PHL - 224 Biochemistry II · PHL - 224 Biochemistry II 3 Introduction to the Laboratory: This course is intended to introduce you to some of the most widely used experimental procedures

PHL - 224 Biochemistry II

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PHL 224 Biochemistry II

Lab. Manual

Academic Year

1438/1439 - 2017/2018

Kingdom of Saudi Arabia

Ministry of Higher Education

Prince Sattam Bin Abdulaziz University

College of Pharmacy

Department of Pharmacology


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Contents

Remarks Name of the Experiment DATE S.NO.

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Introduction to the Laboratory:

This course is intended to introduce you to some of the most widely used experimental procedures in Biochemistry. Prior

to each lab period, you will need to spend some time reading the Laboratory Manual. This reading will provide background

information and an outline of the procedures to be performed. If you do not do this, you will find yourself wasting large

amounts of class time, and annoying both your lab partners and your Instructor.

SAFETY:

Laboratories contain hazards of various kinds. Everyone is required to wear closed-toe shoes, long pants, goggles with side

shields, and a lab coat while performing laboratory work. Students should not work in the laboratory if the instructor is not

present.

COMMON LABORATORY TOOLS & EQUIPMENT USED IN BIOCHEMISTRY:

Plastic & Glass Tubes Used For the Storage of Liquids:


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PRECISE VOLUMETRIC MEASUREMENTS WITH GRADUATED CYLINDERS AND MICROPIPETTES

Centrifuges:

A centrifuge is a laboratory device that is used for the separation of

fluids, gas or liquid, based on density. Separation is achieved by

spinning a vessel containing material at high speed;

the centrifugal force pushes heavier materials to the outside of the

vessel.


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Cuvette:

A cuvette (from French cuvette = "little vessel") is a small tube of

circular or square cross section, sealed at one end, made of plastic,

glass, or fused quartz (for UV light) and designed to hold samples

for spectroscopic experiments.

Spectrophotometer:

A spectrophotometer is a special instrument that measures how

much light a substance absorbs. Every substance will transmit

(reflect back) and absorb light slightly differently.


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PREPARATION OF BUFFER’s

PREPARATION OF BUFFER AND MEASURING pH AIM:

To study the nature of the buffer and prepare a buffer at the required pH

INTRODUCTION AND PRINCIPLE:

Buffers are defined as the solutions that resist changes in the pH of systems. Regulation of the pH of body fluids and tissues

consistent with life and normal functions are obtained by the presence of buffer

The pH meter measures an electrical potential developed by a pair of electrode pins in a solution. For the measurement of pH, an

electrode system sensitive to change in H+ ion concentration of a solution is used.

PROCEDURE:

An acidic buffer is prepared by adding a weak acid to salt of that acid. A basic buffer is prepared by adding a weak base to the

salt of base.

The required pH of two buffers is given. The obtained pH can be adjusted by adding HCl for a more acidic or NaOH for a more

basic solution.


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Buffer pH

Citrate buffer 2.5

Carbonate-bicarbonate buffer 10.2

Carbonate-bicarbonate buffer

Measure out 13.75ml of sodium carbonate in a glass beaker. Add11.25ml sodium bicarbonate solution and make up to 50ml with

distilled water.

Measure the pH with a standardized pH meter and record the result.

Adjust the pH if necessary.

Citrate buffer

Measure out 23.25ml of citric acid in glass beaker. Add 1.75ml sodium citrate solution and make up to 50 ml with distilled water.

Measure the pH with standardized pH meter and record the result.

Adjust the pH if necessary


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RESULTS AND DISCUSSION:

Buffer pH obtained HCl added?

(Yes/No)

NaOH added?

(Yes/No)

REPORT:

_______________________________________________________________________________________________________________

_______________________________________________________________________________


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Qualitative analysis


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Identification of Carbohydrates

Carbohydrates

Carbohydrates: Are aldehyde or free ketone derivatives of polyhydric alcohols.

Classification of Carbohydrate

Monosaccharaides: can't hydrolyzed into simpler. They may classified as:

trioses, tetroses, pentoses, hexoses or heptoses depending on the number of carbon atoms.


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And aldoses (e.g glucose, galactose and mannose) or ketoses (e.g fructose) depending upon whether they have an aldehyde or

ketone group.

1. Disaccharides: are condensation products of two monosaccharaide units e.g. Maltose, lactose and sucrose.

2. Oligosaccharides: are condensation products of three to ten monosaccharaide units.

3. Polysaccharides: are condensation products of more than ten monosaccharaide units ex. Starch, glycogen and dextrin

Reducing sugar is a carbohydrate possessing either a free aldehyde or free ketone functional group.

Glucose: is the most important biological carbohydrates, it is produced by plants during photosynthesis. Glucose is reducing

sugar. It is stored as Starch in plant and as Glycogen in animals. Disaccharide Sucrose is not reducing sugar.


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No. Test Observation Inference

1.

Solubility:

Compound + water

Soluble

Insoluble

Presence of Mono and/or

disaccharides

Presence of Polysaccharides

2.

Molisch test:

Procedure: Mix 2ml of sugar sample with 5

drops of Molisch's Reagent in a test tube. Add

gently through the side by tilting the tube, about

2 ml of concentrated H2SO4 so as to form a

bottom layer.

Violet/purple ring at the junction of two liquids

Presence of carbohydrates

3.

Benedict’s test:

Procedure: Take 5 ml of Benedict's qualitative

reagent, add 8 drops of sugar solution. Boil over

a flame for 2 minutes or place in boiling water

bath for 3 minutes. Allow to cool.

Green, to brick red precipitate is observed

Presence of reducing sugar


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4. 44

4

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Fehling’s test:

Procedure: 2 ml of Fehling's solution A + 2ml of

Fehling's solution B + 2 ml of Sugar solution

Boil.

Yellow or brick red precipitate

Presence of reducing sugar

5.

Barfoed’s test

Procedure: Sugar solution + Barfoed’s reagent,

heat for 3 min. on boiling water bah

Red Precipitate

Scanty brick red precipitate

Presence of monosaccharide

6.

