carbohydrates 2
DESCRIPTION
CarbohydratesTRANSCRIPT
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Carbohydratesand
their Structures
BIOCHEM LABORATORY
Maria Salvacion A. Esmalla, M. Sc.University of Santo Tomas
Faculty of PharmacyBiochemistry Department
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What are carbohydrates?What are carbohydrates?
BIOCHEM LABORATORY
- 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
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According to Number of Sugar UnitsAccording to Number of Sugar Units
BIOCHEM LABORATORY
Monosaccharide
Disaccharide
Polysaccharide
OH
OH
H
OH
H
OHH
OH
CH2OH
H
O
OHOH
OH
CH2OHO
OOH
OH
CH2OH
OH
O
OOH
OH
CH2OHO
OOH
OH
CH2OHO
OOH
OH
CH2OHO
OOH
OH
CH2OH
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According to Functional GroupAccording to Functional Group
BIOCHEM LABORATORY
Aldose Ketose
CH
C
CH2OH
O
OHH
CH2OH
C
CH2OH
O
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According to Number of CarbonsAccording to Number of Carbons
BIOCHEM LABORATORY
pentose
hexose
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According to Number of CarbonsAccording to Number of Carbons
BIOCHEM LABORATORY
Triose
Tetrose
CHO1
C2
CH2OH3
OHH
CHO1
C2
C3
OHH
CH2OH4
H OH
CHO1
C2
C3
OHH
C4
H OH
CH2OH5
H OH
CHO1
C2
C3
OHH
C4
H OH
C5
H OH
CH2OH6
H OH
PentoseHexose
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BIOCHEM LABORATORY
Fischer ProjectionFischer Projection
D-Glucose
C1
C2
OHH
C3
HHO
C4
OHH
C5
OHH
CH2OH6
OH
CH2OH1
C2
O
C3
HHO
C4
OHH
C5
OHH
CH2OH6
D-Fructose
two-dimensional representation of the stereochemistry of three-dimensional molecules
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BIOCHEM LABORATORY
D, L NotationD, 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.
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BIOCHEM LABORATORY
D NotationD Notation
C
C
CH2OH
OHH
OHH
CO
H
Right = D
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BIOCHEM LABORATORY
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BIOCHEM LABORATORY
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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
D-Glyceraldehyde
C OHH
CHO
CH2OH
C HHO
CHO
CH2OH
L-Glyceraldehyde
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BIOCHEM LABORATORY
What are enantiomers?
D-Glyceraldehyde
C OHH
CHO
CH2OH
C HHO
CHO
CH2OH
L-Glyceraldehyde
molecules that are mirror images but not superimposable
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BIOCHEM LABORATORY
What are diastereomers?
nonsuperimposable, non-mirror image stereoisomers
C
C OHH
C OHH
CH2OH
OHC
C HHO
C OHH
CH2OH
OH
D-ThreoseD-Erythrose
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BIOCHEM LABORATORY
What are epimers?
C
C OHH
C OHH
C
OH
CH2OH
OHH
C
C OHH
C HHO
C
OH
CH2OH
OHH
D-Ribose D-Xylose
diastereomers that differ from each other in configuration at only one chiral carbon
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BIOCHEM LABORATORY
What are epimers?
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BIOCHEM LABORATORYBIOCHEM LABORATORY
C
C
CH2OH
H OHC
OHH C
HHO
H OH
C OH
D-Glucose
C
C
CH2OH
H OHC
OHH C
HHO
O
CH2OH
D-Fructose
Important MonosaccharidesImportant Monosaccharides
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Important MonosaccharidesImportant Monosaccharides
D-galactose
C
C
C
C
C
CH2OH
OH
OHH
HO H
HHO
OHH
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BIOCHEM LABORATORY
Does monosaccharide forms cyclic structure?
C1
C2
OHH
C3
HHO
C4
OHH
C5
OHH
CH2OH6
OH
D-Glucose
C5 O
C4
C3
C1
C 2
OH
H
H
OH
CH2OH6
H
OHO
H
H
HC5 O
C4
C3
C 1
C 2
OH
H
H
OH
CH2OH6
H
OH OH
H
H
Hemiacetal form
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BIOCHEM LABORATORY
Does monosaccharide forms cyclic structure?
