YEREVAN STATE MEDICAL UNIVERSITY
M.M. Melkonyan, K.M. Kocharyan
Manual To Laboratory Classes on
Bioorganic Chemistry
YEREVAN-2012
1
YEREVAN STATE MEDICAL UNIVERSITY
M.M. Melkonyan, K.M. Kocharyan
Department of Medical Chemistry
Manual To Laboratory Classes on
Bioorganic Chemistry
2
Laboratory Manual on Bioorganic Chemistry. - Yerevan: Publishing
House of Yerevan State Medical University, 2012
The given manual is designed according to the curriculum on bioorganic
chemistry for the students of the Faculty of Foreign Admissions (general
medicine, pharmacy, dentistry specialties). It is intended to save the student's
time and optimize their practical work.
Written and compiled by:
Head of the Department of Medical Chemistry M.M. Melkonyan, M.D.,
Ph.D. , associate-professor K. M. Kocharyan, Ph.D.
Proof read by Nazaretyan N.R., senior teacher of the Department of Foreign
Languages.
Reviewer:
Head of the Biochemistry Department of YSMU, Professor M.I. Agajanov,
M.D., Ph.D.
The manual is recommended by the Methodic Committee (Methodological
Board (council)) of Foreign Students of YSMU
??Recommended for publishing by the Methodic Committee on NaturalSciencesDisciplines of YSMU
3
Some Rules Of The Laboratory:
1) Work in groups of more than two only by permission of your lab
instructor.
2) If you miss an experiment due to unavoidable absence, don't wait until
your next lab period. It may be impossible to arrange to make it up.
3) No unauthorized experiments are permitted in the laboratory.
4) Never work alone in the laboratory.
5) You are expected to follow all safety rules. Remember, approved safety
goggles and a lab coat or apron are required whenever hazardous materials
are being used in the room.
6) Protective gloves are required for handling corrosive and toxic chemicals.
Open-toed shoes or sandals are not permitted in the laboratory.
7) When you have completed your experiment, use a damp sponge to clean
your bench space. Throw away any waste materials, paper towels, etc.
SAFE LABORATORY PROCEDURES MUST BE
FOLLOWED AT ALL TIMES.
a. All experiments carried out must have prior knowledge and approval of
the instructor supervising the laboratory. Unauthorized experiments will
result in permanent expulsion from the laboratory and the course.
b. Know the hazards associated with all chemicals and procedures in use in
your laboratory. Know the proper means for dealing with these hazards. Do
what is necessary to make all laboratory work safe for yourself and for
everyone else.
No food or drinks may be consumed or stored in the laboratory. Waste
glass and chemicals must be disposed of in proper containers.
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Laboratory work 1.
The oxidation of benzol homologues lateral chains
Facts to Know. Although benzene and arenes are stable toward the usual
oxidizing agents (KMnO 4 , K 2 Cr 2 O 7 , etc.) the benzene ring renders an
aliphatic side chain quite susceptible to oxidation.
R
R
[O]
HOOC
COOH
The side chain is oxidized completely, only a carboxyl group (-COOH)
remaining to indicate the position of the original side chain. In the result of
oxidation each lateral chain in benzene ring form carboxyl group at the end.
Must be noted that oxidation of a side chain is more difficult than oxidation
of an alkene and requires prolonged treatment with hot KMnO 4 .
Chemicals: H 2 O, KMnO 4 -2%, H 2 SO 4 -10%
Experimental procedure:
(a) Take 5 drops of H 2 O, 3 drops of 2% KMnO 4 and 1 drop of 10%
H 2 SO 4 solution in the reaction tube.
(b) Add 1-2 drops of toluene and shake it. After that, heat it. The
reaction is:
toluene
Record what takes place with the primary colour of the solution?
Tasks and Questions.
1. Write the scheme of the reaction of toluene oxidation. Name the
reaction product.
2. Write the scheme of the reaction of 2-ethyl-1-methylbenzene
oxidation. Name the reaction product.
3. In the result of benzene derivative oxidation 1,4-phenylen
dicarboxylic (terephthalic acid) is formed. In which position alkyl
groups were in the initial compound?
Laboratory work 2.
Oxidation of oleic acid with KMnO 4
Facts to Know. Oleic acid like alkenes is oxidized with cold dilute alkaline
or neutral KMnO 4 solution due to presence of double bond in the structure.
The resulting product of oxidation is diol.
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+ MnO 2 + KOH
Chemicals: Oleic acid, Na 2 CO 3 -5%, KMnO 4 -2%
Experimental procedure: Take 2 drops of oleic acid in the reaction
tube, add 2 drops of 5% Na 2 CO 3 solution and 2drops of 2% KMnO 4
solution. Record the changes which take place with primary violet colour of
the solution.
Tasks and Questions.
1. Why do we use reaction with KMnO 4 solution?
2. Write the oxidation reaction between oleic acid and KMnO 4 solution in
alkali medium.
Laboratory work 3.
Formation of glycerol cuprate (II)
Facts to Know. Due to –I effect hydroxyl groups of polyhydric alcohols are
more acidic in comparison with monohydric alcohols. Polyhydric alcohols
with hydroxides of some heavy metals form internal complex compounds
(chelates) with characteristic colour in an alkali medium. The reaction can be
used as qualitative.
glycerol cuprate (II).
This reaction can be used also for monosaccharyde determination.
C
O
C
H
H – C – OH
HO – C – H
H – C – OH
O
O
H C
HO – C – H
H
H – C – OH
HO – C – H
CuSO4, NaOH
H–C–O
H
H – C – OH
Cu O – C – H
O–C–H
CH2OH H
CH2OH
H – C – OH
H–C–O
CH2OH
glucose
diglucosocopper II
Chemicals:
CuSO 4 solution – 2%
NaOH solution – 10%
Glycerol
6
Experimental procedure:
(a) Take 2 drops of 2 % CuSO 4 solution in the test tube and add 2
drops of 10 % NaOH. Formation of blue precipitate of Cu(OH) 2
takes place.