Seliwanoff's Test

Procedure:Sugar solution+ few crystals of

Resorcinol + Equal volume of conc. HCl and

warm on water bath

Rose Red Color

Presence of Keto Sugar


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7-

Bial's test:

Procedure: 2 ml of sample solution is placed in a

test tube and 2 ml of Bial's reagent is added,

solution is heated gently in hot W.B

Bluish product.

All other colors indicate negative

Presence of pentose sugar

8.

Osazone test

Procedure:

1- Add 10 drops of glacial acetic acid to 5 ml of

sugar solution in test tube.

2- Then add a knife point of phenyl hydrazine

hydrochloride and double the amount of

sodium acetate crystals.

3- Mix and warm a little to see that the solids

are dissolved.

4- Filter the solution, and keep the filtrate in a

boiling W.B for 20 min.

5- Cool slowly in water bath not under the tap

to have better crystals.

6- Examine the crystal under microscope.

As under microscope:

Glucosazone: Needle shaped crystals

galactosazone: Rhombic shaped crystals.

Maltosazone: Sun flower shaped crystals lactose:

Powder puff/hedgehog shaped crystals.

Glucose, Fructose, Maltose and

Lactose.

Glucose, fructose and mannose

produce the same osazone

Galactosazone crystals are

formed in 7 min. Maltosazone

crystals are formed in 10-15 min


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9.

Hydrolysis of sucrose (Inversion test)

Principle: sucrose on hydrolysis with HCl is

converted to glucose and fructose.

the prescence of these two monoscharides can be

confirmed by Benedict's and Seliwanoff's tests.

Procedure: add 2 drops of HCl and 1 drop of

thymol blue to 5 ml of sucrose solution.

The development of pink color indicates that the

solution is acidic.

Divide it in to two equal parts

Boil one portion for about one min., cool under

tape water.

Formatison of blue color indicates neutralization .

Perform Benedict's and Seliwanoff's tests.

Boiled portion gives positive

Benedict's and Seliwanoff's


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Neutralize both portions by adding 2% sodium

carbonate drop by drop.

10.

Iodine/Potassium iodide test:

Principle: iodine forms a coordinate complex

between the helically coiled polysaccharide chain

and iodine.

Procedure: Two ml of a sample solution is placed

in a test tube. Two drops of iodine / potassium

iodide solution and one ml of water are added.

Blue-black complex

a) Lugol's Iodine

b) b) Starch solution

c) Starch solution with iodine

Starch confirmed

Results: On the basis of above observations the given sample was found to be

a. __________________________________

b. __________________________________

c. __________________________________ (____________________)


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Monosaccharides

Fructose

Galactose

Glucose

Fructose Date……………….


1.

Solubility:

Compound + water

2.

Molisch test:

Procedure: Mix 2ml of sugar sample with 5 drops

of Molisch's Reagent in a test tube. Add gently

through the side by tilting the tube, about 2 ml of

concentrated H2SO4 so as to form a bottom layer.


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3.

Benedict’s test:


reagent; add 8 drops of sugar solution. Boil over



4.

Fehling’s test:


Fehling's solution B + 2 ml of Sugar solution Boil.

5.

Barfoed’s test

Procedure: Sugar solution + Barfoed’s reagent

(Cu Acetate/Acetic acid), heat for 3 min. on

boiling water bath


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6.

Seliwanoff's Test


Resorcinol + Equal volume of conc.HCl and

warm on water bath

7.

Bial's test:



solution is heated gently in hot water bath

8.

Osazone test

Procedure:

Add 10 drops of glacial acetic acid to 5 ml of sugar

solution in test tube.

Then add a knife point of phenyl hydrazine

hydrochloride and double the amount of sodium

acetate crystals.

Mix and warm a little to see that the solids are

dissolved.


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Filter the solution, and keep the filtrate in a boiling

W.B for 20 min.

Cool slowly in water bath not under the tap to

have better crystals.

Examine the crystal under microscope.


d. __________________________________

e. __________________________________

f. __________________________________ (____________________)


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Galactose Date……………….


1.

Solubility:

Compound + water

2.

Molisch test:

Procedure: Mix 2ml of sugar sample with 5 drops of Molisch's

Reagent in a test tube. Add gently through the side by tilting the

tube, about 2 ml of concentrated H2SO4 so as to form a bottom

layer.

3.

Benedict’s test:

Procedure: Take 5 ml of Benedict's qualitative reagent, add 8

drops of sugar solution. Boil over a flame for 2 minutes or place

in boiling water bath for 3 minutes. Allow to cool.


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4.

Fehling’s test:

Procedure: 2 ml of Fehling's solution A + 2ml of Fehling's

solution B + 2 ml of Sugar solution Boil.

5.

Barfoed’s test

Procedure: Sugar solution + Barfoed’s reagent, heat for 3 min. on

boiling water bath.

6.

Seliwanoff's Test

Procedure:Sugar solution+ few crystals of Resorcinol + Equal

volume of conc. HCl and warm on W.B.

7.

Bial's test:

Procedure: 2 ml of sample solution is placed in a test tube and 2

ml of Bial's reagent is added, solution is heated gently in hot W.B

8.

Osazone test

Procedure:

Add 10 drops of glacial acetic acid to 5 ml of sugar solution

in test tube.


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Then add a knife point of phenyl hydrazine hydrochloride

and double the amount of sodium acetate crystals.

Mix and warm a little to see that the solids are dissolved.

Filter the solution, and keep the filtrate in a boiling water

bath for 20 min.

Cool slowly in water bath not under the tap to have better

crystals.



a. __________________________________

b. __________________________________

c. __________________________________ (____________________)


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Glucose Date……………….


1.

Solubility:

Compound + water

2.

Molisch test:


Reagent in a test tube. Add gently through the side by tilting the tube,

about 2 ml of concentrated H2SO4 so as to form a bottom layer.

3.

Benedict’s test:

Procedure: Take 5 ml of Benedict's qualitative reagent, add 8 drops of

sugar solution. Boil over a flame for 2 minutes or place in boiling

water bath for 3 minutes. Allow to cool.


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4.

Fehling’s test:

Procedure: 2 ml of Fehling's solution A + 2ml of Fehling's solution B

+ 2 ml of Sugar solution and Boil.

