Experiment 5 Guide

Bulang, Del Gallego, Estrella, Guillermo, Jurilla, Lumaban 3BIO-6

Slide # 4 Topic: Carbohydrates

They can be polyhydroxy aldehydes or polyhydroxy ketones because they have either an aldehyde or a ketone group, along with OH substituted carbons in a chain Polyhydroxy aldehydes are called aldoses. Polyhydroxy ketones are called ketoses. The suffix ose means sugar

Slide # 5 Topic: Monosaccharide

Pentose any of the class of simple sugars whose molecules contain five carbon atoms such as xylose Cyclic forms of monosaccharides result from the ability of their carbonyl group to react intramolecularly with hydroxyl group
Hexose any of the class of simple sugars whose molecules contain six carbon atoms D-galactose and D-glucose differ only in the configuration of the OH group and H group on carbon 4 Epimers (diastereomers that differ only in the configuration at one chiral center

D-fructose also known as levulose and fruit sugar

Slide #6 Topic: Disaccharide

A monosaccharide that has cyclic forms (hemiacetal forms) can react with an alcohol to form a glycoside (acetal) = produce a disaccharide In disaccharide, one of the monosaccharide reactants function as a hemiacetal, and the other functions as an alcohol Glycosidic linkage bond in a disaccharide resulting from the reaction between hemiacetal carbon atom OH group of one monosaccharide and and OH group on the other monosaccharide Maltose malt sugar; produced whenever polysaccharide starch breaks down; 2 D-glucose units, one of which must be a D-glucose

Acetal - an organic compound formed by the condensation of two alcohol molecules with an aldehyde molecule

Slide #6 Topic: Disaccharide

The glycosidic linkage between two glucose units is an a(1-4) linkage The two OH groups that form the linkage are attached, respectively to carbon 1 of the first glucose unit and to a carbon 4 of the second Maltose reducing sugar because the glucose unit on the right has a hemiacetal carbon atom. Thus this glucose unit can open and close: it is in an equilibrium with its openchain aldehyde form. This means there are three forms of the maltose molecule: a-molecule, molecule, and the open chain form

In solid state, the -form is dominant Hydrolysis of maltose produces two molecules of D-glucose Acidic conditions or the enzyme maltase is needed for the hydrolysis to occur Lactose made up of a Dgalactose unit and a D-glucose unit joined by a (1-4) glycosidic linkage The glucose hemiacetal center is unaffected when galactosebonds to glucose in the formation of lactose, so lactose is a reducing sugar (the glucose ring can open to give an aldehyde)

Slide # 6 Topic: Disaccharide

Lactose major sugar found in milk; milk sugar Epimerization of glucose glucose yields galactose, and then the (1-4) linkage forms between a galactose and a glucose unit Souring of milk is caused by the conversion of lactose to lactic acid by bacteria in milk
Epimerization a chemical process where an epimer is tranformed into its chiral counter part Epimer - each of two isomers with different configurations of atoms around one of several asymmetric carbon atoms present

Sucrose table sugar; most abundant of all disaccharides; a-Dglucose and -D-fructose

The glycosidic linkage is not a (1-4) linkage, instead it is an a. (1-2) glycosidic linkage The OH group on carbon 2 of Dfructose (the hemiacetal carbon) reacts with the OH group on carbon 1 of D-glucose (the hemiacetal carbon) Sucrose, unlike maltose and lactose is a non-reducing sugar No hemiacetal is present in the molecule, because the glycosidic linkage involves the reducing ends of both monosaccharides

Slide # 6 Topic: Disaccharide

Sucrose exist in only one form there are no a and isomers, and an open chain form is not possible Sucrase enzyme n9eeded to break the a. (1-2) linkage in sucrose Sucrose hydrolysis (digestions) produces an equimolar mixture of glucose and fructose called invert sugar

Slide # 7 Topic: Polysaccharide

Monomer - a molecule that can be bonded to other identical molecules to form a polymer Polysaccharides are also called glycans How to distinguish polysaccharide Based on repeating units Homopolysaccharides starch, glycogen, cellulose, and chitin Heteropolysaccharide Length of the polymer chain Type of glycosidic linkage Degree of branching of the polymer chain

