General Colorimetric Tests for Standard

Amylose, Glycogen, and Cellulose, and

Specific Colorimetric Tests for Xylose,
Glucose, Galactose, Fructose, Maltose,
Lactose, and Sucrose

Austero.Casipit.Enaje.Jung.Lanto
3Bio9
Introduction
 Introduction
● Carbohydrates
○ Also known as:
■ Sugars - monosaccharides and disaccharides
■ Glycans - polysaccharides
○ composed carbon (carbo-) and hydrogen and oxygen (-hydrate)
○ (CH2O)n - general formula
Functions:
● Provide and store energy
● Serve as structural components
 Introduction
● Carbohydrates
○ 3 categories
■ monosaccharides: polyhydroxy aldehydes or ketones containing
3 ≤ carbon atoms
■ oligosaccharides: 2-10 monosaccharide residues
● disaccharides are found most widely in nature
■ polysaccharides: 10< monosaccharide residues
● homopolysaccharides
● heteropolysaccharides
 Introduction
● Monosaccharides - types based on the functional group

○ Aldose - contains an aldehyde

■ Glucose
GLU FRU

■ Galactose
■ Xylose
○ Ketose - contains a ketone
■ Fructose
 Introduction
● Monosaccharides - types based on the number of carbon atoms

○ Pentose - 5 Carbons
■ Xylose
XYL GLU
○ Hexose - 6 Carbons
■ Glucose
■ Galactose
■ Fructose
 Introduction
● Monosaccharides - types based on ring structure

○ Furanose - 5-membered ring

■ Fructose FRU
■ Xylose
○ Pyranose - 6-membered ring
■ Glucose
■ Galactose

GLU
 Introduction
● Hemiacetals and Hemiketals
○ ring structure produced by reacting an aldehyde or ketone with an
alcohol
○ Monosaccharides are cyclic hemiacetals or hemiketals
○ Anomeric carbon = chiral carbon of hemiacetal/hemiketal
 Introduction
● Furfural

○ produced by dehydration of pentoses

● 5 -Hydroxymethyl furfural (5-HMF)

○ produced by dehydration of hexoses
○
XYL GLU GAL FRU
 Introduction
● Disaccharide
○ two monosaccharides linked by a glycosidic bond or acetal linkage
○ formed by condensation of an alcoholic hydroxyl (ーOH of another
monosaccharide) with the hemiacetal (or hemiketal) form of the
carbonyl carbon of a sugar
 Introduction
● Glycosidic bond
○ anomeric carbon of one sugar to a hydroxyl group of another sugar
■ anomers: isomers differing in configuration only at the carbonyl
carbon
 Introduction
Disaccharides
● Maltose = GLU + GLU MAL

○ α(1→4) glycosidic bond

● Lactose = GLU + GAL

LAC
○ ß(1→4) glycosidic bond

● Sucrose = GLU + FRU

○ α(1→2)ß glycosidic bond SUC
MAL SUC

LAC
 Introduction
Polysaccharides
○ Amylose: 1 of 2 components of starch
■ linear and unbranched
■ coiled structure
■ glucose residues joined by α(1→4) glycosidic bonds
 Introduction
Polysaccharides
○ Glycogen:
■ stores energy in animals
■ branched-chain wherein each segment forms an open helix
■ α(1→4) glycosidic bonds with α(1→6) branch points
 Introduction
Polysaccharides
○ Cellulose:
■ structural component in a plant’s cell wall
■ linear and unbranched
■ ß(1→4) glycosidic bonds
 Objectives
● to characterize carbohydrates by subjecting standard
amylose, glycogen, and cellulose to several color reactions

● to identify an unknown sugar, side-by-side with several

standard sugars using a number of specific chemical tests
Methodology
 Methodology - Preliminaries

solid unknown sugar

dissolve half of the solid unknown sugar in 5mL

distilled H2O (to be used for specific chemical tests)

keep other half in locker (to be used for other tests)

prepare water bath on a hot plate

boiling water bath and unknown

solution
General Tests
 Methodology - Molisch Test
10 drops of
standard amylose solution
standard glycogen solution
standard cellulose solution
in separate test tubes

