postlab biochem experiment 4 8b
TRANSCRIPT
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NUCLEOPROTEINS
Experiment No. 4
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HAT ARE NUCLEOPROTEINS? Conjugated proteins, the prosthetic group of which are
nucleic acids
Found abundant in tissues possessing closely packed
cells with big nuclei, like thymus, liver, spleen, kidney
and pancreas
Also found in bacteria, bacteriophages, chromosomes
and constitute the whole makeup of the simple
filterable viruses
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HAT ARE NUCLEOPROTEINS?
Not coagulated by heat but exhibit the precipitation
and color reactions characteristic of protein substances
Have been proven to be desoxyribonucleic acids
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ABORATORYRESULTS: SOLUBILITY
Solvents Results
H2O Insoluble
NaCl Soluble
HCl Insoluble
KOH Soluble
Alcohol Insoluble
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ABORATORYRESULTS: TEST FOR
HOSPHATES AND COLOR REACTION TEST
Tests Results
Biuret Test Purple color
Xanthoproteic Reaction Yellow to orange color
Benedicts Test Brick red precipitate
Molisch Test Violet ring
Test for Phosphates Yellow solution with yellow precipitate
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HEMISTRY OF ENZYMES
Experiment No. 5
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ABORATORYRESULTS: TEST FOR THE
CTIVITY OF CATALASE
Test tube 1 Test tube 2
Production of morebubbles
Production of blue
flame with lighted
matchsticks Production of blue to
green precipitate with
benzidine
Production of lessbubbles
No reaction with
lighted matchsticks
The yellow color wasintensified with
benzidine
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ABORATORYRESULTS: ACTION OF
ANCREATIN ANDAMYLASE
SampleResult and
Observations
Test tube 1(starch solution + iodine solution)
Blue black color
Test tube 2(starch solution + pancreatin solution + heat +
iodine solution)
Violet to Blue color
Test tube 3(starch solution + amylase solution + heat +
iodine solution)
Violet to Blue color
Test tube 4(starch + amylase + ethanol + heat + iodine
solution)
Blue color
Test tube 5(starch + pancreatin + heat + iodine solution)
Violet color
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EXPERIMENT 6A 7MONOSACCHAARIDES AND
DISACCHARIDES
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CARBOHYDRATES:ARE HYDRATES OF CARBON
-are polyhydroxyaldehydes or
polyhydroxyketone, or
substances that give these
compounds on hydrolysis.
(CH2O)n
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Carbohydrates
They have the molecular formulas Cn(H2O)n
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CLASSIFICATION OF CARBOHYDRATES
1. According to type of carbonyl group
aldoses - aldehyde group
ketoses - keto group
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D and L notations are used to describe the
configurations of carbohydrates
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CLASSIFICATION OF CARBOHYDRATES
2. According to the number of carbon atoms
Hexose
Heptose
Octose
Triose
Tetrose
Pentose
FormulaName
C3 H6 O3
C4 H8 O4
C5 H1 0 O5
C6 H1 2 O6
C7 H1 4 O7
C8 H1 6 O8
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CLASSIFICATION OF CARBOHYDRATESThere are only two trioses:
Often aldo- and keto- are omitted and these compounds
are referred to simply as trioses.
Although triose does not tell the nature of the carbonylgroup, it at least tells the number of carbons.
Dihydroxyacetone
(a ketotriose)
Glyceraldehyde
(an aldotriose)
CHO
CHOH
CH2OH
CH2OH
C=O
CH2OH
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CLASSIFICATION OF CARBOHYDRATES
3. According to the number of sugar units
Monosaccharidecompose of one sugar units Simple sugars
Disaccharide- compose of twomonosaccharide units
OligosaccharidesOligo = "a few" - usually2 to 10 monosaccharide units
Polysaccharidesare polymers of the simplesugars usually more than 10monosaccharide units
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EXPERIMENT 6-A
MONOSACCHARIDES(1)
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TEST FOR CARBOHYDRATES
Moores TestTest for the presence ofcarbohydrates in a substance in the
influence of a concentrated alkali
Molisch Testis a general test for
carbohydrates where sugars are mixed with-naphthol, the test tube is inclined and about
1mL of concentrated sulphuric acid is added
along the sides of the tube.
The color formed is due to the reaction of alpha-
naphthol with furfural and/or its derivative
formed by the dehydration of sugars by
concentrated sulphuric acid. All carbohydrates
react positively with this reagent.
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TEST FOR CARBOHYDRATES
Seliwanoffs Testuse to
differentiate ketohexoses from
aldohexoses.