Starch glucose monosaccharide units If excess glucose enters a plant cell, it is converted to starch; when cell cannot get enough glucose, it hydrolyzes starch to release glucose Two different polyglucose polysaccharides can be isolated from most starches: amylose and amylopectin Amylose straight-chain glucose polymer (15%-20%) Amylopectin branched glucose polymer (80%-85%)

Slide # 7 Topic: Polysaccharide

Amyloses non-branched structure, the glucose units are connected by a(1-4) glycosidic linkages Amylopectin high degree of branching in its polyglucose structure; the branch points involve a(1-6) linkages Because of the branching, amylopectin has a larger average molecular mass than the linear amylose All of the glycosidic linkages in starch (both amylose and amylopectin) are of the a type In amylose, they are all (1-4): in amylopectin, both (1-4) and (1-6) linkages are present Because both types of linkages can be broken through hydrolysis within the human digestive tract, starch has nutritional value for humans Iodine is often used to test for the presence of starch in solution Starch-containing solutions turn a dark blue-black when iodine is added As starch is broken down through acid or enzymatic hydrolysis to glucose monomers, the blue-black color disappears

Slide # 7 Topic: Polysaccharide

Glycogen animal starch; structure similar to amylopectin: all glycosidic linkages are of the a type, and both (1-4) and (1-6) linkages are present Glycogen and amylopectin differ in the number of glucose units between branches and in the total number of glucose units present in molecule Glycogenesis formation of glycogen Glycogenolysis - decomposition of glycogen

Structural poysaccharide serves as a structural element in plant cell walls and animal exoskeleton Cellulose unbranched glucose polymer like amylose but differ in in that the glucose residues present in cellulose have a beta-configuration whereas the glucose residues in amylose have an alphaconfiguration. the glycosidic linkages in cellulose are therefeore (1-4) linkages rather than a(1-4) linkages

Slide # 7 Topic: Polysaccharide

The difference in glycosidic linkages type causes cellulose and amylose to have different shapes Amylose tend to have a spiral-like structures whereas cellulose molecules tend to have linear structures The linear (straight-chain) cellulose molecules, when aligned side by side, become water-insoluble fibers because of interchain hydrogen bonding involving the numerous hydroxyl groups present Cellulose is not a source of nutriotion for human beings because humans lack enzymes capable of catalyzing the hydrolysis of (1-4) linkages in cellulose Even animals lack the enzymes for cellulose digestion Intestinal tracts of animals such as horses have bacteria that produce cellulase, enzyme that can hydrolyze cellulose (1-4) linkages and produce free glucose from cellulose

Slide # 8 Topic: Reducing / non-reducing sugars

A reducing sugar is a carbohydrate that gives a positive test with Benedicts solutions Aldoses act as reducing agents in such reactions, they are called reducing sugars Under the basic conditions associated with Benedicts solutions, ketoses are also reducing sugars In this situation, ketose undegoes a structural arrangement that produces an aldose and the aldose then reacts Thus all monosaccharides, both aldoses and ketoses, are reducing sugars

SLIDE # Topic: Molisch Test

Amylose
A linear or non-branches polymer of glucose The glucose units are joined by a a-1-4 glucosidic linkages Exists in coiled form and each coil contains six glucose residues Amylose (unbranched) + amylopectin (branched) = starch

Glycogen
Storage form of carbohydrates in animals Stored in liver and muscles Made up of many unbranched linear chains, each having 10-15 glucose units linked by alpha-1,6-glycosidic linkages to form highly branched glycogen molecule Forms a colloidal solution in water and is non-reducing Gives a red-violet color w/ iodine MORE BRANCHED than starch

Cellulose
Composed of glucose units joined together in the form of the repeating units of the disaccharide cellobiose w/ numerous cross linkages Insoulble structural homoglycan of plant fiber Main constituent of the cell wall of plant cells Made of long, unbranched linear chains of 300-3000 Beta-glucose units, joined by Beta-1,4-glycosidic bonds Is non-reducing and us nit digested by enzymes of the human digestive system

SLIDE # Topic: Molisch Test

To detect the presence of carbohydrates Positive: purple/violet ring interphase b/w the acid and test layers Carbohydrates are dehydrated (loss of water) by conc. H2SO4 to form hydroxymethylfurfural (an aldehyde) will react with (condenses with) two molecules of alpha-naphthol (Molisch reagent) to yield a purple condensation product Molisch reagent
10 % a-naphthol (C10H8OH) dissolved in ethanol (C2H5OH) To test all types of carbohydrates