(b) Add 1 drop of glycerol to the blue precipitate and shake it.
Formation of dark blue solution of Cu-glycolate takes place.
Tasks and Questions:
1. Write the scheme of the reaction between glycerol and Cu(OH) 2
2. Which structural fragment of organic compound is involved in the
reaction?
3. Compare acidity of ethylene glycol and ethanol. Which reactions can
prove difference in their acidity?
This reaction is used to determine the organic compounds, containing diol
fragments.
Laboratory work 4.
Sodium phenoxide formation and its decomposition by acid
Facts to Know. Phenols are converted into their salts by aqueous
hydroxides. The salts are converted into free phenols by aqueous mineral
acids, carboxylic acids or carbonic acid. Phenols must therefore be
considerably stronger acids than water, but considerably weaker acids than
carboxylic acids. Although weaker than carboxylic acids, phenols are
significantly more acidic than alcohols which have K a values 10-16 to 1018
and can react with alkali (NaOH). Simple phenols have K a of the order of
10-10.
ONa + H2O
OH + NaOH
C 6 H 5 ONa + HCl → C 6 H 5 OH↓ + NaCl
The acidic behaviour of phenol can be explained on the basis of resonance.
Hence, phenol has a tendency to form a relatively more stable phenoxide ion
by the release of a proton in basic media.
Objective: sodium phenoxide formation and its decomposition by acid.
Chemicals:
H2O
Phenol-crystals
NaOH-10%
HCl-10%
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Experimental procedure.
(a) Add 3 drops of H 2 O to few crystals of phenol and shake the tube.
(b) To the formed muddy emulsion add 10% of NaOH in dropwise
manner until limpid solution is formed.
(c) Then add several drops of 10% HCl-until phenol is released and
muddy emulsion restored.
Tasks and questions:
1. Write the reaction of sodium phenoxide formation.
2. Why can phenol react with NaOH unlike alcohols?
3. When HCl is added to sodium phenoxide the formation of muddy
solution takes place. Why? Write the scheme of the reaction.
4. What’s the reason of the higher acidity of phenol compared with
alcohols?
Laboratory work 5.
Oxidation of aldehydes
Facts to Know. Aldehydes are easily oxidized to carboxylic acids. Ketones
are not. This difference stems directly from their difference in structure:
aldehyde has a hydrogen atom attached to the carbonyl carbon, and ketone
has not. In the presence of alkali aldehydes undergo oxidation by heavy
metal ions, especially those of silver and copper. In the result of the reactions
ions are reduced. These reactions are used chiefly for detection of aldehydes.
Oxidation by silver requires an alkaline medium, to prevent precipitation of
the insoluble silver oxide.
NaOH + AgNO 3 → AgOH↓ + NaNO 3
AgOH + 2NH 3 → [Ag(NH3 ) 2 ]OH (Tollens’ reagent )
O
O
.
.
C
C
+ 2[Ag(NH 3 ) 2 ]OH 
→R
+ 2Ag↓+ 4NH 3 +H 2 O
R
OH
H
Ammonia oxidation by Tollens’ reagent is used chiefly for detecting
aldehydes, and in particular for differentiating them from ketones. A similar
process takes place with Cu2+.
CuSO 4 + NaOH → Cu(OH)↓ + Na 2 SO 4
I. Formaldehyde oxidation with AgOH (silver mirror reaction).
Chemicals:
AgNO 3 solution-5%.
NaOH-10%
8
NH 4 OH-2,8%.
Formaline-40%
Experimental procedure: Add 5 drops of 10% NaOH to 10 drops of 5%
silver nitrate solution. To the grey precipitate of AgOH add 2,8% NH 4 OH in
drop wise manner until precipitate dissolves. Divide the mixture in two tubes
in equal amounts. Then add 2 drops of 40% formalin solution in the 1-st test
tube and 2 drops of acetone in the 2nd test tube. Metallic silver deposits on
the inner walls of the 1-st tube, producing a beautiful, shiny mirror. In the 2nd
tube no reaction is observed.
AgNO 3 + NaOH → AgOH ↓+ NaNO 3
H-CHO + 4[Ag(NH 3 ) 2 ]OH = 3H 2 O + CO 2 + 4Ag↓ + 8NH 3
Tasks and questions:
1.Write silver mirror reaction.
2.What will it be with the reaction products if oxidizing agent is in excess?
3. Explain why no reaction is observed in the 2nd tube.
II. Oxidation with Cu(OH) 2.
Chemicals:
NaOH-10%
H2O
CuSO 4 -2%
Formaline-40%
Acetone.
Experimental procedure: Take 5 drops of 10% NaOH and H 2 O in each of
the two test tubes, add 1drop of 2% CuSO 4 solution.The precipitate of
Cu(OH) 2 is formed. 3 drops of 40% formalin solution add to the precipitate
in the 1st tube and 3 drops of acetone in the 2nd tube. Heat the flasks carefully
till boiling. In the 1st tube aldehyde converts into a carboxylic acid and yields
a brick red precipitate of Cu 2 O. The reagent solution contains blue copper
(II) ion that is reduced by an aldehyde to give a precipitate of red copper (I)
oxide. In the 2nd tube no reaction is observed.
Change of precipitate colour can be explained by the different oxidation
degree of Cu:
Cu(OH) 2 →CuOH → Cu 2 O → Cu
Blue
yellow
brick red metallic Cu
Tasks and questions:
1. Write the reaction of formaldehyde oxidation by Cu(OH) 2.
2. Compare the oxidation ability of formaldehyde and acetone based on the
experimental data.
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Laboratory work 6. Cannizzaro reaction.
Self oxidation-reduction of formaldehyde in aqueous solutions.
