Experiment 1: Carbohydrates y y polyhydroxy aldehydes or ketones and their derivatives (termed aldoses or ketoses) sugars water soluble and

d sweet Classifications: a. Monosaccharides simple sugars with saccharide group, which cannot be hydrolyzed further b. Oligosaccharides 2 to 10 saccharide groups linked by acetal or ketal linkages can be hydrolyzed to their respective monosaccharides or to two molecules of disaccharides. c. Polysaccharides may be branched or long linear monosaccharides in glucosidic linkages d. homopolysaccharides yield only one kind of monosaccharide upon hydrolysis (starch and glycogen) e. heterosaccharides yield a mixture of monosaccharides and derived products Cellulose and agar-agar polysaccharides with structural function For energy storage dextrin, glycogen, insulin and starch Glycogen animal starch; absent in brain but present in liver, muscles and other tissues - abundant in oysters and other shellfish - upon hydrolysis with amylase maltose - upon hydrolysis with acid glucose - 1,4- and 1,6- alpha glycosidic bonds giving a branched structure. Reagents Na2CO3 Sodium citrate CuSO4 Sample Starch RHO + 2Cu2+ + 4OHGlycogen Solubility of Polysaccharides Schweitzers Test [Cu(NH3)4(H2O)2 ](OH)2 Cellulose Amorphous precipitate + precipitation by addition of dilute acetic acid + reprecipitation of cellulose by 95% ethanol + also soluble in NaOH and CS2 for making viscose (precursor of rayon) + Iodine + polysaccharides colored complexes + alpha-amylose deep blue color + amylopectin (other component of starch), cellulose red brown solution + mono & disaccharides too small for I2 + differentiate helical from non helical polysaccharide + hemicelluloses (GA & A) vs cellulose may be hydrolyzed by dilute mineral acid RCOOH + Cu2 O + 2H2 O Chemical Reactions Visible Results Brick-red precipitate Brick-red precipitate Principle Involved + reducing sugars

y y y

B C 1

Tests Acid Hydrolysis of Polysaccharides Benedicts Test

Cross and Bevans Test

HCl ZnCl2

Cellulose

soluble

Starch

Glycogen

Iodine Test for Polysaccharides

40% NaOH I2 solution

Cellulose Gum Arabic Agar

Deep blue solution without NaOH Wine-red solution Or red precipitate Reddish-brown solution Colorless or yellow solution Colorless or yellow solution Colorless or yellow solution

Glucose

E I Glucose Molisch Test (Naphthol Reaction) Molisch Reagent (a-naphthol in 95% Ethanol) Conc. H2SO4 dehydrates CHO

Qualitative Tests for Carbohydrates General Color Reactions Reddish-violet ring at junction Reddish-violet ring at junction Reddish-violet ring at junction
O OH

Sucrose

Starch

+ H2SO4 dehydrates pentoses to furfural and hexoses to 5hydroxymethylfurfural + glycosidic bonds are hydrolyzed + condensation with anaphthol to produce purple product + intensity dependent on amount of CHO + same principle as above + used in the assay of carbohydrates carried under appropriate conditions + sugars react with 3 mol PhNHNH2 to form 1,2diphenylhydrazones or osazones + Ketoses with configurations identical to aldoses below C-2 give the same osazones e.g. glucose and fructose + sucrose hydrolysis glucose + fructose + lactose tufts of short fine crystals + for reducing sugars with free aldehyde or ketone group + osazones of maltose and lactose are formed upon cooling + has characteristic MP and shape + reducing sugars + free or potentially free ketone or aldehyde substituent + alkali present brings about considerable decomposition of sugar into reactive fragments which may reduce metal ions

Glucose

+
2 Anthrone Test Anthrone reagent (0.2% in H2SO4 ) Sucrose

H 2SO 4
OH O

Blue/bluegreen solution Blue/bluegreen solution Blue/bluegreen solution Yellow crystalline precipitate (5 minutes) Osazone soluble in hot water Yellow crystalline precipitate (glucosazone obtained by heating) (30 minutes) Yellow crystalline precipitate (20 minutes) Brick-red precipitate Brick-red precipitate Brick-red precipitate Brick-red precipitate Brick-red precipitate

Carbohydrate + H2SO4
OH O

Starch

O OH

Glucose

Maltose

Phenylhydrazine Test (Osazone Formation)

Phenylhydrazine HCl NaOAc

Sucrose

The Crystals:

Galactose Glucosazone (broom-shaped) Glucose Fructose 4 Benedicts Test Xylose Arabinose Maltose maltosazone (sunflower-shaped) galactosazone (fluffy ball shaped)

