Methodical Instruction for Students of the 2 Course pharmaceutical Faculty
LESSON № 23 (PRACTICAL – 6 HOURS)
Themes: 1. Disaccharides. Small practicum.
2. Polysaccharides. Small practicum.
Aim: To learn: the structure, classification, nomenclature, isomery, methods of obtaining and chemical properties of oligosaccharides and polysaccharides.
Professional orientation of students.
Oligosaccharides are carbohydrates that contain from two to ten monosaccharide units. Disaccharides are the most common type of oligosaccharide.Disaccharides are carbohydrates composed of two monosaccharide units covalently bonded to each other. Like monosaccharides, disaccharides are crystalline, water-soluble substances. Sucrose (table sugar) and lactose (milk sugar) are disaccharides.
Within the human body, oligosaccharides are often found associated with proteins and lipids in complexes that have both structural and regulatory functions. Free oligosaccharides, other than disaccharides, are seldom encountered in biological systems.
Complete hydrolysis of an oligosaccharide produces monosaccharides. Upon hydrolysis, а disaccharide produces two monosaccharides, а trisaccharide three monosaccharides, а hexasaccharide six monosaccharides, and so on.
Polysaccharides are carbohydrates made up of many monosaccharide units. Polysaccharides, which are polymers, often consist of tens of thousands of monosaccharide units. Both cellulose and starch are polysaccharides. We encounter these two substances everywhere. The paper on which this book is printed is mainly cellulose, as are the cotton in our clothes and the wood in our houses. Starch is а component of many types of foods including bread, pasta, potatoes, rice, corn, beans, and peas.
Methodology of Practical Class (900-1200).
Experiment 1. Barfed reaction (difference between reducing disaccharides and monosaccharides).
Reagents: 1% solution of glucose, 1% solution of lactose, 6% solution of cooper (II) acetate, 1% solution of acetic acid.
Technique of the experiment. To the two test-tubes bring 5 drops of Barfed reagent (6% solution of cooper (II) acetate in 1% solution of acetic acid) add 1 ml of 1% solutions of glucose and maltose. Mixture is heated on a water-bath during 10 minutes. Glucose reduces this reagent to Cu2O, and maltose does not give this reaction. In this reaction important is term of heating (must watch, that there was not maltose hydrolysis).
Experiment 2. Quality reactions on sucrose.
Reagents: 10% sucrose solution, 2% sucrose solution, 5% solution of NaOH, 5% solution of CoSO4, 5% solution of NiSO4, 5% alcoholic solution of α- naphthalene, conc. H2SO4.
Technique of the experiment. To the two test-tubes bring 2 ml of 10% sucrose solution and 1 ml of 5% NaOH solution. Then to the mixture of sucrose and alkali in one test tube add a few drops of 5% solution of CoSO4, and in another tube – a few drops of 5% solution of NiSO4. Observe after in the test tube with cobalt salt – the violet coloring appears, and in a test tube with nickel salt – the green coloring appears.
Experiment 3. The reaction of disaccharides with Felling liquid.
Reagents: 1% solution of maltose, 1% solution of lactose, 1% solution of sucrose, Felling liquid.
Technique of the experiment. The experiment is carried out in three test tubes: in the first test tube add 1 or 2 ml of 1% maltose solution, in the second – 1-2 ml of 1% lactose solution and in the third – 1-2 ml of 1% sucrose solution. Then in every test-tube add 2 ml of Felling liquid and overhead parts of the got reactionary mixtures heat in the flame to the beginning of boiling. The lower layers of solutions do not heat and leave for comparison. Observe after, as in the test tubes which contain solutions of maltose and lactose red sediment of Cu2O forms, and reactionary mixture, which contains sucrose solution, does not change its color during the heating.
Experiment 4. Hydrolysis of sucrose.
Reagents: 5% solution of sucrose, 10% solution of H2SO4, NaHCO3, Felling liquid, Selivanov reagent.
Equipment and materials: test tubes, polarimeter, water-bath, electroplate.
Technique of the experiment. In the 50 ml flask pour 15-20 ml of 5% solution of sucrose and 5-6 ml of 10% solution of sulfate acid. To the got mixture add a few boiling stones. Reactionary mixture is boiled during 10 min., then cooled and used for research. The tube of polyarimetr is rinsed by the got reactionary mixture. Then fill it under the same reactionary mixture. Determine a sign and corner of rotation of solution which contains the products of sucrose hydrolysis.
