Medical Chemistry Lectures & Labs

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Department of Biochemistry
Lithuanian University of Health Sciences
PRACTICALS OF MEDICAL CHEMISTRY

for Students of Medicine and Odontology
Kaunas, 2013
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SCHEDULE OF MEDICAL CHEMISTRY LECTURES

FOR MEDICINE AND ODONTOLOGY 1st YEAR STUDENTS
( Spring semester, 2013)
The lectures take place at 800–930
N Date Title of lectures Location

1 March 28 1. Water and solutions. Prof Ramunė Morkūnienė Room 427
2 March 29 2. Properties of buffers. Prof Ramunė Morkūnienė Room 427
March 30 - Easter holiday
April 7
3 April 8 3. Basics of colloidal chemistry. Prof Laima Ivanovienė Room 427
4 April 9 4. Heterogenic processes. Prof Artūras Kašauskas Room 427
5 April 10 5. Coordination compounds. Prof Artūras Kašauskas The Small auditorium
6 April 11 6. Thermodynamics. Prof Artūras Kašauskas Room 427
7 April 12 7. Chemical kinetics and catalysis. (for Medical students only) Room 427
Prof Laima Ivanovienė
April 13
April 14
8 April 15
April 16 CONTROL TEST I
9 April 17 8. Classes of organic compounds. Prof Ramunė Morkūnienė The Small auditorium
10 April 18 9. Isomers. (for Medical students only) Prof Artūras Kašauskas Room 427
11 April 19 10. Alcohols, aldehydes, carboxylic acids. Room 427

Prof Artūras Kašauskas
April 20
April 21
12 April 22 11. Carbohydrates. Prof Ramunė Morkūnienė Room 427
13 April 23 12. Nucleotides and nucleic acids. Prof Artūras Kašauskas Room 427
14 April 24 13. Amino acids and proteins. Prof Laima Ivanovienė The Big auditorium
15 April 25 14. Fatty acids and lipids. Prof Laima Ivanovienė Room 427
April 26 CONTROL TEST II
April 27
April 28
April 29
April 30 FINAL EXAM
Head of the Biochemistry department Prof. L. Ivanovienė

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SCHEDULE OF MEDICAL CHEMISTRY LABORATORY WORK

FOR 1st YEAR MEDICINE AND ODONTOLOGY STUDENTS
(Spring semester, 2013)
Laboratory practicals take place in laboratory124
945–1145 for groups 31, 32, 36 MF R.Morkūnienė/S.Dubickienė
1200–1400 for groups 29, 33 MF L.Ivanovienė
1400-1600 for groups 30, 34, 35 MF A.Kašauskas/S.Dubickienė
1600-1800 for groups 13-15 OF students A.Kašauskas/S.Dubickienė
N Date Title of laboratory practicals
1 March 28 1. Principles of safe work in a chemical laboratory.
Preparation of solutions. Percent concentration.
2 March 29 2. Preparation of molar solutions: molar concentration.
March 30 - Easter holiday
April 7
3 April 8 3. Volumetric analysis. Acid-base titration.
4 April 9 4. Preparation of buffer solutions.

Buffering capacity and its determination.
5 April 10 5. Preparation of colloidal solutions.
Effect of ions on coagulation of colloidal solutions.
6 April 11 6. High-molecular mass compounds and their properties: jellynation.
7 April 12 7. Coordination-compound based determination of water hardness.
April 13
April 14
8 April 15 8. Last minute arrangements.
April 16 CONTROL TEST I.
9 April 17 9. Chemical catalysis and thermodynamics of chemical processes. Examples of reaction

rate changes, effect of a catalyst, equilibrium shift.
Determination of Michaelis-Menten constant of an enzyme from experimental data (Dry
practical). (This day laboratory works are for Medical students only)
10 April 18 10. Carbonyl compounds – aldehydes, ketones and acids. Their properties – specific
reactions of functional groups. Esters and lipids. Determination of products of lipid
peroxidation.
11 April 19 11. Basics of organic compounds. Some chemical properties of alcohols, phenols and
amines.
April 20
April 21
12 April 22 12. Chemical properties of mono- and polysaccharides.
13 April 23 13. Nucleotides and nucleic acids: characteristic reactions. Specific reactions for products
of nucleic acid hydrolysis.
14 April 24 14. Specific chemical reactions for amines, amino acids and proteins. Estimation of
molecular mass of a protein from the gel chromatography data (Dry practical). Estimation
of molecular mass of a protein by the electrophoresis method (Dry practical).
15 April 25 15. Last minute arrangements.
April 26 CONTROL TEST II
April 27
April 28
April 29 Repetition of control tests if failed
April 30 FINAL EXAM
Head of the Biochemistry department Prof. L. Ivanovienė

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Questions for the control test N1 - 2013
1. Characteristics of water molecule. Distribution of charges.

2. Hydrophylic, hydrophobic and amphipatic compounds.
3. Types of real solutions. Electrolytes and non-electrolytes.
4. Calculation of percent concentration. Calculation of molarity.
5. Osmolarity of real solutions. Isotonic, hypertonic and hypotonic solutions.
6. Electrolytic dissociation of weak electrolytes. Principle of Le Chatelier.
7. Dissociation of water - pH. Calculation of pH as the function of -log[H+]. Calculation of pOH
8. pK - meaning and a way of its determination.
9. Buffering capacity of buffers. What compounds can act as buffers in solutions.
10. Bicarbonate buffer system of human blood. Know how to calculate its efficiency using
Henderson-Hasselbalch equation.
11. Properties of colloidal solutions. Composition of micelle.
12. Coagulation of colloidal solutions. What factor is responsible for resistance of colloidal solution
against coagulation.
13. Properties of biopolymer solutions.
14. Solubility and solubility product. Heterogeneous equilibrium. What factors promote shifting of the
heterogeneous equilibrium?
15. Adsorption and absorption. Factors which influence adsorption process.
Ion-exchange adsorption.
16. Coordination compounds, their properties.
17. Complexones. Characteristic of complexones and some examples of them (EDTA, Trilon B).
18. Systems in the thermodynamics.
19. First law of thermodynamics.
20. Second law of thermodynamic.
Questions to the 2nd control test on Medical Chemistry - 2013
1. Functional groups.
2. Sigma and pi bonds.
3. Important reactions of organic compounds
4. Level of organic compound oxidation
5. Naming of organic compounds by IUPAC
6. Chemical properties of alcohols and phenols.
7. Reactions for identification of aldehyde group.
8. Saponification.
9. Classification of monosaccharides.
10. Structure of main monosaccharides and disaccharides. Anomeric C atom.
11. Polysaccharides: structure and types of bonds.
12. Physical and chemical properties of saturated fatty acids. Structural formulas of palmitic and stearic
acids.
13. Physical and chemical properties of unsaturated fatty acids. Omega-classification. Structural formulas
of linolic and linolenic acids.
14. Fats and oils. Structure of glycerophospholipids.
15. Properties of cholesterol
16. Formation of peptide bond.
17. Acid-base properties of amino acids.
18. Nitrogen bases, nucleosides and nucleotides: their nomenclature and chemical structure.
19. Chemical structure of nucleic acids and its relation to the appropriate biological function.
20. Hydrogen bonds between molecules of organic compounds. Solubility in water.
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QUESTIONS FOR FINAL EXAMINATION ON MEDICAL CHEMISTRY

2012-2013 academic year
1. Characteristics of water molecule. Hydrogen bond.

2. Hydrophylic, hydrophobic and amphipatic compounds.
3. Electrolytes and non-electrolytes.
4. Percent concentration (m/m %, v/v %, m/v %). Molar concentration.
5. Colligative properties. Osmotic pressure, osmolarity.
6. Electrolytic dissociation of weak electrolytes. Le Chatelier's principle.
7. Index of acidity (pH).
8. Buffers. Buffering capacity. pH calculations with buffers: the Henderson-Hasselbalch equation.
9. Carbonic acid-bicarbonate buffer system and phosphate buffer system: principles of their action.
10. Real solutions, colloidal solutions and low dispersion systems.
11. High-molecular mass compounds, properties of high molecular mass compound solutions.
12. Coagulation of colloidal solutions: its kinetics and factors, affecting coagulation.
13. Heterogeneous processes. Heterogeneous equilibrium. Examples of the heterogeneous processes occurring in the
human body.
14. Adsorption and absorption. Adsorption when adsorbent is liquid substance: surface-active substances, aeroembolism
(decompression sickness).
15. Coordination compounds, the structure of coordination compounds.
16. Dissociation of coordination compounds, instability constant of coordination compounds.
17. Chelates, their structure. Complexones.
18. Hess’s law.
19. Entropy and enthalpy.
20. Gibbs free energy.
21. Exergonic and endergonic reactions.
22. Reaction rate. Rate law.
23. Effect of temperature on reaction rate.
24. Effect of catalysts on chemical reaction: on activation energy, reaction rate and equilibrium constant.
25. Sigma bonds in organic compounds
26. Pi bonds in organic compounds
27. Functional groups.
28. Level of organic compounds oxidation.
29. Structural isomers.
30. Cis- and trans-isomers.
31. Chiral carbon atom.
32. L and D-isomers.
33. Classification of alcohols, with examples.
34. Chemical properties of alcohols and phenols.
35. Identification of phenols.
36. Identification of aldehyde group.
37. Chemical properties of carboxylic acids.
38. Monosaccharides: aldoses and ketoses; be able to draw structural formulas of an aldose and a ketose.
39. Anomeric C atom.
40. Mutarotation.
41. Glycosidic bond in di- and polysacharide. Types of the bond.
42. Omega series of fatty acids.
43. Chemical properties of triacylglycerols. Acidic and alkaline hydrolysis: reaction equations with formulas of initial and
final reactants.
44. Structure and properties of cholesterol.
45. Classification of amino acids (know formulas of all 20 amino acids)
46. Zwitterions. Isoelectric point.
47. Specific chemical reactions for amino acids and peptides (from lab works).
48. Structures of nucleosides and nucleotides; their nomenclatures.
49. Types of bonds in nucleosides and in nucleotides.
50. Chemical structure of nucleic acids. Types of nucleic acids.
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MEDICAL CHEMISTRY. REQUIREMENTS FOR STUDY:
Practical work
1. The attendance of practical work is obligatory. Missing practical work is not allowed.
2. The practical work descriptions together with the report forms have to be printed out and bond into a
folder.
3. The practical work has to be defended during the time allotted for the particular laboratory
assignment. Students have to fill in the report form, complete additional tasks and prepare to answer
questions provided in the description of the practical work. Only the defended practical work will be
accepted.
4. Practical work will make 10 of the final assessment (up to 1 point).
5. For the credit a student must have all practical work completed and defended.
Control tests
1. Students will take 2 control tests during the cycle on the material delivered during the lectures before
the test.
2. The tests will be prepared as MCQs. To pass, a student needs to collect 40% of the correct answers.
There will be one day to pass the test failed (only one test of the two by students choice).
3. The tests are obligatory.
4. For the credit students have to pass at least 1 control test.
5. The control tests will make 50 (1st – 25, 2nd – 25) of the final assessment (up to 5 points).
Final examination
1. The exam is a multiple-choice test of 50 questions.