5.

Barfoed’s test


boiling water bath

6.

Seliwanoff's Test

Procedure:Sugar solution+ few crystals of Resorcinol + Equal volume

of conc. HCl and warm on water bath.

7.

Bial's test:

Procedure: 2 ml of sample solution is placed in a test tube and 2 ml of

Bial's reagent is added, solution is heated gently in hot W.B

8.

Osazone test

Procedure:

Add 10 drops of glacial acetic acid to 5 ml of sugar solution in


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test tube.

Then add a knife point of phenyl hydrazine hydrochloride and

double the amount of sodium acetate crystals.


Filter the solution, and keep the filtrate in a boiling W.B for 20

min.

Cool slowly in water bath not under the tap to have better

crystals.



a. __________________________________

b. __________________________________

c. __________________________________ (____________________)


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Disaccharides

Lactose Date……………….

No

.

Test Observation Inference

1. Solubility:

Compound + water

2.

Molisch test:


Reagent in a test tube. Add gently through the side by tilting the tube,

about 2 ml of concentrated H2SO4 so as to form a bottom layer.


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3.

Benedict’s test:

Procedure: Take 5 ml of Benedict's qualitative reagent; add 8 drops of

sugar solution. Boil over a flame for 2 minutes or place in boiling

water bath for 3 minutes. Allow to cool.

4.

Fehling’s test:

Procedure: 2 ml of Fehling's solution A + 2ml of Fehling's solution B

+ 2 ml of Sugar solution Boil.

5.

Barfoed’s test


boiling water bath.

6.

Seliwanoff's Test

Procedure:Sugar solution+ few crystals of Resorcinol + Equal volume

of conc. HCl and warm on water bath

7.

Bial's test:

Procedure: 2 ml of sample solution is placed in a test tube and 2 ml of

Bial's reagent is added, solution is heated gently in hot W.B


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8.

Osazone test

Procedure:

Add 10 drops of glacial acetic acid to 5 ml of sugar solution in test tube.

Then add a knife point of phenyl hydrazine hydrochloride and double the amount of sodium acetate crystals.

Mix and warm a little to see that the solids are dissolved. Filter the solution, and keep the filtrate in a boiling W.B for 20

min. Cool slowly in water bath not under the tap to have better

crystals. Examine the crystal under microscope.


a. __________________________________

b. __________________________________

c. __________________________________ (____________________)


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Maltose Date……………….


1.

Solubility:

Compound + water

2.

Molisch test:

Procedure: Mix 2ml of sugar sample with 5 drops of Molisch's Reagent in a test

tube. Add gently through the side by tilting the tube, about 2 ml of

concentrated H2SO4 so as to form a bottom layer.

3.

Benedict’s test:

Procedure: Take 5 ml of Benedict's qualitative reagent, add 8 drops of sugar

solution. Boil over a flame for 2 minutes or place in boiling water bath for 3

minutes. Allow to cool.


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4.

Fehling’s test:

Procedure: 2 ml of Fehling's solution A + 2ml of Fehling's solution B + 2 ml of

Sugar solution Boil.

5.

Barfoed’s test

Procedure: Sugar solution + Barfoed’s reagent , heat for 3 min. on boiling

water bath

6.

Seliwanoff's Test

Procedure: Sugar solution+ few crystals of Resorcinol + Equal volume of conc.

HCl and warm on water bath.

7.

Bial's test:

Procedure: 2 ml of sample solution is placed in a test tube and 2 ml of Bial's

reagent is added, solution is heated gently in hot water bath.


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8. Osazone test

Procedure:

Add 10 drops of glacial acetic acid to 5 ml of sugar solution in test tube.

Then add a knife point of phenyl hydrazine hydrochloride and double

the amount of sodium acetate crystals.


Filter the solution, and keep the filtrate in a boiling W.B for 20 min.

Cool slowly in water bath not under the tap to have better crystals.



a. __________________________________

b. __________________________________

c. __________________________________ (____________________)


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Sucrose Date……………….


1.

Solubility:

Compound + water

2.

Molisch test:





bottom layer.


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3.

Benedict’s test:





4.

Fehling’s test:



Boil.

5.

Barfoed’s test


heat for 3 min. on boiling water bath.


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6.

Seliwanoff's Test

Procedure: Sugar solution+ few crystals of


warm on water bath

7.

Bial's test:





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8.


Procedure:

Add 2 drops of HCl and 1 drop of thymol blue to

5 ml of sucrose solution.


solution is acidic.

Divide it in to two equal parts Boil one portion

for about one min.

cool under tape water.

Neutralize both portions by adding 2% sodium

carbonate drop by drop.

9.

Osazone test

Procedure:

Add 10 drops of glacial acetic acid to 5 ml of

sugar solution in test tube.

Then add a knife point of phenyl hydrazine

hydrochloride and double the amount of sodium


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acetate crystals.

Mix and warm a little to see that the solids are

dissolved.

Filter the solution, and keep the filtrate in a

boiling W.B for 20 min.

Cool slowly in water bath not under the tap to

have better crystals.



a. __________________________________

b. __________________________________

c. __________________________________ (____________________)


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Polysaccharides Starch

Date……………….


1.

Solubility:

Compound + water

2.

Molisch test:


Reagent in a test tube. Add gently through the side by tilting

the tube, about 2 ml of concentrated H2SO4 so as to form a

bottom layer.


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3.

Benedict’s test:

Procedure: Take 5 ml of Benedict's qualitative reagent; add 8



4.

Fehling’s test:



5.

Barfoed’s test

Procedure: Sugar solution + Barfoed’s reagent, heat for 3 min.

on boiling water bath.

6.

Seliwanoff's Test

Procedure: Sugar solution+ few crystals of Resorcinol + Equal

volume of conc. HCl and warm on water bath


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7.

Bial's test:


ml of Bial's reagent is added, solution is heated gently in hot

water bath.

8.


Procedure: 2 ml of a sample solution is placed in a test tube.

Two drops of iodine / potassium iodide solution and one ml of

water are added.

9.

Hydrolysis of starch

Procedure: Prepare starch solution using warm water Take 3 ml

of starch solution and 3 ml of hydrochloric acid, mix the solution

and boil gently. Remove the sample after every five minute on a

glass rod and touch on some iodine on the surface of a tile.