Before hydrolysis sucrose solution has a sign of rotation “+”, after hydrolysis – “-“. This shift of the solution right rotation on left rotation due to hydrolysis is called inversion (from Lat. Inversia – rotation). The phenomenon of inversion in this case is cased by the heating with sulfate acid sucrose solution hydrolyzes into glucose and fructose:
To the test tube bring 2 ml of solution which contains the products of sucrose hydrolysis, and to this solution during the mixing add small portions of NaHCO3 (powder). (Watch out! The liquid frothed over the allocation of CO2). After the neutralization (when CO2 will stop separating) to solution in a test tube add 1 ml of Felling liquid and heat got reaction mixture.
Heat 2 ml of 5% sucrose solution with 1 ml of Felling liquid. Sure that the sucrose solution before hydrolysis does not change color of Felling liquid and has, as before heating, the blue color. What does it means?
During the hydrolysis sucrose forms glucose, so the reaction mixture after hydrolysis reduces Felling liquid. In addition to glucose also fructose forms during the sucrose hydrolysis. How can it be determined?
To the test-tube bring 1 ml of solution, which contains the products of sucrose hydrolysis, and 2 ml of Selivanov reagent. The got reactionary mixture is heated on the boiling water-bath. The bright red coloring appears which confirms presence of fructose in the reaction mixture.
Write the equations of reactions.
Experiment 5. The reaction of starch and glycogen with iodine.
Reagents: 1% starch solution, 1% glycogen solution, Lughole solution.
Technique of the experiment. To 2-3 ml of 1% starch solution add 2-3 drops of Lughole solution. Solution becomes blue. Content of the tube is heated and then cooled. Explain the changes. To the test tube bring 2 ml of glycogen solution and add 1-2 drops of Lughole solution. How to explain the difference in color compared to starch.
The qualitative test for starch is iodine water solution. If to the starch solution add iodine solution – blue color complex has been formed, because iodine molecules are adsorbed on the starch molecules. This complex is broken, if this mixture is heated.
It is believed that starch forms with iodine compounds-inclusions (clathrates), painted in distinctive colors – blue (l max = 620-680 nm) for amylose and red (lmax = 520-555 nm) for amylopectin. Molecules of amylose in these complexes form spiral around the iodine molecule, each turn of which consists of 6 glucose remainders. At the heating of the colored solution of starch with iodine color disappears and appears again after the cooling, due to the spiral untwisting.
What is special in the structure of glycogen that determines its color with iodine?
Experiment 6. Acidic hydrolysis of starch.
Reagents: 1% starch solution (starch paste), 10% solution of sulfate acid, iodine in KI, 10% solution NaOH, Felling liquid.
Equipment and materials: glass, electroplate.
Technique of the experiment. In glass bring 20-30 ml of 1% solution of starch and 10 ml of 10% solution of sulfate acid. Mixture boils during 5-10 minutes.After 4-5 minutes pour 0,5 ml of the mixture and add a drop of iodine solution. If the blue color does not appear, the hydrolysis is finished. If you have a blue color heating should be continued. Solution is cooled and neutralized by the dry calcium carbonate. After the neutralizing solution is filtered and conduct reaction with Felling liquid (or Trommer’s liquid) at the heating. What do you observe?
So, during hydrolysis starch forms carbohydrates, which reduces Feling’s (or Tromer’s) reagent.
Experiment 7. Dissolution of cellulose.
Reagents: Schweitzer’s reagent, conc. sulfate acid.
Equipment and materials: test-tubes, filtration paper, syringe (medical); cup of Petri; porcelain cup.
Technique of the experiment. To the test tube bring 5 ml of copper-ammonium solution (Schweitzer’s reagent), immerse filtration paper and constantly mix with glass stick to the complete dissolution of cellulose. In a glass pour 100 ml of warm water, then add 2-3 ml of concentrated sulfate acid and pour there solution of cellulose. Cellulose separates from solution in the form of threads.
In a small medical syringe (capacity 2 ml) gain 1 ml of cellulose in Schweitzer’s reagent and slowly squeeze out it in a Petri cup or in a porcelain cup, in which previously poured 5% solution of sulfate acid. Cellulose separated from solution in the form of white thread.
The ability of cellulose to dissolve in Schweitzer’s reagent and then separate in a solution of sulfate acid from solution with Schweitzer reagent in the form of threads is used in the production of copper-ammonium silk.
Copper-ammonium reagent proposed by Schweitzer, contains complex base [Cu(NH 3) 4] 2 +(OH –)2. Cellulose molecule (C 6 H 10 O 5) n or [C 6 H 7 O 2 (OH)3 ] n is built from a large number of interconnected residues of b-D-glucopyranose, each of theme has three hydroxyl groups, hydrogen atoms from these groups at the adding of Schweitzer’s reagent are replaced on the copper atom (as in a polyhydric alcohols) and form a water soluble complex of copper cellulosate.