2. To pass, a student needs to collect 50% of the correct answers.
3. The exam will make 40 of the final assessment (up to 4 points).
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SAFETY INSTRUCTIONS FOR WORK IN THE MEDICAL CHEMISTRY LAB
I. GENERAL REQUIREMENTS
1. A student is only allowed to work after listening to the safety instructions and signing in the
registration book: a student must know the character of possible accidents and be able to provide first-aid.
2. A student must know:

- Location of water and gas taps;
- location of electrical switches;
- location of fire extinguishers;
- location of the first-aid kit;
and be able to use them properly in an emergency situation.
3. In the lab it is not allowed:

- to be without a lab coat;
- to disturb others by unnecessary talking and walking round;
- to eat and drink;
- to work in the absence of the teacher or technician;
- to do things not related to the laboratory work;
- to carry out experiments not included in the teaching plan;
- to use damaged equipment and unfamiliar substances.
II. REQUIREMENTS BEFORE THE WORK
- to understand the task which is going to be carried out;

- to know properties of the reagents to be used and properties of the reaction products, and to
know how to handle them safely;
- to know how to work properly and safely with the equipment and glassware required for that
particular laboratory work;
- the lab bench, on which you are going to do the experiment, must be clean and in order, without
unnecessary equipment, glassware or personal belongings.
III. REQUIREMENTS DURING THE WORK
1. Avoid noisy behaviour, keep everything clean and tidy.
2. During laboratory work it is not allowed:

- to leave working electrical equipment and burning gas burners unattended;
- to leave the laboratory without teacher’s permission;
- to work with damaged equipment or glassware;
- to work with unknown substances;
- to remove reagents from the places they have to stay;
- to overload the lab bench with books, unnecessary reagents, glassware, etc.;
- to leave gas burners and electrical equipment switched on when they are not needed;
3. Carefully follow the description of the laboratory work
4. When working with concentrated acids, bases, flammable substances or reagents with unpleasant
odour, all the work must be done under a fume hood.
5. When heating over a flame, a test tube has to be inclined at an angle of 45 and pointed away from the
working person and other students.
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6. Spilt reagents and broken glassware have to be cleaned immediately with appropriate precautions and
under supervision of the technician.
IV. REQUIREMENTS AFTER THE WORK.
- turn off equipment, water and gas taps;

- wash the glassware and clean the work surface;
- place all the equipment, reagents and glassware in appropriate places.
V. FIRST-AID.
1. First-aid to the injured person has to be immediate and correct. All people present in the room must be
ready to help.
2. If chemical compound gets on the face, eyes, hands or clothes, they have to be washed immediately
with large amounts of water.
3. Every accident must be reported to the teacher.
4. In the case of more serious injuries, intoxications or burns, an ambulance must be called immediately.
5. In the case of electric shock, the power must be turned off immediately. If the victim is unconscious,
cardiac massage and artificial respiration should be started right away.
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The example of the laboratory work report
Name Family name
Group N Year Faculty
Laboratory work N …..
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Principle of the lab-work
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Results and calculations
Conclusion
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1. Preparation of solutions. Percent concentration
Background. Solution is a homogenous mixture containing two or more components. One of them,
solvent, generally is present in the largest amount. All the remaining components are called solute and are
equally distributed in the solvent. The physical state of solvent does not change. If solute is an ionic
compound, it dissociates into ions when dissolved in the ionic solvent (water).
The amount of solute per amount unit of solution (mass or volume unit) is called concentration,
abbreviated as [C]:
[C] = amount of solute/amount unit of solvent
Depending on the units used to express the amount of solute and solvent there are several ways to indicate
concentration. Thus, there is percentage concentration, molar concentration, molar concentration of
equivalent and a few others.
A widely used type of concentration is the percentage concentration. The term percent literally means
number of parts in the total of one hundred parts. Consequently, the percentage concentration means
number of solute parts in one hundred parts of solution. There are several types of the percentage
concentration:
mass / mass – indicates number of solute mass units per 100 solution mass units and is denoted in
parenthesis as (mass/mass), (m/m), (w/w). For example, 5 % NaCl (mass/mass) means that there are 5
grams of NaCl in 100 g of the solution;
volume / volume – indicates number of solute volume units per 100 solution volume units and is
denoted in parenthesis as (vol./vol.) or (v/v). 10 % (vol./vol.) ethanol means that there are 10 ml of pure
ethanol in 100 ml of the solution;
mass / volume – indicates the number of solute grams per 100 ml of solution. 3 % NaCl
(mass/vol.) means 3 grams of NaCl in 100 ml of the solution.
If the percentage concentration is shown without an indication in parenthesis, generally it means grams of
solute per 100 g of solution. In clinical trials sometimes the obsolete milligram percentage concentration
mg% is used, which indicates milligrams of solute in 100 grams of solution.
Experimental part:
Prepare 250 g of CaCl2 solution in water of the following concentration:
A 3%
B 5%
C 7%
D 9%
E 11%
F 13%
1.Calculate the amount of salt and water needed.
Example: Prepare 250 g of 20 % CaCl2 solution in water.

20 % solution will contain 20 g CaCl2 in 100 g of solution;
x g should be in 250 g of solution;
then x = 20g  250g/100g = 50 g CaCl2
250g – 50g = 200g H2O
Thus, 250 g of the solution will be made of 50 g of CaCl2, and the rest 200 g will be water.
2. Weigh CaCl2 on a piece of paper and transfer it to the flask.

3. Since the density of water is approximately1g/cm 3= 1g/mL, the weight of water will be equal to its
volume. Use a graduated cylinder to measure the needed amount of water.
4. Pour the water into the flask with the salt and dissolve the salt by mixing.
5. Transfer the obtained solution into the cylinder and measure its density (d) with the aerometer. Find the
real % concentration of this solution from the density tables. Indicate how much it differs from the
theoretical value. Explain the possible error.
General questions
1. What systems are called solutions?

2. What is a solvent and what is a solute?
3. Describe the structure of water molecule in short.
4. What is solution concentration?
5. What types of concentration do you know?
6. What is a percentage concentration? What types of percentage concentration are there?
7. What does it mean: mass/mass, volume/volume, mass/volume concentration?
8. How to determine the density of the solution and what density units are there?
9. How can aerometer be used to determine the density of solution?
10. What is the density of water and when is it the highest?
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Laboratory work N 1
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Conclusion
2. Preparation of molar solutions: molar concentration
Background. Molar concentration - the method of expressing concentration that indicates how many moles of
solute are present per unit volume of solution. The concentration of solution can be varied by using more or less
solute or solvent, but in any case the molarity of solution is the number of moles of solute per liter of solution.
The abbreviation for molarity is M.
Molarity = number of moles of solute/ number of liters of solution,
Or using abbreviations: M = mol / L
It can be calculated dividing the amount of solute in moles (mol) by the volume of solution in liters (L):
For example, if we have 4 moles of NaCl in 2 liters of solution, the molar concentration will be
4 mol / 2 L = 2 M (= mol/L).
To remind you, one mole of a compound is numerically equal to the sum of atomic masses of all elements
making up that particular compound.
A concentration lower than 1M can be expressed using prefixes which mean:
milli- = 10-3; 1 mmol = 1 10-3 mol; 1mM = 1 10-3 M (mol/L)
micro- = 10-6; 1 mol = 1 10-6 mol; 1M = 1 10-6 M (mol/L)
nano- = 10-9; 1 nmol = 1 10-9 mol; 1nM = 1 10-9 M (mol/L)
pico- = 10-12; 1 pmol = 1 10-12 mol; 1 pM = 1 10-12 M (mol/L)
Here are some examples of calculations using molar concentration:

Example 1.
What is the molarity of NaOH solution, if it contains 16.0 grams of NaOH in 2 liters of water solution?
Molecular mass of NaOH = 23 + 16 + 1 = 40, thus, 1 mol of NaOH = 40 g
Number of moles of NaOH = grams of NaOH in solution / grams of 1 mol = 16 g / 40 g = 0.4 mol
Molarity of this solution = number of moles of solute / volume of solution =
= 0.4 mol / 2 L = 0.2 mol/L= 0.2 M
Example 2.
24.5 mL of 1.5 M NaOH is added to the 20.5 mL of 0.85 M NaOH.
What is the molarity of the final solution?
It is equal to the amount of NaOH in moles in the initial portions of both solutions divided by the final volume
of the mixture (in liters!)
moles of NaOH in the first solution:
in (1 liter) 1000 mL there is -- 1.5 mol NaOH
in 24.5 mL -- x mol
x = 24.5 mL  1.5 mol / 1000 mL = 0.0368 mol
moles of NaOH in the second solution:
in 1000 mL -- 0.85 mol
in 20.5 mL -- x
x = 20.5 mL  0.85 mol / 1000 mL = 0.0174 mol
the total number of moles of NaOH in both solutions = 0.0368 + 0.0174 = 0.0542 mol
the final volume of the solution in liters = 24.5 mL + 20.5 mL = 45 mL = 0.045 L
the molarity of NaOH in the final solution:
there is 0.0542 mol NaOH in 0.045 L of solution
x -- in 1 L
x = 0.0542 mol  1L / 0.045 L = 1.2 M
The following example is of calculation is used in everyday lab work. Solutions usually are stored as stock-
solutions of relatively high concentration (they are more stable), and working solutions are made from them by
an appropriate dilution with water or other solvent. Such dilution is based on the fact that the number of moles
of solute does not change during dilution:
moles of solute = molar concentration  volume of solution; thus:
Mbefore dilution  Vbefore dilution = Mafter dilution  Vafter dilution
Example 3.
What volume of 1M KNO3 has to be diluted with water in order to get 250 mL of 0.2 M KNO3?
Put the numbers given into the equation above:
1 M  x = 0.2 M  0.250 L
then x = 0.2 M  0.250 L / 1 M = 0.05 L = 50 mL
Experimental: Prepare 250 ml of CaCl2 solution in water of following concentration:
A 0.5M
B 0.8M
C 0.9M
D 1.3M
E 1.5M
F 1.2M
Find the percent concentration (w/w and w/v) of this solution.
Procedure:
1. Calculate the amount of the salt needed.
Example: Prepare 250 ml of 2M CaCl2 solution in water. Calculate its % (weight/volume) concentration.
2.0 M CaCl2 means that in 1 liter (1000 ml) there are needed 2.0 moles of CaCl2;
then in 250 ml there should be X moles of CaCl2
X = 250  2.0/ 1000 = 0.5 moles CaCl2 is needed for making 250 ml of 2.0M solution.
1 mol of CaCl2 amounts to 40 + 2×35.5 = 111 g;