Appearance (color) with iodine

Starch solution only

After 5 mins of boiling




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10.

Molisch test:


Reagent in atest tube. Add gently through the side bytilting the

tube, about 2 ml of concentrated H2SO4 so as to form a bottom

layer.

11.

Benedict’s test:

Procedure: Take 5 ml of Benedict's qualitative reagent; add 8



12.

Fehling’s test:



13.

Barfoed’s test

Procedure: Sugar solution + Barfoed’s reagent, heat for 3 min.

on boiling water bath.


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14.

Seliwanoff's Test

Procedure:Sugar solution+ few crystals of Resorcinol + Equal

volume of conc. HCl and warm on water bath.

15.

Bial's test:


ml of Bial's reagent is added, solution is heated gently in hot

water bath.


a. __________________________________

b. __________________________________

c. __________________________________ (____________________)


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(UNKNOWN) Date……………….


1.

Solubility:

Compound + water

2.

Molisch test:





bottom layer.


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3.

Benedict’s test:





4.

Fehling’s test:



Boil.

5.

Barfoed’s test


heat for 3 min. on boiling water bath.

6.

Seliwanoff's Test




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warm on water bath.

7.

Bial's test:




8.


Procedure: add 2 drops of HCl and 1 drop of

thymol blue to 5 ml of sucrose solution.


solution is acidic. Divide it in to two equal parts

Boil one portion for about one min., cool under

tape water.Neutralize both portions by adding

2% sodium carbonate drop by drop.


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9.


Procedure: Two ml of a sample solution is placed

in a test tube. Two drops of iodine / potassium

iodide solution and one ml of water are added.

10.

Hydrolysis of starch

Procedure: Prepare starch solution using warm

water Take 3 ml of starch solution and 3 ml of

hydrochloric acid, mix the solution and boil

gently. Remove the sample after every five

minute on a glass rod and touch on some iodine

on the surface of a tile.

Color

starch solution only

after 5 mins of boiling




a. __________________________________

b. __________________________________

c. __________________________________ (____________________)


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AMINO ACIDS & PROTEINS

What are amino acids?

Amino Acids are the building blocks of proteins. Proteins are polymers of amino acids linked together by what is called “Peptide

bond”.

20% of the human body is made up of protein. There are about 300 amino acids occur in nature. Only 20 of them occur in proteins.

PROPERTIES OF AMINO ACIDS:

Physical Properties of Amino acids:

Amino acids have a tetrahedral structure.

Zwitterion: At pH 7 amino group is protonated (-NH3+) and carboxylic group is ionized (COO-)

Solubility: Most of the amino acids are usually soluble in water and insoluble in organic solvents.

Melting points: Amino acids generally melt at higher temperatures, often above 200°C.

Taste: Amino acids may be sweet (Gly, Ala, Val), tasteless (Leu) or bitter (Arg, lle). Monosodium glutamate is used as a flavoring agent

in food industry, and Chinese foods to increase taste and flavor.


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Importance of amino acids:

Amino acids have an influence on the function of organs, glands, tendons and arteries. They are furthermore essential for healing

wounds and repairing tissue, especially in the muscles, bones, skin and hair as well as for the removal of all kinds of waste deposits

produced in connection with the metabolism.

Essential Amino Acids: (cannot be synthesized by an organism and must be obtained in the diet) Histidine, Isoleucine, Leucine, Lysine,

Methionine, Phenylalanine, Threonine, Tryptophan, and Valine.

Non-essential Amino Acids: Alanine, Asparagine, Aspartic Acid, Glutamic Acid.

Conditional Amino Acids: Arginine (essential in children, not in adults), Cysteine, Glutamine, Glycine, Proline, Serine, and Tyrosine.


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In protein formation, the condensation of amino group of one α-amino acid with the carboxyl group of another molecule of same or

different α-amino acid with the elimination of a water molecule forms an amide linkage between molecules. This amide linkage (-C

(=O)NH-) between two α-amino acids is termed as peptide bond or peptide linkage. The dimer formed due to this linkage is called

as dipeptide.


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1- Solubility Tests:

Principle:

The solubility of amino acids and proteins is largely dependent on the solution pH. The structural changes in an amino acid or

protein that take place at different pH values alter the relative solubility of the molecule. In acidic solutions, both amino and

carboxylic groups are protonated. In basic solutions, both groups are deprotonated.

Amino acids are essentially soluble in water. Their solubility in water dilute alkali and dilute acid vary from one compound to the

other depending on the structure of their side chains. Apply this test to glycine, tyrosine, glutamic acid and cysteine.

Procedure:

1- Note the solubility of amino acids in water and alcohol by placing a small amount in a test tube, adding a few mL of solvent and

warming if necessary.

2- Determine the amino acid solution is acidic or basic by using a litmus paper while testing the solubility in water.

3- Repeat the solubility test using dilute HCl and dilute NaOH.


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Experimental Procedure:

No. Test Observation Inference &Interpretation

I. General Test for amino acid and protein (Amine groups in proteins, peptones and amino acids)

1.

Ninhydrin test:

Principle: In the pH range of 4-8, all α- amino acids

react with ninhydrin (triketohydrindene hydrate), a

powerful oxidizing agent to give colored product

(diketohydrin) termed Rhuemann’s purple.

Procedure: To 1ml solution add 5 drops of 0.2%

ninhydrin solution in acetone. Boil over a water bath for

2 min. Allow to cool.

N.B: Avoid spilling ninhydrin solutions on your skin, as

the resulting stains are difficult to remove.

a- Blue color formed(primary

amine)

b. yellow color is formed

(secondary amine)

a- Protein or Amino acid present

detect alpha-amino acids and also free

amino and carboxylic acid groups on

proteins and peptides

Ninhydrin is most commonly used as a

forensic chemical to detect

“fingerprints”, as amines left over from

proteins sloughed off in fingerprints react

with ninhydrin giving a characteristic

purple color.

b- Presence of amino acid Proline


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II. Tests for protein

1. Biuret’s test

Principle: Biuret test is Specific for Proteins

differentiate between Proteins (+ve) and Amino Acids

(-ve). The biuret reagent (copper sulfate in a strong

base) reacts with peptide bonds in proteins to form a

violet complex known as “Biuret complex”.

