postlab biochem experiment 4 8b

7/25/2019 POstlab Biochem Experiment 4 8B

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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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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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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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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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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


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


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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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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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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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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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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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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Data and ResultsA. Reduction Tests

EXPT.6-B THE MONOSACCHARIDES

(PART 2)


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


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.


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


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


Color produced black black red


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E. Mucic Acid Test

-only lactose can produce white sandy crystals

F. Seliwanoffs Test


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


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


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


Hydrolyzed Amyloid Solution

Fehlings 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


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


Hydrolyzed Agar-Agar

BenedictsTest Brick red

Fehlings Test Brick red


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CELLULOSEVS. STARCH

linkage

4,1

linkage

4,1


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IODINE TEST FOR POLYSACCHARIDES

Glucose: Product upon hydrolysis


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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

postlab biochem experiment 4 8b

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