Workplace number _____ Name ____________________________

58-th INTENATIONAL MENDELEEV OLYMPIAD

PRACTICAL EXAMINATION

Shenzhen – 2024

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General Directions

• Safety rules: follow the conventional rules of carrying out chemical experiments; when
in the lab, you are required to wear the lab coat and safety goggles (or your prescribed glasses); no
eating or drinking in the lab.
• Violating safety rules: you get one warning, offend again: you are disqualified.
• Time: the total duration of the experimental exam is 5 h. You will have 15 min to read the
text. You are not allowed to start working during this period (you can look at the sampling
pipette and other equipment).
• The lab is equipped with clocks. Once the STOP command is given, you must immediately
stop working and deliver all sheets.
• Besides chemicals, labware and equipment, use only calculator and pen.
• Approach your lab assistant, if you got questions on lab safety or you need a toilet break.
• Dispose liquid waste in the plastic container labelled “Liquid waste”. You will find it
at your table.
• Write down answers only in the designated places; answers written elsewhere will not be
marked. Give calculations where appropriate.
• You can re-use glassware. Wash these carefully.
• The reagents given are limited. Spilled or totally used solution will be substituted
with penalty.
• Using burettes: you are given burettes with open stopcocks. Close it before use. If there is
a bubble left in the tip when filling the burette, position it vertically and carefully shake. If the
bubble is still there, seek for help from your lab assistant. The burette may leak. If so, fasten the
plastic stopcock adaptor. Always place a beaker under the burette.
• You can use the backside of the exam sheets as draft paper.
• Keep your workspace in order. There are tissue rolls at each table. Besides, there are rolls
of paper towels in each lab.
• If you have broken an item, approach your lab assistant, who will help you to fix the case
and provide you with a replacement item.
• You will be using a sampling pipette with the range of volumes from 100 up to 1000 μL.
Adjust the required volume by turning the ring. Pressing the piston to the first stop allows filling
the tip with the adjusted volume, pressing the piston to the final stop position releases the entire
volume. Be sure not to suck the liquid inside the pipette itself, which may destroy it. Use a separate
tip for each individual solution.
• You are expected to use a 3-valve bulb to fill glass pipettes. It is completely prohibited
to fill the pipettes by mouth. The pipette filler has three valves (glass balls): А for air draining
when pressing the bulb, S for sucking a solution into the pipette, Е for draining the pipette. The
valves are difficult to press; please apply power!

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Spirulina platensis – superfood

Spirulina, very popular in China and a number of neighboring countries, is a blue-green

microalgae with a unique composition. It contains up to 70% of valuable protein, which is easily
absorbed by the body, as well as many vitamins, microelements and other useful substances.
Spirulina is rich in unsaturated fatty acids, such as linoleic and linolenic acids, which play a key
role in the status of human cardiovascular system. Spirulina is rich in polysaccharides and
photosynthetic pigments, including phycocyanin, chlorophyll and phycoerythrin, which have
a positive effect on metabolism and functioning of the digestive system. Thus, spirulina is
a multifunctional means of maintaining health and improving functioning of various body systems.

Part А. Conformations of phycocyanin

Spirulina is rich in protein phycocyanin, which is beneficial to humans as it promotes
the production of red blood cells, white blood cells and platelets, as well as the growth
and differentiation of stem cells. Although proteins are composed of the same amino acids,
some proteins are colored, and some are not. Phycocyanin has a bright color due to a chromophore
called phycocyanobilin.
Reagents:
Reagent Label Placed in
Phycocyanin solution Phycocyanin 10 mL vial
Buffer solution (pH 7.0) Buffer 10 mL vial
Water H2 O Wash bottle
Sodium chloride solution NaCl Eppendorf tube
Guanidinium chloride solution Guanidine Eppendorf tube
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Sodium lauryl sulfate solution SDS Eppendorf tube
Urea solution, 8 M Urea 8 М 10 mL vial
Sodium hydroxide solution, 1 M NaOH 1 M Eppendorf tube
Hydrochloric acid solution, 1 M HCl 1 M Eppendorf tube
Acetone Acetone Eppendorf tube
Ethanol Ethanol Eppendorf tube
Ammonium sulfate solution (NH4)2SO4 Eppendorf tube
Equipment: cuvettes, photometer, sampling pipette, tips, Eppendorf tubes with rack.

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There may be precipitate in the solution, which does not interfere the experiment. One can reduce the amount of
the precipitate by heating and shaking the solution.

