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Test for Carbohydrates: Mono-, Di-,
and Polysaccharides
Carbohydrates
• Learning objectives:
• Use chemical tests to distinguish between
mono-, di-, and polysaccharides
Review of carbohydrates:
Carbohydrates
• Carbon, hydrogen, and oxygen
• The basic formula -
• Example:
C3H6O3
I
(CH2O)n or H - C - OH
I
Classification of carbohydrates
• Monosaccharides
• Trioses, tetroses, pentoses, hexoses
• Oligosaccharides
• Di, tri, tetra, penta, up to 9 or 10
• Most important are the disaccharides (sucrose)
• Polysaccharides or glycans
Monosaccharides
Aldoses (e.g., glucose)
have an aldehyde group at
one end.
Ketoses (e.g., fructose) have
a keto group, usually at C2.
C
C OHH
C HHO
C OHH
C OHH
CH2OH
D-glucose
OH
C HHO
C OHH
C OHH
CH2OH
CH2OH
C O
D-fructose
Most of the names of
carbohydrates end in -ose
• Glucose-What plants make
• Maltose- used in making beer
(disaccharide)
• Fructose – found in fruit (monosaccharide)
• Sucrose- Table sugar (disaccharide)
• Lactose – In milk (disaccharide)
D vs L Designation
D & L designations
are based on the
configuration about
the single C with
four different groups
around it in
glyceraldehyde.
CHO
C
CH2OH
HO H
CHO
C
CH2OH
H OH
CHO
C
CH2OH
HO H
CHO
C
CH2OH
H OH
L-glyceraldehydeD-glyceraldehyde
L-glyceraldehydeD-glyceraldehyde
Glucose can be found in a ring
structure or linear structure
• In Water
Haworth projection
Pentoses and
hexoses can
cyclize as the
ketone or aldehyde
reacts with a
distal OH.
Cyclic glucose
forms as the C1
aldehyde & C5
OH react, to form
a 6-member ring.
H O
OH
H
OHH
OH
CH2OH
H
OH
H H O
OH
H
OHH
OH
CH2OH
H
H
OH
-D-glucose -D-glucose
23
4
5
6
1 1
6
5
4
3 2
H
CHO
C OH
C HHO
C OHH
C OHH
CH2OH
1
5
2
3
4
6
D-glucose
(linear form)
Fructose forms
 a 5-member furanose ring, by reaction of
the C2 keto group with the OH on C5.
CH2OH
C O
C HHO
C OHH
C OHH
CH2OH
HOH2C
OH
CH2OH
H
OH H
H HO
O
1
6
5
4
3
2
6
5
4 3
2
1
D-fructose (linear) -D-fructofuranose
Cyclization of glucose produces a new carbon where the
OH can be up or down. The 2 structures are called
anomers,  & .
 (OH below the ring)
  (OH above the ring).
H O
OH
H
OHH
OH
CH2OH
H
-D-glucose
OH
H H O
OH
H
OHH
OH
CH2OH
H
H
OH
-D-glucose
23
4
5
6
1 1
6
5
4
3 2
Carbohydrate Tests
Benedict’s Test For Reducing
Sugars
Distinguish reducing from non-reducing sugars.
Reducing sugar …
Sugars that contain aldehyde groups that are oxidized to
carboxylic acids
In order for oxidation to occur, the cyclic form must first ring-
open to give the reactive aldehyde.
Benedict’s Test For Reducing
Sugars
Benedict’s reagent is a mild oxidant with CuSO4, Cu (II)
sulfate, as one of the reagents.
Copper (II) sulphate, Sodium carbonate and Sodium citrate.
Benedict’s Test For Reducing
Sugars
oxidized reduced
In the presence of a reducing sugar, the blue solution of
Cu (II) or Cu+2, is changed to a brick red/brown
precipitate of Copper (I) or Cu+1 oxide,Cu2O.
Formation of a brick red/brown precipitate is indicative
of the presence of reducing sugars.
precipitate
Benedict’s Test For Reducing
Sugars
All mono- and most disaccharide's will reduce
CuSO4, producing a precipitate of copper (I)
oxide on heating, so they are called reducing
sugars.
 A precipitate indicates reducing sugars
 Original Pale Blue = no reducing sugar
 Brown/Red = reducing sugar
Benedict’s Test For Reducing
Sugars
• Ketoses can also be reducing sugars
because they can isomerise to aldoses:
•
Benedict’s Test For Reducing
Sugars
Why sucrose is non-reducing sugar…
sucrose has a anomeric carbon which is not free since the
carbon links glucose and fructose and fructose does not
have free -OH group to undergo reducing reaction and to
open the ring so sucrose is non-reducing.
