You are on page 1of 81

BIOCHEM LABORATORY

Carbohydrates
and
their Structures
Maria Salvacion A. Esmalla, M. Sc.
University of Santo Tomas
Faculty of Pharmacy
Biochemistry Department

BIOCHEM LABORATORY

What are carbohydrates?


- a biological compound containing carbon,
hydrogen, and oxygen that is an important source
of food and energy
- the formula of most of these compounds may be
expressed as Cm(H2O)n
the most abundant organic compounds found in
nature
-

BIOCHEM LABORATORY

According to Number of Sugar Units


Monosaccharide
Disaccharide
OH

OH

Polysaccharide

CH2OH
O
OH

CH2OH
O
OH
OH

CH2OH
O
OH

CH2OH
O
OH

OH

OH

CH2OH
O
OH

CH2OH
O
OH
OH

OH

OH

BIOCHEM LABORATORY

According to Functional Group


Aldose

Ketose

CH2OH

CH
H

OH

CH2OH

CH2OH

BIOCHEM LABORATORY

According to Number of Carbons

pentose

hexose

BIOCHEM LABORATORY

According to Number of Carbons


1
1
1
1

CHO
H
C

H
C

OH

OH
H

CH2OH

Triose

CHO

H
C

OH

CH2OH

Tetrose

CHO

OH

OH

OH

H
H
H

CHO
C

OH

OH

OH
OH

CH2OH

Pentose

CH2OH

Hexose

BIOCHEM LABORATORY

Fischer Projection
two-dimensional representation of the stereochemistry of
three-dimensional molecules
H

H
HO
H
H

CH2OH

OH
H
OH
OH

CH2OH

D-Glucose

HO
H
H

3
4
5

OH

OH

CH2OH

D-Fructose

BIOCHEM LABORATORY

D, L Notation
D,L tells which of the two chiral isomers we
are referring to.
If the OH group on the next to the bottom
carbon atom points to the right , the isomer
is a D-isomer; if it points left, the isomer is L.
The D form is usually the isomer found in
nature.

BIOCHEM LABORATORY

D Notation

H C

H C OH
H C OH
CH2OH

Right = D

BIOCHEM LABORATORY

BIOCHEM LABORATORY

BIOCHEM LABORATORY

What are optical isomers?


also called stereoisomers
molecules that differ from each other only in
configuration of structures
usual source of optical isomerism is a chiral carbon

CHO
H

CHO

OH

CH2OH
D-Glyceraldehyde

HO

CH2OH
L-Glyceraldehyde

BIOCHEM LABORATORY

What are enantiomers?


molecules that are mirror images but not superimposable

CHO
H

CHO

OH

CH2OH
D-Glyceraldehyde

HO

CH2OH
L-Glyceraldehyde

BIOCHEM LABORATORY

What are diastereomers?


nonsuperimposable, non-mirror image stereoisomers
O

H
C

C
H

OH

HO

OH

OH

CH2OH
D-Erythrose

CH2OH
D-Threose

BIOCHEM LABORATORY

What are epimers?


diastereomers that differ from each other in configuration
at only one chiral carbon
O

H
C

C
H

OH

OH

OH

HO

OH

OH

CH2OH
D-Ribose

CH2OH
D-Xylose

BIOCHEM LABORATORY

What are epimers?

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Important Monosaccharides
H

CH 2OH
C

OH

HO

HO

OH

OH

OH

OH

CH 2OH
D-Glucose

CH 2OH
D-Fructose

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Important Monosaccharides
H

O
C

H C OH
HO C H
HO C H
H C OH
CH 2OH
D-galactose

BIOCHEM LABORATORY

Does monosaccharide forms cyclic structure?


H
O

H
HO
H
H

OH
6
6

CH2OH

OH
OH

C
OH

CH2OH

D-Glucose

C
OH
H

H
O
H

C2

OH

H
1

C
O

CH2OH

C
OH
H

C
OH

O
H

C2

OH

Hemiacetal form

H
C

OH

BIOCHEM LABORATORY

Does monosaccharide forms cyclic structure?