Facts to Know. In the presence of concentrated alkali, aldehydes containing
no α-hydrogens undergo self-oxidation and reduction to yield a mixture of
an alcohol and a salt of a carboxylic acid. This reaction is known as
Cannizzaro reaction.
H–C
O
H
+ H–C
O HO
2
H
H–C
O
OH
+ CH3OH
Objective: self oxidation-reduction of formaldehyde in aqueous solutions.
Chemicals:
Formaline-40%
Methyl red
Experimental procedure: Add 1 drop of 0,2% methyl red indicator to 2-3
drops of 40% formalin solution. Formation of red-colour solution shows
acidic reaction of the medium. (In the acidic medium the colour of the
methyl red indicator is red).
Tasks and questions:
1. Write the reaction of self oxidation-reduction.
2. What is the mechanism of this reaction?
Laboratory work 7.
Preparation of Formaldehyde 2,4-dinitrophenylhydrazone
Facts to Know. The formaldehyde reacts with 2,4-dinitrophenylhydrazine
with corresponding phenylhydrazone formation. The reaction is rapid and
the product is quite stable.This reaction is used for detection of aldehydes.
Experimental procedure: Add 1-2 drops of 40% formalin to 5 drops of 2,4dinitrophenylhydrazine until yellow sediment of formaldehyde 2,4dinitrophenylhydrazone formation:
Laboratory work 8.
Iodoform test
Facts to Know. All ketones containing the acetyl group, i. e., methyl
ketones as well as acetaldehyde undergo haloform reactions. This reaction is
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best carried out by dissolving the compound in dioxane, adding dilute
sodium hydroxide, followed by the addition of slight excess of iodine in KI
solution. If the compound contains the acetyl group, yellow precipitate of
iodoform is formed. The iodoform reaction is the basic chemical test for
methyl ketones (CH 3 COR, CH3 C R ) and acetaldehyde determination.
O
This reaction is used in clinical laboratory and has practical significance for
diagnostics of diabetes.
Chemicals:
KI-solution
NaOH-10%
Acetone.
I 2 + 2NaOH → NaOI + NaI + H 2 O
H
3NaOI + H – C – C – CH3
H
O
I3C – C – CH3 + NaOH
I
3NaOH + I – C – C – CH3
I
O
CHI3↓ + CH3 – C – ONa
O
O
Iodoform
Experimental procedure: (a) Add drop by drop 10% NaOH solution to
the test tube containing iodine solution until the solution becomes colourless.
(b) To the colourless solution add 1 drop of acetone. (c) Stopper the test tube
and shake vigorously. (d) In slight heating yellow precipitate of iodoform
separates. Formation of solid iodoform (yellow) is a positive test. (Iodoform
can be recognized by its odor and yellow color and, more securely, from the
melting point 119o-123oC). This reaction can be used for diabetes mellitus
diagnosis (detection of acetone in urine).
Tasks and questions :
1. Write the reaction of iodoform formation.
2. Which compounds can be recognized by iodoform test?
3. What type of structural fragments must they contain?
Laboratory work 9.
Determination of acetic acid. Acetic acid is one of the important
intermediates in the metabolic transformations in living systems. Mainly
present in active form as acetyl-CoA (a thioester of acetic acid and the thiol
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group of coenzyme A). It is one of the sources of carbon atoms for the
synthesis of biomolecules in biological systems.
CH 3 – COOH + NaOH → CH3 – COONa + H 2 O
3CH 3 – COONa + FeCl 3 → CH3 – COO) 3 Fe + 3NaOH 
CH
CH 3 – COO) 3 Fe + H 2 O →FeOH
3 – COO) 2 ↓ + CH 3 – COOH
Chemicals:
H2O
Litmus.
NaOH-10%
CH 3 COOH
FeCl 3 -1%
Experimental procedure: (a)Take 3 drops of acetic acid in reaction tube.
Check the medium of this solution by lithmus. (b) Add 2-3 drops of 10%
NaOH to this solution till CH 3 COOH is neutralized. (c)Then add 2-3 drops
of 1% FeCl 3 . Formation of yellow-red acetate iron (III) takes place. (d) Heat
the solution till boiling. Formation of brown red precipitate of Fe (III)
hydroxyacetate ((FeOHCH3 – COO) 2 ) takes place.
Tasks and questions:
1. Write the scheme of the dissociation of CH 3 COOH.
2. Write the scheme of the reaction of CH 3 COOH with NaOH. How can we
determine neutralization of CH 3 COOH in the experiment?
3. Write the scheme of the reaction of acetate iron (III) formation.
4. Write the structural formula of diacetate iron (III) hydroxide
Laboratory work 10.
Formation of insoluble calcium salts of higher fatty acids
Facts to Know. By modern soap manufactures hydrolysis of glycerides (a
fat) yields salts of the carboxylic acids and glycerol. Ordinary soap today is
simply a mixture of sodium salts of long-chain fatty acids. Soap solutions are
represented as spherical clusters called micelles, each of which may contain
hundreds of soap molecules. A soap molecule has a polar end –COO-Na+
(water-soluble, hydrophilic), non-polar end, the long carbon chain of 12 to
18 carbons (water-insoluble, hydrophobic).
Fig. Soap micelle.
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The polar —COO groups dissolve in water. Similarly charged micelles
repel each other. Hard water contains calcium and magnesium salts which
react with soap to form insoluble Ca and Mg carboxylates (the “ring” in the
bathtub).
Chemicals. Soap, CaCl 2
Experimental procedure: Put 5 drops of soap solution in the reaction tube
and add 1 drop of CaCl 2 solution and shake it. The appearance of white
sediment is observed.
Tasks and Questions.
1. Write the scheme of the calcium salt of stearic acid formation.
2. Which compounds are called soaps?
Laboratory work 11.
Determination of oxalic acid.