0.5 moles is Xg
X = 0.5  111/ 1 = 55.5 g
So, to make the required solution we need 55.5 g of CaCl2
2. Weigh a salt on a piece of paper, and then transfer it into the flask.
3. Use a graduated cylinder to measure approximately 150-200 ml of water.
4. Pour the water into the flask; dissolve all the salt by stirring with a magnetic stirrer.
5. Transfer the solution of salt back into the cylinder. Adjust the volume of the solution to 250 ml. Now you
have the right amount of salt in the required volume of solution. However, the solution is not yet homogenous –
you may notice disturbances and flows in the cylinder, indicating zones of various concentrations.
6. Transfer the solution to the flask and mix it. The solution is ready.
7. Calculate % (weight/volume) concentration of this solution:
Example: Solution has 55.5 g of salt in 250 ml
Xg in 100 ml
X = 55.5  100/ 250 = 22.2 % (w/vol) concentration
8. Determine the % (weight/weight) concentration:
Transfer the solution into the graduated cylinder and measure its density with an aerometer. Find the real %
(w/w) concentration to which the density of the obtained solution corresponds.
General questions
1. What is the mole?
2. How many elementary units does the mole contain?
3. What kind of concentration is called molar concentration (molarity)?
4. What is the dilution of solution?
5. What principal works when solutions are diluted?
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Laboratory work N 2
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Conclusion
3.Volumetric analysis. Acid-base titration

Background. In chemical analysis, it is often necessary to determine the concentration of ions in
solution. For this purpose, a technique called titration is often used. It is based on the measurement of the
volume of one reactant required to react with a measured mass or volume of another reactant in the
chemical reaction. For this, a standard solution of a titrant (a reactant of known concentration) is added
drop by drop to the measured volume of the solution of unknown concentration until the reaction is
stoichiometrically complete. The task of titration is to determine what volume exactly of the titrant is
needed to complete the reaction. The reaction is completed when all the particles of the substance have
reacted with the particles of the titrant – this is called reaction endpoint or equivalence point. To make
this point visible we use indicators. An acid-base indicator is a weak acid or a weak base. The
undissociated form of the indicator has a different colour from its ionic form.
Ind-H <-------> H+ + Ind-
(colour (1) <-----> different colour (2))
The colour change occurs over a range of hydrogen ion concentrations. This range is called colour change
interval and is expressed as a pH range.
Examples of the most widely used indicators:
Methylorange -- red (pH 3.2) <<>> yellow (pH 4.4)
Phenolphtalein -- colourless (pH 8.2) <<>> pink (pH 10.0)
Problem. Determine the amount of NaOH in solution of unknown concentration by titration with
0.1 M HCl.
Procedure
Reaction which will take place NaOH + HCl ----> NaCl + H2O
To neutralize 1 mol of NaOH needs 1 mol of HCl
1. Fill the burette with 0.1 M HCl (titrant). Burette is a long tube, graduated
in mL and tenths of mL, at the bottom it has a stopper which allows
dripping of the solution. Mark the initial volume of the solution in the
burette.
2. Dilute the sample. Take a volumetric flask X with NaOH solution of
unknown concentration (provided by the technician), and add distilled
water up to the 100 mL mark. Close the flask and mix the contents
thoroughly by turning over several times.
3. Pipette exactly 10 mL of diluted NaOH solution into a 100 ml
Erlenmeyer flask; add 2 drops of phenolphthalein indicator. The solution
colours pink.
4. Put a magnetic bar in the flask and place it on the magnetic stirrer. While
opening the stopper of the burette with one hand, slowly add HCl acid into
the reaction flask. If titrating by hand swirl flask contents with another hand
continuously. Near the endpoint slow the rate of addition to drops; the last
few drops should be added at 3 -5 second intervals.
5. Titrate until the colourless endpoint, which indicates that the
neutralization reaction is complete. You need to catch the first moment of
the colour change otherwise there would be too much titrant added. Record
the final volume of the burette. The difference between the initial and final volumes is the volume of HCl
required neutralizing all NaOH in the investigated sample.
6. Repeat the titration once more. If the second measurement is close to the first, go to step 7, if not -
titrate for the third time. Find the average of the closest two measuring.
7. Calculate the concentration of the given NaOH solution from the obtained titration data:
-- reaction is completed when number of HCl (acid) moles added is equal to the number of
NaOH (base) moles in the flask:
molesNaOH = molesHCl ;
moles = Molarity  Volume
Molarity NaOH  VolumeNaOH = Molarity HCl  VolumeHCl
Molarity NaOH = Molarity HCl  VolumeHCl / VolumeNaOH
8. Calculate the amount of NaOH in the flask X in grams.
General questions
1. What is the method of volumetric analysis based on?

2. What is the method of volumetric analysis (titration) used for?
3. What solution is called “titrant”?
4. What process is called “titration”?
5. What is the equivalence point and what is its pH?
6. What substances are acid-base indicators? How to choose a suitable one?
7. What is the colour change interval of the indicator?
8. At what pH does Methylorange change its colour?
9. What colour does Phenolphtalein have when the pH is less than 8.2, and the pH is higher
than 10?
10. What is the “titration curve”?
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Laboratory work N 3
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Conclusion
4. Buffer solutions
Theory: Solutions of substances (usually in water) which can resist against changes of pH are called
buffer solutions or buffer systems. The buffer solutions are made from a weak acid and its salt with a
strong base (as the acetic acid/sodium acetate buffer) or a weak base and its salt with a strong acid (as
ammonium hydroxide/ammonium chloride buffer). As dissociation of weak acids or weak basis is
extremely low, amounts of conjugated basis of conjugated acids are also low.
Preparations of buffers solutions.
1. Selected weak acid is mixed with a salt containing common ion in the structure. This common ion
acts as a base conjugated to selected acid. For example, acetic acid (CH 3-COOH) and sodium
acetate (CH3-COONa) have acetate (CH3-COO-) as a common ion. Wen acetic acid solution is
mixed with sodium acetate solution, obtained buffer is known as acetate buffer.
2. Salts of different acidities can also be used for preparation of buffers. For example, NaH 2PO4 (acts
as an acid) and Na2HPO4 (acts as a base), which dissociation results in ions of different acidities:
NaH2PO4  Na+ + H2PO4-
Na2HPO4  2Na+ + HPO42- (base)
H2PO4- ion contains 2H+ ions and acts as a weak acid, HPO 42- ion has a single H+ therefore it acts
as a base conjugated to H2PO4-.
pH of buffer solutions.
Let consider a case when buffer comes from an acid HA and a salt of the acid MeA. In water, an acid
undergoes ionisation by this equation (for simplicity take dissociation):
HA  H+ +A-
Constant of acidity for such dissociation is described by an equation:
[H+]x[A-]
Ka=
[HA]
Concentration of protons (H+ ions) can be calculated from re-arranged equation:

[HA]
H+ = Ka x
[A-]
If we add salt in the solution of such acid, dissociation of the acid will be depressed by increased amounts
of the ion A-. It means, that concentration of non-dissociated acid [HA] will be equal to concentration of
the acid added at the beginning. Therefore H + concentration in such acidic buffer can be calculated by the
formula:
[acid]
H+ = Ka x [salt]
We can calculate pH of such a buffer solution using Henderson-Hasselbalch equation:
[salt]
pH= -lg[H+]= lg - lgKa
[acid]
We can also prepare a buffer solution if we mix different volumes of solutions of an acid and a base. In
this case Henderson-Hasselbalch equation is:
[salt] x Vsalt
pH= -lg[H+]= lg
[acid] x Vacid - lgKa
In Henderson-Hasselbalch equation, Vsalt is a volume of salt solution

Vacid is a volume of acid solution
[salt] is a concentration of salt solution taken for preparation of a buffer
[acid] is a concentration of acid solution taken for preparation of a
buffer
Mechanism of buffer action. H+ concentration and pH of buffer solution do not change (or change very
little) when acid or base is added to them. This phenomenon comes from interaction of buffer components
with added acid or base. Suppose we add HCl (strong hydrochloric acid) into acetate buffer (composition
was considered above):
CH3-COONa + HCl CH3-COOH + NaCl

CH3-COO- + H+  CH3-COOH
As CH3-COOH (acetic acid) undergoes very low dissociation, pH is little changed. Little change of pH
comes from alterations in the ratio between salt and acid amounts.
If a strong base is added to acetate buffer, it combines with acidic component of the buffer:
CH3-COOH + NaOH CH3-COONa + H2O

CH3-COOH + OH- CH3-COO-+ H2O
This example demonstrates increasing in salt amount in the buffer solution.
Buffer capacity. A buffer solution can keep pH stable only up to a certain amount of acid or base added.
After reaching this threshold pH of a buffer solution changes if extra acid/base is added, as it does in the
case of regular solutions. Therefore, every buffer solution is characterized by the buffer capacity (B)
which indicates how many moles of a strong acid or base should be added to 1 litre of the buffer in order
to change its pH by 1 pH unit:
B = C / (pHafter addition of acid or base - pHinitial); where B - buffer capacity, C - acid or base concentration
mol/L
The buffer capacity depends on the nature and concentration of the buffer components as well as on
the ratio of these concentrations:
- The buffer capacity increases when the concentration of buffer components increases;
- The capacity for both acid and base is highest in a buffer where ratio of concentrations equals 1.
Experimental
Prepare acetate/sodium acetate buffer. Calculate its pH and buffer capacity. Each pair of students has to
prepare and analyse only one buffer solution.
Procedure
1. Take 2 flasks and, mixing the appropriate volumes of two components -- 0.1 M acetic acid and
0.1 M sodium acetate in each flask prepare 2 identical acetic buffers (one - for determining buffer
capacity for acid, and the other - buffer capacity for base). The volumes are indicated in the table.
Draw the table in your notebook and fill it in with the data of your own experiment and the
experiments of other students:
Vbuffer=VCH3COOH pH pH V (titrant) Buffer

+VCH3COO-Na capacity
=10 ml
N CH3COOH, CH3COONa, Initial After After pHacid pHbase Vacid Vbase Bacid Bbase
mL mL pHi titration titration
with with
acid base
1 2 8 3.4 6.3
2 3 7 3.4 6.3
3 4 6 3.4 6.3
4 5 5 3.4 6.3
5 6 4 3.4 6.3
6 7 3 3.4 6.3
2. Calculate the initial pH of the prepared buffer using Henderson-Hasselbalch equation:

pH = lg ([salt] / [acid]) - lg Ka;
Ka - ionization constant for the acetic acid, Ka= 1.85  10-5, and pKa=-lg (1.85  10-5) = 4.7.
Since the concentrations of components in the buffer are equal, the volumes in mL (used in preparation of
the buffer) can be used instead of the concentration:
pH = lg ( volume of salt / volume of acid ) + 4.7
2. Add 3--4 drops of methyl-orange indicator to one flask. Titrate this flask with 0.1 M HCl until the
pink-orange colour appears (indicating pH = 3.4 – that is pH after titration with acid). Use the
titration volume of HCl (Vacid) to calculate the buffer capacity for acid Bacid:
Bacid = (Vacid  Cacid / pHacid  Vbuffer)
Vbuffer - volume of buffer in ml,
pHacid - change of pH after titration with acid.
3. Add 3-4 drops of methyl-red indicator to the second flask. Titrate this flask with 0.1 M NaOH
until the yellow colour starts to appear (pH = 6.3 – the pH after titration with base). Use the
titration volume of NaOH (Vbase) to calculate the buffer capacity for base Bbase:
Bbase = (Vbase  Cbase / pHbase  Vbuffer)
pHbase -- change of pH after titration with base.
5. Copy the results of other students who were analysing different buffers.
6. Draw the graphs of Bacid and Bbase dependence on pH initial. The best buffer capacity for both acid and
base you should get around pKa value (pH=4.7).
General questions
1. What solutions are called “buffers”?