1

CH2OH

HO
H
H

O
H
OH
OH

CH2OH

D-Fructose

C
4

CH2OH

CH2OH O
C

OH C

OH

CH2OH

CH2OH O
C
H

H
C

OH

OH C
C

OH

Hemiketal form

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Mutarotation: A small amount of open


chain is in equilibrium with the cyclic
forms.
The most stable form of glucose is -Dglucose .
-D-glucose

(36%)

D-glucose (open)

(trace)

-D-glucose

(64%)

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Mutarotation

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Mutarotation

BIOCHEM LABORATORY

Are there new features in the cyclic structure of


monosaccharides?
6

H
4

C
OH

CH2OH

C
OH
H

C
H

O
H
C2
OH

H
C

Anomeric
carbon
5

OH

CH2OH

CH2OH O
C
H

H
C

OH

OH C
C

OH

Anomeric
carbon

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Are there new features in the cyclic structure of


monosaccharides?
6

H
4

C
OH

C
OH
H

CH2OH
O
H

C2

OH

-D-Glucose

H
C

H
1

OH

C
OH

CH2OH

C
OH
H

O
H

C2

OH

-D-Glucose

OH
C
H

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Haworth Projection
perspective representation of the cyclic forms of sugars
O

C
C

C
C

Furanose structures

Furan

C
C

Pyranose structures

Pyran

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Fischer to Haworth Projection


H
H
HO
H
H

OH

OH
H

CH2OH

OH
OH

H
OH
3

H
1

OH

5
4

OH

-D-Glucose

CH2OH
D-Glucose

O
1

OH
3

OH

OH

CH2OH

(-D-Glucopyranose)

OH

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Conformation

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Haworth to Chair Conformation

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Haworth to Chair Conformation


6

CH2OH
5

H
OH

OH

O
H

H
1

OH

OH

-D-Glucose
(-D-Glucopyranose)

Draw the Fischer, Haworth and Chair conformation of


-D-mannopyranose and -D-galactopyranose

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

How are glycosidic bonds formed?

CH2OH

CH2OH

O
+

OH
OH

OH
OH

Glycosidic bond

CH3OH

OH
OH

OCH3
OH

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Glycosidic linkage among sugars


CH2OH

CH2OH
O

CH2OH

OH

OH

OH
O

OH

OH

OH

OH

OH

H2
C

OH

OH

OH

(1-4) Glycosidic bond

(1-6) Glycosidic bond


OH
OH

CH2OH
O
OH

OH
O

O
CH2OH

OH
OH

,(1-1) Glycosidic bond

OH
OH

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Important Disaccharides
Maltose = Glucose + Glucose

Lactose = Glucose + Galactose

Sucrose = Glucose + Fructose

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Important Disaccharides
CH2OH

CH2OH

CH2OH

OH

OH
O

OH

OH

D-Lactose

CH2OH

CH2OH
O

OH

OH

O
OH

OH

(1-4) glycosidic bond

CH2OH
O

OH

OH
OH

,(1-2) glycosidic bond

OH

OH

Sucrose

CH2OH

CH2OH

OH

OH

OH

CH2OH

OH
OH

O
O

OH

OH
OH

-D-Maltose

D-Cellobiose

(1-4) glycosidic bond

(1-4) glycosidic bond

OH

OH

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Sucrose
CH 2OH

o
OH
OH

, -1,2-glycosidic
bond

OH
CH 2OH

O
OH
OH

CH 2OH

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Lactose
(1-4) glycosidic bond

CH2OH

CH2OH
O

OH

O
O

OH

OH
OH

OH

OH

D-Lactose

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Maltose
(1-4) glycosidic bond
CH2OH

CH2OH
O

OH

OH
O

OH
OH

OH
OH

-D-Maltose

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Cellobiose
(1-4) glycosidic bond
CH2OH

CH2OH
O

OH

OH

OH

OH
OH

D-Cellobiose

OH

Learning Check
Identify the monosaccharides in each of the
following:
A. lactose
(1) glucose
(2) fructose (3) galactose
B. maltose
(1) glucose

(2) fructose (3) galactose

C. sucrose
(1) glucose

(2) fructose (3) galactose


39

Solution
A. lactose
(1) glucose and (3) galactose
B. maltose
(1) glucose and (1) glucose
C. sucrose
(1) glucose and (2) fructose

40

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Important Polysaccharides
Starch
Amylose
Amylopectin
Glycogen
Cellulose

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Polysaccharides
Polymers of D-glucose
6

CH2OH

H
4

C
OH

C
OH
H

O
H

C2

OH

H
C

OH

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Amylose
Polymer with -1,4 bonds
CH2OH