Facts to know. Oxalic acid COOH-COOH is a dicarboxylic acid and occurs
naturally in a number of plants including sorrel and begonia. Salts of oxalic
acid are called oxalates. Many metal ions form insoluble precipitates with
oxalate, a prominent example being calcium oxalate, the primary constituent
of the most common kind of kidney and bladder stones.
The sodium salt used as an antidote for metal poisoning, serves as an
anticoagulant.
Chemicals.
Oxalic acid,
CaCl 2 -5 % aqueous solution.
Experimental procedure. (a) 4-5 drops of H 2 O add to few crystals of
oxalic acid till complete dissolution. (b) Take 1 drop of the solution onto the
glass slide and add 1 drop of CaCl 2 solution. Formation of the sediment of
Ca-oxalate is observed.
Tasks and Questions.
1. Write the scheme of Ca-oxalate formation.
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Laborator work 12.
Determination of two carboxylic group presence in tartaric acid.
CuSO 4 + 2NaOH → Cu(OH) 2 ↓ + Na2SO4
CúúK
2
CH – OH
CH – OH
CúúK
H
+ Cu(OH)2
H–C–O
H–C–O
CúúK
CúúK
CúúK
Cu
O – CH
O – CH
H
CúúK
blue
Chemicals. Tartaric acid –15%, KOH –5%.
Experimental procedure:
(a) Take 1 drop of 15% tartaric acid in reaction tube, 2 drops of 5% KOH
solution and shake it. The formation of white sediment (acidic potassium salt
of tartaric acid) takes place.
(b) Add 4-5 drops of KOH solution to the sediment. The formation of
neutral potassium salt of tartaric acid takes place.
Tasks and Questions:
1. Write the scheme of hydrotartrate and potassium tartrate formation.
2.What shows the formation of both salts of tartaric acid.
Laboratory work 13.
Determination of hydroxyl groups in tartaric acid.
Chemicals: CuSO4 –2%, NaOH –10%
Experimental procedure: (a) Take 2 drops of 2% CuSO4 solution and 2
drops of 10% NaOH solution in two reaction tubes. Formation of blue
precipitate of Cu(OH)2 takes place. This blue solution is known as Felling
reagent. (b) Add the potassium tartrate solution prepared in the previous
experiment in the first reaction tube. The precipitate in the first reaction tube
dissolves with transparent blue solution formation. (c) Heat both reaction
tubes till boiling.
In the first tube the liquid colour doesn’t change, the second tube blue
sediment of Cu(OH)2 changes into black CuO. Felling reagent is used for
glucose determination in urine.
Tasks and Questions:
1. Write the scheme of the reaction between Cu(OH)2 and potassium
tartrate. The presence of which structural fragment is determined by the
reaction?
2. Why doesn’t change the colour of the solution in the 1st test tube?
3. Why is changed the colour of the solution in the 2nd test tube?
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Laboratory work 14.
Decomposition of citric acid.
Facts to Know. Decomposition of citric acid at heating in the presence
of H2SO4 takes place according to the mechanism of decomposition of αhydroxycarboxylic acids. Subsequent transformations of citric acid lead to
final products – H2O, CO2 and acetone formation. Determination of CO2
and acetone formation will prove the mechanism of decomposition.
citric acid
aceton
Ba(OH)2 + CO2 → BaCO3↓+ H2O
CH3 – C – CH3
I2 + NaOH
CHI3↓
+
CH3COONa
O
Iodoform
Chemicals. Citric acid, concentric H2SO4, Ba(OH)2 – saturated
solution, I2 in KI, NaOH- 5 %.
Experimental procedure. In dry tube, supplied with gas-holder
place citric acid on a trowel and 10 drops of concentric H2SO4 and heat. The
end of gas-holder immerse in first tube with 5 drops of Ba(OH)2 solution.
Then, when the solution becomes muddy in the first tube, transfer gas-holder
into the second tube containing 2 drops of I2 in KI and after that add few
drops of 10 % NaOH solution. In slight heating yellow precipitate of
iodoform separates, which indicates the presence of acetone.
Tasks and Questions:
1. Which product of citric acid decomposition is determined in the first
tube?
2. Which product of citric acid decomposition is determined in the second
tube? Write the scheme of the reaction.
Laboratory work 15.
Amino acids and peptides
Facts to Know. Proteins are the most abundant class of organic
compounds in the human body and amino acids are the building blocks of
proteins. Almost all of the naturally occurred amino acids in proteins are Lα-amino acids. Amino acids have a variety of chemically reactive groups.
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The reactions for side chains, α-amino, and α-carboxyl groups can be used to
characterize both free amino acids and proteins.
Ninhydrin reaction
Ninhydrin is a chemical substance mostly used in biochemical
laboratories as a reagent for α-amino acids, as well as in criminalistics to
detect finger prints and faint blood stains. α-Amino acids can be readily
detected and quantified by reaction with ninhydrin. Ninhydrin react with
amino acids, producing a colored solution. Ninhydrin, or triketohydrindene
hydrate, is a strong oxidizing agent and causes the oxidative deamination of
α-amino function. The products of the reaction are resulting aldehyde,
ammonia, carbon dioxide, and hydrindantin. The ammonia produced in this
way can react with the hydrindantin and another molecule of ninhydrin to
yield a purple product (Ruhemann's Purple) that can be quantified
spectrophotometrically at 570 nm. α-imino acids, such as proline and
hydroxyproline, give bright yellow ninhydrin products with absorption maxima
at 440 nm, allowing these to be distinguished from α-amino acids.
C
C
O
C
O
OH
OH
ninhydrin
Glycine reaction with ninhydrin
The universal qualitative reaction of α-amino acids is ninhydrin reaction.
O
O
O
C
C
C
2
C
C
O
OH
OH
C= N – C
+ NH 2 CHCOOH
R
O
+
C
C
O
OH
RCH
CO2
H2O
ninhydrin
amino acid glycine
The reaction product has blue-violet colour with maximum absorption at
570nm. Hence the reaction with ninhydrin can be used for visual detection of
α-amino acids on chromatographs.
