=1$ h at a current of $I=1$ A.
Record the dependence of the volume of oxygen released $V_{\rm O_2}$ on time $t$. Take at least 10 measurements. Plot the resulting dependence and draw an approximation curve.
After electrolysis, stir the solution remaining in the electrolyzer. In accordance with the G2 instructions, filter approximately 20-25 mL of the stirred solution after electrolysis.
We will refer to the filtered solution as “solution A4.” Pour 5 ml of solution A4 into Answer tube A4.
In this part, you need to determine the concentration of copper ions in solution A4 based on how much it absorbs certain light. The blue color of the solutions under investigation is entirely determined by the concentration of $\rm Cu^{2+}$ ions.
Prepare five solutions of $\rm CuSO_4$, each with a volume of 4 mL, in optical cuvettes.
Cuvette number Molar concentration $[\rm Cu^{2+}], ~M$ 1 0.0400 2 0.0350 3 0.0300 4 0.0250 5 0.0200
Note that the initial solution A2 has very strong absorption, so in this task you calculate its dilution by a factor of 10 or more.
Using the calculations made in the previous task, prepare five solutions in optical cuvettes. In accordance with instruction G1, measure the absorption spectrum of each of the five solutions.
Save the measured spectra in the “Results/B2” folder on your desktop under the names “B2.{cuvette number}.txt” (for example, “B2.3.txt”).
In an optical cuvette, prepare 4 mL of a 10-fold diluted solution of A4.
Save the measured spectrum in the “Results/B5” folder on your desktop under the name “B5.txt”.
In this part of the problem, you need to determine the $\rm pH$ in solutions A2 and A4 using indicators that change color depending on the $\rm pH$ of the solution.
You are familiar with and know how to work with colored $\rm pH$ indicators such as methyl orange or bromothymol blue. These indicators change color when the acid form transits to the base form. And typically by the color change one determines that the $\rm pH$ of the solution has changed to the desired degree. However, using a spectrometer, it is possible to determine the $\rm pH$ quantitatively (this is possible near the transition point).
Let's consider how to determine the $\rm pH$ using the example of the cresol red indicator. Several cuvettes with different $\rm pH$ values (from 0.5 to 1.3) were prepared. The same amount of indicator was added to each cuvette. As a result, a color transition from red to orange/yellow is observed (Fig. 4).
Then, the absorption spectra for each of these cuvettes were measured and plotted together on a single graph (Fig. 5).
What features can be observed in the graph:
absorption at $\lambda^{CR}_{peak}\approx 520$ nm, the value of which changes significantly with pH (this peak decreases with increasing $\rm pH$);
absorption at 430 nm, the value of which changes much less significantly with $\rm pH$ changes (this peak increases as pH increases);
absorption at $\lambda^{CR}_{iso}=475$ nm, the value of which remained constant with pH changes (all spectra intersected at this point, which is called the isosbestic point).
Based on the absorption at the isosbestic point, it is possible to calculate the concentration of the indicator, since the absorption at this point does not depend on $\rm pH$. It is clear that if the concentration of the indicator is doubled, for example, then all absorption values will also double, but the ratio $A_{peak}/A_{iso}$ will remain constant and will correspond to a certain $\rm pH$.
The resulting graph is universal and can be used as a calibration graph, since the values plotted on the axes do not depend on the initial concentration of the indicator. It can be used to determine the $\rm pH$ of solution A4.
Prepare 500 µL of undiluted solution A4 in two 2 mL test tubes. Add 25 µL of cresol red solution to one of them. Mix the solution with the indicator thoroughly.
This step uses a thin glass cuvette with an adapter. Following the G1 instructions for thin cuvettes, obtain the absorption spectrum of undiluted solution A4 without indicator. Save the measured spectrum to the “Results/C2” folder on your desktop under the name “C2.txt”.
This step uses a thin glass cuvette with an adapter. Following the G1 instructions for thin cuvettes, obtain the absorption spectrum of the undiluted A4 solution with the indicator. Save the measured spectrum to the “Results/C3” folder on your desktop under the name “C3.txt”.
In the previous step, you determined the final $\rm pH$, i.e., the concentration of hydrogen ions in the solution after electrolysis. Next, you need to determine the initial $\rm pH$ of the solution before electrolysis. The $\rm pH$ of solution A2 is in the range from 3.00 to 5.00. In this range, the bromophenol blue indicator changes color well. Your task will be to obtain the same family of spectra for it as the authors did for cresol red (Fig. 5).