2. How are buffer solutions prepared?
3. What does the pH of buffer solutions depend on?
4. What equation is used to calculate the buffer pH?
5. How does acetic acid/acetate buffer maintain solution pH at almost the same level if a small
amount of strong acid is added? Write the equations.
6. How does acetic acid/acetate buffer maintain solution pH at almost the same level if a small
amount of strong base is added? Write the equations.
7. What is called the buffer capacity?
8. How can buffer capacity for acid be calculated? Write the formula.
9. How can buffer capacity for base be calculated? Write the formula.
10. What does buffer capacity depend on?
11. Why do biological fluids have a stable pH?
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Laboratory work N 4
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Conclusion
5. Colloidal solutions
Background. Colloidal solutions are disperse systems where disperse phase particles are 1-100 nm in
size. Such solutions are stable over time. The solution where disperse phase is solid and medium is liquid
is called sol. To prepare a colloidal solution, the particles of disperse phase have to be made of the size of
colloidal particles. There are two ways to do this: condensation (to make the colloidal size particles from
smaller ones) and dispersion (to disperse larger particles into colloidal size particles).
Dispersion methods:
- Colloidal mill (a mechanical way to grind large particles);
- Ultrasound;
- Peptization (disaggregates large particles by chemical agents, which increase repulsion of
particles).
Condensation methods (produce compounds of relatively low solubility from soluble ones):
- Exchange of solvent (by another solvent in which the same substance has lower solubility);
-Oxidation (obtains neutral chemical elements (mainly non-metals) from their ionic forms);
- Reduction (obtains neutral elements (mainly metals) from their ionic forms);
- Hydrolysis reaction (makes compounds of lower solubility);
-Exchange reactions (makes insoluble compounds).
Exchange reaction: AgNO3(aq) + KCl(aq) → AgCl↓(s) + KNO3(aq).
Micelle when it is excess of AgNO3: {m(AgCl)nAg+(n-x)NO3-}x+NO3- .

Micelle when it is excess of KCl : {m(AgCl) nCl-(n-x)K+}x-K+.
Experimental
A. Prepare colloidal Fe(OH)3 solution (sol) by chemical condensation (hydrolysis reaction):
1. Put 1 mL of 2% FeCl3 into a test tube, add 10 mL of distilled water.
2. Mix thoroughly and boil the mixture until brown transparent Fe(OH)3 sol. is formed.
3. Write the hydrolysis reaction and micelle of Fe(OH)3.
Reaction of hydrolysis: FeCl3 + H2O→……………………………………………………………..
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Micelle: {m(Fe(OH)3●●●●●. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Preparation of colloidal colophony (pine resin) sol by the exchange of solvent
1. Add several drops of 2% colophony/ ethanol solution to 10 mL of distilled water.
2. Mix the mixture. Milky sol should be obtained.
3. Explain, how and why this sol was formed.
C. Stability and coagulation of colloidal solutions.

The coagulation of colloidal solutions usually occurs when an electrolyte solution is added to it.
The coagulation means that the particles became larger (more than 100 nm), and these newly
formed particles easily sediment.
Prepare 5 test tubes for each electrolyte (one pair of students experiments with one electrolyte),
and fill them accordingly to the table:
Number of the test tube 1 2 3 4 5

Fe(OH)3 sol (prepared by the technician), mL 5 5 5 5 5
H2O, mL 4.5 4 3 2 1
Electrolyte, mL
1) 3 M KCl 0.5 1 2 3 4
2) 0.01 M K2SO4 0.5 1 2 3 4
3) 0.001 M K3[Fe(CN)6] 0.5 1 2 3 4
Leave the test tubes for 30 min, and then observe where the colloidal solution has coagulated. The
test tube with the lowest concentration of electrolyte where coagulation was observed has
concentration called coagulation threshold.
Coagulation threshold is the lowest electrolyte concentration (mmol/L) which induces
coagulation of the sol. Calculate the coagulation thresholds:
C electrolyte (mmol / L) xVelectrolyte

C thr   (mmol / L)
Velectrolyte  Vsol  Vwater
1
The opposite number of the coagulation threshold is called the coagulation power: P  .
C thr
Write the results in the table given below. Discuss the results with other students who have done the
experiment with different electrolytes. Make a conclusion of which electrolyte is a better coagulation
agent and explain why.
Electrolyte Coagulating ion Coagulation threshold Coagulation power
KCl
K2SO4
K3[Fe(CN)6]
General questions
1. What is called the disperse phase and the dispersion medium?

2. What solutions are called “colloidal solutions”?
3. What systems are called “sol”?
4. Provide some examples of disperse systems in living organisms.
5. What properties should chemical substances have, so that they can be used to prepare
colloidal solutions?
6. What methods of colloid preparation are there?
7. What method of preparation of colloids is called peptization?
8. What is micelle and what is it made of?
9. What is called coagulation, coagulation threshold and coagulation power?
10. How can electrolytes cause coagulation?
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Conclusion
11.
6. High molecular mass compounds and their properties.

Swelling and gelatinization of high-molecular mass compounds
Background. High-molecular mass compounds are organic compounds with molecular mass as
big as hundreds of thousands or even millions of Daltons. By structural features, they usually are
polymers two origins:
1. Cellular origin high-molecular mass compounds are called biopolymers, e.g. nucleic acids,
proteins and polysaccharides.
2. Synthetic/artificial high-molecular mass compounds.
Sizes of particles of high molecular mass compounds are similar to colloidal particles, but they are water-
soluble. Therefore solutions of high molecular mass compounds share some common properties with
colloidal solutions and with real solutions. They also have very specific properties.
Properties of solutions of biopolymers:
1. Properties that are common with colloidal solutions:
 Size of molecules of biomolecules is similar to the size of colloidal particles.
 Solutions of biopolymers have slow rate of diffusion.
 Molecules of biopolymers cannot pass through semi-permeable membrane, what implies that their
solutions have low osmotic pressure. The osmotic pressure of those solutions depends only on number
of biopolymer molecules.
2. Properties of biopolymer solutions, that are common with those of real solutions:
 Do not form micelles, i.e. they are solutions of a single phase (homogeneous systems).
 Solutions of biopolymers with linear structure do not show Tindall effect.
 Solutions of biopolymers are stable systems. They do not show sedimentation phenomenon.
3. Specific properties of biopolymer solutions.
 Biopolymers swell before dissolving. Under swelling solvent surrounds the molecule of biopolymer,
then it moves into the empty spaces of molecule, so that the volume of the molecule increases.
Swelling of high-molecular mass compounds. Swelling of a high-molecular mass compound results in

increasing of both mass and volume of a high-molecular mass compound. There are two sorts of swelling:
the limited swelling and unlimited swelling where a solution of high-molecular mass compounds is
formed. Swelling depends on the properties of the solvent.
The swelling degree (Q) is the main characteristic of swelling. It shows how much the volume of
the high molecular mass compound enlarges during the swelling. The swelling degree shows how many
cm3 of the solvent can be absorbed by 1 cm3 of a high-molecular mass compound:
Q = V2 – V1
V1
V1 is the volume of a high-molecular mass compound before swelling,

V2 is the volume of a high-molecular mass compound after swelling.
The swelling degree (Q) can be estimated by weighing of a high molecular mass compound before its
swelling and after swelling.
The swelling is affected by:
1. Temperature. As swelling is an exothermic process, increasing in temperature results in decreasing of

the swelling degree.
2. Pressure. Increasing in pressure results in increasing of the swelling degree.
3. Electrolytes and pH of solution. Swelling is little at isoelectric point, at which molecules can stick
together and entering of water is slow.
4. Degree of dispersion. High dispersion degree favours to swelling.
5. Time.
Jellification (gelatinization). Solutions of high-molecular mass compounds can form a jelly. It is a net-
like structure formed in the solution when hydrophobic regions of the molecules interact with each other
and the hydrophilic regions are highly hydrated, water molecules filling in the gaps between the
molecules. In chemistry, the jelly is called gel. The process when solutions of high-molecular mass
compounds lose their fluidity is called jellification. It depends on the following factors:
1. Concentration of high-molecular mass compound solutions. Jellification can occur in concentrated
solutions of high-molecular mass compounds.
2. Size and shape of molecules of a high-molecular mass compound. Thread-like molecules can easily
form gel, but the ball-like ones can hardly do this.
3. Temperature. Low temperatures favour to jellification.
4. Presence of electrolytes and pH. Anions are the most important for the jellification. According to the
efficiency of the effect, the anions make a line:
SO42- >citrate>CH3-COO- >Cl- >NO3- ->Br- >I- >SCN-
Jellification is promoted by highly hydrated ions. Starting with Cl - the anions diminish the jellification
(gelatinization). These ions are adsorbed on the surface of the macromolecules. They give charge to
polymers and prevent macromolecules from jellification.
Lab. Procedure
Effect of solvent on swelling.
1. Take 4 test tubes. Put 2ml of water in the tubes N1 and N2. Put 2 ml of benzene into the tubes N3 and
N4.
2. Add one piece of agar (polysaccharide) into the tube N1 and another piece of the same size into the
tube N3.
3. Add one piece of synthetic rubber into the tube N2 and another piece of the same size into the tube N4.
4. After 20 min., compare the sizes of the agar and rubber. Make a conclusion about the effect of solvents
on the swelling.
Effect of electrolytes on jellification (gelatinization)
1. Take 6 test tubes and put 1.5 ml of 1 M solutions of the electrolytes as indicated in the table:
Number of test tube 1 2 3 4 5 6
Electrolyte K2SO4 CH3COOK KCl KI KSCN H2O
Beginning of gelatinization t1
End of gelatinization t2
Time required for complete
gelatinization t2 - t1
2. Add 1.5 ml of 6% hot gelatine solution into each test tube and mix thoroughly.
3. Place the test tubes into the hot water bath (50 - 60º C) for 10 minutes.
4. Remove the test tubes from the bath and place them in cold water. Mark the time t1.
5. Periodically check the fluidity of the solution by inclining the test tubes. If you observe that solution is
no longer fluid, mark the end time of gelatinization.
6. Fill in the table. Make a conclusion about which ions favour gelatinization and which do not.
General questions
1. What substances are called the high molecular mass compounds (HMMC)? Give
some examples.
2. How are the solutions of HMMC similar to the colloidal solutions?
3. How are the solutions of HMMC similar to the real solutions?
4. What properties are specific to the high molecular mass compounds?
5. What process is called swelling?
6. What effect on the swelling does the solvent have?
7. What does the swelling degree show?
8. What process is called the jellification?
9. How does the gel form and what can affect its formation?
10. What effect do the electrolytes have on jellification?
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Laboratory work N 6
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Conclusion
7. Coordination compound based determination of water hardness