Chemicals: Glycine – 1%, Ninhydrin – 0,1%
Experimental procedure:
(a) Take 4 drops of 1% glycine solution in the reaction tube and add 2
drops of 0,1% ninhydrin solution.
(b) The content of the tube heat carefully till blue-red colour appears.
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Tasks and Questions:
1. Write the scheme reaction between glycine and ninhydrin.
2. What is the characteristic feature of the reaction between α-amino acid
and ninhydrin?
3. How can the reaction between α-amino acid and ninhydrin be used in
practice? For which purposes?
Laboratory work 16.
Sorensen’s reaction. Glycine reaction with formalin.
Facts to Know. The mentioned reaction is used to determine the quantity
of α-amino acids in the solutions. Interaction of α-amino acids with
aldehydes lead to replaced imines (Shiff’s basis) formation:
- H 2O
R–CH–COOH + O=CH–R' → R–CH–COOH → R–CH–COOH
NH2
NH–CH–R'
OH
N=CH–R'
imine
The obtained N-methyl derivatives containing free -COOH group are
then titrated by alkali.
Chemicals: Glycine – 1%, Methylene red, formalin
Experimental procedure:
(a) Take 5 drops of 1 % glycine solution in the reaction tube and add 1
drop of methylene red indicator. The solution is coloured in yellow
(neutral medium).
(b) Formaldehyde (methanal) is then added to the sample and reacts with
amino acids amino groups, liberating –COOH groups (red colour of
solution).
(c) Than titrate sample with NaOH (the reaction has a 1:1 mole ratio).
The solution is coloured in yellow (neutral medium). The amount of
H+ ions reacted represents the amount of amino acid present in the
sample since each amino acid liberates one H+ ion.
Tasks and Questions:
1. Write the reaction scheme between glycine and formalin.
2. What is the cause of the indicator colour change?
3. What kind of practical application has the reaction between α-amino
acid and formalin?
Laboratory work 17. Fohl`s reaction
Facts to Know. Fohl`s reaction is used for determination of S-containing
amino acids. Cysteine and methionine are sulfur containing amino acids, but
17
they are different as cysteine can be oxidized in alkaline solution to form a
disulfide bond linking two molecules to form cystine, which reacts with lead,
while methionine has a methyl group on the sulfur making it less reactive
toward lead. So only –SH group containing amino acids can be determined
by this reaction. Specific reaction for detection of α-amino acids containing
“S” is based on the formation of black precipitate of PbS in the reaction of
protein with plumbum acetate. A black or brown color indicates the presence
of S-containing amino acids.
CH2 – SH
CH2 – OH
CH – NH2 + 2NaOH
CH – NH2 + Na2S + H2O
COOH
COOH
(CH3COO)2Pb + 2NaOH → Pb(ONa)2 + 2CH3COOH
Na2S + Pb(ONa)2 + 2H2O → PbS↓ + 4NaOH
Protein + (CH3COO)2Pb → PbS↓
Chemicals: Albumen, NaOH - 10 %, (CH3COO)2Pb – 10%
Experimental procedure:
(a) Take 10 drops of albumen solution and twice as much of NaOH
solution in the test tube.
(b) Heat the test tube till boiling (1-2min.)
(c) Add 5 drops of 10% (CH3COO)2 Pb solution to alkali solution in
the test tube and boil it again. Formation of gray-black precipitate
takes place.
Tasks and Questions:
1. Write the scheme reaction between protein and (CH3COO)2Pb.
2. Which α-amino acids can be revealed in protein structure by the
given qualitative reaction?
Laboratory work 18. Biuret Test
Biuret Test is used for detecting the presence of peptide bonds. It relies on
the reduction of copper(II) ions to copper(I), the latter form a complex with
the nitrogens of the peptide bonds in an alkaline solution. A violet color
indicates the presence of proteins. It is possible to use the biuret reaction to
determine the concentration of proteins because (for most proteins) peptide
bonds occur with approximately the same frequency per gram of material.
The intensity of the color, and hence the absorption at 540 nm, is directly
proportional to the protein concentration.
Chemicals:
2% CuSO4
18
1% NaOH
Determination of peptide bonds presence in biuret and proteins
The Biuret reagent is made of sodium (NaOH) and hydrated copper(II)
sulfate, together with potassium sodium tartrate. The biuret test is a
chemical test used for detecting the presence of peptide bonds (not less than
two).
Biuret
Despite its name, the reagent does not in fact contain biuret ((H2N-CO)2NH).
The test is named so because it also gives a positive reaction to the peptidelike bonds in the biuret molecule. Biuret is obtained by heating urea. Biuret
contains amide bonds similar to those in proteins. Biuret reacts with copper
(II) ions (blue) in basic solution to form a purple complex ion.
1.Biuret formation and peptide bond determination
Biuret formation:
(a) Take 0.5 g of urea in the test tube and carefully heat the tube. The
urea is dissolved, releasing ammonia. Heating is stopped by the time
of new solid (biuret) formation.
(b) After freezing the tube up to till room temperature add 2-3 ml of
warm water, carefully shake until dissolving of solid (formation of
biuret solution)
(c) Then add 3-4 drops of alkali solution (until the solution becomes
transparent) and 1 drop of CuSO4 solution. In the presence of
peptides, copper (II) ion forms a pink colored coordination
complexes when combined with short-chain polypeptides.
2. Biuret reaction with proteins : In the test tube take 5-6 drops of protein
solution than add 3-4 drops of alkali solution and 1 drop of CuSO4 solution.
In the presence of peptides, copper (II) ion forms a violet-colored
coordination complexes in an alkaline solution.
Tasks and Questions:
1. How is the formation of coloured complex explained?
2. Write the reaction of peptide bond formation between three aminoacids.
19
3. Is it possible to determine presence of dipeptide in solution using biuret
reaction?