At the dissolution of cellulose in copper-ammonium complex two hydrogen atoms of hydroxyl groups are replaced by copper atom and hydrogen atom of the third OH-group is ionized. Therefore glucose residues in cellulose keep their linkages and form anions (C 6 H 7 O 5 Cu) –, which combine with ammonia. This leads to the dissolution of cellulose.
Experiment 8. Extraction and properties of cellulose nitrates.
Reagents: conc. sulfate acid, conc. nitrate acid, a mixture of ethanol and diethyl ether (1:3).
Equipment and materials: conical flask, cups, porcelain cup, water-bath, cotton wool.
Demonstration research. (A study conducted under the ventilation cupboard).
Technique of the experiment. To the conical 50 ml flask pour 4 ml of concentrated nitrate acid and carefully add by small amounts 8 ml of concentrated sulfate acid. mixing solution becomes warm. Heat mixture is cooled to the room temperature. In the cooled mixture of acids bring small glomerulus of cotton, which are periodically mixed in the nitric mixture for 15-20 minutes. Then, using glass stick, cotton is removed from a mixture of acids and washed in a glass with water, and then – in a jet of water under the tap. Good washed from acids nitrated cellulose is wring out from water, dried in a cotton cloth, mellow, eliminated glomerulus, and dried in a porcelain cup on the boiling water-bath. (Be careful! Do not lean over a porcelain cup, as at the drying nitrated cellulose can catch fire!).
In the conditions of this experiment dinitrates and trinitrates of cellulose are obtained.
Dried cellulose nitrate is deviated into three parts. One part of cellulose nitrate is placed on asbestos grid and set on a fire. Nitrated cellulose burns extremely quickly. set on a fire a piece of nonnitrated cellulose (cotton). Cotton burns slowly, and at the end of burning putrefies.
The second part of nitrated cellulose is contained with the usage of glass stick in the dry test tube, well pressed to the bottom of the tube, and then heated in the flame of gas-burner or spirit lamp. (Be careful! Hole of the tubes must be directed from yourself and others!). Nitrated cellulose during the heating decomposes with explosions and crashes of test tubes with simultaneous catch fire of the air.
The third part of nitrated cellulose is contained in a mixture of ethanol and diethyl ether (1:3). Observe, as cellulose nitrate dissolves in a mixture of alcohol and ether and forms a viscous solution, called collodium.
Experiment 9. Extraction and properties of cellulose triacetate.
Reagents: conc. sulfate acid, ice acetic acid, acetic anhydride, ethanol, acetone.
Equipment and materials: conical flask, test tubes, Buchner funnel, glasses, water -bath, cotton wool, glass plate (10x10 cm).
Demonstration research. (A study conducted under ventilation cupboard).
Technique of the experiment. In the conic flask (capacity 50 ml) mix 5 ml of acetic anhydride, 5 ml of ice acetic acid and 1-2 drops of concentrated sulfate acid. Then in flask bring a small piece of cotton wool. The obtained reaction mixture is heated in a water-bath to complete dissolution of cotton. Obtained solution is poured into 250 ml of water. Sediment of cellulose acetate falls out, then filtrate this sediment on Buchner funnel, wash several times with water and then with alcohol.
In the conditions of this experiment mixture of cellulose diacetate and triacetate are formed.
Part of the obtained cellulose acetates is contained in a dry test tube and then add 2 ml of acetone. Cellulose diacetate is very good dissolved in acetone and cellulose triacetate is more difficult dissolved in acetone. So, full acetylated cellulose is not soluble in acetone.
Obtained acetone solution of cellulose acetate with sediment is poured on a glass plate (10x10 cm) and left in a horizontal position for acetone evaporation (under a ventilation cupboard). On a glass plate film of cellulose acetate is formed. To remove it, carefully pour a glass with water. Then the film is dried between the sheets of filtration paper and piece of it with help of pincers bring it in the flame of gas burner or spirit lamp. Cellulose acetate catches fire very difficult.
Experimental task
1. Three test tubes contain glucose, fructose and lactose. How to identify which carbohydrates in each test tube? Conduct laboratory experiments.
2. Four test tubes contain glucose, formaldehyde, sucrose and lactose. Propose ways of these substances identification.
Individual Students Program.
1. Oligosaccharides. Classification, structure and nomenclature of disaccharides. Reducing disaccharides (maltose, lactose, cellobiose) and nonreducing disaccharides (trehalose, sucrose). Conformation construction of the maltose and cellobiose.