It would be possible to prepare solutions of different $\rm pH$ values, add the same amount of indicator, and measure their spectra. However, adding the same amount of indicator is difficult, and large errors will arise in the spectra. Therefore, you can choose the following approach:
take a cuvette and pour $V_0=4$ mL of solution with pH = 5.0 into it;
add 30 µL of bromophenol blue solution to the cuvette and mix well;
measure the absorption spectrum of the indicator;
add a certain volume of $\Delta V$ of acid solution ${\rm HCl}$ to the cuvette and mix (you will be provided with 10 and 100 mM acid solutions), which will lower the $\rm pH$;
measure the absorption spectrum of the modified solution;
continue adding concentrated acid and repeat steps 4 and 5 until we reach pH = 3.0 or lower.
The total volume of acid added will be small, so the change in the total volume of the solutions can be neglected.
To construct a graph similar to Fig. 5 and task C1, the $\rm pH$ values of the solution after each addition of concentrated acid are missing. The table on the answer sheets shows the initial concentration of ${\rm HCl}$, the volume $\Delta V$ of concentrated acid added at each step, and the concentration $C_{\rm HCl}$ of the acid used. Your task is to calculate the $\rm pH$ values (to two decimal places) that the solution will have at each step of acid addition.
Step $V_0, \rm{mL}$ $C_{\rm HCl},~{\rm mM}$ $\Delta V,~\mu{\rm L}$ 0 4.0 - - 1 4.0 10 +4.0 2 4.0 10 +8.0 3 4.0 10 +16.0 4 4.0 10 +30.0 5 4.0 100 +6.0 6 4.0 100 +12.0 7 4.0 100 +25.0
Perform the experiment described above, adding the specified amount $\Delta V$ of acid with concentration $C_{\rm HCl}$ at each step. Measure and save the absorption spectrum at each step according to instruction G1. Save the measured spectra in the folder on your desktop named “Results/C7” under the names “C7.{step number}.txt” (for example, “C7.2.txt”). You should end up with 8 spectra. Pour the remaining solution after obtaining all spectra into Answer tube C7.
Next, do the same as in steps C2 and C3, but with a different indicator. Pour 500 µL of undiluted solution A2 in two 2 mL test tubes. Add 20 µL of bromophenol blue solution to one of them. Mix the solution with the dye thoroughly.
This step uses a thin glass cuvette with an adapter. Following the G1 instructions for thin cuvettes, obtain the absorption spectrum of the undiluted A2 solution without indicator. Save the measured spectrum to the “Results/C10” folder on your desktop under the name “C10.txt”.
This step uses a thin glass cuvette with an adapter. Following the G1 instructions for thin cuvettes, obtain the absorption spectrum of the undiluted A2 solution with indicator. Save the measured spectrum to the “Results/C11” folder on your desktop under the name “C11.txt”.
Based on the measurements in questions C10-C11, calculate the absorption $A'_{peak}$ at wavelength $\lambda^{BB}_{peak}$ caused only by the absorption of the indicator. What is the absorption $A'_{iso}$ at a wavelength of $\lambda^{BB}_{iso}$ nm caused only by the absorption of the indicator?
No. Statement True False 1 Some of the oxygen produced escapes through the open surface of the solution. 2 During electrolysis, molecular oxygen can form at the cathode. 3 The oxygen formed at the anode can react with the graphite electrode. 4 During the experiment, other gases besides oxygen may form on the electrodes. 5 A significant portion of copper sulfate does not dissociate in solution. 6 Replacing the graphite cathode with a copper one will not allow the charge to be correctly determined by the change in ${\rm pH}$ of the solution. 7 Replacing the graphite anode with a copper one will make it impossible to correctly determine the charge by the concentration of copper ions.
$Q_{\rm O_2}$ $Q_{\rm Cu}$ $Q_{\rm pH}$
Photosynthesis is a key process occurring in our planet's biosphere, allowing organisms to use the energy of sunlight to sustain life. Due to photosynthesis, more than 99% of organic substances are formed, which are used to feed organisms at all levels of the food chain. However, there are many different types of metabolism based on the use of light energy. If carbon dioxide is the source of carbon in photosynthesis, i.e., it is fixed with the subsequent formation of organic matter, this is referred to as carbon dioxide fixation, and such organisms are called photoautotrophs. Photosynthesis can also occur without carbon dioxide fixation, using various organic substances instead of carbon dioxide; such organisms are called photoheterotrophs.
