Practical Biochemistry

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PRACTICAL BIOCHEMISTRY
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Babylon university College of pharmacy practical biochemistry
Prof. assist Dr. abdulhussien al-jebory and tamadhur al-salman
groups important in biochemistry

Alcohols:
Many biochemical compounds such as sugars certain lipids and amino acid are
alcohols. They have both polar hydroxyl and non polar alkyl character. Water
solubility decreases with increasing length of the carbon chain i.e. with increasing
non polar character. Primary secondary and tertiary alcohols have respectively
one, two , three alkyl groups attached to the carbon bearing the --OH group.
Both monohydric (one –OH ) and polyhydric alcohols are of physiological
significance, sugars are derivatives of polyhydric alcohols. Their highly polar
character makes them far more water soluble than corresponding monohydric
alcohols with equivalent numbers of carbon atoms.
Chemical reactions :-
1- Oxidation:-
Primary alcohols are oxidized by strong oxidizing agents to aldehydes and
acids, where as secondary alcohols are oxidized to ketones.
[O]
R-CH2OH ------------------→ RCHO + RCOOH ( primary )
[O]
R1R2CHOH ----------------→ R1R2CO (Secondary)
Tertiary alcohols can not be oxidized (dehydrogenated) without rupture of
a C-C bond.
2- Esterification :-
An ester is formed when water is split out between an alcohol and an acid.
O O
II II
R-C- OH + HO-R -------------→ R-C-O-R/ + H2O
/
1
The acid may be organic or inorganic . Esters of H3PO4 ( phosphorylated sugars

and phospholipids) and H2SO4 , are of significance in biochemistry . many lipids
contain carboxylic ester linkages.
3- Ether formation:-
Ether are derivatives of alcohol in which the hydrogen of the hydroxyl
group is replaced by an alkyl group ( R- O –R/). The ether linkage is
comparatively uncommon in living tissues.
Sulphur which is in the same group of the periodic table as oxygen, forms
compounds. Thioalcohols ( thiols, mercaptans, thioester, and thioethers) all
occur in nature.
R-CH2-SH R-C – S – R/ R-S-R/
Thioalcohols thioesters thioether
In addition , the disulfides ( left) and peroxides ( right)
R-S-S R/ R-O-O-R/
Play an important role in protein structure and in prostaglandins biosyntheses

respectively.
Aldehydes and ketones:-

Aldehydes and ketones possess the strongly reducing carbonyl group > C= O .
aldehydes have one and ketones two alkyl groups.
R- CH = O RR/- C = O
Aldehyde Ketone
Sugars , being polyhydric alcohols are also either aldehydes or ketones.
2
1- Oxidation :-
Aldehyde yields corresponding carboxylic acid ,ketones are not readily
oxidized, since like tertiay alcohols, they can not lose hydrogen with out
rupture of a C – C bond .
H
I [O]
R – C = O ----------------------→ R - COOH
2- Reduction :-
Aldehyde yields corresponding primary alcohol. Ketones yield
corresponding secondary alcohol.
H
I [ 2H+ ]
R – C = O -------------------------------→ R – CH2 – OH
[ 2 H+ ]
RR/ C = O --------------------------------→ RR/ CH - OH
3- Hemi acetal and acetal formation:-

Under acidic conditions, aldehydes can combine with one or two of the
hydroxyl groups of an alcohol, forming respectively , a hemi acetal or an
acetal . OR/
I
/
R – CH = O + R - OH --------------------→ R – CH – OH
A hemiacetal
OR/
H2O I
R – CH = O + 2 R/ - OH --------------------→ R – CH – OR/
An acetal
3
The carbonyl and alcohol functions can be part of the same molecule as in an aldo
sugar forming internal hemiacetal in solution . analogous structures hemiketal
and ketals are formed from alcohols and ketones . aldehydes may form
thiohemiacetals and thioacetals with thioalcohols.
4- Aldol condensation :-
In alkali , aldehydes and to a lesser extent ketones undergo condensation
between their carbonyl and their α – carbon atom to form aldols or β –
hydroxyaldehydes or ketones .
β – hydroxyacids derived from these are of importance in fatty acid

metabolism .
CH3CH = O + CH3CH = O -------- [ OH- ]-----------→ CH3CHOH – CH2 – CH = O
Carboxylic acid
Carboxylic acid have both a carbonyl group and a hydroxyl group at the same
carbon atom. They are typical weak acids.
1- Reduction :-
Complete reduction yields the corresponding primary alcohol.
R – COOH ------ [ 4H+ ] ---------→ R – CH2OH