add 2 drops Molisch reagent to each tube and mix

thoroughly

incline the tubes and allow 10 drops of concentrated

H2SO4 to flow down the side of the tube

note the color formed at the interphase of the two

liquids and record results

condensation product with

interphase
 Methodology - Anthrone Test
4-5 drops of
standard amylose solution
standard glycogen solution
standard cellulose solution
in separate wells of a spot plate

place 10 drops of anthrone solution

note color and record results

bluish-green complex
 Methodology - Iodine Test
10 drops of
standard amylose solution
standard glycogen solution
standard cellulose solution
in separate test tubes

add 1 drop of iodine solution to each tube and

record observations

heat tubes in boiling water bath for up to 3 minutes

and note changes produced

remove tubes from the bath and cool the solutions

record observations

solution
Specific Tests
 Methodology - Mucic acid Test
10 drops of
unknown sugars
7 standard sugars
in separate medium-sized test tubes

add 10 drops of concentrated HNO3 to each tube

plug tubes with cotton and heat in boiling water

bath for 1 hour

let stand until next laboratory period placed in a

locker or refrigerator

note which standard sugars and UNK produce

crystals

solution
Methodology - Benedict’s Test
10 drops of
Benedict’s reagent
in separate test tubes

place 5 drops of the unknown sugars and the 7

standard sugars in their respective tubes already
containing the reagent

heat the tubes in a water bath up to 5 minutes or

until a muddy green suspension is observed,
settling as brick red precipitate

immediately remove tubes from bath, cool on the

rack, and record results

cool on the rack and record results

solution
 Methodology - Barfoed’s Test
10 drops of
Barfoed’s reagent
in separate tubes

place 10 drops the unknown sugars and the 7

standard sugars in their respective tubes already
containing the reagent

heat tubes in a water bath up to 5 minutes or until a

brick red precipitate is observed

note the time when the precipitate is observed

immediately remove the tubes from the water bath

and record the results

solution
 Methodology - Bial’s Orcinol Test
5 drops of
unknown sugars
7 standard sugars
in separate small-sized test tubes

add 10 drops of Bial’s orcinol reagent to each tube

heat the tubes in a boiling water bath up to 5

minutes or until a blue-green solution is observed
and note the time when it is observed

record colors formed during heating

remove tubes from water bath and record the

results

solution
Methodology - Seliwanoff’s Test
10 drops of
Seliwanoff’s reagent
in separate test tubes

place 5 drops of the unknown sugars and the 7

standard sugars in their respective tubes already
containing the reagent

immerse the tubes in a boiling water bath

heat up to 5 minutes or until a cherry red solution is

observed then remove tubes from water bath

note the time when the cherry red solution is

produced

solution
Results
General Tests
 Results
Table 1. Molisch Test
Group Number
Sample
1 2 3 4 5 6 7 8 9

yellow- light
purple peach purple purple light pink purple purple
Amylose green pink
interphase interphase interphase interphase interface interphase interphase
interface interface

light dark light

colorless purple purple violet purple light purple
Glycogen peach purple pink
interphase interphase interphase interface interphase interphase
interphase interphase interface

dark violet/ dark dark

purple violet purple violet purple
Cellulose violet dark pink purple purple
interphase interface interphase interphase interface
interphase interphase interphase interphase
 Results
Table 2. Anthrone Test
Group Number
Sample
1 2 3 4 5 6 7 8 9

blue-
green
blue- blue- dark green yellow- dark blue- dark
blue outer,
Amylose green green center w/ green green green green
yellow ring solution yellow
solution solution solution solution solution middle
solution
solution

yellow light blue

light blue green light
light light center outer, blue-
light blue blue- center w/ blue-
Glycogen light yellow
green green blue outer yellow green
solution green green middle
ring solution solution ring solution
solution solution solution
solution

blue-
light blue yellow
light green
green light blue center dark
blue blue- green outer,
Cellulose solution green green blue outer green
solution green with yellow solution yellow
solution solution ring middle
solution
solution ring solution solution
 Results
Table 3. Iodine Test
Group Number
Sample
1 2 3

after adding I2 blue solution blue solution dark blue liquid

Amylose during heating colorless liquid colorless liquid clear colorless liquid

after cooling clear purple liquid colorless liquid clear light violet liquid

clear light yellow

after adding I2 colorless liquid clear yellow solution
liquid
Glycogen
during heating colorless liquid colorless solution clear colorless liquid