In the concentrated HCl, ketones
undergo dehydration to yield furfuralderivatives more rapidly than do
aldoses. These derivatives form
complexes with resorcinol to yield deep
red color.
It is a timed color reaction specific for
ketohexoses.
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TEST FOR CARBOHYDRATES
Phenylhydrazine
The ketoses and aldoses react with phenyl-
hydrazine to produce a phenylhydrazone
which in turn reacts with another two
molecules of phenylhydrazine to form theosazone.
Glucose, fructose and mannose produce
needle-shaped yellow osazone crystals.
Whereas lactosazone in mushroom-shaped.
Different osazones show crystals of
different shapes. Maltose produces star-
shaped (flower-shaped) crystals.
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Data and ResultsA. Macroscopic Appearance
B. Solubility
EXPT. NO. 6 CARBOHYDRATES: THE
MONOSACCHARIDES (1)
Glucose Fructose Galactose
Color White White White
Odor Odorless Odorless Odorless
Form Crystalline Crystalline Crystalline
Glucose Fructose Galactose
Water FreelySoluble
FreelySoluble
FreelySoluble
10% NaCl Soluble Soluble Soluble
0.25% HCl Soluble Soluble Soluble
95%Ethylalcohol
Slightlysoluble
Slightlysoluble
Slightlysoluble
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C. Moores Test
D. Molisch Test
Glucose Fructose GalactoseColor Change brown brown Brown
Odor produced Caramel like Caramel like Caramel like
Glucose Fructose Galactose
Color produced
at the junction
of the two
liquids
Purple to red
violet
Purple to red
violet
Purple to red
violet
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PHENYLHYDRAZINE REACTION
Glucosazone Fructosazone Galactosazone
yellow osazone
crystals are
formed
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POST LAB QUESTIONS
1. The three simple sugars are most soluble in water.2. In Molisch Test. what is the principle behind the
production of color at the junction of two liquids?
- The color formed is due to the reaction of alpha-
naphthol with furfural and/or its derivative formedby the dehydration of sugars by concentrated
sulphuric acid
3. In phenylhydrazine reaction, what is the color of the
crystals formed? - yellow
4. In Moores Test. what is responsible for production of
color at the junction of two liquids?
- presence of concentrated alkali (NaOH)
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EXPERIMENT 6-B
MONOSACCHARIDES(2)
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TEST FOR CARBOHYDRATES
Fehlings Test
The blue alkaline cupric hydroxide present
in solution, when heated in the presence
of reducing sugars, gets reduced to yellow
or red cuprous oxide and it getsprecipitated. Hence, formation of the
colored precipitate indicates the presence
of reducing sugars in the test solution.
Cu(OH)2 Cu2O + H2O
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FEHLINGS TEST
Reaction:
Rochelle salt
CuSO4 + KOH Cu(OH)2 + K2SO4
reducing sugar
2 Cu(OH)2 Cu2O + H2O + [O]
brick red ppt
Note:
Fehlings A and B have to be kept in separate containers to prevent the
formation of Cu(OH)2 immediately.
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TROMMERS TEST
Reaction:
CuSO4 + 2 NaOH Cu(OH)2 + Na2SO4
reducing sugar
2 Cu(OH)2 Cu2O + H2O + [O]
brick red ppt
Cu(OH)2 + H2O CuO + H2Oblack ppt
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NYLANDERS TEST
Reaction:
Rochelle salt
Bi(OH)2NO3 + KOH Bi(OH)3 + KNO3
reducing sugar
2Bi(OH)3 2Bi + 3H2O + 3[O]black ppt
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TEST FOR CARBOHYDRATES
Benedicts Test
As in Fehlings test, the reducing sugar
because of having potentially free
aldehyde or keto group reduce cupric
hydroxide in alkaline solution to redcolored cuprous oxide.
Formation of red, yellow or green
color/precipitate.
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BENEDICTS TEST
Reaction:
Na2CO3 + 2H2O 2NaOH + CO2 + H2O
sodium citrate
NaOH + CuSO4 Cu(OH)2 + Na2SO4
reducing sugar
2 Cu(OH)2 Cu2O + H2O + [O]brick red ppt
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TEST FOR CARBOHYDRATES
Barfoeds Test
Only monosaccharides answer this test.
Since Barfoeds reagent (Copper acetate)
is weakly acidic, it is reduced only by
monosaccharides.Formation of brick-red precipitate.
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TEST FOR CARBOHYDRATES
Mucic Acid Test
Formation of crystals
The both end carbon groups are
oxidized to carboxylic groups. Theresultant saccharic acid of galactose is
called mucic acid which is insoluble in
water.