Hemiketal formD-Fructose
CH2OH1
C2
O
C3
HHO
C4
OHH
C5
OHH
CH2OH6
O
5C
4C
3
C
1
C
2
H
OH
OH
H
CH2OH6
H
O
CH2OHH
O
5C
4C
3
C
1
C
2
H
OH
OH
H
CH2OH6
H OH
CH2OH
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Mutarotation: A small amount of open chain is in equilibrium with the cyclic
forms.The most stable form of glucose is β-D-
glucose .
-D-glucose D-glucose (open) β-D-glucose
(36%) (trace) (64%)
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Mutarotation
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Mutarotation
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BIOCHEM LABORATORY
C5 O
C4
C3
C 1
C 2
OH
H
H
OH
CH2OH6
H
OH OH
H
H
Anomeric carbon
O
5C
4C
3
C
1
C
2
H
OH
OH
H
CH2OH6
H OH
CH2OHAnomeric
carbon
Are there new features in the cyclic structure of monosaccharides?
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BIOCHEM LABORATORYBIOCHEM LABORATORY
C5 O
C4
C3
C 1
C 2
OH
H
H
OH
CH2OH6
H
OH OH
H
H
C5 O
C4
C3
C 1
C 2
OH
H
H
OH
CH2OH6
H
OH H
OH
H
α-D-Glucose β-D-Glucose
Are there new features in the cyclic structure of monosaccharides?
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BIOCHEM LABORATORYBIOCHEM LABORATORY
O
CC
CO
C
O C
C
C C
C
O
Furanose structures
Pyranose structures
O
O
Furan
Pyran
perspective representation of the cyclic forms of sugars
Haworth Projection
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BIOCHEM LABORATORYBIOCHEM LABORATORY
C1
C2
H OH
C3
OHH
C4
HO H
OHH
C5
H
CH2OH6
O
5
4
3 2
1
OH
CH2OH6
H
OH
OH
H
H
OH
H
OH
5
4
3 2
1
OCH2OH
6
OH
OHOHOH
D-Glucose
α -D-Glucose
(α-D-Glucopyranose)
Fischer to Haworth Projection
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Conformation
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Haworth to Chair Conformation
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Haworth to Chair Conformation
5
4
3 2
1
OH
CH2OH6
H
OH
OH
H
H
OH
H
OH
α -D-Glucose
(α-D-Glucopyranose)
?
Draw the Fischer, Haworth and Chair conformation of α-D-mannopyranose and α-D-galactopyranose
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BIOCHEM LABORATORYBIOCHEM LABORATORY
How are glycosidic bonds formed?
+ CH3OH
OCH2OH
OH
OHOHOH
OCH2OH
OH
OHOCH3OH
Glycosidic bond
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Glycosidic linkage among sugars
OCH2OH
OH
OH
OOH
OCH2OH
OH
OHOH
OCH2OH
OH
OH
OH
O
H2C
OH
OHOHO
OCH2OH
OH
OH
OH
O
OH
CH2OH
OH
OH
O
α(1-4) Glycosidic bond α(1-6) Glycosidic bond
β,β(1-1) Glycosidic bond
OH
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Important Disaccharides
Maltose = Glucose + Glucose
Lactose = Glucose + Galactose
Sucrose = Glucose + Fructose
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Important Disaccharides
OCH2OH
OH
OH
OH O CH2OH
CH2OH
OH
OHO
OCH2OH
OH
OH
OOH
OCH2OH
OH
OHOH
Sucrose
,β(1-2) glycosidic bond
OCH2OH
OH
OH
OH OCH2OH
OH
OHOH
O
–D-Lactose
β(1-4) glycosidic bond
-D-Maltose
(1-4) glycosidic bond
β–D-Cellobiose
β(1-4) glycosidic bond
OCH2OH
OH
OHOH
OCH2OH
OH
OH
OH
O
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Sucrose
O
CH2OH
OH
OH
CH2OH
oCH2OH
OHOH
OH
O
α, -1,2-glycosidic bond
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Lactose
OCH2OH
OH
OH
OH OCH2OH
OH
OHOH
O
–D-Lactose
β(1-4) glycosidic bond
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Maltose
OCH2OH
OH
OH
OOH
OCH2OH
OH
OHOH
-D-Maltose
(1-4) glycosidic bond
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Cellobiose
β–D-Cellobiose
OCH2OH
OH
OHOH
OCH2OH
OH
OH
OH
O
β(1-4) glycosidic bond
β
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39
Learning CheckIdentify 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
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40
Solution
A. lactose
(1) glucose and (3) galactose
B. maltose
(1) glucose and (1) glucose
C. sucrose
(1) glucose and (2) fructose
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Important Polysaccharides
Starch
Amylose
Amylopectin
Glycogen
Cellulose
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Polysaccharides
C5 O
C4
C3
C 1
C 2
OH
H
H
OH
CH2OH6
H
OH OH
H
H
Polymers of D-glucose