after cooling clear yellow liquid light yellow solution clear colorless liquid

after adding I2 white turbid solution turbid yellow solution turbid white liquid

during heating colorless liquid colorless solution colorless turbid liquid

Cellulose
turbid solution with white colorless solution with turbid liquid with white
after cooling
precipitate white precipitate precipitate
 Results
Table 3 continuation. Iodine Test
Group Number
Sample
4 5 6

after adding I2 dark blue solution. dark blue solution dark blue solution

Amylose during heating clear colorless liquid clear colorless solution colorless solution

after cooling clear light purple liquid clear colorless solution colorless solution

after adding I2 colorless liquid clear colorless solution clear yellow solution

Glycogen during heating clear colorless solution clear colorless solution colorless solution

after cooling clear colorless solution clear colorless solution yellow solution

after adding I2 turbid white solution turbid solution turbid white solution

during heating clear colorless solution clear colorless solution turbid solution
Cellulose
turbid white with white
after cooling turbid white solution turbid solution
precipitate
 Results
Table 3 continuation. Iodine Test
Group Number
Sample
7 8 9

after adding I2 dark blue solution clear dark blue solution dark blue solution

Amylose during heating colorless solution colorless solution colorless solution

after cooling light purple solution clear solution colorless solution

clear pale yellow

after adding I2 pale yellow solution clear light yellow solution
solution
Glycogen
during heating colorless solution colorless solution colorless solution

after cooling colorless solution clear solution colorless solution

after adding I2 turbid solution; white ppt turbid yellow solution turbid white solution

yellowish solution with

Cellulose during heating turbid solution; white ppt turbid white
white precipitate

after cooling turbid solution; white ppt turbid jelly-like substance turbid white
Specific Tests
 Results
Table 4. Mucic acid Test
Group Number
Sample
1 2 3 4 5 6 7 8 9

colorless clear, colorless no clear

colorless colorless colorless colorless
Xylose liquid; no colorless liquid; no crystals colorless
solution solution solution solution
crystals liquid crystals formed solution

colorless clear, colorless no clear

colorless colorless colorless colorless
Glucose liquid; no colorless liquid; no crystals colorless
solution solution solution solution
crystals liquid crystals formed solution

colorless colorless
colorless colorless colorless colorless colorless
colorless solution solution
liquid, solution; liquid; crystal solution'w solution
Galactose liquid; no with with
white produced produced formation ith white with
crystals white white
crystals crystals crystals crystals crystals
crystals crystals

no clear
colorless clear, colorless
colorless colorless crystals colorless colorless colorless
Fructose liquid; no colorless liquid; no
solution solution formed solution solution solution
 Results
Table 4 continuation. Mucic acid Test
Group Number
Sample
1 2 3 4 5 6 7 8 9

colorless clear, colorless no clear

colorless colorless colorless colorless
Maltose liquid; no colorless liquid; no crystals colorless
solution solution solution solution
crystals liquid crystals formed solution

colorless colorless colorless colorless

colorless colorless colorless
with solution liquid, solution solution
solution; liquid; crystal solution
Lactose white with white with with
produced produced formation with
crystals white crystals white white
crystals crystals crystals
crystals crystals crystals

clear
colorless clear, colorless no
colorless colorless colorless colorless colorless
Sucrose liquid; no colorless liquid; no crystals
solution solution solution solution solution
crystals liquid crystals formed
 Results
Table 5. Benedict’s Test
Group Number
Sample
1 2 3 4 5 6 7 8 9

brick red brick red brick red brick red brick red brick red brick red brick red brick red
Xylose
ppt ppt ppt ppt ppt ppt ppt ppt ppt

brick red brick red brick red brick red brick red brick red brick red brick red brick red
Glucose
ppt ppt ppt ppt ppt ppt ppt ppt ppt

brick red brick red brick red brick red brick red brick red brick red brick red brick red
Galactose
ppt ppt ppt ppt ppt ppt ppt ppt ppt

brick red brick red brick red brick red brick red brick red brick red brick red brick red
Fructose
ppt ppt ppt ppt ppt ppt ppt ppt ppt
 Results
Table 5 continuation. Benedict’s Test
Group Number
Sample
1 2 3 4 5 6 7 8 9

brick red brick red brick red brick red brick red brick red brick red brick red brick red
Maltose
ppt ppt ppt ppt ppt ppt ppt ppt ppt