Background. Some amino-polycarbonic acids and their salts can form stable coordination compounds with most
of the cations. These compounds are called complexones (chelating agents). An example of such complexones is
EDTA (ethylendiaminetetraacetic acid):
Most often the disodium salt of EDTA is used, which has a trivial name of Trilon B. This compound reacts with
the divalent ions (such as Ca2+, Mg2+, Ba2+) forming a rather stable colourless coordination compound. One
molecule of Trilon B binds one ion:
Na2[H2EDTA] + Ca2+ <-----> Na2[CaEDTA] + 2H +

[H2EDTA] 2- + Ca2+ <-----> [CaEDTA] 2- + 2H +
The term “total water hardness” means a total amount of Ca2+ and Mg2+ ions dissolved in water. Due to the ability
to bind these ions and to form a coordination compound Trilon B is used for the quantitative determination of these
ions measuring the water hardness. Since the compound of Trilon B with Ca2+ and Mg2+ ions is colourless, an
indicator is needed to pinpoint the equivalence point. The most suitable for this purpose is Eriochrom Black T (for
convenience abbreviated as H3Ind). Depending on pH this indicator has different colours: pH 6 – red;
pH 7-11 – blue; pH 12 – yellow-orange
H3Ind ↔ H2Ind- + H+;
pH = 6
red
H2Ind- ↔ HInd2- + H+;

pH = 7-11
blue
HInd2- ↔ Ind3- + H+;

pH = 12
yellow-orange
Eriochrom Black T forms a red-violet coordination compound with Ca 2+ and Mg2+ (at pH>7), which is less stable
than the one formed by Trilon B. While titrated with Trilon B, all Ca 2+ and Mg2+ ions from the solution are
coordinated by it, then this red-violet compound decomposes and more stable compound of Ca 2+ and Mg2+ with
Trilon B forms. The free anions of Eriochrom Black T make solution blue-coloured at the equivalence point:
[MgInd] - + [H2EDTA]2- ------> [MgEDTA]2- + HInd2- + H+

Red-violet colourless colourless blue
Water hardness values:
< 0.75 mmol/L – soft water
1.5 – 2.7 mmol/L – medium soft/hard water
2.7 – 5.35 mmol/L – hard water
> 5.35 mmol/L – very hard water
Examples: rainwater – 0.05 mmol/ L; sea water – 65 mmol/ L.
Experimental
Procedure
1. Dilute the given solution X (solution of MgCl2 whose hardness has to be determined) to the 100 ml in
a volumetric flask (add distilled water up to 100 ml)
2. Put 10 mL of this diluted solution X into the flask, add 2 mL of the buffer solution (1 M
NH4Cl/NH4OH) and 4 drops of the indicator –Eriochrom Black T. Mix well.
3. Titrate the mixture with the solution of 0.05 M Trilon B (mix well during titration) until the red colour
starts changing into violet (blue).
4. The titration is over when after one single drop of Trilon B the solution becomes blue (You need to
catch the first moment of the change in the initial colour). Therefore the last portion of Trilon B should be
added drop-by-drop. Record the volume (mL) of Trilon B used for titration.
5. Repeat the titration three times, and take the mean volume of the three titrations.
6. Calculate the total water hardness. One mole of Trilon B can bind one mole of Ca 2+ or/and Mg2+ ions.
Use the equation:
HT = (MB  VB / Vwater )  1000 mmol/L,
where HT -- total water hardness in mmol/L;
MB – molarity of the Trilon B solution;
VB – volume of the Trilon B used for titration;
Vwater – volume of water used for titration.
7. Calculate how many milligrams of MgCl2 are in your X solution (in 100 ml of solution).
General questions
1. What compounds are called coordination compounds? Give some examples.

2. What compounds are called complexones (chelating agents)? Give some examples.
3. Draw the formulas of EDTA and disodium salt of EDTA.
4. What is the trivial name of the disodium salt of EDTA?
5. What does the term “total water hardness” mean?
6. Why is the indicator Eriochrom Black T used when total water hardness is determined?
7. Write down the equation of the reaction between Eriochrom Black T, Trilon B and metal ions.
Explain why the colour of the solution changes at the equivalence point.
8. Write down the formula which is used to calculate the total water hardness.
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Laboratory work N 7
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Conclusion
9. Chemical kinetics
Experimental. A. Rate of chemical reaction determined by change of reaction product concentration.

This is the reaction of sodium thiosulphate decomposition:
Na2S2O3 + H2SO4 -------> Na2SO4 + S + SO2 + H2O
One of the products is insoluble in water – it is elementary sulphur S. The increased turbidity of the
reaction mixture provides a pretty good way to evaluate the reaction rate. The increased turbidity means
that more sulphur was formed, and the sooner this process occurs the higher is the rate of the above
mentioned reaction.
Procedure
1. Take a small beaker and fill it according to the table (draw it in your notebook) as N 1.
As it is obvious from the table’s second column, the following flasks will have
increasing sodium thiosulphate concentrations, as the total volume in each of them will
be the same:
Number 0.25 M H2O, Time, Rate of

Na2S2O3; mL s (seconds) reaction,
mL 1/s
N1 2 4
N2 4 2
N3 6 0
2. Add 2 mL of 0.25 M H 2SO4, (use a little graduated cylinder for that, not a pipette), stir
carefully with a glass rod. Start time countdown from the moment of adding the acid. Immediately put
this flask on the piece of paper lined in squares. While slowly stirring the contents of the flask, observe
when the lines first become obscured by the layer of reaction mixture. Record the time elapsed from the
moment of H2SO4 addition. Calculate the reaction rate.
3. Wash the beaker carefully and repeat the same procedure with the other concentrations of
sodium thiosulphate N 2 and N 3.
4. Draw the graph of the dependence of the time needed for the reaction (in seconds) on the
volume of substrate added (in mL), (mL should be on the X axis, and seconds -on the Y axis).
B. Effect of catalyst on the reaction rate

Catalyst is the substance which changes the reaction rate when present in the reaction mixture,
but remains unaltered itself. The reaction rate changes because catalyst decreases the activation energy of
that particular reaction. This is the reaction of hydrogen peroxide decomposition:
2 H2O2 ------> 2 H2O + 2 [O], then
2[O] -------> O2
The catalyst for this reaction is MnO 2. In living organisms this reaction is catalysed by the enzyme
catalase. Decomposition of hydrogen peroxide at the room temperature occurs very slowly, and the
bubbles of formed oxygen are not visible. When MnO2 is added, the reaction rate increases significantly,
and this can be noticed by vigorous bubbling.
Procedure
Put 1 mL of 3% H2O2 solution into the test tube. There are no bubbles. Add a little of MnO 2 powder and
cover the test tube with your finger while the reaction proceeds. At that time get a wooden splint burning
and blow the flame out. Stick a glowing wooden splint inside the test tube. Observe what happens and
explain the result.
C. Equilibrium shift.
A chemical reaction consists of direct and reverse reactions, going on in opposite directions. The
term “Equilibrium of chemical reaction” means that equilibrium between the direct and reverse reaction
has been reached, and the rate of direct reaction is equal to the rate of reverse reaction. Equilibrium of the
chemical reaction can be disturbed by variety of factors among which is change of:
1) Concentration of reactants or reaction products;
2) Temperature;
3) Pressure;
4) Volume of the system.
In living organisms the main factor affecting equilibrium of chemical reaction is the change of substrate
or product concentration, since temperature, pressure and volume are kept constant. The effect of various
factors on the equilibrium of chemical reaction is described by Le Chatelier’s principle:
If a system in equilibrium has been disturbed by the outside factors (as concentration,
temperature or pressure) then the equilibrium of this system shifts in the direction which tends to
decrease the effect of outside factors.
I.
CoCl2 dissociates when dissolved in water:

CoCl2 ↔ Co2+ + 2Cl-
Then the ions formed make a coordination compound where Co (II) has additional bonds with water:
Co2+ + 2 Cl- + 6 H2O ↔ [Co (H2O)6]Cl 2 ↔ 2 Cl- + [Co(H2O)6]2+ (red colour).
When HCl is added, the red-coloured coordination compound transforms into another blue-coloured:
(Red colour) [Co (H2O)6]2+ + 2 Cl- + 2 Cl- ↔ 6 H2O + [CoCl4]2- (blue colour)
Keq = [[CoCl4] 2-] [H2O]6 /[ [Co(H2O)] 2+]  [Cl-]4
When HCl is added, the equilibrium of the reaction shifts towards formation of [CoCl4]2-. If extra water is
added the equilibrium shifts in the opposite direction.
Procedure
1. Dissolve a crystal of CoCl2 in a test tube with few drops of water.
2. Add several drops of concentrated HCl until the solution becomes blue.
3. Dilute this solution with water until reappearance of less intense red colour.
4. By heating and cooling the test tube find what is the effect of temperature on this equilibrium.
II.
FeCl3 reacts with NH4SCN and red solution of the iron rhodanide forms:
FeCl3 + 3NH4SCN ↔ Fe(SCN)3+ 3NH4Cl.
This reaction is a reversible one. The shift of the equilibrium could be seen by observing the intensity of
the red colour in the solution. The colour intensity depends on the concentration of the iron rhodanide –
Fe(SCN)3. When the concentration of the Fe(SCN)3 in the solution is high, the red colour of solution is
intense. When the concentration of the Fe(SCN)3 is low the solution is faded red. It is possible to shift
the equilibrium by adding more reactants or products.
Procedure
1. Pour into a beaker 20 mL of water and add 3 drops of the saturated solution of the FeCl3 and 3
drops of the saturated solution of the NH4SCN.
2. Divide the solution into four test tubes.
3. Add 2 drops of the FeCl3 into the first test tube.
4. Add 2 drops of the NH4SCN into the second test tube.
5. Add some crystals of the NH4C l into the third test tube.
6. Stir the tubes and compare colour intensity in these test tubes.
7. Describe and explain the observed differences.
Calculation of Michaelis-Menten constant and maximal reaction rate
Michaelis-Menten equation describes the relationship between reaction rate and substrate concentration in
enzyme-catalysed reaction.
V  [S]
V0  max
K m  [S]
Vmax is the maximum velocity of the reaction – when all enzyme molecules are fully active. K m –
Michaelis-Menten constant – is the concentration of substrate at Vmax/2.
Task: calculate Michaelis-Menten constant and maximal reaction rate for the given enzyme-catalysed
reactions.
Procedure:
amount of product formed
1. Calculate reaction rates (v) with different substrate concentrations. v
reaction time
mol/min.
2. Plot 1/[S] on x axis and 1/v – on y axis.
3. Find the Km and Vmax values for the given reactions from Lineweaver-Burk plot.
Version A. Assay conditions were following:

1 mg of the enzyme was incubated with the indicated concentration of substrate [S] for 5 minutes, after which the
amount of formed product was determined and is presented in the table:
Substrate concentration (μM) 0.63 0.83 1.25 3.3
Amount of product formed per 5 min (μmol) 29 36.2 47.2 76.9
Version B. Assay conditions were following:

1 mg of the enzyme was incubated with indicated concentration of substrate [S] for 5 minutes,
after which the amount of formed product was determined and is presented in the table:
Amount of product formed per 5 min (μmol) 37 43.5 53 62
Version C. Assay conditions were following:

1 mg of the enzyme was incubated with indicated concentration of substrate [S] for 5 minutes, after
which the amount of formed product was determined and is presented in the table:
Amount of product formed per 5 min (μmol) 116 147 200 250
Calculation of Km and Vmax values

Lineweaver-Burk Plot (1)
0.5
0.4
0.3
1/v
0.2
0.1
-2.0 -1.5 -1.0 -0.5 0.5 1.0 1.5 2.0
-0.1
1/[S]
0.5
0.4
0.3
1/v
0.2
0.1
-2.0 -1.5 -1.0 -0.5 0.5 1.0 1.5 2.0
-0.1
1/[S]
0.5
0.4
0.3
1/v
0.2
0.1
-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.5 1.0 1.5 2.0 2.5 3.0
-0.1
1/[S]
General questions
1. What does chemical kinetics study?