2- Ester and thioester formation :-
See alcohols above.
3- Acid anhydride formation:-
A molecule of water is split ourt between the carboxyl groups of two acid
molecules
R – CO – O – H + HO – CO – R/ ------- - H2O ------→ R – CO – O – CO –R/
When both acids are the same , a symmetric anhydride is produced .

molecules of different acids yields mixed anhydrides found in nature
4
include this of phosphoric acid ( in ATP ) and the mixed anhydrides formed
from phosphoric acid and a carboxylic acid e.g:
CH3 – CO – O – PO (OH)2 Acetyl phosphate
4- Salt formation:-
Carboxylic acids react stoichiometrcally ( equivalent to equivalent ) with
bases to form salts . Na+ and K+ salts are 100% dissociated in solution .
5- A mide formation:-
Splitting out a molecule of water between a carboxylic acid and ammonia
or an amine forms an amide. Particularly important amides are peptides.
Formed from the amino group of one amino acid and the carboxyl group of
anther.
CH3 – CO – OH + H- NH2 ------- - H2O -----→CH3 – CO – NH2
Acetic acid acetamide
5
General reactions of carbohydrates

Introduction:-
Carbohydrate is a very general term that applies to a very large number of
materials covering a wide spectrum of chemical structure and biological function.
Carbohydrates are found in all living cells in plants, animals and microorganisms.
Carbohydrates may be defined as polyhydroxy aldehydes or ketones, or

substances that yield one of these compounds on hydrolysis. In most organisms,
carbohydrate material, largely in the form of simple sugar glucose, is the primary
foodstuff, providing most of the energy and carbon required in the biosynthesis of
proteins, nucleic acids, lipids and anther carbohydrates. Some carbohydrates have
a structural role such as cellulose in plants, others have a carbon and energy
storage role, such as starch in plants and glycogen in animals and bacteria.
Carbohydrates are classified into three main classes:-
1- Monosaccharides(simple sugar molecules)

can be classified according to the number of carbon atoms in the chain.
Those found most commonly in humans include the following :-
a- Trioses :- composed of three carbon atoms like glyceraldehydes.
b- Tetroses :- composed of four carbon atoms like erythrose
c- Pentoses:- composed of five carbon atoms like ribose,xylose,ribulose
and arabainose.
d- Hexoses:- composed of six carbon atoms like
 Aldohexoses :- contain aldehyde group as glucose, galactose,
mannose.
 Ketohexoses:- contain ketone group as fructose .
6
2- Disaccharides:-
Composed of two molecules of simple sugar linked together by glycosidic
linkages like lactose , sucrose and maltose which divided into :-
a- Reducing sugars:-
they contain free aldehyde or ketone groups that are capable of
reducing an oxidizing agent . examples are maltose, that is composed of
two molecules of glucose , and lactose, that is composed of glucose and
galactose.
b- Non- reducing sugars:-
They lack the free aldehyde or ketone groups , an example is sucrose

which is composed of one molecule of α- glucose and one molecule of β-
fructose linked together by β-1-2 glycosidic linkage.
3- Oligosaccharides:-
Composed of three to twelve glucose units.
4- Polysaccharides:-
They are polymer of glucose units linked together by glycosidic linkages like
a- Starch:-
Is a storage form of glucose in plants. It is composed of both amylase
and amylopectin. Amylase is a linear polymer of glucose units, whereas
amylopectin is a branched chain of glucose units.
b- Dextrins:-
Is a mixture of branched fragments produced by partial hydrolysis of
amylopectin.
c- Glycogen:-
Is the storage from of glucose in animals and is present mainly in the
liver and muscles. It is composed of branched of glucose units linked by
α- 1- 4 and α- 1 – 6 glycosidic linkage . it has even more branching than
amylopectin does, therefore glycogen molecules are more spherical in
shape.
7
d- Cellulose:-
It is composed of a linear polymer of glucose units, like amylase, but the
strands form fibers . it is distributed in the cell walls of plants and thus it
provides the rigidity of plants.
Because they are derivatives of polyhydroxy aldehydes or ketones, their
chemical reactions are characteristic of the primary functional groups (
hydroxyl, aldehyde, or ketone).
Experimental practical
Part ((1))
1 - Effects of acids on carbohydrates
Treatment of carbohydrates with strongly acid solution hydrolyzes the glycosidic
linkages in disaccharides and polysaccharides to yield Monosaccharides . these
Monosaccharides are subjected to a large number of color reactions more or less,
characteristic for these sugars . there is something in common in these reactions (
Molisch, Anthrone, Bial’s and Seliwanoff’s tests) they all involve:-
1- Heating with strong acids causes the hydrolysis of disaccharides and