Procedure: To 1 mL of protein solution (Albumin –

Casein – Gelatin – Peptone) in a test tube, add 1 mL of

10% sodium hydroxide solution and 2-3 drops of 1%

copper sulfate solution. Mix well

Violet color is obtained with

albumin, casein & gelatin

and a pinkish violet color with

peptone

Protein

Two peptide bonds are at least required

for the formation of this complex, this is

why amino acids give negative results

with Biuret test.

2. Heat coagulation test

Principle: protein coagulated by heating, acetic acid is

added, if coagulation persists, its protein.

Procedure: Place about 5 ml of egg-white solution

(albumin solution) in a test tube and heat the top part

cloudy and a flocculent precipitate

of coagulated protein is produced Protein


56

of the solution only.

3. Picric acid test

Procedure To 3 ml of gelatin solution in a test tube,

add 2ml of saturated picric acid solution

a

yellow gelatinous precipitate

Protein

4. Precipitation by salts of heavy metal:

Principle: protein precipitate by heavy metals, where

positively charged metal ion neutralize the negatively

charged protein molecule resulting in

Metal proteinate.

A-Procedure: to protein solution add mercuric

chloride drop by drop.

B-Procedure: to protein solution add lead acetate soln

drop by drop.

White precipitate Protein

5. Precipitation by acids

Principle: protein precipitate by acids, where

positively charged acid neutralize the negatively

White precipitate

Protein


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charged protein

A - Procedure: to protein solution add sulphasalicylic

acid drop by drop.

B - Procedure: to protein solution add tannic acid

drop by drop.

A light brownprecipitate

6. Esbach's test

Principle: protein precipitate by Esbchs reagent, where

positively charged reagent neutralize the negatively

charged protein

Procedure: to protein solution add 5 ml of

esbachsreagent drop by drop.

yellow precipitate

Protein

III. Test for α -amino-acid glycine

1. p-nitrobenzoyl chloride & pyridine test:

Principle: Glycine in quantities as small as 0.5 g

detected as an orange-red color by reaction with p-

nitrobenzoylchloride & pyridine. The procedure can

be used both as a qualitative spot test and as a sensitive

Orange-red to maroon color

develops immediately, varying in

shade with the concentration of

glycine.

Glycine

N.B.As little as 0.5 g. of glycine develops

a perceptible orange-red color. All other

amino acids fail to react in concentrations

below 0.5 mg with larger quantities, a


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quantitative method, the color is due to aziactone

formation.

Procedure: A few crystals of powder sample, on a filter

paper strip or a glass slide, and approximately 1 mg. of

solidp-nitrobenzoyl chloride is placed on top. One to

three drops of pyridine are then added to wet the

mixture.

The color is soluble in polar solvents such as

chloroform, dichioroethylene, tetrachloroethane,

ethylacetate, and also in excess pyridine.

pale bluish color, in contrast to colorless

blanks, develops with some amino acids.

Acetylglycine, glycyiglycine,hippuric

acid, and salicyluric acid do not react in

the cold, but on warming, a yellow color

is produced with milligram quantities.

IV. Test for amino-acids contain: activated benzene rings ( Tyrosine and Tryptophan)

1.

Xanthoproteic test:

Principle: Aromatic amino acids, such as Phenyl

alanine, tyrosine and tryptophan, respond to this test.

In the presence of concentrated nitric acid, the aromatic

phenyl ring is nitrated to give yellow colored nitro-

derivatives. At alkaline pH, the color changes to

Yellow color formed

Amino acid present

(tyrosine, tryptophan)


59

orange due to the ionization of the phenolic group.

Procedure: To 2ml of solution in a boiling test tube,

add an equal volume of conc. HNO3. Heat over a flame

for 2 min and observe the color. Now COOL

THOROUGHLY under the tap and CAUTIOSLY run

in sufficient 40% NaOH to make the solution strongly

alkaline.

V. Test for amino-acids contain: Indol group

1. Hopkins-Cole Test:

Principle:This test is specific test for detecting

tryptophan. The indole moiety of tryptophan reacts

with glyoxilic acid in the presence of concentrated

sulphuric acid to give a purple colored product.

Glyoxilic acid is prepared from glacial acetic acid by

being exposed to sunlight.

Procedure: To a few ml of glacial acetic acid containing

glyoxylic acid, add 1-2 drops of the amino acid

solution. Pour 1-2ml concentrated H2SO4 down the

purple color at the interface

Amino acid present

Tryptophan


60

side of the sloping test tube to form a layer underneath

the acetic acid.

2. Ehrlich's test:To 0.5ml of the amino acid solution, add

2ml Ehrlich reagent

A colored complex formed

Amino acid present

Tryptophan

VI. Test for : phenolic amino acid

1. Millon’s test:

Principle: Phenolic amino acids such as Tyrosine and

its derivatives respond to this test. Compounds with a

hydroxybenzene radical react with Millon’s reagent to

form a red colored complex. Millon’s reagent is a

solution of mercuric sulphate in sulphuric acid.

Procedure: To 2ml of amino acid solution in a test tube,

add 1-2 drops of Millon’s reagent. Warm the tube in a

boiling bath

A brick red color is a positive

reaction.

(red– pink colour )

Amino acid present

phenolic amino acid as (tyrosine)

N.B: A yellow precipitate of HgO is NOT

a positive reaction but usually indicates

that the solution is too alkaline.


61

VII. Test for amino-acids contain: Sulfhydryl group –SH (cystine&Cysteine)

1. Nitroprusside test:

PrincipleIt is specific for Amino Acids or Proteins

containing sulfur, -SH (in cysteine & cystine)

Procedure: Add 2ml of the amino acid solution into

test tubes. Add 0.5ml fresh sodium nitroprusside

solution and shake thoroughly. Add 0.5ml ammonium

hydroxide.

Red color formed

Amino acid present

(cystine, cysteine)

The nitroprusside test is specific for

cystine & cysteine, the only amino acid

containing sulfhydryl group (-SH).