At the end of the lab…
• You should…
• Distinguish between reducing and non-reducing
sugar
• Understand the theory for Benedict’s test
• Know which of the tested carbohydrates are
positive or negative for Benedict’s solution and
why
Seliwanoff’s Test for Ketoses
Distinguishes between ketohexoses (ketone)
and aldohexoses (aldehyde) sugars.
Seliwanoff’s Test for Ketoses
Rapid appearance of a dark red solution indicates
the presence of a ketose
Aldohexoses react slower and the appearance of
the red solution takes longer
• Distinguishes polysaccharides and other
carbohydrates
Iodine Test for Polysaccharides
• Polysaccharides will react with iodine to
form a blue, red, violet, or purple
product which is a positive indicator of
the presence of a polysaccharide.
• Monosaccharides and disaccharides
are too small to trap iodine molecules
and do not form any dark color with
iodine
Iodine Test for Polysaccharides
Some examples of polysaccharides…
Disaccharides and polysaccharides can
be hydrolyzed in acidic solution into
their component monosaccharides, and
then submitted to chemical tests like
Benedict's test.
Hydrolysis Test for Di- and
Polysaccharides
• Starch and sucrose do not give positive
test with Benedict’s. After hydrolysis,
sucrose produces fructose and glucose
that give a positive Benedict’s test.
• Sucrose + H2O -> Glucose + Fructose
Hydrolysis Test for Di- and
Polysaccharides
Negative
Benedict’s test
Positive
Benedict’s test
This test distinguishes carbohydrates that
undergo fermentation and those that do not.
Yeast is used as a source of an enzyme, zymase.
The products are CO2 and ethanol.
C6H12O6 + yeast  C2H5OH + CO2
Formation of bubbles of carbon dioxide is used to
confirm the fermentation process
Fermentation Test
Benedict’s Test For Non-
Reducing Sugars
Non-Reducing sugars do not reduce copper sulphate.
However, if it is first hydrolysed to its constituent
monosaccharides, it will then give a positive Benedict’s
Test.
First test a sample for reducing sugars, to see if there are any
present before hydrolysis.
Then using a separate sample,
 Boil the test solution with dilute hydrochloric acid for a few
minutes to hydrolyse the glycosidic bond.
 Neutralise, by adding small amounts of solid sodium
hydrogen carbonate until it stops fizzing.
 Perform the Benedict’s test
 A positive result indicates the presence of simple non-
reducing sugar.

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Carbspowerpoint 150602233036-lva1-app6892

  • 1. Test for Carbohydrates: Mono-, Di-, and Polysaccharides
  • 2. Carbohydrates • Learning objectives: • Use chemical tests to distinguish between mono-, di-, and polysaccharides
  • 4. Carbohydrates • Carbon, hydrogen, and oxygen • The basic formula - • Example: C3H6O3 I (CH2O)n or H - C - OH I
  • 5.
  • 6. Classification of carbohydrates • Monosaccharides • Trioses, tetroses, pentoses, hexoses • Oligosaccharides • Di, tri, tetra, penta, up to 9 or 10 • Most important are the disaccharides (sucrose) • Polysaccharides or glycans
  • 7.
  • 8. Monosaccharides Aldoses (e.g., glucose) have an aldehyde group at one end. Ketoses (e.g., fructose) have a keto group, usually at C2. C C OHH C HHO C OHH C OHH CH2OH D-glucose OH C HHO C OHH C OHH CH2OH CH2OH C O D-fructose
  • 9. Most of the names of carbohydrates end in -ose • Glucose-What plants make • Maltose- used in making beer (disaccharide) • Fructose – found in fruit (monosaccharide) • Sucrose- Table sugar (disaccharide) • Lactose – In milk (disaccharide)
  • 10. D vs L Designation D & L designations are based on the configuration about the single C with four different groups around it in glyceraldehyde. CHO C CH2OH HO H CHO C CH2OH H OH CHO C CH2OH HO H CHO C CH2OH H OH L-glyceraldehydeD-glyceraldehyde L-glyceraldehydeD-glyceraldehyde
  • 11. Glucose can be found in a ring structure or linear structure • In Water Haworth projection
  • 12. Pentoses and hexoses can cyclize as the ketone or aldehyde reacts with a distal OH. Cyclic glucose forms as the C1 aldehyde & C5 OH react, to form a 6-member ring. H O OH H OHH OH CH2OH H OH H H O OH H OHH OH CH2OH H H OH -D-glucose -D-glucose 23 4 5 6 1 1 6 5 4 3 2 H CHO C OH C HHO C OHH C OHH CH2OH 1 5 2 3 4 6 D-glucose (linear form)
  • 13. Fructose forms  a 5-member furanose ring, by reaction of the C2 keto group with the OH on C5. CH2OH C O C HHO C OHH C OHH CH2OH HOH2C OH CH2OH H OH H H HO O 1 6 5 4 3 2 6 5 4 3 2 1 D-fructose (linear) -D-fructofuranose
  • 14. Cyclization of glucose produces a new carbon where the OH can be up or down. The 2 structures are called anomers,  & .  (OH below the ring)   (OH above the ring). H O OH H OHH OH CH2OH H -D-glucose OH H H O OH H OHH OH CH2OH H H OH -D-glucose 23 4 5 6 1 1 6 5 4 3 2
  • 16. Benedict’s Test For Reducing Sugars Distinguish reducing from non-reducing sugars. Reducing sugar … Sugars that contain aldehyde groups that are oxidized to carboxylic acids In order for oxidation to occur, the cyclic form must first ring- open to give the reactive aldehyde.