polysaccharides to yield Monosaccharides ( simple sugars ).
2- Ring formation due to loss of water molecules ( dehydration ) from
Monosaccharides . in this process , pentoses lose three molacules of water
to yield a cyclic compound called furfural, whereas hexoses yield
hydroxymethyl furfural and on long heating other decomposition products
( levulnic acid)
O
H OH -3H2O
H OH O
H OH
acid
O
OH
pentose furfural
8
H OH -3H2O
HO H O
H OH acid O
H OH HO
OH
Hexose hydroxymethyfurfural
3- These furfurals are colorless compounds which undergo condensation

reactions with phenolic substances.
This is the second thing common with these four tests. The phenolic
substance is different for each test. These phenolic substances are all
derivatives of phenol and they have the same basic structure . the
following are examples of these compounds:-
OH OH
O
Phenol a - naphthol Anthrone
CH 3 OH
HO OH OH
Orcinol Resorcinol
The condensation reactions yield colored complexes. The actual nature of these
complexes has not been established for the majority of these condensation
reactions.
9
Exp. 1
1- Molisch’s test:-
This is a very sensitive general test for all carbohydrates because they all
give furfural or hydroxymethyl furfural upon heating with a strong hot
acidic solutions.
It is performed by mixing a few drops of an alcoholic solution of α-
Naphthol (Molisch’s reagent ) with the sugar solution in the presence of
concentrated sulfuric acid to yield a purple condensation colored complex .
the intensity of the color is proportional to the amount of sugar present in
the solution.
Furfural OH
+ +
H H
sugar + Purple colored complex
OH- Methyl-furfural
Procedure :-
1- Add 2ml of 1% solutions of glucose, sucrose and starch into three separate
test tubes.
2- Add 2 drops of Molisch’s reagent into each test tube and mix well by
shaking.
3- Pour slowly and carefully about 3 ml of concentrated Sulpharic acid down
the side of the tubes to form a layer below the sugar solution. A reddish –
violet ring at the junction disappears and the color distributed all over the
solution.
4- Report your observations and explain your results.
To prepare Molisch’s reagent :-

Dissolve 10 gm of α- naphthol in 100 ml of ethyl alcohol 95% .
10
Exp. 2
2- Anthrone test:-
It is also a general qualitative test for all carbohydrates. It can also be used for
quantitative determination of carbohydrates because the intensity of the color
is proportional to the amount of carbohydrate tested ; the more sugar you add,
the darker the color ,and this can be determined spectrophotometry.
It is performed by mixing acidic anthrone reagent ( contains H2SO4) solution
with the sugar solution to yield a dark green complex.
O
H2SO4 +
H
sugar furfural or hydroxy- + Dark green colored complex
methyl furfural
Procedure:-
1- Add 0.2 ml of 0.1 solutions of glucose , sucrose and 1% starch into three
separate test tubes.
2- Carefully add 2 ml of anthrone reagent ( 0.2 % anthrone in conc. H2SO4)
and mix the tubes thoroughly.
3- Heat the test tubes in boiling water bath for 3 minutes.
4- Cool the tubes under tap water and observe the color . record your
observations.
5- Repeat the test with a few pieces of filter paper and record your
observations.
Anthrone reagent:-
Dissolve 2 gm of anthrone in 1 liter conc. H2SO4

11
Exp. 3
3- Bial’s Orcinol test:-

It is a sensitive specific test reaction for the detection of pentoses, and other
biochemical molecules containing these pentoses in their structure, such as ribose
in RNA ( ribonucleic acid ) and Deoxyribose in DNA ( deoxyribonucleic acid). Thus,
this test can be used to distinguish pentoses from other sugars ( hexoses ). It is
performed by mixing few milliliters of Bial’s Orcinol reagent with the sugar to
yield a blue-green colored complex in the presence of pentoses, within a short
time. Hexoses also react with this phenolic substance, but the product is yellow to
brown in color.
CH3
+
H
pentose C C CHO + Blue condensation product
O HO OH
furfural orcinol
Procedure:-
1- Add 3 ml of bial’s reagent into three separate test tubes.