2. How is the rate of chemical reaction expressed?
3. What factors can make an impact on the rate of chemical reaction?
4. How does the reaction rate depend on the concentrations of reactants?
5. How is it possible to determine the reaction rate of this reaction?
Na2S2O3 + H2SO4---> Na2SO4 + S+ SO2 + H2O
6. What does a catalyst do when it is present in a reaction mixture? How does it work?
7. Which catalyst can catalyze the reaction of hydrogen peroxide decomposition?
8. What does “the equilibrium of chemical reaction” mean?
9. What factors can influence the equilibrium of chemical reaction?
10. Describe “the Le Chatelier’s principle”.
11. Why and how can the CoCl2 solution change its colour?
12. What factor determines the intensity of colour of iron rhodonide solution?
13. What does the Michaelis-Menten equation describe? Draw this equation.
14. What does „the Lineweaver-Burk plot“ mean? Draw this plot.
15. How could the Michaelis-Menten constant and maximal reaction rate for the given enzyme-
catalysed reaction be calculated?
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Conclusion
10. Chemical properties of carbonyl compounds

Esters and lipids
Carbonyl compounds are organic compounds containing carbonyl (oxo) group in a molecule. The
compounds fall into two big groups – aldehydes and ketones. In aldehydes, the carbonyl group has both
hydrogen atom and hydrocarbon radical attached. In ketones, the group is bound with two hydrocarbon
radicals. According to hydrocarbon radical present, aldehydes are grouped into aliphatic ones (a),
alicyclic ones (b), and aromatic ones (c):
O O
C C
O
H H
H3C C
H
ethanal cyclohexanecarbaldehyde benzaldehyde
a b c
Ketones are also grouped into aliphatic (a), alicyclic, and aromatic.
H3C C CH3
O
propanone
(acetone)
a
Aldehydes and ketones with up to 4 C atoms in molecules are volatile liquids of specific odour. They are
soluble in water and in organic solvents. Solubility in water decreases with an increase of a number of C
atoms in a chain. Aldehydes containing 8-10 C atoms in a chain have odour of flowers and are used in
perfumery.
Chemical properties of carbonyl compounds
Properties of carbonyl compounds are determined by chemical properties of both the carbonyl group and
hydrocarbon radical.
1. Reduction producing alcohols (both aldehydes and ketones):
H2 H
H3C C CH3 H3C C CH3
O OH
propanone
(acetone)
2. Oxidation producing acids (only aldehydes):
O O
=
H-C-H H-C-OH
Formaldehyde Formic acid
In this laboratory work you will prove chemical properties of carbonyl compounds.
Experimental
A. Oxidation of aldehydes (12)

Free aldehyde groups can be oxidized to carboxyl easily by mild oxidizing agents – for example, Cu ++
in alkaline solution:
R-CHO + 2 Cu++ + 5 OH- + heating  R-COO- + Cu2O + 3H2O
Blue solution Red precipitate
Trommer’s reagent: mix of CuSO4 and NaOH;

Fehling’s reagent: copper is coordinated with sodium potassium tartrate (Fehling-I – CuSO4,
Fehling-II – basic sodium potassium tartrate)
Procedure 1: Oxidation by Trommer’s reagent

1. Take 2 test tubes and add 6 drops of 2 M NaOH, 6 drops of H 2O and 2 drops of 0.1 M CuSO4
solutions into each tube. The blue Cu (OH)2 precipitate should form in them.
2. Add a few drops of formalin (the solution of formaldehyde) to the 1st tube and a few drops of
acetone to the 2nd tube and shake.
3. Heat the upper part of the solution in the tubes over the flame until the reaction mixture changes
colour from blue (Cu++) to the yellow (CuOH) or red (Cu2O).
Procedure 2: oxidation by Fehling’s reagent
1. Take 2 test tubes; put 5 drops of formalin into the 1st tube and acetone into the 2nd tube.
2. Add 10 drops of Fehling-I and 10 drops of Fehling-II reagents into each tube.
3. Heat the upper part of the solution in the tubes over the flame until the colours of reduced
copper compounds appear.
B. Reaction of acetone with nitroprusside

Sodium nitroprusside is a coordination compound Na2[Fe(CN)5NO] which forms with acetone the
complex compound of intense red colour:
CH3-CO-CH3 + Na2[Fe(CN)5NO] + 2NaOH → Na4[Fe(CN)5NO=CH-CO-CH3] + 2H2O
Procedure:
1. Add 1 drop of 0.25M sodium nitroprusside solution, 5 drops of water and 1 drop of acetone in
the test tube.
2. Add 1 drop of 2M NaOH – solution becomes red. Add 1 drop of 2M CH 3COOH – the colour
becomes more intense – almost cherry.
C. Determination of products of lipid peroxidation

Plant oils, containing high amount of unsaturated fatty acids, during prolonged storage in the light
place at room temperature can gain bitter taste and unpleasant odour. It happens due to lipid
peroxidation. Peroxidation involves free radical formation and results in the fragmentation of
unsaturated chain, forming products with shorter carbon chain – aldehydes and acids. Reactions for
determination of peroxides in oil:
[O] (from peroxides in oil) +2KI +2CH3COOH  2CH3COOK +H2O +I2

I2 + starch  blue complex compound
The reaction of the formed iodine with sodium thiosulfate can be used to determine the degree of lipid
peroxidation in oil:
I2 + 2Na2S2O3  2NaI +Na2S4O6
Procedure:
1. Take 2 test tubes. Add 2 drops of fresh oil into the 1 st tube and add 2 drops of old rancid oil into
the 2nd tube.
2. Add 10 drops of acetic acid and chloroform mixture to the each tube, then add 5 drops of 0.5 M
KI to the each tube and shake.
3. Add 2 drops of 0.5 starch solution into each tube. Observe the colour change.
4. Take the tube with the blue solution and drop-by-drop (count the drops added) add 0.05 M
sodium thiosulfate until the blue solution becomes colourless. Record the number of thiosulfate
drops required to get the colour change.
General questions
1. What compounds are called “carbonyl compounds”? What groups of them are there?
2. What chemical properties do carbonyl compounds have?
3. Why aldehydes can react with copper sulfate, and ketones cannot?
4. What principle of chemical reaction works between aldehyde and copper sulfate (CuSO4)?
5. Draw the equation of reaction between formaldehyde and CuSO4. Explain why the colour of the
solution changes.
6. What chemical substances occurs compose Fehling’s reagent?
7. What type of reaction does occur when acetone reacts with Na2[Fe(CN)5NO]?
8. What processes do take place in plant oils during prolonged storage in a light place at room
temperature?
9. What reactions are used for the identification of peroxides in oil?
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Conclusion
11. Chemical properties of alcohols, phenols and amines
Background. Alcohols are chemical substances which contain one or more hydroxyl group (-OH). The
possession of this group provides some specific physical and chemical properties. Alcohol can form
hydrogen bonds. As a result of this they are soluble in water. The hydroxyl group gives some acidic
properties. Alcohols can react with active metals:
2CH3-CH2-OH + 2Na → 2CH3-CH2-ONa + H2
When alcohol contains more than one -OH group it can form blue coloured coordination compound with
the basic Cu(OH) 2:
2-
CH2 OH H2C O O CH2
+ Cu(OH)2 + 2 KOH H2C O
Cu
O CH2
2K+ + 4H2O
CH2 OH
blue colour water soluble
precipitate blue complex compound
The oxidation of primary alcohols results in the formation of aldehydes (1), and then aldehydes undergo
further oxidation to carboxylic acids.
[O] O [O] O
R CH2 OH R C R C
-H2O H OH
Under the oxidation of secondary alcohols, ketones are produced (2). Ketones are more resistant to
oxidation.
[O]
R CH R1 R C R1
-H2O
OH O
Alcohols can be oxidized using strong oxidizers. Mild oxidizers as Trommer’s and Fehling’s reagents
cannot oxidize alcohols.
1. 3CH3-CH2-OH + K2Cr2O7 + 4H2SO4 →3CH3-CHO + Cr2(SO4)3 +K2SO4 +7H2O.
2. 5CH3-CHOH-CH3 + 2KMO4 + 3H2SO4 →5CH3-CO-CH3 + 2MnSO4 + K2SO4 +8H2O.
Alcohols form esters with both mineral and organic acids:

R1 CH2 OH + H2SO4 R1 CH2 O SO3-
O
R1 CH2 OH + R COOH R C O CH2 R1
Phenols, like alcohols, have hydroxyl groups in theirs structures. The difference is that hydroxyl group in
alcohols is bonded to aliphatic structure when hydroxyl group in phenols is bonded to aromatic cycle:
Because of this fact phenols have more acidic properties than alcohols. Phenols react with metals and
bases forming phenolates:
OH ONa
NaOH
-H2O
phenol sodium phenolate
Phenols can form esters with organic and inorganic acids. When the acetylsalicylic acid is produced the
salicylic acid reacts as phenol and forms ester with the acetic acid:
Phenols are characterized with one specific reaction. They can form coloured compounds reacting
with ferric trichloride. In such reaction phenols form coloured coordination compounds - ferric
phenolates:
FeCl3 + 6C6H5OH = [Fe(OC6H5)6]3- + 6H+ + 3Cl-.
The reaction with iron(III) chloride solution can be used as a test for phenol. Solutions of various
phenols in this reaction acquire their characteristic colour. Phenols will typically yield dramatic purple,
blue, red or green colour. This type of reactions takes place when substances containing a phenol
group as well as substances containing an enol group react. The enol group is a chemical structure in
which there is OH group side-by-side with double bound:
OH
C C
This structure can be found both in aromatic compounds (phenols) and in aliphatic compounds. The
reaction with FeCl3 can be used for qualitative determination of all compounds containing OH group
attached to the phenol ring as well as for the enol group.
Amines are organic compounds containing one or more amino group. There are aliphatic and aromatic
amines:
CH3-CH2-NH2 NH2
Ethylamine
Aniline
They are nonpolar and slightly soluble in water substances. Amines show basic properties. Aliphatic
amines are more basic than aromatic ones. They can form salts reacting with acids. These salts are
polar and soluble in water:
CH3-CH2-NH2 + HCl → CH3-CH2-NH3+Cl-
Experimental
A. Oxidation of ethanol by potassium dichromate or permanganate in the acidic medium