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When phycocyanin folds around phycocyanobilin, a bright blue color appears, indicating
whether the protein is folded or unfolded. Various substances that change the conformation can be
used to test the conformational state of the protein.
In a 10 mL vial you are given a solution of phycocyanin in a buffer solution. Using the
sampling pipette, pour 100 µl of phycocyanin solution into 11 Eppendorf tubes. Then add 900 µl
of analyzed reagents to each Eppendorf tube. Close the tube and shake it several times. Equilibrium
in the systems is established within 5 minutes.
Determine how the color of the solution has changed using the following notation:
“B” is for deep blue, take the color of phycocyanin in the buffer solution as a reference;
“L” is for light blue;
“P” is for purple;
“O” for colorless;
“X” for any other coloring.

А1. Place the Eppendorf tubes according to their numbers in the Sheet 5. Record your observations
according to the notations provided. Get your lab assistant’s signature.

А2. Which reagent is most efficient in unfolding the protein according to the analysis performed?

A3. Changes in what factors do lead to protein denaturation?

Temperature
Ionic strength
Medium acidity
Protein concentration

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Write down the

Example color code: 1. Buffer 2. H2O 3. NaCl 4. Guanidine 5. SDS
B/L/P/O/X

Place Eppendorf tube

here for photographing

6. Urea 7. NaOH 8. HCl 9. 11.

10. Ethanol
8M 1M 1M Acetone (NH4)2SO4

Lab assistant’s signature __________________________________

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Workplace number _____ Name ____________________________

Many proteins perform their functions in the form of quaternary structures, the energy
of which differs from that of the linear biopolymer. Estimating the difference in energy between
protein structures is not an easy task, but colored proteins can serve as a convenient model for such
calculations.
To calculate the Gibbs free energy of the protein unfolding process, prepare 7 phycocyanin
solutions with a total volume of 2 mL each, using different volumes of 8 M urea solution according
to the table below. Using the sampling pipette, add 200 μL of phycocyanin and the corresponding
volume of the urea solution to plastic cuvettes (for measuring optical density), then add
the calculated volume of the buffer solution and mix well (you can do this by multiple insertion
of the solution into the pipette tip). Label the cuvettes with a marker. Wait approximately
20-30 min to establish equilibrium. During this time, you can fulfil other parts of the exam set.

Cuvette number 1 2 3 4 5 6 7

V(Phycocyanin), μL 200 200 200 200 200 200 200

V(Urea 8 М), μL 0 750 900 1100 1300 1400 1800

V(Buffer), μL

А4. Calculate the concentration of urea in the numbered test tubes. Record your results in the table.
Cuvette number 1 2 3 4 5 6 7

с(Urea), M

After the given time, record the transmittance (T, %) of each solution using
a monochromatic light transmittance sensor (photometer) located next to the computer.
If the device is switched off, then allow it to warm up for about 30 s after switching on
before starting measurements. To carry out measurements, open the lid and place the cuvette
in the cuvette holder with the arrow pointing towards the screen. Measurements are carried out
at the wavelength of 650 nm (Red(650nm) and light transmittance are displayed on the device
screen). It is important to carry out all measurements using the same device!
The transmittance T is related to the absorbance (A) by the following relationship:

𝑇, %
𝐴 = −lg & ,
100

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А5. Indicate the number of the photometer you are using. Record the measured transmittance
values in the table. Calculate the absorbance values for each sample.

The number of the photometer: __________

Cuvette number 1 2 3 4 5 6 7

T, %

Use the next formula to calculate the equilibrium constant for protein unfolding:
[unfolded] 𝐴# − 𝐴
𝐾!" = = ,
[folded] 𝐴 − 𝐴$
where Keq is the equilibrium constant, Af is the absorbance of the folded protein (in the buffer
system), Au is the absorbance of the unfolded protein (at the highest urea concentration),
А is the absorbance of the solution for which the constant is calculated.

А6. Calculate the equilibrium constant and Gibbs free energy (20°C) of the protein unfolding in
accordance with the cuvette number.
Cuvette
2 3 4 5 6
number
Keq

ΔrG°

А7. On graph paper (next sheet), plot the dependence of Δ% 𝐺 ∘ on the urea concentration and draw
the trend line by hand. Indicate the dimensions along the axes.

А8. Calculate the Gibbs free energy in the aqueous solution (𝛥% 𝐺'∘ ! ( ) from the calibration plot,
assuming the following form of the dependence:
Δ% 𝐺 ∘ = Δ% 𝐺'∘ ! ( − 𝑚 ∙ 𝑐,
where m is a certain coefficient, c is the concentration of urea.