brick red brick red brick red brick red brick red brick red brick red brick red brick red
Lactose
ppt ppt ppt ppt ppt ppt ppt ppt ppt

blue clear olive/

blue- muddy blue-
green green blue- green yellow muddy
Sucrose green green green
solution turbid green solution solution green
solution suspension solution
solution solution suspension
 Results
Table 6. Barfoed’s Test
Group Number
Sample
1 2 3 4 5 6 7 8 9

brick red brick red brick red brick red brick red brick red brick red brick red brick red
Xylose ppt ppt ppt ppt ppt ppt ppt ppt ppt
(35 secs) (44 secs) (33 secs) (44 secs) (33 secs) (40 secs) (25 secs) (37 secs) (50 secs)

brick red brick red brick red brick red brick red brick red brick red brick red brick red
Glucose ppt ppt ppt ppt. ppt ppt ppt ppt ppt
(29 secs) (1 min) (36 secs) (43 secs) (39 secs) (1:10 min) (50 secs) (43 secs) (50 secs)

brick red
brick red brick red brick red brick red brick red brick red brick red brick red
ppt
Galactose ppt ppt ppt. ppt ppt ppt ppt ppt
(1:30
(30 secs) (39 secs) (54 secs) (43 secs) (1 min) (55 secs) (42 secs) (58 secs)
mins)

brick red
brick red brick red brick red brick red brick red brick red brick red brick red
ppt
Fructose ppt ppt ppt ppt ppt ppt ppt ppt
(2:08
(20 secs) (46 secs) (26 secs) (54 secs) (36 secs) (45 secs) (43 secs) (58 secs)
mins)
 Results
Table 6 continuation. Barfoed’s Test
Group Number
Sample
1 2 3 4 5 6 7 8 9

small
brick red brick red brick red brick red amount brick red
brick red brick red brick red
ppt ppt ppt ppt brick red ppt
Maltose ppt ppt ppt
(2:40 (4:14 (2:43 (4:01 ppt (5
(2 mins) (1 min) (1:45 min)
mins) mins) mins) min) (4:26 minutes)
mins)

remained clear blue brick red remained clear

blue clear blue blue no brick
Lactose blue sol’n ppt blue light-blue
solution solution solution red ppt
solution (5 mins) (5 min) solution solution

remained clear blue remained clear

blue clear blue clear blue blue no brick
Sucrose blue sol’n blue light-blue
solution solution solution solution red ppt
solution (5 mins) solution solution
 Results
Table 7. Bial’s Orcinol Test
Group Number
Sample
1 2 3 4 5 6 7 8 9

blue- dark blue blue- blue- blue- blue- blue-

dark green blue-green
green green green green green Green green
Xylose solution solution
solution solution solution solution solution solution solution
(30 secs) (30 secs)
(16 secs) (33 secs) (31 secs) (31 secs) (30 secs) (18 secs) (40 secs)

dark
orange dark dark dark brown dark dark
brown dark brown brownish
Glucose solution brown brown solution brown brown
solution solution solution
(17 secs) solution solution (5 mins) solution solution
(5 mins)

dark dark
orange dark dark brown dark
yellow- brown dark brown brown brownish
Galactose solution brown solution brown
green solution solution solution solution
(33 secs) solution (5 mins) solution
solution (5 mins)

dark dark dark brown dark

dark dark brown
brown dark red brown solution brown brown brownish
Fructose brown solution
solution solution solution with dark solution sol’n with solution
solution (5 mins)
(25 secs) (5 mins) brown ppt black ppt
 Results
Table 7 continuation. Bial’s Orcinol Test
Group Number
Sample
1 2 3 4 5 6 7 8 9

dark
orange yellow- dark dark dark brown dark
brown brown brownish
Maltose solution green brown brown solution brown
solution solution solution
(22 secs) solution solution solution (5 mins) solution
(5 mins)

dark dark
orange dark dark dark brown dark
yellow- brown brown brownish
Lactose solution brown brown solution brown
green solution solution solution
(33 secs) solution solution (5 mins) solution
solution (5 mins)

dark
dark dark
dark dark dark dark brown brown
brown brown brown brownish
Sucrose brown brown brown solution solution
solution solution solution solution
solution solution solution (5 mins) with black
(17 secs) (5 mins)
ppt
 Results
Table 8. Seliwanoff’s Test
Group Number
Sample
1 2 3 4 5 6 7 8 9