2. Chemical properties of disaccharides. Sucrose inversion.
3. Polysaccharides. Structure. Classification. Homopolysaccharides: starch (amylose, amylopectine), glycogen, dextran, cellulose. Hydrolysis of polysaccharides.
4. Connection of the cellulose structural features with mechanical properties and chemical resistance. Complex and simple ethers of polysaccharides (nitrates, acetates, xanthogenates), their relation to hydrolysis.
5. Cellophane. Carboxymethylcellulose. Inulin, pectin, their finding in the nature. Polysaccharides of the bacteria cellular walls (muramine).
6. Conception of heteropolysaccharides (hyaluronic acid, heparin, chondroitin sulfate, vegetable gums and idea about their structure).
7. Biologically important oligo- and polysaccharides
Seminar discussion of theoretical issues (1230 – 1400 hour).
Test evaluation and situational tasks.
Home task. 1, 4, 7.
1. Write structural formulas of the following compounds: 1) a-maltose; 2) methyl–a-D-lactoside; 3) cellobionic acid; 4) fragment of amylose, amylopectine and cellulose that contain 4 monosaccharides (show 1,4- and 1,6-glycosidic bonds).
2. Give the name of the following connections:
3. What compounds are reducing and nonredusing disaccharides? Provide examples. How these disaccharides will react with methyl iodide, methanol in the presence of HCl. Write the appropriate reaction schemes.
4. Write the schemes of sucrose, maltose and lactose hydrolysis. Give the complete name to these disaccharides.
5. Carry out transformation. Name the intermediate and eventual products of reactions:
lactose? ?? ?
sucrose ??+ ethylene glycol С6Н12О3
maltose?? Х
cellobiose?? SN С6Н10О4
6. After the lactose oxidation by bromine water D-lactobionic acid forms, which by methylation forms octa-o-methyloctabionic acid. As a result of hydrolysis of the last compound 2,3,4,6-tetra-o-methyl-b–D-galactose and 2,3,5,6-tetra-o-methylgluconic acid form. Write the appropriate reactions.
7. Is it possible to distinguish the following compounds by Trommer’s sample: 1) sucrose from its products of hydrolysis; 2) lactose from its products of hydrolysis; 3) lactose from sucrose? Write the appropriate reactions.
8. Write the schemes of reactions of maltose with such reagents:
[H]; 2) [Ag(NH3)2]OH; 3) HCN; 4) NH2OH; 5) excess of C6H5NHNH2; 6) CH3OH (HCl(gas); 7) excess of CH3I; 8) excess of (CH3CO)2O. Name the products.
9. Methylcellulose is used for the preparation of medical forms. Will methylcellulose products of hydrolysis show reducing properties? Write the schemes of appropriate reactions.
10. What disaccharides will form after the partial hydrolysis of cellulose and starch? Which from these two polysaccharides does hydrolyze easier? Write the reaction of hydrolysis of cellulose and starch with formation of all intermediate products of hydrolysis. For cellulose write the schemes of reactions of triacetate and trinitrate formation.
11. Why does free D-glucose give a positive Trommer’s test and D-glucose remainder in sucrose molecule do not?
12. Write the structural formula of trisaccharide, which consists of three remainders of α-D-glucopyranose, that are connected in position 1,4.
13. What application do the following di- and polysaccharides find in the medical practice: sucrose, lactose, maltose, starch, cellulose, cellobiose, dextrans, agar and others?
14. Determine the type of polysaccharides of the following compounds: hyaluronic acid, heparin, chondroitin sulfate and vegetable gums?
15. Carry out the followings schemes of transformations and name the products of reactions:
16. What disaccharide will give reaction with Tollens reagent?
A. Sucrose
B. Maltose
C. Amylose
D. Amylopectin
E. Cellulose
17. What monosaccharide is a product of starch hydrolysis?
A. Glucose
B. Fructose
C. Mannose
D. Ribose
E. Galactose
18. Nonreducing disaccharide is the following one:
A. Cellobiose
B. Maltose
C. Sucrose
D. Lactose
E. Xylose
19. Disaccharide form by hydrolysis of polysaccharides. What disaccharide will form after the hydrolysis of starch?
A. Pyrogalose
B. Lactose
C. Cellobiose
D. Sucrose
E. Maltose
Correct answers of test evaluations and situational tasks:
16. B; 17. A; 18.C;19 E.
Individual student work (1415-1500 hour) are checked by solving situational tasks for each topic, answers in test evaluations and constructive questions (the instructor has tests & situational tasks).
Student should know:
– structure, classification, nomenclature, isomery, methods of getting and chemical properties of oligo- and polysaccharides .