2- Add 0.5 ml of 1% solutions of xylose, glucose and lactose into the test tubes
respectively and mix well.
3- Heat the test tubes in a boiling water bath until a color develops.
4- Report your observations as to what color developed, and record the time
required for the formatiom of the corresponding color.
Bial’s reagent:-
Dissolve 300 mg of orcinol in 100 ml of conc.HCL and add 0.25 ml of ferric chloride
solution ( 10 g/ 100 ml)
12
Exp. 4
4-Seliwanoff’s Resorcinol test:-

This test is given by ketoses. Thus it is used to distinguish ketoses ( fructose) ,
from aldoses ( glucose and mannose). Seliwanoff’s is a solution of resorcinol in
hydrochloric acid . fructose gives a deep red color in a short time compared to a
light pink color produced by glucose. When dehydrated ( removal of water
molecules) in concentrated acid , both ketihexoses and aldohexoses yield
hydroxymethyl furfural and other decomposition products. They both yield the
same products except that the relative amounts of the products vary depending
on whether one works with aldoses or ketoses. Only small amount of
hydroxymethyl furfural are formed from aldohexoses. For this reason Seliwanoff’s
test can be used to distinguish between ketoses and aldoses.
OH
HOCH2 C C CHO + Red condensation prpduct

OH
O
hydroxymethylfurfural Resorcinol
Procedure:-
1- Add 3 ml of Seliwanoff’s (Resorcinol) reagent into each of four separate test
tubes.
2- Add 3 drops of 1% solutions of fructose, glucose, sucrose and starch , one
sugar solution per tube . mix the contents of the tubes.
3- Heat the tubes in a boiling water bath for 10 minutes.
4- Report your observations and accurately record the tine required for the
color formation in each tube.
13
Seliwanoff’s reagent :-
Dissolve 50 mg of resorcinol in 33 ml of conc. HCL and dilute to 100 ml with H2O .
Test Phenolic Acid Sugar Color

substance
Molisch’s α- naphthol H2SO4 All purple
Anthrone Anthrone H2SO4 All Dark – green
Bial’s orcinol HCL Pentoses ˃˃Hexoses Blue
Seliwanoff’s resorcinol HCL Ketoses ˃˃ Others red
Exp. 5
5-Mucic aci test:-

The oxidation of some carbohydrates with concentrated nitric acid HNO3 ,
produces disaccharic acid or called dicarboxylic acid which differs in solubility.
The dicarboxylic acid produced by galactose is insoluble ( white precipitate ).
Thus galactose can be distinguished from other carbohydrates. The nitric acid
oxidizes both the aldehyde and the primary alcohol groups of
monosaccharides to yield dicarboxylic acid.
14
CHO COOH
COOH
H C OH OH
H C OH H C
HO C H H
HO C H HO C
H C OH HNO3 HNO3
H C OH H C OH
H C OH H OH
H C OH C
H C OH COOH
H C OH
H
H
D- glucose D- gluconic acid D- glucosaccharic acid
CHO COOH
H C OH OH
H C
HO C H H
HO C
HO C H HNO3
HO C H
H C OH H OH
C
H C OH COOH
H
D-galactose D-galactosaccharic acid ( Mucic acid)
Procedure:-
1- Place 10 ml of 1% glucose, galactose and lactose solution into three test
tubes separately.
2- Add 1 ml of H2O and carefully 3 ml of conc. HNO3 into each tube.
3- Place the tibes in a boiling water bath in a fume hood for 1.30 to 2.00
hours.
4- Cool the tubes in an ice bath. Scratch carefully the inner walls of the tubes
with a glass rod to facilitate crystallization. Record your observations.
15
2- effects of alkaline on carbohydrates