CH2OH
O

CH2OH
O

OH

OH

OH

O
OH

CH2OH
OH

O
OH

O
OH

-1,4 bonds

OH

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Amylopectin
Polymer with -1,4 and -1,6 bonds branches
-1,6 bond
CH2OH

CH2OH
O

CH2OH
O

OH

OH

OH

OH

OH
CH2OH

OH
CH2OH

CH2
O

OH

OH

OH

O
OH

CH2OH
OH

O
OH

-1,4 bonds

O
OH

OH

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Cellulose
Polymer with -1,4 bonds

CH2OH
O
OH

CH 2OH
O

OH

OH

CH2OH
O

O
OH

OH

O
OH

-1,4 bonds

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Glycogen

Learning Check
Identify the polysaccharide in each as:
1) starch 2) glycogen 3) cellulose
A.

B.

C.

47

Solution

3) cellulose

1) starch

2) glycogen

48

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Physical Properties
of
Carbohydrates

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Physical Properties
Carbohydrates are solid at room temp
Mono- and disaccharides are white crystalline substances
and soluble in H2O
Starches are amorphous and slighlty soluble in H2O
Cellulose is fibrous and insoluble in H2O

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Chemical Properties
of
Carbohydrates

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Reducing Property
1.

All aldoses are reducing sugars because they contain free


aldehyde functional group.
The aldehydes are oxidized by a mild oxidizing agent
(e.g. Benedicts or Fehlings reagent) to the
corresponding carboxylates.

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Reducing Property
2.

All ketoses are reducing sugars because they have a


ketone functional group next to an alcohol functional
group.
The reactivity of the -hydroxyketone is due to the
formation of -hydroxyaldehyde in basic media.

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Reducing Property
3.

Carbohydrates with hemiacetal functional groups can


reduce mild oxidizing agent because hemiacetals can
form aldehydes through the following equilibrium
equation.

- Sucrose is a non-reducing sugar because it does not contain a


hemiacetal functional group.
-

Starch is a non-reducing sugar because the effect of the


hemiacetal
functional group is insignificant in a very large starch molecule.

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Hydrolysis of acetal groups


1.

Disaccharides and polysaccharides can be converted into


monosaccharide by hydrolysis

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Isolation
of
Carbohydrates

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Glycogen from Chicken liver


- energy storage polysaccharide in animals
- a branched-chain polysaccharide (homoglycan)
- consists entirely of linear chains of glucose units connected by 1,4-glycosidic bonds and branched points every 1 in 10 glucose units
linked by -1,6-glycosidic bonds

Homogenization disrupts membranes


0.1% acetic acid removes protein via precipitation
Glycogen is insoluble in ethanol.
Method: Protein denaturation and solubility of glycogen

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Starch from Potato


- energy storage polysaccharide in plants
- a homoglycan mixture of amylose and amylopectin

1. Amylose linear homoglycan of glucose connected by -1,4


linkages

2. Amylopectin branched homoglycan with approximately 1 in 25


glucose units linked in both -1,4 and -1,6 fashion

Starch is slightly soluble in H2O


Method: Selective dissolution

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Hydrolysis
of
Carbohydrates

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Acid Hydrolysis
- 2 N HCl
- complete hydrolysis
- efficient method to obtain monosaccharides
starch/glycogen

glucose

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Enzymatic Hydrolysis

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Enzymatic Hydrolysis
- salivary amylase (-amylase)
- very specific
- an endoglycosidase hydrolyzing -1,4 linkages at random
to yield a mixture of glucose, maltose, dextrin & other
hydrolysates
starch/glycogen

dextrin, amylodextrin, maltose,glucose

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Thin layer chromatography


of
Carbohydrates

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Thin Layer Chromatography


based on adsorption phenomenon
normal phase chromatography
stationary phase: silica G-250
mobile phase: butanol: acetic acid:ether:H2O (9:6:3:1)
visualizing agent: p-anisaldehyde
appeared as dark green spots
standards: dextrin, maltose and glucose

Rf

polar

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Quantitative Determination
of
Carbohydrates