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TEST FOR CARBOHYDRATES
Picric Acid Test test for the presence of reducing sugars
Picric acid is a chemical compound formally
called 2,4,6-trinitrophenol (TNP).
a toxic yellow crystalline solid and one of the
most acidic phenols.
It reacts with metal to form metal picrates.
formation of MAHOGANY RED SOLUTION
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TEST FOR CARBOHYDRATES
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Data and ResultsA. Reduction Tests
EXPT.6-B THE MONOSACCHARIDES
(PART 2)
Glucose Fructose Galactose
Trommers Test Brick red Brick red Brick red
Fehlings Test Brick red Brick red Brick red
Benedicts Test Brick red Brick red Brick red
Nylanders Test Black Black BlackBarfoeds Test Brick red Brick red Brick red
Picric acid Test Mahogany red Mahogany red Mahogany red
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B. Phloroglucinol-HCl Test
C. Mucic Acid Test
Glucose and Fructose- no crystals formed
Galactose- white sandy crystals
Glucose Fructose Galactose
Color produced brown brown red
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D. Seliwanoffs Test
Post-Lab Questions1. a. What difference have you observed when
Trommers test was carried out using distilled
water instead of sugar solution?- formation of brick red ppt. is not observed indistilled water, carbohydrate is not present.
Glucose Fructose Galactose
Time required for the
development of color
Color of the dissolved
precipitate in alcoholyellow Red color Yellow
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1.b) What is the role of the sugar solutions in thechanges that have taken place?
- sugars act as reducing agents; sugars reducedalkaline metals present in the solution/reagent
2.a. What is responsible for the production of thecolored precipitate? Write the reactioninvolved.
- free monosaccharides are responsible for theproduction of the colored precipitate
b. Why are Fehlings solution kept in separate
containers?- to avoid formation of complex compound from thesubstances present in Fehlings A and B that mayinterfere Fehlings test
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3. a. What are the differences between Fehlings
and Benedicts solutions?
Ans. Benedicts soln. is made of only one solution whileFehlings is made of two. Fehlings is not specific to
aldehydes.
b. Between Benedicts and Fehlings solution,
which is more sensitive? Why?
Ans. Benedicts. Because it uses only one solution and
it undergoes a series of change in color when exposed to
heat.
4. What is the name of the colored precipitateformed in Nylanders test?
- Metallic bismuth
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5. a. In phloroglucinol-HCl test, is there a development
of red color in all sugar solutions?
- No
b. Phloroglucinol-HCl test is a specific test for what
sugar?- Galactose and Pentoses (like Ribose)
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DISACCHARIDES
-COMPOSED OF TWO MONOSACCHARIDE SUBUNITS HOOKEDTOGETHER BY AN ACETAL LINKAGE
1. MALTOSE-D-glucose linked to -D-glucose by an
(1-4) glycosidic bond
- A reducing sugar- repeating disaccharide unit of starch
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DISACCHARIDES
Maltose
Present in malt, the juice from sprouted barley andother cereal grains.
Maltose is a reducing sugar.
OHOHO
OH
OOHO OH
OH
CH2OH
CH2OH
O
OH
O
OH
HO
OOH
HO
OH
CH2OH
HOCH2 1
4
-1,4-glycosidicbond
1 4
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2. LACTOSE
-D-galactopyranosyl-(1-4) -D-glucopyranoside
Made up of -D-galactose linked to -D-glucose
by a (1-4) glycosidic bond (galactose: brain sugar)
A reducing sugar
Lactose is the principal sugar present in milk; it
makes up about 5 to 8 percent of human milk and 4to 6 percent of cow's milk
D
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DISACCHARIDES
Lactose
O
OH
HO
OH
O
CH2OH
O
HOOH
OH
CH2OH
OOH O
OH
OH
CH2OH
O OH
OH
OH
CH2OH
1
1
4
4
-1,4-glycosidic bond
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3. SUCROSE
Made up of -D-fructose linked to an -D-glucose by a (2-1) glycosidic bond
A non-reducing sugar (Why?)
Table sugar
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Sucrose (table sugar) Sucrose is the most abundant disaccharide in the
biological world; it is obtained principally from the juice of
sugar cane and sugar beets.
Sucrose is a nonreducing sugar.
