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YEREVAN STATE MEDICAL UNIVERSITY

M.M. Melkonyan, K.M. Kocharyan

Manual To Laboratory Classes on Bioorganic Chemistry

YEREVAN-2012

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YEREVAN STATE MEDICAL UNIVERSITY M.M. Melkonyan, K.M. Kocharyan

Department of Medical Chemistry

Manual To Laboratory Classes on Bioorganic Chemistry

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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 Natural-SciencesDisciplines of YSMU

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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 (KMnO4, K2Cr2O7, etc.) the benzene ring renders an aliphatic side chain quite susceptible to oxidation.

[O]R R COOHHOOC

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 KMnO4.

Chemicals: H2O, KMnO4-2%, H2SO4-10% Experimental procedure: (a) Take 5 drops of H2O, 3 drops of 2% KMnO4 and 1 drop of 10%

H2SO4 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 KMnO4

Facts to Know. Oleic acid like alkenes is oxidized with cold dilute alkaline or neutral KMnO4 solution due to presence of double bond in the structure. The resulting product of oxidation is diol.

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+ MnO2+ KOH

Chemicals: Oleic acid, Na2CO3-5%, KMnO4-2% Experimental procedure: Take 2 drops of oleic acid in the reaction

tube, add 2 drops of 5% Na2CO3 solution and 2drops of 2% KMnO4

solution. Record the changes which take place with primary violet colour of the solution.

Tasks and Questions. 1. Why do we use reaction with KMnO4 solution? 2. Write the oxidation reaction between oleic acid and KMnO4 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.

H – C – OH

HO – C – H

H – C – OH

H – C – OH

CH2OH

CO

H

CuSO4, NaOH H

H

Cu

H – C – OH

HO – C – H

H – C – O

H – C – O

CH2OH

CO

H

HO – C – H

H – C – OH

O – C – H

O – C – H

CH2OH

CO

H

glucose diglucosocopper II Chemicals: CuSO4 solution – 2% NaOH solution – 10% Glycerol

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Experimental procedure: (a) Take 2 drops of 2 % CuSO4 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 Ka values 10-16 to 10-

18 and can react with alkali (NaOH). Simple phenols have Ka of the order of 10-10.

OH + NaOH ONa + H2O

C6H5ONa + HCl → C6H5OH↓ + 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 H2O 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 + AgNO3 → AgOH↓ + NaNO3 AgOH + 2NH3 → [Ag(NH3)2]OH (Tollens’ reagent )

R.CO

H + 2[Ag(NH3)2]OH → R.

OHC

O+ 2Ag↓+ 4NH3 +H2O

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+. CuSO4 + NaOH → Cu(OH)↓ + Na2SO4

I. Formaldehyde oxidation with AgOH (silver mirror reaction). Chemicals: AgNO3 solution-5%. NaOH-10%

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NH4OH-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% NH4OH 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.

AgNO3 + NaOH → AgOH ↓+ NaNO3

H-CHO + 4[Ag(NH3)2]OH = 3H2O + CO2 + 4Ag↓ + 8NH3 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 CuSO4-2% Formaline-40% Acetone. Experimental procedure: Take 5 drops of 10% NaOH and H2O in each of the two test tubes, add 1drop of 2% CuSO4 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 Cu2O. 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 → Cu2O → 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

HH – C

O

H+ H2O 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 2,4-dinitrophenylhydrazine until yellow sediment of formaldehyde 2,4-dinitrophenylhydrazone 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 (CH3COR, CH3 C R

O

) and acetaldehyde determination.

This reaction is used in clinical laboratory and has practical significance for diagnostics of diabetes. Chemicals: KI-solution NaOH-10% Acetone. I2 + 2NaOH → NaOI + NaI + H2O

H – C – C – CH3H

H

O

3NaOI + 3NaOH + I – C – C – CH3I

I

OI3C – C – CH3 + NaOH

O

CHI3↓ + CH3 – C – ONa

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.

CH3 – COOH + NaOH → CH3 – COONa + H2O 3CH3 – COONa + FeCl3 → CH3 – COO)3Fe + 3NaOH

CH3 – COO)3Fe + H2O → FeOH CH3 – COO)2↓ + CH3 – COOH Chemicals: H2O Litmus. NaOH-10% CH3COOH FeCl3-1% 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 CH3COOH is neutralized. (c)Then add 2-3 drops of 1% FeCl3. Formation of yellow-red acetate iron (III) takes place. (d) Heat the solution till boiling. Formation of brown red precipitate of Fe (III) hydroxyacetate ((FeOHCH3 – COO)2) takes place. Tasks and questions: 1. Write the scheme of the dissociation of CH3COOH. 2. Write the scheme of the reaction of CH3COOH with NaOH. How can we

determine neutralization of CH3COOH 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, CaCl2 Experimental procedure: Put 5 drops of soap solution in the reaction tube and add 1 drop of CaCl2 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, CaCl2 -5 % aqueous solution. Experimental procedure. (a) 4-5 drops of H2O 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 CaCl2 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.

CuSO4 + 2NaOH → Cu(OH)2↓ + Na2SO4

+ Cu(OH)22

CúúK

CH – OH

CH – OH

CúúK

CuH – C – O O – CH

O – CH

H

H – C – O

H

CúúK CúúK

CúúK 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 α-hydroxycarboxylic 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 – CH3I2 + NaOH

O

CHI3↓ + CH3COONa

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 L-α-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

O

COH

OH

ninhydrin Glycine reaction with ninhydrin The universal qualitative reaction of α-amino acids is ninhydrin reaction.

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.

C C

C

O

O

= N –

C C

C

O

OH

+ NH 2 CHCOOH

R

OH

OH C C

C

O

O

2 CO2

H2O

RCH

O

+

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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:

R–CH–COOH + O=CH–R' → R–CH–COOH → R–CH–COOH

NH2

OH

NH–CH–R'

-H2O

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

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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

CH – NH2

COOH

+ 2NaOH

CH2 – OH

CH – NH2

COOH

+ Na2S + H2O

(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

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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 peptide-like 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.

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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.

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(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.

- H2O+ C – C

úH

H ú

úNH H

CH2 – CH – COOH

NH2NH

COOH

CH – NH2

CH2

NH

COOH

CH – NH2

CH2

CH COOH

2

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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.

Cu

HC – OH

HOC – H

CH2úH

CHú

HC – O

HC – OH

+ 2H2O

HO – C – H

H – C – OH

CH2úH

O – C – H

O – C – H

CHú

H2

HC – OH

HOC – H

CH2úH

CHú

HC – OH

HC – OH

+ Cu(OH)2

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.

23

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:

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(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 5-hydroxymethylfurfural 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,

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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.

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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?

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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.

(C6H10O5)nHOH(H+) (C6H12O5)m H2O,H+

(C12H22O11)H2O,H+

(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.

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R E F E R E N C E S

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.

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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

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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

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Detection of starch ---------------------------------------------------------------- 25

Laboratory work 30. ------------------------------------------------------------ 26

Starch hydrolysis in acidic medium --------------------------------------------- 26