Reason for obtaining a positive result: alpha-naphthol is not a selective compound and it would react to every sugar

Positive for: Monosaccharides give a rapid positive test. Disaccharides and polysaccharides react slower. This purple color is produced by almost all carbohydrates, with the exception of sugar alcohols (alditols) and 2-amino-2-deoxy sugars. The conc. H2SO4 hydrolyzes the glycosidic bonds that are present in oligosaccharides and polysaccharides. The resulting monosaccharides are then dehydrated to produce the furfural derivatives. The furfural compounds condense with alpha-naphthol, creating the purple color. Pentoses and hexoses form five member oxygen containing rings (furfural) on dehydration Positive = presence of carbonyl grp Furfural derived from the dehydration of pentoses 5-hydroxymethyl furfural produces from hexoses and hexosans

SLIDE # Topic: Molisch Test

A green ring alone is seen even in the absence of carbohydrate and it indicates an excess of alpha-naphthol Molisch test universal test for carbihydrates and their derivatives Conc. H2SO4 not only catalyses the dehydrations of sugars but also brings about the hydrolysis of glycosidic bonds of oligosaccharides and polysaccharides

SLIDE # Topic: Molisch Test

SLIDE # Topic: Anthrone Test

For the determination of nonreducing sugars Reagent: 2% Anthrone reagent (0.3 g of Anthrone to 100mL conc Sulfuric acid) Anthrone
Tricyclic aromatic ketone

Concentrated sulphuric acid hydrolyses the glycosidic bonds of carbs and produces monsaccharides which are dehydrated by the concentrated acid and form furfural (liquid aldehyde C5H4O2) and their derivatives which react with anthrone to form a blue-green complex Carbohydrates + concentrated sulphuric acid -> furfurals + anthrone -> blue-green complex

SLIDE # Topic: Iodine Test

To test for the presence of starch Reagent: 0.01 M iodine solution (180 mg of KI in 100 mL water. 130 mg of iodine) Starch
Polysaccharide used for energy storage Composed of amylose (straight chain forms helices where iodine can assemble) and amylopectin (branched, shorter helices iodine cannot assemble), polymers of alpha-D-glucose

Reaction of Iodine with starch Deep blue/violet color: reaction of starch (amylose helices) and iodine > formation of adsorption complex (adsorbent [starch] + adsorbate [iodine]), formation of poly iodide chains During heating: clear colorless solution - breaking of the adsorption complex with heat Cooling: deep blue/violet color reformation of adsorption complex; remained colorless - absence of reformation; mono and disaccharides are too small to trap the iodine molecules; do not show color change in presence of iodine Color depends on length and organization of chains

SLIDE # Topic: Iodine Test

The blue color arises when the electrons of the entrapped iodine molecules interact with the electrons of the starch molecule and the resulting complex absorbs visible light (appears dark) Amylose a linear chain component of starch gives a deepblue color Amylopectin a branched component of starch gives a purple color Glycogen gives a reddish brown color Cellulose gives no color w/ iodine

SLIDE # Topic: Mucic Acid Test

Procedure Fumes generated while boiling acid fumes from nitric acid

SLIDE # Topic: Mucic Acid Test

Principle: Monosaccharides, upon treating with strong oxidizing agents such as nitric acid, yield saccharic acids. The saccharic acid obtained after the oxidation of galactose is insoluble and separates as gritty crystals. The acid derivative is known as mucic acid. Mucic Acid Test: to identify galactose
Galactose is classified as a monosaccharide, an aldose, a hexose, and is a reducing sugar.
Galactose is a monosaccharide. When combined with glucose, through a dehydration reaction, the result is the disaccharide lactose

Glucose is defined as the -OH on C # 4 in a horizontal projection in the chair form, (down in the Haworth structure) Galactose is defined as the -OH on C # 4 in a upward projection in the chair form

Reagent: HNO3 (nitric acid)

The position of the -OH group on the carbon (#4) is the only distinction between glucose and galactose

Oxidizes primary functional groups to carboxylic groups

SLIDE # Topic: Mucic Acid Test

Oxidation of terminal groups of aldoses
aldehyde primary alcohol groups are converted to carboxylic groups