Sucrose

Blue solution

Starch

Blue solution

Glucose Fructose 5 Barfoeds Test Barfoeds Reagent (Cu(CH3COO)2 Acetic Acid) Maltose

Brick-red precipitate Brick-red precipitate Brick-red precipitate (slow) Blue solution Yellow to faint pink color Cherry-red solution Green solution Cherry-red solution Presence of Pentoses Green, brown or reddish brown solution Green, brown or reddish brown solution Blue-green solution Blue-green solution
H3 C
OH

Sucrose Glucose

Seliwanoffs Test

Seliwanoffs Reagent (6M HCldeH2O agent Resorcinol)

Fructose Xylose Sucrose

+ has quantitative version since amount of precipitate is the basis of the test + di and polysacc. may be hydrolyzed to monosacc. + sucrose the only nonreducing sugar + carried out at pH 4.6 + differentiates di and monosaccharides + monosaccharides are more readily oxidized + reducing disaccharides (slow) lactose but not sucrose + 2-3 mins. for monosaccharides + differentiating for aldohexoses and ketohexoses + pentoses green (-) + keto cherry-red + aldo - yellow to pink + ketohexoses are dehydrated faster + boil for NMT 20-30s to avoid false + results

F Glucose Orcinol (m-methylresorcinol) Condenses furfural to colored compounds FeCl3 in HCl decomposition to furfural

Fructose

Bials Test (Orcinol Test)

Xylose

+ condensation reaction

Arabinose Glucose 2 Tollens Phloroglucinol Test Phloroglucinol Fructose Xylose Galactose

Pentoses
,

OH HO
HO OH

CHO (

HO

CH 3

HO

Colorless solution Colorless solution Colorless solution Red solution

+ condensation reaction

Glucose

HO
Lactose 3 Mucic Acid Test Conc. HNO3 Galactose

OH H H H O H OH OH
HNO 3 (

HO H HO H

COOH
OH

H HO

H
COOH

Colorless solution Colorless Crystalline precipitate Colorless Crystalline precipitate

OH

Fermentation Test (d-glucose, d-maltose, d-fructose)

Bakers yeast

Effervescence

Zymase + H3PO4 + coenzyme phosphorylation of sugar forming hexose PO4 CO2 + ethanol hexose hexose diphosphoric acid phosphor-glyceric acid pyruvic acid acetaldehyde ethanol

+ Mucic Acid = galactaric acid + differentiating test from fructose and glucose + galactose is [O] by HNO3 to mucic acid (sandy crystals) + lactose reducing disacc. gluc + galac + yeast ferments only the naturally occurring dsugars. Some yeasts do not ferment lactose or dgalactose at all. + Einhorn saccharometer determine amount of CO2 evolved + zymase enzyme in yeast which produces fragmentation of sugar molecule + 10-15% ethanol because it inhibits action of yeast + invertase sucrose to glucose and fructose + addition of PO43- salts catalyzes by buffering action + CHI3 hexagonal crystalline shape under the microscope

Test for Ethanol

Lugols solution (I2-KI solution) NaOH

2NAOH + I2 NaIO + NaI + H2O C2H5OH + NaIO CH3CHO + NaI + H2O CH3CHO + 3NaIO I3CCHO + 3NaOH I3CCHO + NaOH CHI3 + HCOONa

Yellow precipitate with foul odor

Test for CO2

CaCl2 TS

CaCl2 + H2O + 2CO2

CaCO3 + HCl

White precipitate

Classification Procedure:

Structure Sugar Fisher projection

O H HO H H OH H OH OH OH OH

Haworth projection
OH H HO H
H H H HO OH OH H
OH O OH H OH H H OH H H

Classification

Simpler fragments

Glucose

H OH H

O OH H

Mono

Hexose

Red

OH
O OH

Fructose

HO H H

O H OH OH OH O

OH

Mono

Hexose

Red

Galactose

H HO HO H

OH H H OH OH

HO

Mono

Hexose

Red

Sucrose

Di

Non-red

Glucose, Fructose

Maltose

Di

Red

Glucose

Lactose

Di

Red

Galactose, Glucose

Cellulose Starch Glycogen

O

Poly See [D] Poly Poly

H O OH H H OH HO H OH H H H H HO H OH H OH H O OH

Non-red Non-red Non-red

Glucose: -1,4 Glucose: -1,4 linkages Analogous to amylopectin

Arabinose

H HO H

OH H OH OH
O

Mono

Pentose

Red

Xylose

H HO H

OH H OH OH

Mono
H

Pentose

Red