1- Benedict’s test
2- Fehling’s test
3- Barfoed’s test
These tests are based on the most important chemical property of sugars; the
reducing property. Benedict’s and Fehling tests are used to determine the
presence of reducing sugars while Barfoed’s test is used more specifically to
distinguish monosaccharides from disaccharides. The ability of sugar to reduce
oxidizing agent depends on the availability of free aldehyde or ketone group for
this reaction. All monosaccharide are reducing sugars. Now you may ask how it is
possible since you have learned that, in solution , the higher monosaccharides
( pentoses and hexoses) exist primarily in the cyclic hemiacetal forms in which the
carbonyl group is tied up.
O OH
R- C-H + R-OH R-C-H
OR
Aldehyde Alcohol Hemiacetal
Similarly, an aldehyde group reacts with an alcohol group forming a hemiacetal.
16
H C OH CH2OH
H C O
H C OH
H C OH C O
HO C H O
HO C H
H H
C OH
C OH H or C
H C OH H
H C OH
H C OH
C OH HO C
H C
H C OH
H H OH
H
Glucose Internal hemiacetal structure
Even if the carbonyl group is tied up, these pentoses and hexoses are easily
oxidized due to the fact that the cyclie forms are in equilibrium with a small
amount of open chain form .
α- form ↔ open chain form ↔ β- form
Oxidation product ( leaves the equilibrium)
When solutions of pentoses or hexoses are treated with an oxidizing agent, the
open chain aldehyde form is oxidized and removed from the equilibrium, mixture.
Some of the α- and β- forms are converted to more open chain from to re-
establish the equilibrium which in turn is further oxidized. Eventually all molecules
are oxidized. Disaccharides have glycosidic linkages which in some cases involve
bonding between the free aldehyde or ketone groups. In such cases there is no
reducing group in the sugar. For example sucrose is a non- reducing sugar for this
particular reason. Sucrose is composed of α- glucose and β- fructose.
17
CH2OH
O
OH
OH OH
OH CH2OH
O
Glucose HOCH3 O
- H2O
+ OH
O OH O HO
OH CH3OH
HOCH2 OH
OH
HO
CH2OH
OH
Fructose sucrose
There is no possibility for the presence of the open – chain form in sucrose.
Maltose on the other hand is composed of two molecules of glucose. In solution it
exists in an equilibrium mixture of α- , β- and the open – chain form. Again ,
because of the presence of a small amount of open – chain form in the second
glucose unit, maltose is a reducing sugar which gives a positive Benedict’s and
Fehling’s tests. Those disaccharides which contain a free hemiacetal groups are
reducing sugars although milder than the monosaccharides. Polysaccharides are
long molecules and have so very few of these open – chain “ endings “ , that non
of polysaccharides have reducing properties. Reducing sugars can react with
many different oxidizing agents. Benedict’s and Barfoed’s tests both have cupper
ion ( Cu+2 ) as oxidizinf agent, so does the widely used Fehling’s test. The alkaline
solution favors the aldehyde form the sugar. In these reactions, the ( Cu+2 )
oxidizing the aldehyde group to a carboxyl group whereas the metal ion is
reduced to cuprous ( Cu+1) ion which forms a red cuprous oxide precipitate.
RCHO [ or α- hydroxyl ketone ] + 2 Cu+2 -----------→RCOOH + Cu2O + 3H2O
18
H C COONa
O
H C OH H OH
C
HO C H HO C H
+ Cu++ + citrate + Na2CO3 Cu2O
H C OH H C OH +
H C OH H C OH
CH2OH CH2OH
sodium gluconate and red ppt
salts of other sugar acids
Glucose
The experimental conditions of these three tests vary :-
In Benedict’s and Fehling’s tests a hot alkaline solution favors open – chain forms
of sugars [ i.e aldehyde forms] increasing their susceptibility to oxidation. The
solution called Benedict’s reagent, in addition to being alkaline, contains also
citrate ions whereas the solution called Fehling’s reagent contains also tartarate
ions. The purpose of either citrate or tartarate ions are to prevent cupric ion
(Cu+2) from precipitating as the insoluble Cu(OH)2 before any oxidation reduction
has occurred.
Citrate and tartarate form soluble complexes with Cu+2 [ i.e stabilize the cupric
ions]. The Fehling’s reagent is prepared by mixings, before use, two solution; one
containing copper sulfate , CuSO4 and one containing sodium potassium tartarate
[ NaKC4H4O6.4H2O+, and potassium hydroxide, while Benedict’s reagent composed
also from copper sulfate , sodium citrate and sodium carbonate Na2CO3.
Depending on the concentration of sugars in a solution , heating with the
Fehling’s or Benedict’s reagents gives a yellowish orange to red colored solution
or precipitate.
On the other hand the reagent in Barfoed’s test has an acidic PH * contains cupric
acetate and acetic acid ]. Therefore Barfoed’s test is used specifically to
19
distinguish between monosaccharides and disaccharides by the speed of

formation of cuprous oxide. Copper acetate and acetic acid are used in the
reaction and produce cuprous oxide from disaccharides at a slower ratwe than
from monosaccharides.
Exp. 6
Procedure of Benedict’s test:-