dark
dark green dark dark dark green dark
green dark green green dark green
Xylose solution green green solution green
solution solution solution solution
(5 mins) solution solution (1:30 min) solution
(5 mins)

red red red red

red red
solution solution solution orange solution red blood red
Glucose solution solution
(1:30 (3:03 (3:30 solution (2:05 solution solution
(1:39 min) (3:23)
min) mins) mins) mins)

red red red dark red light red

red dark
solution solution solution solution red solution blood red
Galactose solution orange
(4:06 (2:00 (4:06 (2:50 solution (4:17 solution
(5 mins) solution
mins) mins) mins) mins) mins)

cherry red
cherry red red cherry red cherry red cherry red cherry red
solution cherry red cherry red
Fructose solution solution solution solution solution solution
(23 solution solution
(26 secs) (33 secs) (55 secs) (31 secs) (37 secs) (32 secs)
seconds)
 Results
Table 8 continuation. Seliwanoff’s Test
Group Number
Sample
1 2 3 4 5 6 7 8 9

red red red light red

red red dark
solution solution solution red solution apple red
Maltose solution solution orange
(3:49 (2:47 (2:45 solution (3:30 solution
(2:32) (5 mins) solution
mins) mins) mins) min)

dark red
red red light red
red red solution
solution solution orange red solution apple red
Lactose solution solution (4:20
(3:35 (3:50 solution solution (4:49 solution
(2:30) (5 mins) mins)
mins) mins) min)

cherry cherry cherry cherry

red cherry red cherry cherry
red red red red cherry red
Sucrose solution solution red red
solution solution solution solution solution
(40 secs) (38 secs) solution solution
(42 secs) (30 secs) (51 secs) (53 secs)
 Results
Table 9. Results of Unknowns for Specific Tests
Test

Unknown Mucic acid Benedict’s Barfoed’s Bial’s Orcinol Seliwanoff’s

brick red
with few small brick red orange solution red solution
1 precipitate.
white crystals precipitate (15 seconds) (3:49 minutes)
(2 minutes)

brick red remained blue orange solution red solution

2 white crystals
precipitate solution (20 seconds) (3:55 minutes)

dark brown cherry red

with few small remained blue
3 clear blue liquid solution solution
white crystals solution
(31 seconds) (42 seconds)

brick red dark brown cherry red

colorless liquid; no brick red
4 precipitate. solution solution
crystals precipitate
(48 seconds) (25 seconds) (30 seconds)

dark green
brick red brick red ppt dark green
5 colorless solution solution
precipitate (36 seconds) solution
(30 seconds)
 Results
Table 9. Results of Unknowns for Specific Tests
Test

Unknown Mucic acid Benedict’s Barfoed’s Bial’s Orcinol Seliwanoff’s

yellow-green
brick red brick red precipitate red solution
6 colorless solution solution
precipitate (1:30 mins) (3:50 mins)
(50 seconds)

dark yellow-green
colorless solution brick red brick red precipitate red solution
7 solution
with white crystals precipitate (56 seconds) (3:50 mins)
(1:07 mins)

cherry red
brick red brick red precipitate dark red solution
8 colorless solution solution
precipitate (56 seconds) (37 seconds)
(20 seconds)

dark brown
brick red clear blue solution red solution
9 clear, colorless liquid solution
precipitate (3:04 mins) (2:07 mis)
(5 mins)

dark brown
clear blue green clear blue solution red solution
10 clear, colorless liquid solution
solution (5 mins) (38 secs)
(5 mins)
 Results
Table 9 continuation. Results of Unknowns for Specific Tests
Test

Unknown Mucic acid Benedict’s Barfoed’s Bial’s Orcinol Seliwanoff’s

dark brown
colorless liquid white brick red clear blue solution red solution
11 solution
crystals precipitate (5 mins) (3:41 mins)
(5 mins)

clear blue solution dark blue green dark green

brick red
12 clear, colorless liquid with brick red ppt. solution solution
precipitate
(41 secs) (33 secs) (5 mins)

brick red brick red ppt. dark brown cherry red sol’n
13 colorless solution
precipitate (40 secs) solution (3 min)

colorless solution brick red brick red ppt. dark brown cherry red sol’n
14
with white crystals precipitate (40 sec) solution (4 min)

brick red ppt. dark brown cherry red sol’n

15 colorless solution brick red ppt.
(40 secs) solution (3 min)
 Results
Table 9 continuation. Results of Unknowns for Specific Tests
Test