VIII. Test for amino-acids contain: Sulfur

1. Lead acetate test:

Principle: cysteine and cystine, the sulfur containing

amino acid react with lead acetate under alkaline

conditions to form a brown precipitate.

Procedure: Everything needed to carry out this test

will be in the hood and should not remove anything

from the hood. A toxic, stinky gas will be made (in

small, but immensely smelly quantities) and you don’t

brownish-black precipitate

Amino acid containing sulphur present

(cystine, cysteine and methionine)


62

want to smell it. Dispense about 0.5mL of the amino

acid solution only into a clean test tube (found in the

hood, where you will leave it when you are done).

Add 0.5mL of 20% NaOH and insert the test tube in a

boiling water bath for 1 min. Add 2 drops of lead (II)

acetate solution.

IX. Test for amino-acids contain: Guanidium group

1. Sakaguchi's test:

Principle: Arginine, containing guanidine group

reacts with α -naphthol under alkaline conditions to

produce red color.

Procedure: 1ml NaOH and 3ml of the arginine solution

is mixed and 2 drops of α-naphthol is added. Mix

thoroughly and add 4-5 drops Bromine solution

Red color formed

Amino acid containing guanidine

present

(Arginine)


63

X. Test for Histadine amino-acids

1. Pauly’s test:

Principle: This test is specific for the detection of

Histidine. The reagent used for this test contains

sulphanilic acid dissolved in hydrochloric acid.

Sulphanilic acid upon diazotization in the presence of

sodium nitrite and hydrochloric acid results in the

formation a diazonium salt. The diazonium salt

formed couples with either tyrosine or histidine in

alkaline medium to give a red colored chromogen (azo

dye).

Procedure: Into clean test tube, dispense 1mL of 1%

sulphanilic acid and 2 drops of 5% sodium nitrite. Mix

for 1 min. Add about 0.5mL of amino acid solution.

Yellow product formed Amino acid present

Histidine

Results: The protein or amino acids were found to be present in given sample.


64

Summary

AMINO acid Ninhydrin

Test

p-nitrobenzoyl

chloride & pyridine

Test

Xanthoproteic

Test

Hopkins-

Cole Test

Ehrlich's

Test Millon's Test

Lead acetate

Test

Glycine +ve +ve

Alanine +ve

Phenylalanine +ve

Glutamic acid +ve

Tyrosine +ve +ve +ve

Tryptophan +ve +ve +ve +ve

Methionine +ve

+ve


65

Date……………….

No. Test Observation

Inference

&Interpretation

1. General Test for amino acid and protein (Amine groups in proteins, peptones and amino acids)

1.

Ninhydrin test:

Procedure: To 1ml solution add 5 drops of 0.2%

ninhydrin solution in acetone. Boil over a water bath

for 2 min. Allow to cool.

N.B: Avoid spilling ninhydrin solutions on your

skin, as the resulting stains are difficult to remove.

A. Blue color formed (primary amine)

B. yellow color is formed (secondary amine)


66

III. Tests for protein

1. Biuret’s test

Procedure: To 1 mL of protein solution (Albumin –

Casein – Gelatin – Peptone) in a test tube, add 1 mL

of 10% sodium hydroxide solution and 2-3 drops of

1% copper sulfate solution. Mix well

Violet color is obtained with albumin, casein &

gelatin

and a pinkish violet color with peptone

2.

Heat coagulation test

Procedure: Place about 5 ml of egg-white solution

(albumin solution) in a test tube and heat the top

part of the solution only.

cloudy and a flocculent precipitate of coagulated

protein is produced

3.

Picric acid test

Procedure To 3 ml of gelatin solution in a test tube,

add 2ml of saturated picric acid solution

a yellow gelatinous precipitate


67

4.

Precipitation by salts of heavy metal:

A-Procedure: to protein solution add mercuric

chloride drop by drop.

B-Procedure: to protein solution add lead acetate

solution drop by drop.

White precipitate

5.

Precipitation by acids

A - Procedure: to protein solution add

sulphasalicylic acid drop by drop.

B - Procedure: to protein solution add tannic acid

drop by drop.

White precipitate

A light brown precipitate

6.

Esbach's test

Procedure: to protein solution add 5 ml of esbachs

reagent drop by drop.

yellow precipitate

III. Test for α -amino-acid glycine

p-nitrobenzoyl chloride& pyridine test:

Procedure: A few crystals of powder sample, on a

filter paper strip or a glass slide, and approximately

1 mg. of solid p-nitrobenzoyl chloride is placed on

Orange-red to maroon color develops immediately,

varying in shade with the concentration of glycine.


68

1.

top. One to three drops of pyridine are then added

to wet the mixture.

The color is soluble in polar solvents such as

chloroform, dichioroethylene, tetrachloroethane,

ethylacetate, and also in excess pyridine.

IV. Test for amino-acids contain: activated benzene rings ( Tyrosine and Tryptophan)

1.

Xanthoproteic test:

Procedure: To 2ml of solution in a boiling test tube,

add an equal volume of conc. HNO3. Heat over a

flame for 2 min and observe the color. Now COOL

THOROUGHLY under the tap and CAUTIOSLY

run in sufficient 40% NaOH to make the solution

strongly alkaline.

Yellow color formed

V. Test for amino-acids contain: Indol group

1.

Hopkins-Cole Test:

Procedure: To a few ml of glacial acetic acid

containing glyoxylic acid, add 1-2 drops of the

amino acid solution. Pour 1-2ml concentrated

purple color at the interface


69

H2SO4 down the side of the sloping test tube to

form a layer underneath the acetic acid.

2. Ehrlich's test: To 0.5ml of the amino acid solution,

add 2ml Ehrlich reagent

A colored complex formed

VI. Test for : phenolic amino acid

1.

Millon’s test:

Procedure: To 2ml of amino acid solution in a test

tube, add 1-2 drops of Millon’s reagent. Warm the

tube in a boiling bath

A brick red color is a positive reaction.

(red– pink colour )


70

VII. Test for amino-acids contain: Sulfhydryl group –SH (cystine&Cysteine)

1.