1- To a series of five test tubes, add 5 ml of Benedict’s reagent.
2- To each tube add 8 drops of 1% solution of glucose, sucrose, xylose and
starch respectively.
3- Mix the contents of each tube and place them in a boiling water bath for 3
minutes.
4- Cool the tubes under tap water, positive test is denoted by the formation
of a yellow , green or red precipitate.
5- Report your observations and compare their appearance the size of the
particles and the amount of precipitate .
Benedict’s reagent:-
Dissolve 17.3 gm of CuSO4.5 H2O in 100 ml of hot water .
Dissolve separately with heating 173 gm of sodium citrate and 100 gm of Na2CO3
IN 100 ml of water, allow to cool.
Add the citrate carbonate solution with mixing to the copper sulfate solution,
dilute to 1 liter with water.
20
Exp. 7
Procedure of Fehling’s test:-

1- Mix 1 ml of Fehling’s solution A with 1 ml of Fehling solution B in each of
three test tubes.
2- Add equal amounts of 1% solution of glucose, sucrose and lactose to each
test tubes respectively.
3- Mix the contents of each tube and place the tubes in a boiling water bath
for 5 minutes.
Fehling’s reagent:-
1- Fehling’s solution A :- dissolve 125 gm potassium hydroxide KOH and 173
gm sodium potassium tartarate ( NaKC4H4O6.4H2O) in distilled water and
dilute to 500 ml with D.W.
2- Fehling’s solution B:- dissolve 34.65 gm copper sulfate ( CuSO4) in distilled
water and dilute to 500 ml with D.W.
Exp. 8
Procedure of Barfoed’s test:-
1- Add 3 ml of Barfoed’s reagent to three separate test tubes.
2- Add 2 ml of 1% solution of glucose, lactose and starch to the test tubes
respectively.
3- Mix well and put them in a boiling water bath
4- Observe the tubes during the first five minutes remove any tube that
becomes cloudy or changes color.
5- Continue heating the tubes for another 5 to 10 minutes.
21
6- Report your observations and notice the time of positive reaction in each
tubes.
Barfoed’s reagent :-
Dissolve 13.3 gm of cupric acetate in 200 ml of D.W then add 1.9 ml of glacial
acetic acid CH3COOH.
Exp. 9
Iodine test
Iodine test is used to distinguish polysaccharides from other carbohydrates and

starch from other polysaccharides. Polysaccharides such as amylase,
amylopectine, glycogen, starch and dextrin from characteristic color complexes
when treated with iodine. The amylase in starch is responsible for the intense
blue color with iodine.
The linear chains of glucose molecules in amylase may be coiled in a helix that
contains six glucose units per turn. The inner space of the helix accommodates an
iodine molecule, which is thought to be held in position by hydrogen atoms of the
glucopyranose rings. Polysaccharides with branched chains of glucose molecules
[ i.e amylopectin and glycogen] do not readily form liner helixes and yield less
intensely colored iodine complexes. So dextrin gives a red color, glycogen
produces a red – brown color. Cellulose, monosaccharides and disaccharides give
no color with iodine.
Procedure:-
1- Into six test tubes add about 2 ml of 1% solution of glucose ,sucrose, starch,
glycogen, dextrin and distilled water [serve as a blank ] , each solution in a
separate tubes.
22
2- Add few drops of iodine [1% iodine in 2% potassium iodide ] solution into
each tube.
3- Mix the contents of each tube and compare the color of the blank to that of
the other tubes.
4- Report the color which you observed in each tube.
5- Heat the tubes containing starch and iodine in a boiling water bath for 10 to
15 minutes. If nothing happens , add few milliliters of water to dilute the
solution and continue heating for another five minutes.
Iodine reagent:-
Dissolve 250 mg of iodine in 100 ml of 2% potassium iodide.
Part ((2))
General reactions of carbohydrates
Objectives:-
1- To hydrolyse the sucrose and starch and to test the hydrolysis products.
2- To differentiate between fermentable and non- fermentable sugars.
3- To confirm the identity of reducing sugars, by phenylhydrazine reaction
test.
Introduction
1- Hydrolysis of sucrose and starch:-
Treatment with dilute acid produces hydrolysis of glycosidic linkages in
polysaccharides, oligosaccharides and disaccharides. Since monosaccharides are
the simplest carbohydrates, they cannot be further degraded by hydrolysis.
23
heating
Carbohydrates -------------------------------------→ monosaccharides
Dilute acid
In general, most of the polysaccharides can be hydrolyzed with good yield to their
components, monosaccharides, by dilute acid hydrolysis. Cellulose is the only one
which is rather resistant to this treatment.
From previous experiments recall what happened to carbohydrates in