Unknown Mucic acid Benedict’s Barfoed’s Bial’s Orcinol Seliwanoff’s

cherry red
brick red ppt dark brown
16 colorless solution brick red ppt solution
(35 secs) solution
(3 min)

colorless liquid; no brick red

17
crystals precipitate

colorless liquid; brick red

18
produced crystals precipitate

colorless liquid; no
19 blue solution
crystals

colorless liquid; brick red

20
produced crystals precipitate
 Results
Table 9 continuation. Results of Unknowns for Specific Tests
Test

Unknown Mucic acid Benedict’s Barfoed’s Bial’s Orcinol Seliwanoff’s

cherry red
brick red ppt dark brown
21 no crystals formed brick red ppt solution
(30 secs) solution
(53 secs)

brick red ppt dark brown red solution

22 no crystals formed brick red ppt
(35 secs) solution (2 mins)

cherry red
clear blue-green dark brown
23 no crystals formed blue-green sol’n solution
solution solution
(40 secs)

clear blue-green dark brown red solution

24 crystal formation brick red ppt.
solution solution (2:30 mins)

dark brown blue solution with

25 colorless solution solution with brick red ppt black solution red solution
brick red ppt (4:40 min)
 Results
Table 9 continuation. Results of Unknowns for Specific Tests
Test

Unknown Mucic acid Benedict’s Barfoed’s Bial’s Orcinol Seliwanoff’s

blue solution; brick

orange solution cherry red
26 colorless solution red ppt black solution
with brick red ppt solution
(30 secs)

dark brown blue solution with

blue-green solution dark green
27 colorless solution solution with brick red ppt
(22 secs) solution
brick red ppt (27 secs)

dark brown blue solution with

colorless solution
28 solution with brick red ppt black solution red solution
with white crystals
brick red ppt (1:05 min)

small amt of brick

dark brown red solution
29 colorless solution brick red ppt red ppt in light blue
solution (2:54 min)
solution

brick red ppt in

dark brown red solution
30 colorless solution brick red ppt light blue solution
solution (2:56 min)
(1:30 min)
 Results
Table 9 continuation. Results of Unknowns for Specific Tests
Test

Unknown Mucic acid Benedict’s Barfoed’s Bial’s Orcinol Seliwanoff’s

dark brown cherry red

aqua green
31 colorless solution light-blue solution solution with black solution
solution
ppt (48 secs)

brick red ppt in dark brown cherry red

brick red
32 colorless solution light blue solution solution with black solution
precipitate
(1:21 min) ppt (37 secs)

colorless solution orange solution

33 no brick red ppt brownish solution apple red solution
with white crystals with brick red ppt.

clear, colorless orange solution brick red ppt. blue-green solution dark green
34
solution with brick red ppt. (40 seconds) (40 seconds) solution

colorless solution; orange solution brick red ppt.

35 brownish solution blood red solution
with crystals with brick red ppt. (40 seconds)
Discussion
General Tests
 Discussion
Molisch Test
● shows positive test for: all carbohydrates; monosaccharides
give a rapid positive test, disaccharides react slower
● positive test indicated by: formation purple interphase
● reagents: Molisch reagent [α-naphthol], H2SO4
 Discussion
Molisch Test
● principle: furfural/furfural derivative formation
○ Hydrolysis of polysaccharides into simpler
monomeric units (Glucose in samples used)
○ Dehydration of Glucose into 5-HMF
○ Condensation of 5-HMF with α-naphthol

Carbohydrates are dehydrated with concentrated sulfuric acid to form furfural or

5-hydroxymethylfurfural (5-HMF) which reacts with α-naphthol giving a purple
product.
 Discussion
Anthrone Test
● shows positive test for: carbohydrates
● positive test indicated by: bluish-green complex
● reagents: Anthrone reagent [anthrone, concentrated H2SO4]
 Discussion
Anthrone Test
● principle: furfural/furfural derivative formation
○ Hydrolysis of polysaccharides into simpler
monomeric units (Glucose in samples used)
○ Dehydration of Glucose into 5-HMF
○ Condensation of 5-HMF with anthrone