Nitroprusside test:

Procedure:Add 2ml of the amino acid solution into

test tubes. Add 0.5ml fresh sodium nitroprusside

solution and shake thoroughly. Add 0.5ml

ammonium hydroxide.

Red color formed

VIII. Test for amino-acids contain: Sulfur

1.

Lead acetate test:

Procedure: Everything needed to carry out this test

will be in the hood and should not remove anything

from the hood. A toxic, stinky gas will be made (in

small, but immensely smelly quantities) and you

don’t want to smell it. Dispense about 0.5mL of the

amino acid solution only into a clean test tube


71

(found in the hood, where you will leave it when

you are done). Add 0.5mL of 20% NaOH and insert

the test tube in a boiling water bath for 1 min. Add

2 drops of lead (II) acetate solution.

brownish-black precipitate

IX. Test for amino-acids contain: Guanidium group

1. Sakaguchi's test:

Procedure: 1ml NaOH and 3ml of the arginine

solution is mixed and 2 drops of α-naphthol is

added. Mix thoroughly and add 4-5 drops Bromine

solution

Red color formed


72

X. Test for Histadine amino-acids

1. Pauly’s test:

Procedure: Into clean test tube, dispense 1mL of 1%

sulphanilic acid and 2 drops of 5% sodium nitrite.

Mix for 1 min. Add about 0.5mL of amino acid

solution.

Yellow product formed

RESULT:

THE GIVEN SAMPLE IS A

1. ______________________________

2. ______________________________

3. ______________________________


73

LIPIDS

IDENTIFICATION OF LIPIDS

Lipids: are organic compounds formed mainly from alcohol and fatty acids combined together by ester linkage.

Properties of Lipids:

Lipids are insoluble in water, but soluble in fat or organic solvents (ether, chloroform, benzene, acetone).

Lipids include fats, oils, waxes and related compounds.

They are widely distributed in nature both in plants and in animals. Classification of Lipids:


74

Why Analysis and Identification of fats and oils (Fat Constants) is needed?

• Fat constants or numbers are tests used for:

1. Checking the purity of fat for detection of adulteration. 3. To quantitatively estimate certain properties of fat. 2. To identify the biological value and natural characteristics of fat. 4. Detection of fat rancidity and presence of toxic fatty acids.

IDENTIFICATION OF LIPIDS by using Sudan III reagent


Sudan III TEST

To a test tube, add equal parts of test

liquid and water to fill about half full.

Add 3 drops of Sudan III stain to test

tube. Shake gently to mix.

A red-stained oil

layer will separate

out and float on the

water surface.

Presence of Lipids

Results: On the basis of above observations the given sample was found to be A LIPID.


75

REPORT

Unknown

Date……………….


Sudan III TEST

To a test tube, add equal parts of test

liquid and water to fill about half full.

Add 3 drops of Sudan III stain to test

tube. Shake gently to mix.

Results: The given sample was found to be ______________________.


76

QUANTITATIVE ANALYSIS

ENZYMES

Definition: Enzymes are highly specific biologic catalysts that greatly speed up the rate of a chemical reaction occurring in living

cells. Enzymes are found in low concentration in body fluids.

The enzyme activity is expressed in the international unit (I.U).

Classification of Enzymes:

Oxidoreductases:

There is a hydrogen donor and a hydrogen acceptor.

a. Aerobic oxidases: use O2 as H-acceptor forming H2O e.g. Tyrosinase.

b. Aerobic dehydrogenases: use O2 as H-acceptor forming H2O2 e.g. Glucose oxidase.

c. Anaerobic dehydrogenases: use co-enzyme as H-acceptor e.g. LDH

Transferases:

Transfer a group from one organic compound to another (CH3, NH2, and Phosphate etc.) e.g. Aminotransferases,

Kinases, Transketolases.

Hydrolases: hydrolyse the substrate

Lyases: Remove groups without hydrolysis leaving a double bond e.g. Decarboxylases.

Isomerases: convert one pair of isomers into another e.g. Racemases.

Ligases = Synthetases: linking two molecules together coupled with the breakdown of phosphate bond.


77

How enzymes work

Factors affecting enzymatic reaction:

1. Substrate concentration. 2. Enzyme concentration. 3. Product concentration. 4. PH. 5. Temperature. 6. Activators and Co-enzymes. 7. Inhibitors. 8. Specificity of enzymes.


78

Date……………….

1. Estimation of Serum Lactate Dehydrogenase Activity (LDH)

Type: anaerobic dehydrogenase enzyme.

Occurrence: Heart > liver > skeletal muscle> erythrocytes > pancreas

Introduction

A lactate dehydrogenase (LDH) test measures the amount of LDH in the blood.

Lactate dehydrogenase is an enzyme that the body uses during the process of turning sugar into energy for your cells to use.

LDH is found in many of the body's tissues and organs, including the muscles, liver, heart, pancreas, kidneys, brain and

blood cells. The LDH test is mainly used to help identify the location and severity of tissue damage in the body. It's also

sometimes used to monitor how far certain conditions have progressed including:

kidney disease

liver disease

some types of cancer

LDH catalyzes the oxidation of lactate to pyruvate in the presence of NAD, which is subsequently reduced to NADH. The

rate of NADH formation measured at 340 nm is directly proportional to serum LDH-L activity

http://www.nhs.uk/conditions/Kidney-disease-chronic/Pages/Introduction.aspx

http://www.nhs.uk/conditions/Cancer/Pages/Introduction.aspx


79

Lactic acid + NAD+ ——LDH →pyruvate + NADH

Procedure:

Pipette into the cuvettes as shown in the table below:

HDL Reagent 1mL

Sample 100µL

Mix and incubate for 1 minute, then read absorbance (at 340 nm against Blank) every minute for three minutes and determine

ΔA/min

Results and Discussion:

Time Absorption

Start

1 min

2 min

3 min

Calculations:

ΔA after 1 minute (start – 1min)

ΔA after 2 minutes (1min – 2min)



80

The LDH concentration in the sample is calculated using following formula;

ΔA/min × 8095= ------------------UL

Mean change in absorbance (ΔA/min):

LDH Concentration:

Normal values:

Adults: 135 -240 U/l Children (2 -15 years old): 120 -300 U/l

Report:

----------------------------------------------------------------------------------------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------------------------------------

Clinical significance:

LDH activity indicates:

a. Myocardial infarction. b. High value is seen in pernicious anemia. c. Moderate increase in viral hepatitis and skeletal muscle disease.