O
HO
OH
OH
CH2OH
O
OH
HO
O
CH2OH
HOCH2
OHOHO
O
OH
CH2OH
OH
HO
O
CH2OH
HOCH2
1
1
2
1
2
1
a unit of -D-glucopyranose
a unit of -D-fructofuranose
-1,2-glycosidic bond
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THE DISACCHARIDES
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Data and Results
A. Moores test
EXPT. 7 THE DISACCHARIDES
Sucrose Maltose Lactose
Color
change
colorless brown brown
Odor
produced
Odorless Caramel
like
Caramel
like
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PHENYLHYDRAZINE REACTION
Sucrose Maltosazone Lactosazone
No crystals
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C. Reduction Test
D. Phloroglucinol-HCl Test
Sucrose Maltose Lactose
Trommers Test colorless Brick red ppt. Brick red ppt.
Fehlings Test Blue soln. Brick red ppt. Brick red ppt
Benedicts Test Blue soln. Brick red ppt. Brick red ppt.
Nylanders Test colorless Black ppt. Black ppt.
Barfoeds Test Blue soln. Blue soln. Blue soln.
Picric Acid Test Yellow solution Mahogany red Mahogany red
Sucrose Maltose Lactose
Color produced black black red
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E. Mucic Acid Test
-only lactose can produce white sandy crystals
F. Seliwanoffs Test
Sucrose Maltose Lactose
Time required for
the development
of red color
Color of thedissolved
precipitate in
alcohol
red No red color No red color
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G. Inversion of Sucrose
Phenylhydrazine Test
Ans. Formation of yellow
osazone crystals
Test Visible Result (with thefiltrate)
a. Reduction Test
Fehlings test Brick red ppt
Benedicts test Brick red ppt
Barfoeds test Brick red ppt
Picric Acid test Mahogany red
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EXPERIMENT 8-A & B
The Polysaccharides
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POLYSACCHARIDES
Homopolysaccharides
Yield one kind of monosaccharide upon
hydrolysis
Heteropolysaccharides
Yields mixtures of monosaccharides and
derived products
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HOMOPOLYSACCHARIDES
Starch (C6H10O5)x Found abundantly in plant kingdom
Most important constituent of human diet
A granule is made up of 98% amylose (300 glucose
units in unbranched chain)
Starch (amylose) maltose
Acid
Glucose
Enzyme
(Amylase)
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HOMOPOLYSACCHARIDES
Starch (C6H10O5)x
The ratios of the two molecules amylose
and amylopectin, are different
depending on the plant which madethem High-amylose corn starch is 85%
amylose, while waxy corn is more than
99% amylopectin
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STARCH COMPONENTS
Amylose. The simple glucose molecules have a
bond between the 1 carbon in the ring and the 4
carbon in the ring. They are joined by an oxygen.
Amylopectin is another component of starch and it
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Amylopectin is another component of starch and it
also consists entirely of repeating chains of glucose. It
is different from amylose because it has two different
types of linkages between the glucose subunits:the 1C-4C and the 1C-6C. The 1:6 linkage makes a
branched structure instead of a linear structure. The
branches occur every 24 to 30 glucose units. The
molecules of Amylopectin are composed of between
2000 and 200,000 units of glucose.
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STAGES IN STARCH HYDROLYSIS
Reaction with iodine Benedicts testStarch Blue
Soluble starch Blue
Amylodextrin Purple
Erythrodextrin Red +
Achrodextrin Colorless ++
Maltose Colorless +++
2 Glucose Colorless ++++
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HOMOPOLYSACCHARIDES
Inulin (C6H10O5)x Found in bulb of onions and garlic
White, odorless, tasteless powder; soluble in hot
water, slightly soluble in cold water and insoluble
in 60% alcohol
Optically active
Yields fructose upon hydrolysis
Glycogen (C6H10O5)x
Animal starch, found mostly in liver as storagematerial; in muscles as source of energy
Enzymatic (amylase) hydrolysis yields maltose
Acid hydrolysis yields glucose
C
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CARBOHYDRATE AND COLOR IT YIELDS
WITH IODINE
Carbohydrate Color
Starch Blue
Amylodextrin Purple
Erythrodextrin Red
Archrodextrin Colorless
Glycogen/Amylopectin
Faint red
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Data and Results
Starch
A. Preparation of Potato Starch:
B. Microscopic Study
EXPT. 8-A THE POLYSACCHARIDES
(STARCH, INULIN AND GLYCOGEN)
Potato starch Cassava starch Corn starch Arrow root starch
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Data and Results
Macroscopic Appearance
EXPT. 8-A THE POLYSACCHARIDES
(STARCH, INULIN AND GLYCOGEN)
Potato
starch
Corn
starch
Arrow
root
starch
Cassava
starch
Color White White White White
Odor Odorless Odorless Odorless OdorlessForm Amorphous Amorphous Amorphous Amorphous
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D. Solubility
E. Iodine Test on Starch Powder
- Blue black or dark blue
- the blue black color is due to starch-iodine
complex
Solvent Solubility Color reaction withiodine solution
Water Insoluble Yellow
Hydrochloric acid Soluble Blue
Sodium hydroxide Slightly soluble Colorless
Alcohol Insoluble Yellow
F I di t t St h P t
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F. Iodine test on Starch Paste
- Blue black or dark blue
G. Hydrolysis of Starch
1. Reduction Tests
Test Performed Visible Result withHydrolyzed starch
Fehlings Test Brick red ppt
Benedicts Test Brick red ppt
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2. Phenylhydrazine Test
- Formation of glucosazone crystals
H. Influence of Tannic acid
Yellow to brown solution with white jelly-like ppt.