Water can be used as an electron donor for the electron transport chain in photosynthetic membranes. In this case, one of the products of photosynthesis is oxygen, and this type of photosynthesis is called oxygenic. Organic or reduced inorganic compounds can also be electron donors. In this case, the products of photosynthesis will not be gaseous, and this type of photosynthesis is called anoxygenic.
Phototrophic organisms are extremely diverse: they differ in cell structure, types of photosynthetic pigments, and many other characteristics. Phototrophic organisms often form complex communities where they occupy different niches. In this task, you are asked to study the characteristics of photosynthesis in two microorganisms, $A$ and $B$, and draw conclusions about their physiology and ecology.
Part E. Determination of spectral efficiency of photosynthesis in cultures of two microorganisms (6.4 points)
In this task, you will determine the presence and effectiveness of oxygen release by cultures of two microorganisms, A and B, when illuminated with light of different wavelengths. Three LED arrays serve as the light source: blue, green, and red. Each array consists of three LEDs connected in series (Fig. 6).
For each LED battery, measure the voltage across the 3 LEDs connected in series and calculate the voltage across single LED when the power source is turned on. Fill in the table in the answer sheets.
The figure below shows the volt-ampere characteristics of LEDs.
The figure below shows the dependence of the light power $P$ emitted by LEDs on the current $I$ flowing through them.
The internal cross-sectional area of the tubes is $S_0=1.77$ mm². Using the setup for studying the spectral sensitivity of photosynthesis one may measure the volume of oxygen $V$, which is released by microorganisms when exposed to light of different colors.
For this task, use microorganism $A$. Prepare the setup for measurements according to the G3 instructions. Turn on the light source and start timing.
If no oxygen release is observed 30 minutes after the start of the experiment, record zero values for $V_{O_2}$ in the table in the answer sheets.
If oxygen release is observed 30 minutes after the start of the experiment, continue the experiment for another 1.5 hours. Record the volume of oxygen $V_{O_2}$ released when illuminated by different colors of light in the table on the answer sheet.
Fill in the table in the answer sheets.
To compare the photosynthetic efficiency $E$ of different microorganisms, it is necessary to obtain the average efficiency per cell and per incident radiation power:
$$E = \frac{V_{\rm O_2}}{N\cdot P},$$
where $V_{\rm O_2}$ is the volume of oxygen released during exposure to light, $N$ is the number of cells exposed to light, and $P$ is the radiation power.
According to instruction G4, use Goryaev's chamber to count the number of cells in the four small squares $n_A$ and $n_B$ of microorganisms $A$ and $B$.
The edge of the large square of the Goryaev chamber is 0.2 mm, the depth of the chamber is 0.1 mm, and the large square consists of 16 small squares. Count the total number of cells $N_A$ and $N_B$ of microorganisms $A$ and $B$ inside a 20 ml syringe. Write down the calculation formula showing how $n_A$ and $N_A$ are related.
Microorganism Red Green Blue \(A\) \(B\)
True False It cannot be concluded from the experiment Organism $A$ is unable to perform photosynthesis when Organism $B$ is capable of moving toward more optimal conditions for photosynthesis. Organisms $A$ and $B$ perform the same type of Organism $B$ performs photosynthesis more efficiently Organism $B$ performs anoxygenic photosynthesis.
The pigment composition largely determines the ability of photosynthetic organisms to absorb different spectral intervals of solar radiation. Pigment composition may vary among organisms of different systematic groups. The main photosynthetic pigments are chlorophylls and carotenoids. They differ in chemical structure and physical properties. The physical properties of the most important pigments, such as absorption maxima and retention factor, are shown in the table below.
Pigments Absorption maxima, nm $R_f$ Chlorophyll a 430, 660 0.48 Chlorophyll b 450, 660 0.38 Bacteriochlorophyll 605, 780 0.42 Bacteriofeofetin 550, 750 0.45
Characteristics of absorption spectra and retention factor ($R_f$) of chlorophyll pigments on a chromatogram
Pigment Absorption maxima, nm $R_f$ Beta-carotene 430, 460, 490 0.98 Lutein 420, 450, 480 0.35 Carotenoid 1 480, 500, 520 0.83 Carotenoid 2 480, 500, 520 0.62 Carotenoid 3 480, 500, 520 0.54
Characteristics of absorption spectra and retention factor ($R_f$) of carotenoid pigments on a chromatogram
The retention factor value is calculated using the formula:
$$R_f=Z_x/Z_f,$$
where $Z_x$ is the distance between the start line and the center of the pigment spot, and $Z_f$ is the distance between the start line and the solvent front.