Heating of HNO3 with an aldose sugar gives a dicarboxylic acid

Formation of mucic acid/galactaric acid

Mucic Acid
Melts at 213 C Insoluble in cold aqueous solution and forms crystals Saccharic acid THREE principal derivatives of Aldoses:
Aldonic Acids( alcohol Acids)-the aldehyde is oxidized Uronic Acid-those in which the primary alcohol group is oxidized Saccharic Acids-both the aldehyde and primary alcohol groups are oxidized

Oxidation of most monosaccharides by nitric acid yields soluble dicarboxylic acids. However, oxidation of galactose yields an insoluble mucic acid.

Results: crystals formed in galactose and lactose

Lactose will also yield a mucic acid, due to hydrolysis of the glycosidic linkage between its glucose and galactose subunits

SLIDE # Topic: Benedicts Test

Procedure: After heating, cool the contents in a test tube rack. Do not hasten cooling by immersion in cold water. WHY?!?

SLIDE # Topic: Benedicts Test

Benedicts test: to detect reducing sugars
reducing sugars: meaning that a molecule of this sugar can react with other molecules by giving electrons to them reduction

The sugar sucrose is a non-reducing sugar. It is formed in a condensation reaction making a glycosidic bond between a glucose molecule and a fructose molecule.
Difference in the glycosidic bond formed in sucrose prevents the sucrose from reacting with the reagent

Benedicts reagent contains: Copper sulphate dissociate to give sufficient cupric ions (as cupric hydroxide) for reduction reactions to occur; furnishes curpic ions (Cu++) in solution Sodium citrate keeps hydroxide in solution w/o getting precipitated; prevents the precipitation of cupric ions as cupric hydroxide by forming a loosely bound cupric-sodium citrate complex w/c on disassociation gives a continuous supply of cupric ions Sodium carbonate (Na2CO3) make the pH of the medium alkaline

Brick red ppt = copper (I) oxide Benedicts Reagent

A copper compound that will oxidize only aldehyde groups (aldoses) and not alcohols If you consider cyclic forms of carbihydrates, hemiacetals give positive tests while acetals give negative tests. The reason for this is that the cyclic form interconverts (is in equilibrium) with the linear form that contains an aldehyde

SLIDE # Topic: Benedicts Test

Carbohydrates with a free aldehyde (RCOH) or keto (RCOR) group have the ability to reduce various metallic ions Cupric ions are reduced to cuprous ions by the enediols from the sugars in the alkaline medium of the reagent
Benedicts reagent provides an alkaline/basic medium Enediol: powerful reducing agents; reduce blue cupric hydroxide to insoluble yellow to red cuprous oxide

Principle: Carbohydrates with free aldehyde or ketone groups have reducing properties. The mild alkali, sodium carbonate, converts glucose into enediol.This enediol reduces copper sulphate to cuprous hydroxide that is unstable and decomposes on boiling to cuprous oxide.

SLIDE # Topic: Benedicts Test

Sensitive up to 0.1-0.15 gm% of sugar solution
gm% - grams percent or grams per deciliter

For negative results, the nonreducing sugar test can be done

Sucrose is broken down into monosaccharides Boil the sample with hydrochloric acid this hydrolyses any sucrose present, splitting sucrose molecules to give glucose and fructose Cool the solution and neutralise it by adding sodium carbonate solution (an alkali solution) Carry out the reducing sugar test (Benedicts test) again

Color of precipitate depends on the concentration

Blue absence of reducing sugar Green up to 0.5 gm% Yellow >0.5 to 1.0 gm% Orange 1.0 to 2.0 gm% Brick Red 2 gm%

Semi-quantitative test
Because it gives an idea about the sugar solutions concentration

If you test a sample for both reducing and non-reducing sugars and:
the colours produced are the same: there are no non-reducing sugars If the colour in the non-reducing sugar test is more towards the red end of the spectrum: non-reducing sugars are present

All monosaccharides (glucose, fructose, galactose, mannose) will give a positive reaction Disaccharides such as lactose and malose will give a positive reaction

SLIDE # Topic: Barfoeds Test

Is there a difference between Benedicts test and Barfoeds Test? Yes, yes there is. Barfoeds test happens in a more acidic medium while Benedicts test happens in an alkaline medium. In an acidic medium monosaccharides enolize much more readily than disaccharides. If monosaccharides reacts faster, does this mean disaccharides also react but in a slower pace? Yes. Disaccharides react too but monosacchrides and disaccharides have different time frames since disaccharides are more complex sugars, also, disaccharides are weaker reducing agents (element or compound in a reductionoxidation (redox) reaction that donates an electron to another species) compared to monosaccharides. In about 1-3 minutes the monosaccharides will react forming the brick red ppt while disaccharides react at around 10 minutes.