Write down your answer:

𝚫𝐫 𝑮∘𝐇𝟐 𝐎 =

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Workplace number _____ Name ____________________________

Part B. Titrimetric determination of fatty acids

using the Margosches method
In addition to the protein, spirulina contains unsaturated fatty acids. Determination
of the iodine value is behind one of the main ways to find the unsaturation of fats. The iodine value
expresses the mass of I2 (in grams) required to completely saturate the fatty acids contained
in 100 g of fat. The Margosches method allows determining the iodine value, based on the
oxidation of a double bond with iodine, followed by titration of the unreacted iodine. You will have
to determine the mass of the oil sample given to you based on the titration results and the iodine
value.

Reagents:
Reagent Label Placed in
Sodium thiosulfate solution, 0.09621 M Na2S2O3 250 mL bottle
Margosches solution (ethanolic solution
I2 100 mL bottle
of iodine, 0.1 M)
Indicator
Starch solution, 1% 30 mL drop-bottle
(starch)
C2H5OH
Alcohol mixture ethanol-chloroform
CHCl3 250 mL bottle
(10:1 vol.)
(10:1)
Equipment: 100 mL volumetric flask with a sample of oil (1 pc.), 25 mL burette (1 pc.),
small glass funnel for the burette (1 pc.), bulb with three valves (1 pc.), 100 mL graduated cylinder
(1 pc.), 10 mL graduated pipette (3 pcs.), 250 mL titration flask with ground stopper (3 pcs.),
50 mL beaker (3 pcs.).

Pour the standard sodium thiosulfate solution into the burette. Using the beaker, add 40 mL
of the mixture of ethanol and chloroform (10:1) (hereinafter referred to as the “alcohol mixture”)
in the volumetric flask with the sample of oil and mix vigorously. Do not turn the flask upside
down. Make sure the sample has dissolved. Bring the solution to the mark with the alcohol mixture,
mix vigorously again, turning the flask upside down.

Reference experiment. To carry out a reference experiment, add 10.00 mL of the mixture
of ethyl alcohol and chloroform (10:1) and 5.00 mL of Margosches solution into a 250 mL conical
flask using a pipette. Then add 40 mL of distilled water, stopper, shake, open the stopper slightly
to release pressure in the flask and leave for 5 min. Then carefully shake the flask with the closed
stopper 2–3 times, holding the stopper, and open the stopper slightly to release the pressure
in the flask. Stir the contents of the flask vigorously for 3 min, periodically releasing the pressure.
Stirring vigorously, titrate the resulting mixture with the sodium thiosulfate solution
until a light yellow color appears (make sure there is no dark precipitate left at the bottom
of the flask), then add 3-4 drops of starch and titrate until the blue color disappears.

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В1. Record the volume of sodium thiosulfate spent in the reference experiment (Vref):
Titration No 1 2 3
The initial volume, mL

The final volume, mL

The volume spent for the titration, mL

The accepted volume, Vref: _____________ mL

Determination of the iodine value of the oil mixture. The procedure described
for the reference experiment must be carried out for the solution of oil in the alcohol mixture.
Transfer a 10.00 mL aliquot of the oil solution from the volumetric flask into a titration flask. Then
add 5.00 mL of Margosches solution to the analyzed sample using a pipette. Add 40 mL of distilled
water, stopper the flask, shake, open the stopper slightly to release the pressure in the flask
and put the flask aside for 5 min. Then gently shake the flask with the stopper closed and open
the stopper slightly to release the pressure in the flask. Stir the contents of the flask vigorously
for 2–3 min, releasing the pressure from time to time. Stirring vigorously, titrate the resulting
mixture with the solution of sodium thiosulfate until the color changes from brown to light yellow.
Then add the indicator (1% starch solution) and continue titration until the blue color disappears.

В2. Record the volume of sodium thiosulfate spent on titrating the sample with oil (V):
Titration No 1 2 3
The initial volume, mL

The final volume, mL

The volume spent for the titration, mL

The accepted volume, V: _____________ mL

B3. Write the equation for the reaction of an unsaturated compound ( ) with the Margosches
reagent in the presence of water. Note that vicinal diiodides are unstable and are not formed
in this reaction.

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B4. Derive a formula relating the iodine value of the oil given to you, the mass of the oil in your
volumetric flask m (mg), the volume of the solution in the 100.0 mL volumetric flask, 10.00 mL
volume of the aliquot, and the volumes (mL) obtained during titrations (Vref for the reference,
V for the actual determination), the concentration of thiosulfate c(Na2S2O3), and the mass of the
iodine equivalent (M = 126.9 g/mol).
Your work:

Iodine value =

В5. Calculate the mass of oil in the sample given to you if its iodine value is 86.