The microbial culture box contains concentrated extracts of these microorganisms in small test tubes. These will be needed for chromatography and absorption spectral measurements. The volume of the extracts provided in the test tubes is approximately 150 µL. Therefore, before opening them, shake the liquid down.
According to instruction G5, perform thin-layer chromatography of extracts of microorganisms $A$ and $B$.
Immediately after completing the chromatography and drying the plate, analyze the table and carefully mark the spots corresponding to chlorophylls with an “X” and the spots corresponding to carotenoids with an “O” on the plate with a pencil.
Raise the HELP sign so that an assistant can come to you and photograph the plate.
Place the marked plate in Answer tube F1.
Take three plastic cuvettes. Pour 3 mL of ethanol into each of them. Use one of the cuvettes to establish the baseline, and add 50 µL of extracts of microorganisms $A$ and $B$ to the others. Mix the diluted extracts thoroughly.
In accordance with instruction G1, obtain the absorption spectrum of extracts from microorganisms $A$ and $B$.
Save the measured spectra in the folder on the desktop “Results/F2” under the names “F2.A.txt” and “F2.B.txt” for microorganisms $A$ and $B$, respectively.
Pour 3 mL of the microorganism extract solutions you measured into Answer tube F2.A and Answer tube F2.B.
True False Chlorophylls will have two maxima in the red and blue regions of the absorption spectrum. Carotenoids can be found on the chromatogram of organism $B$. Carotenoids are more polar than chlorophylls. Carotenoids in this experiment can be clearly identified only by their mobility, since their absorption spectra are similar. On the chromatogram of organism $A$ extract, chlorophylls have the highest mobility. Bacteriochlorophyll absorbs the longer wavelength part of the spectrum than chlorophylls. Carotenoids participate in electron transfer along the photosynthetic electron transport chain.
True False Organism $B$ can use the longer wavelength part of the spectrum for photosynthesis. The pigment compositions of organisms $A$ and $B$ are identical. In mixed communities, organism $B$ is found in deeper layers than organism $A$. The carotenoid sets of both organisms are identical.
In small water bodies, the community of microorganisms is distributed according to physiological characteristics. In small water bodies with poor water mixing, a zone of oxygen concentration shift is formed, when anaerobic conditions are created in the deep layers. In the absence of oxygen, phototrophic organisms can use compounds other than water as electron donors. In this case oxygen is not released and this type of photosynthesis is called anoxygenic, unlike the oxygenic photosynthesis of cyanobacteria and green algae.
In the water column, phototrophic microorganisms are distributed according to their relationship to oxygen (aerobic and anaerobic) and their ability to absorb light in different parts of the spectrum.
It is known, for example, that among anoxygenic phototrophic bacteria, green bacteria are anaerobic and many of them are well adapted to using low-intensity light, while purple bacteria are resistant to oxygen.
True False Green algae are aerobic microorganisms Purple and green bacteria perform oxygenic photosynthesis. Phototrophic bacteria can use reduced sulfur compounds as electron donors in anoxygenic photosynthesis. Cyanobacteria mainly live in anaerobic conditions.
The figure shows a diagram of a small pond with poor water circulation. Identify the zones of the pond (A-D) where the following microorganisms will live.
Write the numbers of the organisms in the table on the answer sheet.
Pond zone Microorganisms A B C D
Microorganisms Pond zone Microorganism \(A\) Microorganism \(B\)
True False Microorganisms can move within the microbial mat. In the upper layer of the cyanobacterial mat, bright light increases the risk of photodamage to the photosynthetic apparatus of cells. Non-phototrophic microorganisms can only exist deep within the cyanobacterial mat. Phototrophic microorganisms with different sets of pigments can change position in microbial mat depending on changes in the wavelength range of sunlight. At sunrise and sunset, bacteria capable of absorbing light in the shorter wavelength region of the spectrum will photosynthesize most actively.