concentrated hot acids. Carbohydrates were not only, hydrolyzed but they were
completely dehydrated as well and produced furfural and hydroxymethylfurfural
compounds.
heating
Carbohydrates ----------------------------------→ hydrolysis & dehydration
Conc. Acid
Disaccharides may be hydrolyzed to yield two monosaccharide molecules. So

when sucrose is hydrolyzed we end up with fructose and glucose.
Heating
Sucrose --------------------------------→ fructose + glucose
Dilute HCI
We also say that dilute acid has resulted in inversion of sucrose due to the fact
that optical rotation changes upon hydrolysis of sucrose. Polysaccharides are
polymers and their complete hydrolysis yields hundreds of molecules of
monosaccharides.
24
When starch is completely hydrolyzed the end product is glucose, while it’s partial
hydrolysis yields mixture of sugars such as dextrin , maltose and glucose.
Complete hydrolysis
Starch ---------------------------------------------→ glucose
With dilute acid
Partial hydrolysis
Starch -------------------------------------------------→ dextrin , maltose and glucose
With dilute acid
Products resulting from complete hydrolysis of some of the common

carbohydrates are listed below:-
carbohydrate Hydrolysis products

Disaccharides
Maltose α- glucose & α- glucose
Lactose β – galactose & β - glucose
Sucrose β – fructose & α - glucose
Polysaccharides
Starch α – glucose
cellulose β- glucose
In this experiment, you will use the tests of the previous experiment ( part 1 ) to
determine the products of hydrolysis. If hydrolysis of starch has noh progressed
very far,the solution contains mainly dextrins which are the first intermediate
products of hydrolysis of starch. Dextrins give red to red – brown colors with
iodine . as the hydrolysis proceeds, the products are mainly maltose and glucose
which will give no color with iodine. Also a positive result of the Benedict’s test is
25
not necessarily a proof of the production of monosaccharides end product, since

the disaccharide maltose is also a reducing sugar. We must add some alkali
( NaOH) before testing the hydrolysis products with Benedict’s reagent to
suppress ring formation. This leads to susceptibility to oxidation reactions much
more so than at neutral or acidic PH. Reduction of cupric ion to insoluble cuprous
oxide occurs now more redily.
Exp. 10
Experimental procedure:-
Hydrolysis of sucrose:-
1- Add 4 ml of sucrose solution into each of three labeled test tubes.
2- Add 0.5 ml of concentrated HCI to tube number 1 and 0.5 ml of D.W. to
tubes number 2 and 3 to keep the volumes nearly equal.
3- Place tubes 1 and 2 in a boiling water bath for 20 -25 minutes , where tube
number 3 should remain at room temperature.
4- Cool the heated tubes to room temperature and then cautiously add 10%
NaOH drop by drop to tube number 1 . check the PH of the solution with PH
paper and continue adding NaOH until the solution is just alkaline ( record
the amount of NaOH added ). A similar amount of water should be added
to tubes number 2 & 3.
5- Divide the contents of each tube into two tubes labeled 1A,1B,2A.2B,3A
and 3B.
6- To those tubes labeled A( 1A,2A,3A) add 5 ml of Benedict’s reagent and to
those tubes labeled B( 1B,2B,3B) add 5 ml of Seliwanoff’s reagent and place
all tubes in boiling water bath.
26
7- Observe the tubes conatanlly in the water bath and record in which tube
the color developed fastest.
Notes:-
*With Seliwanoff’s test timing is crucial !
* eventually all three test tubes are going to give a positive result due to
the fact that Seliwanoff’s reagent contains strong acid which results in
hydrolysis of sucrose upon heating.
8- record your results in a table form.
Exp. 11
Hydrolysis of starch:-
1- Add 4 ml of starch solution into each of three labeled test tubes.
2- Add 0.5 ml of conc. HCI to tube number 1 and 0.5 ml of D.W. to tubes 2 and
3 to keep the volumes nearly equal.
3- Place tubes 1 and 2 in a boiling water bath for 20 -30 minutes , whereas
tube 3 should remain at room temperature.
4- Cool the heated tubes to room temperature and then cautiously add 10 %
NaOH drop by drop to tube number 1 . check the PH of solution with litmus
paper and continue adding NaOH until the solution is just alkaline ( record
the amount of NaOH added . A similar amount of water should be added to
tubes No. 2 & 3.
5- Remove 2 drops of solution from each tube and test with iodine test.
6- Add 5 ml of Benedict’s reagent to each of the three test tubes and place
them in a boiling water bath form five minutes.
7- Record your observation in a table form.
27
2- fermentation of carbohydrates :-
The purpose of this experiment is to differentiate between fermentable sugar (
can be fermented by yeast) and non- fermentable sugar (can not fermented by
yeast). Glucose , fructose and mannose ( monosaccharides) sucrose and maltose
(disaccharides) are fermentable sugars, while lactose , galactose , all pentoses,
and polysaccharides are non-fermentable sugars.
Conversion of carbohydrates to ethanol and carbon dioxide is called alcoholic