INULIN
A. Solubility
Solvents Solubility of Inulin
Hot Water Soluble
Cold water Slightly soluble
Alcohol Insoluble
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B. Iodine Test on Inulin Powder- yellow
C. Iodine test on Inulin Solution - yellow
D. Benedicts Test- blue color
E. Hydrolysis of Inulin
Test Performed Visible Result with
Hydrolyzed Inulin
Benedicts Test Brick red ppt
Seliwanoffs Test Brick red ppt
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HYDROLYSIS OF INULIN
Inulin
acid/ inulase
Fructose
Gl
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Glycogen
A. Preparation and Test of Glycogen
B. Hydrolysis of Glycogen
a. Benedicts Test brick red ppt.
b. Hydrolyzed glycogen contains reducing sugar
c. End product of glycogen hydrolysis is GLUCOSE
Test Performed Visible Result with
Glycogen Solution
Benedicts Test Blue
Iodine Test Red
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HYDROLYSIS OF GLYCOGEN
Glycogen
amylase
Maltose
acid
2 Glucose
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Data and Results
Cellulose
A. Solubility
EXPT. 8-B: THE POLYSACCHARIDES
( CELLULOSE AND HEMICELLULOSE)
Solvents Solubility of Cotton
H2O InsolubleDilute H2 SO4 Insoluble
Concentrated H2 SO4 Soluble
Dilute HCl Insoluble
Concentrated HCl Insoluble95% alcohol Insoluble
Dilute NaOH Insoluble
Concentrated NaOH Insoluble
Iodine Test reddish brown color
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Iodine Test -reddish brown color
Schweitzers Test - white flocculent precipitate
(cellulose acetate)Cross and Bevans Test- white gelatinous ppt
(ethyl cellulose)
Formation of Amyloidblue-black or blue
Hydrolysis of Amyloid
Test Performed Visible Result with
Hydrolyzed Amyloid Solution
Fehlings Test Brick red ppt
Benedicts Test Brick red ppt
Picric Acid Test Mahogany Red
H i ll l
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Hemicellulose
Pentosans: Gum Arabic
A. Macroscopic Appearance
B. Solubility
Gum Arabic
Color Flesh (dirty white)
Odor Odorless
Form AmorphousTaste Tasteless
Solvents Solubility of Gum
Arabic
Hot water Freely soluble
Cold water Soluble
95% alcohol insoluble
C I di T t
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C. Iodine Test
Ans. Yellowish color
D. Hydrolysis of Gum Arabic
ans. Liberation of arabinose D-arabinose
E. Phenylhydrazine Test
ans. Yellow crystals of
arabinosazone
Test Performed Visible Result with
Hydrolyzed Gum Arabic
BenedictsTest Brick red
Fehlings Test Brick red
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Hexosan: Agar-Agar
A. Solubility
B. Iodine Test on Agar-Agar powder- yellow
Solvents Solubility of Agar-Agar
Hot water Soluble forming gel
Cold water Insoluble
95% alcohol Insoluble
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C. Iodine Test on Agar-agar solution
- yellow
D. Hydrolysis of Agar-Agar
E. Mucic Acid Test
ans. White sandy crystals
Test Performed Visible Result with
Hydrolyzed Agar-Agar
BenedictsTest Brick red
Fehlings Test Brick red
-
7/25/2019 POstlab Biochem Experiment 4 8B
78/80
CELLULOSEVS. STARCH
linkage
4,1
linkage
4,1
-
7/25/2019 POstlab Biochem Experiment 4 8B
79/80
IODINE TEST FOR POLYSACCHARIDES
Glucose: Product upon hydrolysis
-
7/25/2019 POstlab Biochem Experiment 4 8B
80/80
REMINDERS
February 23 - Monday
Submit Summary of Scores reflected in the Logbook
(1/4 piece of paper)
Submit BLUE answer sheet
February 24
Quizzes
Midterm Exam