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Nelsons Test
REAGENTS: Nelsons A Na2CO3, NaKtartrate, NaHCO3, Na2SO4
Nelsons B CuSO45H2O, H2SO4
Arsenomolybdate reagent
- CuSO4 serves as the oxidizing agent
- Na2CO3 & NaHCO3 makes sugars more reactive
- NaKtartrate prevents the precipitation of Cu(OH)2
- NaKtartrate would tend to reduce Cu2+ in time
- Arsenomolybdate is added to dissolve Cu2O & is converted into molybdenum blue
- color intensity of molybdenum blue A480

y = mx + b

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Characterization
of
Carbohydrates

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

General Test: Molisch Test


REAGENTS: conc. H2SO4; -naphthol in 95% ethanol
PURPOSE: confirm presence of carbohydrates
POSITIVE RESULT: violet interphase
PRINCIPLE INVOLVED:
hydrolysis, dehydration forming either a furfural (pentoses) or a 5hydroxufurfural (hexoses); condensation with -naphthol
REACTION:

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

General Test: Iodine Test


REAGENTS: iodine reagent
PURPOSE: distinguish starch and glycogen from other polysaccharides
POSITIVE RESULT: Starch gives a blue-black color
Glycogen gives a red color
PRINCIPLE INVOLVED: complexation
REACTION:

I2

I2
I2
I2

HEAT

I2
COOL

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Specific Test: Anthrone Test


REAGENTS: conc. H2SO4; anthrone reagent
PURPOSE: confirm presence of carbohydrates
POSITIVE RESULT: blue-green solution
PRINCIPLE INVOLVED:
Hydrolysis, dehydration forming either a furfural or a 5- hydroxyfurfural;
condensation of anthrone via anthranol intermediate
REACTION:
O

OH
H

CH O

[O]

H
H

OH

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Specific Test: Benedicts Test


REAGENTS: CuSO4; Na3C6H5O7; Na2CO3 (Benedicts reagent)
PURPOSE: detect presence of reducing sugars
POSITIVE RESULT: brick red precipitate
PRINCIPLE INVOLVED:
oxidation in less basic media
REACTION:

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Specific Test: Barfoeds Test


REAGENTS: Cu(Ac)2; Acetic acid (Barfoeds reagent)
PURPOSE: detect reducing sugars; distinguishes between reducing
monosaccharide and reducing disaccharide
POSITIVE RESULT: brick red precipitate
PRINCIPLE INVOLVED:
oxidation of a reducing monosaccharide or disaccharide in acidic
condition
REACTION:

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Specific Test: Bials Orcinol Test


REAGENTS: orcinol in conc. HCl; FeCl3 (Bials reagent)
PURPOSE: detect presence of pentoses
POSITIVE RESULT: blue-green solution
PRINCIPLE INVOLVED:
dehydration and condensation reaction with orcinol
REACTION:

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Specific Test: Seliwanoffs Test


REAGENTS: resorcinol; HCl
PURPOSE: differentiate ketohexoses from aldohexoses
POSITIVE RESULT: rapid formation of cherry red solution
PRINCIPLE INVOLVED:
rapid dehydration and condensation reaction with resorcinol
REACTION:

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

General Test: Mucic acid Test


REAGENTS: conc. HNO3
PURPOSE: detect presence of galactose (moiety)
POSITIVE RESULT: rhombic crystal
PRINCIPLE INVOLVED:
1,6 -oxidation of sugars whereby galactose-containing carbohydrates form
a meso compound (galactaric acid)
REACTION:

H
C
H

HO

HO
H

C
C
H2C

1
2

OH
OH
OH

HO

HNO3

OH
2

HO
O

OH
OH

HO

O
6
5

OH

OH

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Specific Test: Osazone/Phenylhydrazone Test


Sucrose does not form osazone unless hydrolyze

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Specific Test: Osazone/Phenylhydrazone Test


Glucosazone - needle-like/feathery, 4-5min

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Specific Test: Osazone/Phenylhydrazone Test


Fructosazone needle-like/feathery, 2 min

Mannosazone needle-like/feathery, 0.5min

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Specific Test: Osazone/Phenylhydrazone Test


Lactosazone fine needles; grouped in balls; soluble in hot H2O

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Specific Test: Osazone/Phenylhydrazone Test


Galactosazone broad/flat crystals (15-20 min)
Maltosazone broad needle-like crystals; soluble in hot H2O
Xylosazone long, fine needle-like crystals (7 min)

BIOCHEM LABORATORY
BIOCHEM
LABORATORY

Specific Test: Osazone/Phenylhydrazone Test


Starch - does not form osazone unless hydrolyze