Student should be able to:
– on the basis of functional groups to attribute matters to the certain class of organic compounds;
– to give the names organic connections with the help of rational and international (IUPAC) top- level systems;
– to conduct reactions which characterize of chemical properties of oligo- and polysaccharides.
References:
Main:
1. Clayden Jonathan. Organic Chemistry. Jonathan Clayden, Nick Geeves, Stuart Warren // Paperback, 2nd Edition. – 2012. – 1234 p.
2. Bruice Paula Y. Organic Chemistry / Paula Y. Bruice // Hardcover, 6th Edition. – 2010. – 1440 p.
3. Brückner Reinhard. Organic Mechanisms – Reactions, Stereochemistry and Synthesis / Reinhard Brückner // Hardcover, First Edition. – 2010. – 856 p.
4. Moloney Mark G. Structure and Reactivity in Organic Chemistry / Mark G. Moloney // Softcover, First Edition. – 2008. – 306 p.
5. Carrea Giacomo. Organic Synthesis with Enzymes in Non-Aqueous Media / Giacomo Carrea, Sergio Riva // Hardcover, First Edition. – 2008. – 328 p.
6. Smith Michael B. March’s Advanced Organic Chemistry. Reactions, mechanisms, and structure / Michael B. Smith, Jerry March // Hardcover, 6th Edition. – 2007. – 2384 p.
7. Carey Francis A. Advanced Organic Chemistry / Francis A. Carey, Richard A. Sundberg // Paperback, 5th Edition. – 2007. – 1199 p.
8. http://intranet.tdmu.edu.ua/data/kafedra/internal/zag_him/classes_stud/pharmaceutical/pharmacy/full_time_study/organic_chemistry/2_course/lesson_23.Di- and polysaccharides.
9. http://intranet.tdmu.edu.ua/data/kafedra/internal/zag_him/presentations/pharmaceutical/pharmacy/full_time_study/organic_chemistry/2_course/lecture_15.Di- and polysaccharides. Terpenes.
Additional:
1. Francotte Eric. Chirality in Drug Research / Eric Francotte, Wolfgang Lindner //
Hardcover, First Edition. – 2006. – 351 p.
2. Quin Louis D. Fundamentals of Heterocyclic Chemistry: Importance in Nature and in the Synthesis of Pharmaceuticals / Louis D. Quin, John Tyrell // Hardcover, 1st Edition. – 2010. – 327 p.
3. Zweifel George S. Modern Organic Synthesis – An Introduction / George S. Zweifel, Michael H. Nantz // Softcover, 1st Edition. – 2007. – 504 p.
4. K. C. Nicolaou. Molecules that changed the World / Nicolaou K. C., Tamsyn Montagnon // Hardcover, First Edition. – 2008. – 385 p.
5. Mundy Bradford P. Name Reactions and Reagents in Organic Synthesis / Bradford P. Mundy, Michael G. Ellerd, Frank G. Favaloro // Hardcover, 2nd Edition. – 2005. – 886 p.
6. Li Jie Jack. Name Reactions. A Collection of Detailed Reaction Mechanisms / Jie Jack Li // Hardcover, 4th Edition. – 2009. – 621 p.
7. Gallego M. Gomez. Organic Reaction Mechanisms / M. Gomez Gallego, M. A. Sierra // Hardcover, First Edition. – 2004. – 290 p.
8. Sankararaman Sethuraman. Pericyclic Reactions – A Textbook / Sethuraman Sankararaman // Softcover, First Edition. – 2005. – 418 p.
9. Tietze Lutz F. Reactions and Syntheses / Lutz F. Tietze, Theophil Eicher, Ulf Diederichsen // Paperback, First Edition. – 2007. – 598 p.
10. Olah George A. Superelectrophiles and Their Chemistry / George A. Olah, Douglas A. Klumpp // Hardcover, First Edition. – 2007. – 301 p.
11. Grossmann Robert B. The Art of Writing Reasonable Organic Reaction Mechanisms / Robert B. Grossmann // Hardcover, 2nd Edition. – 2003. – 355 p.
12. Cole Theodor C.H. Wörterbuch Labor – Laboratory Dictionary / Theodor C.H. Cole // Hardcover, 2nd Edition. – 2009. – 453 p.
The methodical instruction has been worked out by: associate prof. Dmukhulska Ye.B., assistant Medvid I.I., assistant Burmas N.I.
Methodical instruction was discussed and adopted at the Department sitting
25.06.2013. Minutes N 11
Methodical instruction was adopted and reviewed at the Department sitting
27.08.2013. Minutes N 1