Calculations:

The oil content in the volumetric flask:__________________ mg

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Workplace number _____ Name ____________________________

Part С. Identification of vitamins

Spirulina is a unique microalgae containing vitamins and microelements necessary
for humans. Spirulina contains vitamins such as А, В1, В2, В3, В6, В9, С, D, and Е. These vitamins
play an important role in maintaining the health of our skin, hair, nails, immune system,
and general health of the body.
The table below lists some of the vitamins that spirulina may contain.

Thiamine hydrochloride Nicotinamide

(vitamin B1) (vitamin B3)
Riboflavin (vitamin B2) (C6H6N2O)
(С12H17ClN4OS)
(C17H20N4O6)

Ascorbic acid (vitamin

Vikasol Pyridoxine hydrochloride C)
(a vitamin K3 analog) (vitamin B6)
(C11H9NaO5S) (C6H8O6)
(C8H12NO3Cl)

Reagents:
Reagent Label Placed in
Thiamine hydrochloride Eppendorf tube
Riboflavin Eppendorf tube
Nicotinamide Eppendorf tube
Vikasol Eppendorf tube
1-8
Pyridoxine hydrochloride Eppendorf tube
Ascorbic acid Eppendorf tube
Potassium iodide solution Eppendorf tube
Phosphate buffer (NaH2PO4/Na2HPO4) Eppendorf tube
Sodium hydroxide solution, 0.1 M NaOH 0.1 М Eppendorf tube
Sodium nitrite solution, 1 M NaNO2 1 М Eppendorf tube
Iron(III) chloride solution, 0.05 M FeCl3 0.05 М Eppendorf tube

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Sulfanilic acid solution†, 0.06 M Sulfanilic acid

Eppendorf tube
0.06 M
Silver nitrate solution, 0.5 M AgNO3 0.5 М Eppendorf tube
Indicator paper for determination of рН

Equipment: 96-well plate, filter paper, sampling pipette, replaceable tips.

You have at your disposal a set of the following reagents: NaOH, NaNO2, FeCl3, sulfanilic
acid, AgNO3. Using these solutions individually or mixing them and obtaining new reagents, you
can identify vitamins and other substances offered to you. You are given solutions of vitamins:
thiamine hydrochloride, riboflavin, nicotinamide, vikasol, pyridoxine hydrochloride, ascorbic acid
(see the formulas in the table above) in Eppendorf tubes labelled 1–8. In addition, the KI solution
and phosphate buffer are among substances to be identified.
Identification of vitamins is based on the ability of substances to lead to colored products, as
well as to the precipitate formation of and (or) gas release when treated with appropriate reagents.
Also use the provided reagents to create the pH value necessary for the reaction. Use universal
indicator paper to monitor pH.
To successfully complete the experiment, pay attention to the following recommendations:
• carry out reactions in the 96-well plate;
• do not pour too much solution into the cells. It is enough to add 100 μL of the analyzed
solution to carry out the reaction.
When performing identification, consider the following facts:
• Riboflavin in a neutral or slightly acidic solution (pH 6.5−7.2) forms a colored compound
when reacting with AgNO3.
• Vitamin B6 and nicotinamide form colored compounds with FeCl3.
• Diazophenylsulfonic acid is formed as a result of the reaction of sulfanilic acid
with an acidified solution of sodium nitrite.
• Thiamine reacts with diazonium salts in an alkaline environment to form red-orange colored
triazenes. Pyridoxine is also capable of reacting with azo coupling in an alkaline medium to form
red-orange colored products.
• In an alkaline environment (pH ≥ 11), the sulfonate group is removed from the vikasol
molecule, and 2-methyl-1,4-naphthoquinone precipitates.

†
4-Aminobenzenesulfonic acid (C6H7NO3S)

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The table for recording your observations (not graded):

The reagents added

2
with the unknown substances

3
No of the test tube

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С1. Determine the contents of the test tubes. Write your answers in the table.
The solution B1 B2 B3 B6 K3 C KI NaH2PO4
analyzed +Na2HPO4
The test tube
No

С2. Write the equation for the reaction of pyridoxine hydrochloride with sodium hydroxide. Use
structural formulas for organic compounds.

С3. Write the equation for the reaction of pyridoxine hydrochloride with diazophenylsulfonic acid
in an alkaline medium. Use structural formulas for organic compounds.

С4. Write the equation for the reaction of ascorbic acid with iron(III) chloride. Use the molecular
formulas of the compounds.

С5. Write the equation for the reaction of ascorbic acid with sodium nitrite in an acidic medium.
Use the molecular formulas of the compounds.

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С6. Write the equation for the reaction of potassium iodide with sodium nitrite
in an acidic medium.

С7. Write the equation for the reaction of iron(III) chloride with the phosphate buffer.

С8. Write the equation for the reaction of iron(III) chloride with potassium iodide.

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