Laboratory work 19, 20.
Determination of tyrosine (Xanthoprotein reaction)
Facts to Know. Xantoprotein reaction is used for detection of α-amino
acids which contain aromatic ring in radical (phenylalanine, tyrosine).
Xanthoproteic test is a test for the detection of proteins in which
concentrated nitric acid reacts with the proteins to form yellow color that is
intensified to orange-yellow by the addition of alkali in the presence of
tyrosine
yellow
orange
Chemicals: Albumen, HNO3 – conc. solution, NaOH –10%
Experimental procedure:
(a) Take 10 drops of albumen and 2 drops of conc. HNO3 solution in the test
tube and carefully heat it. The solution and precipitate is coloured in
yellow.
(b) Cool the tube and carefully add 1-3 drops of 10% NaOH solution till
bright orange colour appears (due to ionization of phenyl –OH- group in
tyrosine).
The same experiment can be done with the samples of phenylalanine and
tyrosine to reveal structural differences in them.
(a) Take 5 drops of phenylalanine in the 1st test tube and 5 drops of
tyrosine solutions in the 2nd test tube.
(b) Interaction of concentric HNO3 with phenylalanine and tyrosine leads
to the yellow nitrocompounds formation.
20
(c) The addition of alkali (ammonium solution) to both tubes yields to the
change of the yellow colour to the orange only in 2nd tube due to
ionization of phenyl –OH- group in tyrosine.
Tasks and Questions:
1. Which α-amino acids can be revealed by xanthoprotein reaction?
2. What is the cause of reaction colour change from yellow to orange in
alkali medium in a case of tyrosine?
Laboratory work 21.
Cooper-glycine coordinate salt formation.
CuCO3 + 2H2O
t
Cu(OH)2↓ + H2O + CO2↑
Chemicals: Glycine – 1%, CuCO3-dry
Experimental procedure: Take 1 ml of 1 % glycine solution in the
reaction tube, add dry CuCO3 on the tip of the trowel and heat the mixture.
The solution is coloured in blue.
Tasks and Questions:
1.Write the scheme reaction between glycine and CuCO3
2.Which colour is characterizer of Cu coordinate salts formations?
Laboratory work 22.
Heterocyclic amino acids. Determination of tryptophan. Adamkevich
reaction.
Facts to Know. Reaction of tryptophan (β,β- indolyl- α-aminopropionic
acid) with glyoxalic acid produces red-coloured compound.
COOH
COOH
CH – NH2 CH – NH2
CH2 – CH – COOH
2
NH
H
NH2
+
H
C–C
ú
úH
- H2O
NH
C
H
21
CH2
CH2
ú
NH
COOH
tryptophan
product of tryptophan interaction with
glyoxalic acid (red colour)
Chemicals: Acetic acid (concentrated), H2SO4 (sulfuric acid, concentrated),
1% solution of ovalbumen, 1 % solution of gelatin.
Experimental procedure:
(a) Take two test tubes. Take 5 drops of 1% solution of ovalbumen in the
1st test tube, 5 drop of 1 % gelatine solution in the 2nd tube.
(b) Add 5 drops of conc. acetic acid to both tubes. Slightly heat both
tubes, and then cool them.
(c) After cooling, add 10 drops of concentrated sulfuric acid carefully,
without mixing, without shaking, slowly, by the wall (slide) of the
tubes, so two nonmixed layers of liquids will be observed. In the 1st
tube, containing ovalbumen, in the border of two layers red-violet
color is observed.
(d) Mild heating can facilitate the color. In the 2nd tube, containing
gelatin no any color is observed. The data prove that there is no
tryptophan amino acid in gelatin.
Carbohydrates
Laboratory work 23.
Determination of hydroxy groups in D-Glucose.
ú
ú
H
C
2
HC – OH
HC – OH
CH2úH
HO – C – H
HOC – H
+ Cu(OH)2
HC – O
HC – O H
CH2úH
H
C
HC – OH
HC – OH
HOC – H
ú
H
C
Cu
H – C – OH
HO – C – H + 2H2O
O–C–H
CH2úH
Blue solution
Chemicals: D-Glucose- 0,5%, NaOH – 10%, CuSO4 –2%
Experimental procedure: (a)Take 1 drop of 0,5% glucose solution and
6 drops of 10% NaOH solution in the test tube. (b)Than add 1 drop of 2%
CuSO4 solution. The blue precipitate of Cu(OH)2 is formed and rapidly
dissolved with a transparent blue colour solution formation.
Tasks and Questions:
1. Which structural fragment of glucose is responsible for dissolving
Cu(OH)2?
2. Write the scheme reaction of Cu (II) with diol fragment of ethylene glycol
which results in coordinate salt formation.
22
Laboratory work 24.
Test for glucose (the Trommer reaction):
1 ml of the researched solution + 0.5 ml of 10 % NaOH + 5 drops of 1 %
CuSO4.
Gluconic acid
Experimental procedure:
(a) Mix 2 ml of sodium hydroxide solution with 3 drops of copper (II)
sulphate solution in the test tube. Pale blue precipitate should be
formed.
(b) While shaking the test tube, add glucose solution until the
precipitate disappears.
(c) Heat the contents of the tube in the boiling water bath - copper (I)
oxide precipitate is formed.
(d) Try to perform the same reaction without glucose.
Tasks and Questions:
Write the reactions (a) and (d) in the chemical equations.
Laboratory work 25.
Reduction of AgOH ammonia solution with glucose.
AgNO3 + NaOH → AgOH ↓+ NaNO3
AgOH + 2NH3 → [Ag (NH3 )2]OH - (Tollence reagent)
Chemicals: AgNO3–5%, NaOH –10%, NH4OH –10%, Glucose –0,5%
Experimental procedure:
23
(a) Take 1 drop of 5% AgNO3 in the test tube and add 2 drops of 10%
NaOH, 3-4 drops 10% aqueous ammonia solution until complete
dissolution of the precipitate.