81

Date……………….

2. Estimation of Aspartate aminotransferase activity (AST) Type: Aminotransferases.

Occurrence: Heart > liver > skeletal muscle > kidney > pancreas

Introduction:

An aspartate aminotransferase (AST) test measures the amount of this enzyme in the blood. AST levels in blood are commonly

used as a marker for liver function. AST levels can occur in response to diseases or injuries in multiple tissues including skeletal

and heart. AST formerly was called serum glutamic oxaloacetic transaminase (SGOT). The amount of AST in the blood is

directly related to the extent of the tissue damage. After severe damage, AST levels rise in 6 to 10 hours and remain high for

about 4 days.

Procedure:

Pipette into a cuvette as shown in the table below

Reagent 1 4 mL

Reagent 2 1mL

Sample 500µL

http://www.webmd.com/hw-popup/enzyme

http://www.webmd.com/heart/anatomy-picture-of-blood

http://www.webmd.com/digestive-disorders/aspartate-aminotransferase-ast


82

Read the absorbance of the sample after 60 seconds. Read again after each minute for a period of 3 minutes at 340nm. Distilled

water serves as blank. Calculate the mean absorbance change per minute (ΔA/min)

Results and discussion:

Time Absorption

Start

1 min

2 min

3 min

Calculations:




Following formula is used to calculate the AST/SGOT enzyme activity

ΔA/min × 1746 = ---------------U/L

Mean change in absorbance (ΔA/min)


83

AST Concentration:

Normal values:

Female up to 21 U/L Male up to 25 U/L

Report:

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------

-------------------------------------------------------------------------------------------------------------------------

Clinical significance

AST activity increases in:

1. Myocardial infarction.

2. Hepatobiliary disease.


84

Date……………….

Estimation of Serum Alanine Amino Transferase (ALT) activity

Type : Aminotransferases.

Occurrence: Liver > Heart > Kidney > skeletal muscle > spleen

Introduction:

An alanine aminotransferase (ALT) test measures the amount of this enzyme in the blood. ALT is found mainly in the liver, but

also in smaller amounts in the kidneys , heart, muscles, and pancreas . ALT was formerly called serum glutamic pyruvic

transaminase (SGPT). ALT is measured to see if the liver is damaged or diseased. Low levels of ALT are normally found in

the blood. But when the liver is damaged or diseased, it releases ALT into the bloodstream, which makes ALT levels go up. Most

increases in ALT levels are caused by liver damage.

Procedure:

Pipette into a cuvette as shown in the table below

Reagent 1 4 mL

Reagent 2 1mL

Sample 500µL

http://www.webmd.com/digestive-disorders/alanine-aminotransferase-alt

http://www.webmd.com/hw-popup/enzyme

http://www.webmd.com/heart/anatomy-picture-of-blood

http://www.webmd.com/digestive-disorders/picture-of-the-liver

http://www.webmd.com/kidney-stones/kidneys

http://www.webmd.com/heart/picture-of-the-heart

http://www.webmd.com/digestive-disorders/pancreas

http://www.webmd.com/hepatitis/ss/slideshow-surprising-liver-damage

http://www.webmd.com/a-to-z-guides/rm-quiz-blood-basics

http://www.webmd.com/hepatitis/rmq-know-your-liver


85

Read the absorbance of the sample after 60 seconds. Read again after each minute for a period of 3 minutes at 340nm. Distilled

water serves as blank. Calculate the mean absorbance change per minute (ΔA/min)

Results and Discussion:

Time Absorption

Start

1 min

2 min

3 min

Calculations:




Following formula is used to calculate the AST/SGOT enzyme activity

ΔA/min × 1746 = ---------------U/L

Mean change in absorbance (ΔA/min)


86

AST Concentration

Normal values:

Males7-55 U/L

Females7-45 U/L

Report:

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------

-------------------------------------------------------------------------------------------------------------------------

Clinical significance:

GPT activity increases in:

a. Liver damage and toxic hepatitis (high level).

b. Myocardial infarction.


87

Date……………….

Estimation of Blood Glucose

Enzymatic colorimetric method:

In medicine, blood sugar is a term used to refer to the level of glucose in blood. Glucose, transported via the bloodstream, is the primary source of energy for the body cells. Blood sugar level (BSL), or serum glucose concentration, is tightly regulated in the human body so that its level remains within a certain limit (70 to 150 mg/dl) throughout the day.

Principle:

Glucose is oxidized by glucose oxidase to gluconate and hydrogen peroxide, hydrogen peroxide reacts in the presence of peroxide with phenol and 4-aminoantipyrine to form quinoneimine dye. The intensity of color formed is proportional to glucose concentration

Sample:

A glucose sample with a concentration similar to blood glucose is given

Standard

Glucose standard 100mg/dl is given.

Procedure:

Pipette into a clean test tubes as indicated in the table below.

Solution added Tube 1 ( Blank) Tube 2 (Sample) Tube 3 (Standard)

Reagent 3 mL 3mL 3mL


88

Sample - 300µL -

Standard - - 300 µL

Mix and let the tube stand for 10 minutes at room temperature or for 5 minutes at 37˚.

Place into cuvette and read the absorbance (A) of the sample and the standard with the spectrophotometer at 500nm

against the reagent blank

Reference values:

Newborn: 30-90mg/dl Hypoglycemia: When blood glucose falls below 60 mg/dl.

Adults: 70-105mg/dl Hyperglycemia: When blood glucose is more than 140 mg/dl

Results and discussion:

Results:

Sample Standard

Absorbance

Calculations:

Absorbance of sample X (C) Standard (100) = mg glucose/dl

Absorbance of standard

Report:

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________


89

PROTEIN AND NUCLEIC ACIDS

PURIFICATION AND EXTRACTION


90

Documents

PHL - 224 Biochemistry II · PHL - 224 Biochemistry II 3 Introduction to the Laboratory: This course is intended to introduce you to some of the most widely used experimental procedures