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Amylose
Polymer with α-1,4 bonds
α-1,4 bonds
OOH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O O
OH
OH
CH2OH
O
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Amylopectin
Polymer with α-1,4 and α-1,6 bonds branches
α-1,6 bond
α-1,4 bonds
OOH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2
O
O O
OH
OH
CH2OH
O
OOH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Cellulose
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2OH
O
Polymer with ß-1,4 bonds
ß-1,4 bonds
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Glycogen
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47
Learning CheckIdentify the polysaccharide in each as:
1) starch 2) glycogen 3) cellulose
A. B. C.
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48
Solution
3) cellulose 1) starch 2) glycogen
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Physical Properties
of
Carbohydrates
BIOCHEM LABORATORYBIOCHEM LABORATORY
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BIOCHEM LABORATORYBIOCHEM LABORATORY
● 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
Physical Properties
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Chemical Properties
of
Carbohydrates
BIOCHEM LABORATORYBIOCHEM LABORATORY
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BIOCHEM LABORATORYBIOCHEM LABORATORY
1. All aldoses are reducing sugars because they contain free aldehyde functional group.
The aldehydes are oxidized by a mild oxidizing agent (e.g. Benedict’s or Fehling’s reagent) to the corresponding carboxylates.
Reducing Property
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BIOCHEM LABORATORYBIOCHEM LABORATORY
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.
Reducing Property
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BIOCHEM LABORATORYBIOCHEM LABORATORY
3. Carbohydrates with hemiacetal functional groups can reduce mild oxidizing agent because hemiacetals can form aldehydes through the following equilibrium equation.
Reducing Property
- 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.
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BIOCHEM LABORATORYBIOCHEM LABORATORY
1. Disaccharides and polysaccharides can be converted into monosaccharide by hydrolysis
Hydrolysis of acetal groups
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Isolation
of
Carbohydrates
BIOCHEM LABORATORYBIOCHEM LABORATORY
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BIOCHEM LABORATORYBIOCHEM 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 membranes0.1% acetic acid – removes protein via precipitationGlycogen is insoluble in ethanol.Method: Protein denaturation and solubility of glycogen
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BIOCHEM LABORATORYBIOCHEM 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 H2OMethod: Selective dissolution
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Hydrolysis
of
Carbohydrates
BIOCHEM LABORATORYBIOCHEM LABORATORY
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Acid Hydrolysis
- 2 N HCl- complete hydrolysis- efficient method to obtain monosaccharides
starch/glycogen
glucose
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Enzymatic Hydrolysis
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BIOCHEM LABORATORYBIOCHEM LABORATORY
- 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
Enzymatic Hydrolysis
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Thin layer chromatography
of
Carbohydrates
BIOCHEM LABORATORYBIOCHEM LABORATORY
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Thin Layer Chromatography
● based on adsorption phenomenon● normal phase chromatographystationary phase: silica G-250mobile phase: butanol: acetic acid:ether:H2O (9:6:3:1)visualizing agent: p-anisaldehyde● appeared as dark green spotsstandards: dextrin, maltose and glucose
Rf polar
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Quantitative Determination
of
Carbohydrates
BIOCHEM LABORATORYBIOCHEM LABORATORY
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BIOCHEM LABORATORYBIOCHEM LABORATORY