(b) Then add 1drop of 0,5% solution of glucose and carefully heat the
test tube till brown colour. Then the reaction proceeds without heating
and metallic silver deposit is observed on the wall, producing a
beautiful shiny mirror.
Tasks and Questions:
1.Write the scheme reaction between glucose and ammonia solution of
AgOH.
2. Which functional group of glucose manifests reducing properties?
Laboratory work 26.
Selivanov’s reaction on fructose
Dehydration of monosaccharides and their condensation with phenols.
Facts to Know. At heating with strong mineral acids (HCl) dehydration
of monosaccharide takes place. Aldopentoses form furfural, aldo- and
ketohexoses form 5-hydroxymethylfurfural. Both furfural and 5hydroxymethylfurfural are able to react with phenols (resorcinol) and
aromatic amines (aniline). Furfural gives red colouring with aniline
(quantitative reaction on pentoses) and 5-hydroxymethylfurfural gives red
colouring with resorcinol.
Chemicals: Resorcin – dry, conc. HCl, Fructose – 0,5%
Experimental procedure:
(a) Take a grain of dry resorcin and 2 drops of conc. HCl in the test
tube.
(b) Add 2 drops of 0,5% fructose solution and heat till boiling.
Gradually the liquid is coloured in red due to formation of unstable
compound – hydroxymethylfurfurol, which with resorcin undergoes
condensation.
Tasks and Questions:
1. Write the scheme reaction of hydroxymethylfurfurol formation from
fructose.
Laboratory work 27.
Sucrose hydrolysis (inversion)
Facts to Know. Sucrose is a nonreducing sugar. Notice that there is no
hemiacetal group. Acetal groups are stable in basic solution. Therefore,
24
under the conditions of Tollen’s or Benedict’s test the acetal linkage will not
revert back to the free aldehyde form and there is no oxidation-reduction
reaction. Hydrolysis of sucrose in acidic medium yields one molecule of
glucose and one molecule of fructose. The 50:50 mixture of glucose and
fructose that results, often referred to as invert sugar (inversion of sign of
optical rotation that occurs). Sucrose is dextrorotatory, the mixture of
monosaccharide’s is levorotatory.
Sucrose
glucose
fructose
Chemicals. Sucrose solution, H2SO4 – dilute solution, NaOH, Trommer
reagent, Selivanov reagent.
Experimental procedure:
(a) Take 4-5 drops of 1 % sucrose in each two test tube,
(b) add 2-3 drops of dilute H2SO4,
(c) heat on aqueous bath 8-10 minute, cool and
(d) neutralize with base.
(e) add few drops of Cu SO4, NaOH in the first tube and heat. Formation
of red colour precipitate takes place. (the positive reaction of
Trommer).
(f) add 3-4 drops of Selivanov’s reagent (mixture of 3 ml resorcinol and
3 ml concentric HCl) in the second test tube, the solution is coloured
into red (quantitative reaction on fructose). After hydrolysis
dextrorotatory sucrose transfers into the mixture of levorotatory
monosaccharide.
Tasks and Questions:
(a) Why sucrose doesn’t undergo oxidation-reduction reaction?
(b) Why the 50:50 mixture of glucose and fructose that results sucrose
hydrolysis, often referred to as invert sugar?
Laboratory work 28.
Reduction ability of lactose.
25
βD- galactopyranosyl-(1,4)-α D- glucopyranose
lactobionic acid
Facts to Know. Lactose (C12H22O11) is reducing sugar. Acidic hydrolysis
converts (+)-lactose into equal amounts of D (+) glucose and D (+)
galactose. (+)-Lactose is evidently a β-glycoside in which glucose unit
contains a “free” aldehyde group and undergoes oxidation to the acid.
Lactose is thus a substituted D-glucose in which D-galactosyl unit is
attached to one of the oxygens; it is galactoside, not glucoside. Lactose is
βD- galactopyranosyl-(1,4)-αD- glucopyranose
Chemicals: lactose -10% , NaOH –10%,CuSO4 –2%
Experimental procedure:
(a) Take 1 drop of 1% lactose solution and 4 drops of 10% NaOH
solution in the test tube.
(b) Add 1 drop of CuSO4. Blue precipitate is formed and after shaking
the tube it is dissolved due to formation of blue cooper coordinate salt
of lactose.
(c) Then add few drops of H2O and heat carefully only the upper part of
the solution till boiling. Leave the lower part of the solution for
control (without heating). The upper part of the solution changes its
colour into yellow-red at heating. Remember, that the same result we
had with D-glucose (positive Tromer’s test) while in the experiment
with sucrose the colouring of the upper part of the solution is
unchanged under the same conditions.
Tasks and Questions:
1. Write lactose structure (using cyclic structure). Which configuration does
the anomer atom of ,,C” have in D-galactopyronose residue?
26
2. Which monosaccharide residue in lactose molecule is responsible for
cyclo-oxotautomerisation?
3. Explain the cause of lactose reducing properties.
Laboratory work 29.
Detection of starch
Chemicals: Starch-0, 5%, I2 –dilute solution
Experimental procedure: Take 5 drops of 0,5% starch solution and 1
drop of dilute I2 solution in the test tube. The solution is coloured in dark
blue. At heating the colour of the solution has disappeared. At cooling dark
blue colour returns.
Tasks and Questions:
1.Which disaccharide is a structural unit of amylose?
2. What type of glycoside linkage exists between D-glucose residues?
3. What conformation has polysaccharide chain of amylose?
4. How can the colour appearance by I2 addition to starch be explained?
Laboratory work 30.
Starch hydrolysis in acidic medium
Facts to Know. Starch, plant nutrient material, is actually a mixture of two
polysaccharides, amylose (which is linear chain of glucose units) and
amylopectin (is a branched polymer). The presence of amylose is proved by
iodine reaction.