  • 17. Benedict’s Test For Reducing Sugars Benedict’s reagent is a mild oxidant with CuSO4, Cu (II) sulfate, as one of the reagents. Copper (II) sulphate, Sodium carbonate and Sodium citrate.
  • 18. Benedict’s Test For Reducing Sugars oxidized reduced In the presence of a reducing sugar, the blue solution of Cu (II) or Cu+2, is changed to a brick red/brown precipitate of Copper (I) or Cu+1 oxide,Cu2O. Formation of a brick red/brown precipitate is indicative of the presence of reducing sugars. precipitate
  • 19. Benedict’s Test For Reducing Sugars All mono- and most disaccharide's will reduce CuSO4, producing a precipitate of copper (I) oxide on heating, so they are called reducing sugars.  A precipitate indicates reducing sugars  Original Pale Blue = no reducing sugar  Brown/Red = reducing sugar
  • 20. Benedict’s Test For Reducing Sugars
  • 21. • Ketoses can also be reducing sugars because they can isomerise to aldoses: • Benedict’s Test For Reducing Sugars
  • 22. Why sucrose is non-reducing sugar… sucrose has a anomeric carbon which is not free since the carbon links glucose and fructose and fructose does not have free -OH group to undergo reducing reaction and to open the ring so sucrose is non-reducing.
  • 23. At the end of the lab… • You should… • Distinguish between reducing and non-reducing sugar • Understand the theory for Benedict’s test • Know which of the tested carbohydrates are positive or negative for Benedict’s solution and why
  • 24. Seliwanoff’s Test for Ketoses Distinguishes between ketohexoses (ketone) and aldohexoses (aldehyde) sugars.
  • 25. Seliwanoff’s Test for Ketoses Rapid appearance of a dark red solution indicates the presence of a ketose Aldohexoses react slower and the appearance of the red solution takes longer
  • 26. • Distinguishes polysaccharides and other carbohydrates Iodine Test for Polysaccharides
  • 27. • Polysaccharides will react with iodine to form a blue, red, violet, or purple product which is a positive indicator of the presence of a polysaccharide. • Monosaccharides and disaccharides are too small to trap iodine molecules and do not form any dark color with iodine Iodine Test for Polysaccharides
  • 28. Some examples of polysaccharides…
  • 29. Disaccharides and polysaccharides can be hydrolyzed in acidic solution into their component monosaccharides, and then submitted to chemical tests like Benedict's test. Hydrolysis Test for Di- and Polysaccharides
  • 30. • Starch and sucrose do not give positive test with Benedict’s. After hydrolysis, sucrose produces fructose and glucose that give a positive Benedict’s test. • Sucrose + H2O -> Glucose + Fructose Hydrolysis Test for Di- and Polysaccharides Negative Benedict’s test Positive Benedict’s test
  • 31. This test distinguishes carbohydrates that undergo fermentation and those that do not. Yeast is used as a source of an enzyme, zymase. The products are CO2 and ethanol. C6H12O6 + yeast  C2H5OH + CO2 Formation of bubbles of carbon dioxide is used to confirm the fermentation process Fermentation Test
  • 32. Benedict’s Test For Non- Reducing Sugars Non-Reducing sugars do not reduce copper sulphate. However, if it is first hydrolysed to its constituent monosaccharides, it will then give a positive Benedict’s Test. First test a sample for reducing sugars, to see if there are any present before hydrolysis. Then using a separate sample,  Boil the test solution with dilute hydrochloric acid for a few minutes to hydrolyse the glycosidic bond.  Neutralise, by adding small amounts of solid sodium hydrogen carbonate until it stops fizzing.  Perform the Benedict’s test  A positive result indicates the presence of simple non- reducing sugar.