Similar to the Molisch test, concentrated H2SO4 dehydrates the carbohydrate forming
furfural, which in turn condenses with anthrone to form a bluish-green complex.
 Discussion
Iodine Test
● used to distinguish helical from non-helical polysaccharides
● reagents: iodine solution
● principle: formation of a complex
● characteristic colors are formed based on chain length
○ blue-black color = starch
○ amylose = blue; glycogen = red;
○ heating causes shift of absorption maximum to UV and
color turns clear
○ returns to blue-black after cooling
Specific Tests
XYL GLU GAL FRU
MAL SUC

LAC
 Discussion
Mucic acid Test
● shows positive test for: galactose and lactose
● positive test indicated by: crystal formation
● reagent: HNO3
 Discussion
Mucic acid Test
● principle: oxidation

Galactose is oxidized with HNO3 to form dicarboxylic acid and mucic acid. Mucic acid is
an insoluble solid that precipitates from the reaction mixture.
 Discussion
Benedict’s Test
● shows positive test for: reducing sugars
● positive test indicated by: formation of brick red precipitate
● reagent: Benedict’s reagent [carbonate-citrate solution &
copper sulfate solution]
 Discussion
Benedict’s Test
● principle: oxidation-reduction

Sample is heated with an alkaline reagent containing cupric copper held in solution.

Potential aldehyde or ketol groups are oxidized, and the copper is reduced to the red
cuprous oxide.
 Discussion
Benedict’s Test
● principle: oxidation-reduction

The aldehyde groups are oxidized to sugar acids

Aldonic acids - the aldose -OH group is oxidized
Xylonic acid - xylose
Gluconic acid - glucose
Galactonic acid - galactose
 Discussion

Ketones (fructose) are non-oxidizable. The ketoses are isomerised to aldoses by

keto-enol tautomerism
 Discussion
Barfoed’s Test
● shows positive test for: reducing monosaccharides
● positive test indicated by: formation of brick red precipitate
● reagent: Barfoed’s reagent [Cu(CH3COO)2]
 Discussion
Barfoed’s Test
● principle: oxidation-reduction

Similar to Benedict’s test with the red cuprous oxide as the visible product, however,
conditions are now slightly acidic.
Both monosaccharides and disaccharides reduce the reagent but monosaccharides act
more rapidly and more vigorously.
Principle is the same as Benedict’s test, so read that again :>
 Discussion
Bial’s Orcinol Test
● shows positive test for: pentoses
● positive test indicated by: blue-green solution
● reagent: Bial’s orcinol reagent [orcinol (C7H8O2), HCl, FeCl3]
 Discussion
Bial’s Orcinol Test
● principle: furfural/furfural derivative formation

Pentose sugars yield furfural on dehydration in acidic solution. Furfural reacts with
orcinol & FeCl3 to give a blue-green condensation product.

Hexose sugars give 5-HMF which reacts with the reagent to yield other colors such as
green, brown, and reddish brown.
 Discussion
Seliwanoff’s Test
● shows positive test for: ketoses
● positive test indicated by: production of cherry red solution
● reagent: Seliwanoff’s reagent [resorcinol (C6H6O2), HCl]
 Discussion
Seliwanoff’s Test
● principle: furfural/furfural derivative formation

A ketohexose reacts rapidly to give 5-HMF, whereas an aldohexose reacts more slowly
to give the same product. Once 5-HMF is produced, it reacts with resorcinol to give a
dark red condensation product.
 Conclusion
● Amylose, glycogen, and cellulose are polysaccharides composed of
hexoses with amylose being the most helical and cellulose the least
● Among the 7 standard sugars, 4 are monosaccharides, XYL, FRU, GAL,
and GLU; 3 are disaccharides, SUC, LAC, and MAL.
○ XYL is the only pentose
○ FRU is the only ketohexose
○ SUC is the only non-reducing disaccharide
○ GAL is an aldohexose that can be oxidized by HNO3
○ LAC is a reducing disaccharide that can be oxidized by HNO 3
○ GLU is an aldohexose that cannot be oxidized by HNO3
○ MAL is a reducing disaccharide that cannot be oxidized by HNO 3
References
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