+
HOH(H )
+
H O,H
+
H O,H
(C6H10O5)n
(C6H12O5)m 2
(C12H22O11) 2
(C6H12O6) m<n)
Starch
dextrin
maltose
glucose
Chemicals. Dilute solution of I2, 10 % NaOH, 2% CuSO4, 2 N H2SO4.
Experimental procedure:
(a) Take 1-2 drops of 0, 5% starch solution in the test tube, then add 2
drops of 2N H2SO4 and
(b) heat during10-15 minute in boiling aqueous bath.
(c) then take 1-2 drops of the reaction mixture and transfer it on the
glass slide and add 1 drop of dilute I2 solution in KI. Absence of
blue colour is the evidence of complete hydrolysis.
(d) then content of the test tube neutralize with 5 drops of NaOH, after
which add 1 drop of CuSO4 solution and heat. Yellow-red
precipitate of Cu2O is formed. The hydrolysis of starch results in
glucose formation, which gives positive Trommer test.
27
REFERENCES
1. Артемьева Н. Н., Белобородое В. Л., Еремин С. К.и др. Руководство
к лабораторным занятиям по биоорганиче ской химии/ Под ред.
Н. А. Тюкавкиной.— М.: Медицина, 1985, 256 с.
2. L. G. Wade, Jr. Organic Chemistry. 4ht edition, Prentice-Hall, New
Jersy, 1999, 1221p.
3. Bhagavan N. V. Medical Biochemistry. 4ht edition, Harcourt Academic
press, Canada, 2002, 1016p.
28
Content
Some Rules Of The Laboratory: ------------------------------------------------- 3
Safe laboratory procedures must be
followed at all times.--------------------------------------------------------------- 3
Laboratory work 1.--------------------------------------------------------------- 4
The oxidation of benzol homologues
lateral chains ----------------------------------------------------------------------- 4
Laboratory work 2.--------------------------------------------------------------- 4
Oxidation of oleic acid with KMnO4---------------------------------------------------------------------- 4
Laboratory work 3. ------------------------------------------------------------- 5
Formation of glycerol cuprate (II) ---------------------------------------------- 5
Laboratory work 4.--------------------------------------------------------------- 6
Sodium phenoxide formation and
its decomposition by acid ------------------------------------------------------- 6
Laboratory work 5.--------------------------------------------------------------- 7
Oxidation of aldehydes ------------------------------------------------------------ 7
Laboratory work 6.--------------------------------------------------------------- 8
Cannizzaro reaction ---------------------------------------------------------------- 8
Self oxidation-reduction of formaldehyde
in aqueous solutions--------------------------------------------------------------- 8
Laboratory work 7.--------------------------------------------------------------- 9
Preparation of Formaldehyde
2,4-dinitrophenylhydrazone. ------------------------------------------------------ 9
Laboratory work 8. ------------------------------------------------------------- 9
Iodoform test ------------------------------------------------------------------------ 9
Laboratory work 9.--------------------------------------------------------------- 10
Determination of acetic acid ------------------------------------------------------ 10
Laboratory work 10. ------------------------------------------------------------ 11
Formation of insoluble calcium
salts of higher fatty acids ---------------------------------------------------------- 11
Laboratory work 11. ------------------------------------------------------------- 12
29
Determination of oxalic acid ----------------------------------------------------- 12
Laborator work 12.--------------------------------------------------------------- 12
Determination of two carboxylic
group presence in tartaric acid---------------------------------------------------- 12
Laboratory work 13. ------------------------------------------------------------- 13
Determination of hydroxyl groups
in tartaric acid. --------------------------------------------------------------------- 13
Laboratory work 14. ------------------------------------------------------------- 14
Decomposition of citric acid------------------------------------------------------ 14
Laboratory work 15. ------------------------------------------------------------- 14
Amino acids and peptides --------------------------------------------------------- 14
Laboratory work 16. ------------------------------------------------------------ 16
Sorensen’s reaction.
Glycine reaction with formalin. ------------------------------------------------- 16
Laboratory work 17.------------------------------------------------------------ 16
Fohl`s reaction. --------------------------------------------------------------------- 16
Laboratory work 18. ----------------------------------------------------------- 17
Biuret Test -------------------------------------------------------------------------- 17
Laboratory work 19. 20. ------------------------------------------------------- 19
Determination of tyrosine (Xanthoprotein reaction) -------------------------- 19
Laboratory work 21.------------------------------------------------------------ 20
Cooper-glycine coordinate salt formation. ------------------------------------- 20
Laboratory work 22. ----------------------------------------------------------- 20
Heterocyclic amino acids.
Determination of tryptophan.
Adamkevich reaction -------------------------------------------------------------- 20
Carbohydrates ---------------------------------------------------------------------- 21
Laboratory work 23.------------------------------------------------------------ 21
Determination of hydroxy groups in D-Glucose ------------------------------- 21
Laboratory work 24.------------------------------------------------------------ 22
Test for glucose (the Trommer reaction)---------------------------------------- 22
Laboratory work 25.------------------------------------------------------------ 22
Reduction of AgOH ammonia solution with glucose ------------------------- 22
Laboratory work 26. ----------------------------------------------------------- 23
Selivanov’s reaction on fructose ------------------------------------------------ 23
Laboratory work 27. ----------------------------------------------------------- 23
Sucrose hydrolysis (inversion) --------------------------------------------------- 23
Laboratory work 28.------------------------------------------------------------ 24
Reduction ability of lactose ------------------------------------------------------ 24
Laboratory work 29.------------------------------------------------------------ 25
30
Detection of starch ---------------------------------------------------------------- 25
Laboratory work 30. ------------------------------------------------------------ 26
Starch hydrolysis in acidic medium --------------------------------------------- 26
31