Whats the principle? This test is based on the reducing nature of sugars due to their having an aldehyde or ketone group which reduce the cupric hydroxide formed in acidic medium to red colored cuprous oxide. This test which is specific for monosaccharides is based on copper reduction, but it differs from Benedicts reactions in term of the reaction medium. Barfoeds test is conducted in acidic conditions, whereas the other test is in alkaline medium. As disaccharides are weak reducing agents, they do not reduce cupric ions in Barfoeds solution under acidic conditions, whereas monosaccharides, being strong reducing agents, are capable of reducing copper ions in acidic conditions. However, on prolonged heating, acetic acid present in the reagent hydrolyzes disaccharides to monosaccharides, which respond to the test.

SLIDE # Topic: Barfoeds Test

Barfoeds reagent Contains cupric acetate in acetic acid Monosaccharides are stronger reducing agents in acidic medium as compared to disaccharides

SLIDE # Topic: Bials Orcinol Test

What happens?

SLIDE # Topic: Bials Orcinol Test

Strong acidity and temperature conditions permits breaking of glycosidic bonds (a type of covalent bond that joins a carbohydrate (sugar) molecule to another group, which may or may not be another carbohydrate) between saccharide units, hence, the latter undergo their penta/hexatomic ring opening The opened ring loses water molecules and convert themselves to furfural or its derivatives Saccharides that contain 5 Carbon atoms or pentoses cannot form polyatomic rings greater than 5 atoms so pentoses generate furfural and give a positive result Furfural acts as an electrophilic agent towards orcinol, it is a better electrophilic agent than its derivatives Among furfural derivatives, you may often meet 5-hydroxymethylfurfural (derivatives depends on what sugar sample is present). It is produced by saccharides having more than 5 carbon atoms but still containing pentatomic rings (i.e. Fructose)

IN SHORT: Pentoses, upon dehydration, forms a product called furfural, which is condensed by orcinol to form a blue-green colored solution. Furanose (a collective term for sugars that have a chemical structure that has a 5 member ring system [4 Carbon atoms and 1 Oxygen atom]) saccharides can either be a pentose or non-pentose. Pentose furanose (xylose) reacts faster and gives off the blue-green solution, thus, a positive result. NOTE: Fructose is a furanose non-pentose. Glucose is a pyranose. What is a furfural? (See figure 1 for structure) It is a heterocyclic aldehyde (meaning it is a cyclic compound that has atoms of at least two different elements as members of its ring). Upon air exposure it turns yellow.

SLIDE # Topic: Bials Orcinol Test

Sensitive test for the detection of pentoses Used to differentiate pentose sugars from hexose sugars
Bials reagent
0.2% orcinol in conc. HCl acid

Slide # 73 Topic: Seliwanoffs Test

Distinguish ketose and aldose via ketone/aldehyde functionality Ketose if a sugar contains ketone Aldose if a sugar contains aldehyde When heated, ketoses are more rapidly dehydrated than aldoses. Acid hydrolysis of polysaccharides and oligosaccharides yields simpler sugars followed by furfural The dehydrated ketose then reacts with the resorcinol to produce a deep cherry red color. Aldoses may react slightly to produce a faint pink color Fructose and sucrose are two common sugars which give a positive test. Sucrose gives a positive test as it is a disaccharide consisting of fructose and glucose.

Slide # 73 Topic: Seliwanoffs Test

Ketose reacts more readily with seliwanoff's test because ketones have 3 carbon atoms whereas aldehyde has 2 hydrogen atoms and one carbon atoms. Sugar loses water on being heated with strong acids and forms furfural derivatives. Thus furfurals form colored complexes with phenolic compounds such as resorcinol