Procedure
1. Put 5 drops of ethanol and 1M H2SO4 mixture (2:3) to the first test tube. Add 2 drops of 0.5 M
K2Cr2O7.
2. Put 5 drops of ethanol and 1M H2SO4 mixture (2:3) to the second test tube. Add 2 drops of 0.1 M
KMnO4.
3. Put 5 drops of ethanol in the third test tube. Add 10 drops of Fehling-I and 10 drops of Fehling-II
to this test tube.
4. Shake the contents of the test tubes
5. Heat the test tubes over the flame (BE CAREFUL – avoid spilling very strong oxidisers!).
The colour in the first test tube changes from orange to dark-green due to the changed oxidation
number of Cr (Cr6+  Cr3+). The violet solution in the second tube becomes colourless and dark MnO 2
precipitate forms (Mn7+  Mn4+). If there is an excess of sulphuric acid, the precipitate might dissolve
forming colourless MnSO4. The presence of a specific odour (similar to that of apples) indicates that
an aldehyde has been formed.
The colour in the third test tube does not change and is blue. Explain the differences which are seen in
the different test tubes.
B. Reaction of phenol with ferric trichloride FeCl3
In such reaction phenols form coloured coordination compounds - ferric phenolates:
FeCl3 + 6C6H5OH = [Fe(OC6H5)6]3- + 3H+ +3Cl-.
Solutions of various phenols in this reaction acquire their characteristic colour. This type of reactions
takes place when react substances containing a phenol group as well as substances containing enol
group.
Procedure
Add one drop of 0.1 N FeCl3 into 3 drops of phenol solution. The solution becomes intensely coloured.
Record the observed colour.
C. Hydrolysis of acetylsalicylic acid

The acetylsalicylic acid is an ester of the salicylic acid, and the -OH group is acetylated. Therefore this
compound when treated with FeCl3 does not acquire a colour characteristic for the phenol -OH group.
The colour appears when the acetylsalicylic acid is hydrolysed and the phenol -OH group forms
instead of the ester group:
COOH + H2O
COOH + CH3COOH
O-CO-CH3
OH
Acetylsalicylic acid Salicylic acid
Procedure
1. Put several crystals of acetylsalicylic acid into a test tube. Add 5-6 drops of water to dissolve them.
2. Add 1 drop of 0.1 M FeCl3. Record the colour.
3. Heat the tube a little, the intense violet colour appears. Explain the change of colour.
D. Formation of salts by aniline

Aniline displays a basic nature and can form salts in reaction with acids. HCl salt is soluble while the
H2SO4 salt has very low solubility. Draw the equations of reactions. Explain these differences.
Procedure.
1. Put 1 drop of aniline and 3 drops of water into a test tube, shake it – the emulsion of aniline in
water is obtained.
2. Immerse a strip of red indicator paper into this emulsion. Record the observed colour.
3. Transfer half of the obtained emulsion into another test tube.
4. 2 M HCl is added drop-by-drop into the first test tube (1 or more drops) until the transparent
aniline hydrochloride is obtained.
5. Add 1 drop of 1M H2SO4 into the second test tube and shake it. The precipitation of aniline
sulphate should form.
General questions
1. What chemical properties have alcohols?

2. What reaction can be used to identify polyhydroxyl alcohols?
3. What products form when primary and secondary alcohols are oxidized? What oxidizers should
be used?
4. What compounds are formed when alcohols react with acids?
5. What are the main differences between alcohols and phenols? Draw the formula of a phenol.
6. What specific reaction is used to identify phenols?
7. What other group of substances can react in the same way?
8. Why does salicylic acid form coloured compound with FeCl3 and acetylsalicylic acid does not?
Explain this difference.
9. Why are amines slightly soluble in water and why are their salts well soluble?
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Conclusion
12. Chemical properties of mono- and polysaccharides

Background. Chemically, carbohydrates are polyhydroxyl aldehydes or polyhydroxyl ketones or
substances that yield these compounds when hydrolyzed. Carbohydrates also are known as saccharides.
Carbohydrates are classified as monosaccharides, disaccharides, oligosaccharides and polysaccharides
according to the number of monosaccharide units linked in a molecule. Monosaccharides usually exist in
a cyclic hemiacetal form. The formation of hemiacetal occurs when the oxogroup of monosaccharide
reacts with the hydroxyl group of the same monosaccharide. The hemiacetals of monosaccharides can
react one with other and form disaccharides and polysaccharides. However, hemiacetals are in
equilibrium with the small amounts of open chain aldehyde. Because of this they can be oxidized.
There are formulas of carbohydrates used in laboratory work:
Maltose Amylopectin
Experimental
A. Reaction of hydroxyl groups of carbohydrates Adjacent –OH groups present in saccharides form
soluble coloured coordination compound with Cu (II) in alkaline solution:
2-
CH OH HC O O CH
2 + Cu(OH)2 + 2NaOH
HC O
Cu
O CH
2Na+ +4H2O
CH OH
blue colour water soluble
precipitate blue complex compound
Procedure:
1. Prepare 4 test tubes.
2. Put 3 drops of 1% carbohydrate solutions into the test tubes:
1st – glucose, 2nd – fructose, 3rd – sucrose and 4th – lactose or maltose.
3. Add 6 drops of 2M NaOH and 1 drop of 0.1 M CuSO4 solution into each tube.
The blue Cu(OH)2 precipitate should form in all the test tubes. After some shaking the precipitate disappears as
the blue-coloured coordination compound between the copper and saccharide is formed.
Leave the tubes for the use in following experiments!
B. Oxidation of the aldehyde group of carbohydrates. Aldoses exist primarily in cyclic hemiacetal
form; however, they are in equilibrium with small amounts of open chain aldehyde. Free aldehyde
groups can be easily oxidized to carboxyl by mild oxidizing agents – for example, Cu ++ in alkaline
solution:
R-CHO + 2 Cu++ + 5 OH- + heating  R-COO- + Cu2O + 3H2O
Blue solution Red precipitate
Trommer’s reagent: CuSO4 and NaOH;

Fehling’s reagent: CuSO4 and basic sodium potassium tartrate.
Procedure 1: Oxidation by Trommer’s reagent

1. Add 5 drops of water to the each test tube from the previous experiment.
2. Heat the tubes in the boiling water bath until the reaction mixture changes colour from blue (Cu ++) to the
yellow (CuOH) or red (Cu2O).
3. Explain the change of colour.
Procedure 2: Oxidation by Fehling’s reagent
1. Take 4 test tubes, put 5 drops of the same carbohydrates as in the A experiment.
2. Add 5 drops of Fehling-I and 5 drops of Fehling-II reagents into each tube.
3. Heat the test tubes in the boiling water bath until the colours of reduced copper compounds appear.
C. Hydrolysis of polysaccharides
Polysaccharides are cleaved gradually: first into oligosaccharides, then – into monosaccharides. This
degradation is catalized by specialized enzymes in vivo, or by acids in vitro. It could be represented by scheme:
Starch (C6H10O5)n  Soluble starch (C6H10O5)n’ (where n’<n)  Dextrins (C6H10O5)n’’(n’’<n’<n) 

Blue violet Violet Brown-red
 Maltose (C12H22O11)  Glucose (C6H12O6)

Yellow Yellow
The colours given below the scheme are of compounds which these hydrolysis products form with iodine
(I2).
Procedure 1: hydrolysis when catalyst is H+:

1. Take two test tubes. Put 10 drops of 0.5% starch solution into the 1st test tube and add 2 drops of 1M H2SO4.
2. 2nd tube will be the control tube: put 10 drops of 0.5% starch solution and add 2 drops of water instead of
acid.
3. Heat both test tubes for approx. 7 min in the boiling water bath.
4. Dilute the iodine solution in another test tube: add 1 drop of iodine solution into 5 ml of water.
5. Take another test tube; put 2 drops of the hydrolyzed starch (1st tube contents) and 1 drop of the diluted
iodine solution. Record the observed colour.
6. Repeat the same with the control (2nd test tube contents).
Compare the both colours. Try to estimate, which stage of the starch hydrolysis was reached in your
experiment.
Procedure 2: hydrolysis when catalyst is enzyme:

Hydrolysis of starch in human beings begins already in the mouth as saliva contains enzyme called amylase.
Thus, your own saliva is a perfect source of this enzyme.
1. Carefully mix 5 drops of starch solution and approximately the same amount of your own saliva in a test-
tube.
3. Leave this test tube at the room temperature for 3-5 min.
4. Analyse it with iodine solution as it was done in the previous experiment.
What conclusions can be made about the effectiveness of the biological catalyst as compared to the non-
biological one?
General questions
1. What substances are called “carbohydrates”?

2. How are carbohydrates classified?
3. What chemical structures usually form monosaccharides?
4. What chemical reaction should be done to prove the existence of hydroxyl groups in
carbohydrates?
5. What oxidizers are usually used to oxidize carbohydrates?
6. What substances can catalyze the hydrolysis of polysaccharides?
7. What enzyme catalyzes the hydrolysis of polysaccharides?
8. What reagents are used to prove the hydrolysis of polysaccharides?
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Conclusion
13. Specific reactions for components of acidic hydrolysis of ribonucleoproteins

Background. Nucleic acids in cells are bound to proteins by non covalent bonds and form conjugated
compounds nucleoproteins. The complex is called deoxyribonucleoprotein if protein is joined to
deoxyribonucleic acid (DNA), and ribonucleoprotein if protein is joined to ribonucleic acid (RNA).
Nucleoproteins are of high molecular mass ranging from 10kDa to several hundred kDa. The complete
acidic hydrolysis of nucleoproteins yields a mixture of substances:
Deoxyribonucleoproteins are found in the nuclei of cells and mitochondria. DNA is associated with basic
proteins called histones. The interaction is maintained by ionic linkages between anionic phosphate
groups of DNA and cationic groups of the side chains of the basic amino acids of histones.
Ribonucleoproteins are composed of RNA and various proteins including enzymes, which participate in
translation. They are located in the different compartments of the cell including cytoplasm and
mitochondria.
Experimental
Yeasts were subjected to hydrolysis for a long time (about 1 h) by boiling with 1M H 2SO4. This
part of laboratory work was done in advance by the technician. Students are to do the reactions with the
prepared yeast hydrolyzate.
Procedure:
A. Biuret reaction for peptides
1. Transfer 5 drops of yeast hydrolyzate to the test tube.
2. Add 10 drops of 10  NaOH and 1 drop of 1 CuSO4 solution.
The violet colour indicates the presence of polypeptides.
B. Specific reaction for ribose – Fehling’s reaction

1. Transfer 10 drops of yeast hydrolyzate to the test tube.
2. Add 10 drops of Fehling’s reagent I
3. Add 10 drops of Fehling’s reagent II
4. Heat over the gas flame until a brown-red precipitate appears.
C. Specific reaction for phosphoric acid.