Nelson’s Test
REAGENTS: Nelson’s A – Na2CO3, NaKtartrate, NaHCO3, Na2SO4
Nelson’s B – CuSO4●5H2O, 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
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Characterization
of
Carbohydrates
BIOCHEM LABORATORYBIOCHEM LABORATORY
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General Test:General Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
Molisch TestREAGENTS: conc. H2SO4; α-naphthol in 95% ethanol
PURPOSE: confirm presence of carbohydrates
POSITIVE RESULT: violet interphase
PRINCIPLE INVOLVED:
hydrolysis, dehydration forming either a furfural (pentoses) or a 5- hydroxufurfural (hexoses); condensation with α-naphthol
REACTION:
5-hydroxyfurfural
O CH2OHH
O
C
OHH
HHO
OHH
OHH
CH2OH
H2SO4
OH
hexose
OH
naphthol
O
OH
OH
HO
purple-colored dye+-
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General Test:General Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
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
I2I2
HEAT
I2
COOL
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Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
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:
OR CHOO OH O
HHO
O
H
R
O
O
OH
R
H+ [O]
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Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
Benedict’s Test
REAGENTS: CuSO4; Na3C6H5O7; Na2CO3 (Benedict’s reagent)
PURPOSE: detect presence of reducing sugars
POSITIVE RESULT: brick red precipitate
PRINCIPLE INVOLVED:
oxidation in less basic media
REACTION:
+ Cu2O
OH
CH2OH
H
H
OH
OH
H
O
O-Na
+
OH
H
O
CH2OH
H
H
OH
OH
H
OH
H
OH
H
Cu+2
Na2CO3, Na citrate
-D-glucose sodium gluconate
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Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
Barfoed’s Test
REAGENTS: Cu(Ac)2; Acetic acid (Barfoed’s 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:
OH
CH2OH
H
H
OH
OH
H
O
OH
OH
H
O
CH2OH
H
H
OH
OH
H
OH
H
OH
H
Cu+2
H+
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Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
Bial’s Orcinol TestREAGENTS: orcinol in conc. HCl; FeCl3 (Bial’s reagent)
PURPOSE: detect presence of pentoses
POSITIVE RESULT: blue-green solution
PRINCIPLE INVOLVED:
dehydration and condensation reaction with orcinol
REACTION:
HO OH
OH
O
O
H
HO
OH
OH
OH
O
O
OH
OH
OH
H+
O
O
O-
O-
O-
Fe+3
[O]
+--
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Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
Seliwanoff’s 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:
C
C
O
C
OHH
C
H OH
CH2OH
- H2O
HO OH
OH
O
HOH2C
OHOH2C
H
HO
OH
OH
OH
OHOH2C
O
OH
OH
OH
H+[O]
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General Test:General Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
Mucic acid Test
HNO3 12
HO
3
OH
4
HO
5
OH
6
O
HO
O
OH
C 1
C2
OHH
C3
OHHO
C4
OH
C5
OHHO
H2C6
H OH
OH
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:
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Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
Osazone/Phenylhydrazone TestSucrose – does not form osazone unless hydrolyze
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Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
Osazone/Phenylhydrazone TestGlucosazone - needle-like/feathery, 4-5min
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Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
Osazone/Phenylhydrazone TestFructosazone – needle-like/feathery, 2 min
Mannosazone – needle-like/feathery, 0.5min
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Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
Osazone/Phenylhydrazone Test
Lactosazone – fine needles; grouped in balls; soluble in hot H2O
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Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
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)
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Specific Test:Specific Test:
BIOCHEM LABORATORYBIOCHEM LABORATORY
Osazone/Phenylhydrazone Test
Starch - does not form osazone unless hydrolyze