1. Transfer 10 drops of ammonium molybdate solution in HNO3 to the test tube.
2. Add 5 drops of yeast hydrolyzate
3. Boil the contents of the test tube for a few minutes. The yellow colour appears in the presence
of inorganic phosphate.
Reaction:
12(NH4)2MoO4 + H3PO4 + 21HNO3  (NH4)3PO4  12MoO3 + 21NH4NO3 + 12H2O
General questions
1. What compounds form nucleic acids with proteins? What types of them are there?
2. What proteins are associated with deoxyribonucleic acids?
3. What substances are produced during the complete hydrolysis of nucleoproteins?
4. What reaction proves the existence of proteins in the hydrolyzate?
5. What reaction proves the existence of carbohydrates in the hydrolyzate?
6. What reaction proves the existence of phosphates in the hydrolyzate?
Name Family name

Title ……………………………………………………………………………………………
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Aim……………………………………………………………………………………………
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Conclusion
14. Reactions of amino acids and proteins

Amino acids are heterofunctional compounds:
They haveAmides
an asymetric C atom, except glycine, so that they have optical isomers. There are L and D
series of optical isomers distinguished. Only amino acids of L-series are present in living organism. In
amino acids, numeration of atoms begins from carboxylic group that has an adjacent amino group. In
carbon atom chain, the second C atom is called -C, and amino group attached to it is called -amino
group. In the relation, such amino acids are referred as to -amino acids.
Classification:
1. According to the shape of C atom chain amino acids fall into aliphatic, heterocyclic and aromatic.
2. According to the type of substituents amino acids are grouped into unsubstituted aliphatic,
hydroxylamino acids, S-containing amino acids (mercapto amino acids), diamino acids, amides of
amino acids, dicarboxylic amino acids, heterocyclic amino acids, and imino acids. In this
classification, the unit:
is considered as the main chain and any group of atoms attached to it is considered as a radical or
substituent.
3. According to polarity of molecule and the charge they fall into:

a) Nonpolar amino acids including aliphatic, imino acid proline and aromatic amino acids;
b) Polar amino acids with non-ionized radical, including mercapto and hydrohyamino acids and
amides of amino acids;
c) Polar negative charged amino acids, they are dicarboxylic amino acids;
d) Polar positively charged amino acids, they are diamino monocarboxylic acids.
It is commonly accepted that names of amino acids are abbreviated using the letter.
Properties of amino acids:
1. Acid-base properties. They depend upon the presence of carboxylic group with acidic properties
and amino group with basic properties. In the relation, amino acids are ionized in solutions:
Such state of molecule is zwitter ion. The

total charge zwitter ion is zero, since
positive charge or amino group is
compensated by negative charge of
carboxylic group.
Ionization ability of functional groups is characterized by pKa

Under pH changing, ionization of amino acids is also changed:
Isoelectric point of amino acids (pI) is pH at which the molecule is electrically neutral. The value of
pI is determined by titration.
2. Condensation reaction. It occurs between amino acids and results in formation of peptide bond:
The product is called peptide. Peptide bond is covalent and nonpolar. It is a half double bond located
in one plane:
Polypeptides are long chains of amino acid residues linked by peptide bonds. The chain of amino acid
residues linked by this bond is the primary structure of proteins. Amino groups of amino acids and
peptide bonds of peptides can be identified by specific reactions. You will do them in laboratory
works.
Practical
A. Coordination compounds of amino acids with copper salts

Amino acids with ions of copper form blue-coloured coordination compounds – chelates. Each
molecule of the formed compound consists of two molecules of amino acid and one copper ion:
NH2 O
R-CH C=O
Cu
O=C HC-R
O NH2
Procedure:
Put one drop of each 0.1 M CuSO4, 4 % glycine and 2 M NaOH solutions into a test tube. The intense
blue colour appears which is characteristic for the copper coordination compounds.
B. Biuret reaction for proteins

In a very strongly basic medium, copper ions with the peptide bonds form a violet coordination
compound. Only peptides containing no less than two peptide bonds and three amino acids can
participate in this reaction. It can be used for the identification of proteins.
O OH
H
Tautomeric forms of a peptide bond: C N C N
OH
...-NH-CHR1-C=N-CHR2-CO-N-CHR3-CO-...
Cu
...-NH-CHR4-CO-N-CHR5-C=N-CHR6-CO-...
OH
Procedure
Put into a test tube 1 mL of the protein solution, 1 mL of 2 M NaOH and 1 drop of 0.1 M CuSO 4. The
violet colour with a red or blue tint appears. If there is not much protein then a larger quantity of
CuSO4 can be carefully poured along the test tube wall trying not to mix it with the contents of the tube
(mixture of protein and NaOH). A violet ring (sometimes called the biuret ring) forms in the test tube.
C. Reaction of proteins with ninhydrine

While treated with ninhydrine, -amino acids become oxidized, deaminated and decarboxylated.
Ninhydrine formed in the reaction, reduced ninhydrine and NH 3 form a blue-coloured compound
containing conjugate bond system.
O O
OH R-CH-COOH t o
H
+ R-C=O + CO + NH
+
OH NH2 2 3
OH H
O O
O O O
O
OH H -3H2O
+ NH3 + =N-
OH OH
O O O O
Ninhydrine Blue-coloured compound

Reduced ninhydrine
If instead of the amino acid a protein is used in the reaction, then the reaction proceeds without CO 2
formation.
Procedure
Into 5 drops of the provided protein solution add 5 drops of the 0.1 % ninhydrine solution. Boil the
mixture for about 5 minutes. The violet colour appears, and when the solution becomes cold, the
colour changes to blue.
This reaction is used for the qualitative determination of proteins and amino acids, in the
chromatography of amino acids and colorimetric analysis.
D. Xantoprotein reaction
When treated with concentrated HNO3, aromatic amino acids within protein molecule react with the
nitric acid, and nitro compounds of yellow colour (xanthos in Greek) are formed. If accidentally some
drop of nitric acid contacts with human skin or nails then due to this reaction they become yellow.
O2N
+ 2 HNO3
HO CH2-CH-COOH HO CH2-CH-COOH
- 2 H2O
NH2 NH2
O2N
Tyrosine
2,4-dinitrotyrosine (yellow)
Procedure
1. Add concentrated HNO3 drop by drop into a test tube with 1mL of undiluted protein solution until
the protein precipitate is formed. Also, 5mL of 10 % protein solution and 3 drops of concentrated
HNO3 could be used.
2. Heat the test tube very carefully. The solution and precipitate gain a yellow colour.
Estimation of molecular mas of a protein by the electrophoresis method
1
2
Estimation of molecular mas of a protein from the gel chromatography data
Calculations of molecular mass of a protein by the electrophoresis method and from

the gel chromatography data
a, mm Rf, a/h, h= 115mm Mw Log of Mw
1 116000 5.064458
2 97000 4.986772
3 66000 4,819544
4 48500 4.685742
5 29000 4.462398
X
a – distance of the line in the gel from the beginning to the centre of the spot
h – distance from the top to the end of gel
Elution volume, ml Mw Log of Mw

1 670000 5.826075
2 4500000 5.653213
3 232000 5.365488
4 158000 5.198657
X
Elution volume is calculated at the peak of absorption
General questions
1. What substances are called “amino acids”?
2. What type of isomers do amino acids form?
3. What are the principals of the classification of amino acids?
4. What happens to amino acids when pH of solution changes?
5. What is the “isoelectric point of amino acids”?
6. What bond is called a “peptide bond”?
7. What compounds do amino acids form with copper salts?
8. What compounds can participate in the biuret reaction?
9. What is the principle of reaction between proteins and ninhydrine? Explain it.
10. What amino acids react with the nitric acids?
11. What is the principle of the estimation of molecular mass of proteins by the method of
electrophoresis?
12. What is the principle of the estimation of molecular mass of proteins using the method of gel
chromatography?
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Conclusion

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1

PRACTICALS OF MEDICAL CHEMISTRY

SCHEDULE OF MEDICAL CHEMISTRY LECTURES

The lectures take place at 800–930

N Date Title of lectures Location

4 April 9 4. Heterogenic processes. Prof Artūras Kašauskas Room 427

5 April 10 5. Coordination compounds. Prof Artūras Kašauskas The Small auditorium

6 April 11 6. Thermodynamics. Prof Artūras Kašauskas Room 427

April 16 CONTROL TEST I

11 April 19 10. Alcohols, aldehydes, carboxylic acids. Room 427

Head of the Biochemistry department Prof. L. Ivanovienė

SCHEDULE OF MEDICAL CHEMISTRY LABORATORY WORK

4 April 9 4. Preparation of buffer solutions.

7 April 12 7. Coordination-compound based determination of water hardness.

April 16 CONTROL TEST I.

9 April 17 9. Chemical catalysis and thermodynamics of chemical processes. Examples of reaction

Head of the Biochemistry department Prof. L. Ivanovienė

Questions for the control test N1 - 2013

1. Characteristics of water molecule. Distribution of charges.

Questions to the 2nd control test on Medical Chemistry - 2013

QUESTIONS FOR FINAL EXAMINATION ON MEDICAL CHEMISTRY

1. Characteristics of water molecule. Hydrogen bond.

MEDICAL CHEMISTRY. REQUIREMENTS FOR STUDY:

1. The exam is a multiple-choice test of 50 questions.

SAFETY INSTRUCTIONS FOR WORK IN THE MEDICAL CHEMISTRY LAB

2. A student must know:

3. In the lab it is not allowed:

II. REQUIREMENTS BEFORE THE WORK

- to understand the task which is going to be carried out;

III. REQUIREMENTS DURING THE WORK

1. Avoid noisy behaviour, keep everything clean and tidy.

2. During laboratory work it is not allowed:

3. Carefully follow the description of the laboratory work

IV. REQUIREMENTS AFTER THE WORK.

- turn off equipment, water and gas taps;

The example of the laboratory work report

Name Family name

Group N Year Faculty

Laboratory work N …..

Principle of the lab-work

Equipment and materials………………………………………………………………………….

Results and calculations

1. Preparation of solutions. Percent concentration

Example: Prepare 250 g of 20 % CaCl2 solution in water.

2. Weigh CaCl2 on a piece of paper and transfer it to the flask.

1. What systems are called solutions?

Group N Year Faculty

Principle of the lab-work

Equipment and materials………………………………………………………………………….

Here are some examples of calculations using molar concentration:

Experimental: Prepare 250 ml of CaCl2 solution in water of following concentration:

1 mol of CaCl2 amounts to 40 + 2×35.5 = 111 g;

Name Family name

Group N Year Faculty

Principle of the lab-work

Equipment and materials………………………………………………………………………….

Results and calculations

3.Volumetric analysis. Acid-base titration

8. Calculate the amount of NaOH in the flask X in grams.

1. What is the method of volumetric analysis based on?

Name Family name

Group N Year Faculty

Principle of the lab-work