fermentation. The purpose of breakdown of carbohydrate molecules in the cell
is the generation of metabolic energy. One of the main energy producing
reaction in all biological systems, from the simplest microorganisms to the most
complex plants and animals, is the oxidation of one molecule of D- glucose to two
molecules of pyruvic acid.
This process, called glycolysis[ the breaking down of a sugar], is a long sequence
of enzymatic reactions. Fermentation process comprises several reactions
catalyzed by the Baker’s yeast enzymes known as “enzymases”.
H C O
H C OH O
HO
C
HO C H enzymatic reactions
+
H C OH
2 C O + 4 H
H C OH CH3
CH2OH
Pyruvic acid
Glucose
This sequence of reactions is very similar in all kinds of cells. However, the fate of
pyruvate in the generation of energy varies depending on the organism. Many
yeasts have ability to form ethanol from pyruvate.
28
COO- NAD+
H
+
CO2 NADH
C O CH3CHO CH3CH2OH
CH3 decarboxylation Reduction
pyruvate Acetaldehyde Ethanol
Notes:- * NADH [ Nicotine Amine Dinucleotide],” reduced from “ and NAD

oxidized form ,” are derived from the vitamin, niacin.
 Some carbohydrates are fermented by Baker’s yeast , but not all.
The net reaction of this anaerobic process is:-
C6H12O6 + 2Pi + 2ADP -------------------→ 2CH3CH2OH + 2CO2 ↑ + 2ATP
Glucose ethanol
Exp. 12
1- For reliable results, Baker’s yeast must be washed with water to remove
any sugar. Which may be present in it, and which could cause formation of
gas and consequently misleading results. To wash the yeast , mix well the
yeast given to you with distilled water and centrifuge for 2 minutes and
then discard the clear solution ( supernatant ) and keep the washed
precipitate ( pellet ) for the test.
2- Add 1 gm of washed yeast and 10 ml of 1% sugar solution you have into
beaker. Mix the content with a glass rod to uniformly suspend the yeast in
the fluid.
3- Fill in the closed arm of the fermentation tube with this yeast suspension ,
and shake the tube long enough for the air bubbles to escape.
4- Place the tube upright in an incubator maintained 37 – 45 0 C for few
minutes.
5- Observe in which tubes gas is produced and record your observations.
29
6- Collect results of all sugars and present them in a table form.

Note :- run the experiment with two sugars ( one represent a fermentable
sugar and the other represent a non – fermentable sugar).
3 – phenylhydrazine reaction [ the formation of Osazone ] :-

This test used to confirm the identity of a reducing sugar. In this a test the free
carbonyl group in a reducing sugar molecule reacts with phenylhydrazine to form
yellow , insoluble Osazone crystals. Formation of Osazone crystals requires an
excess of phenylhydrazine molecules. The net reaction is shown below:-
H NH2
H C O C = N - NH -
H C OH + 3 H2N-NH- + NH3 +
C = N - NH -
R
R
Aldose Phenylhydrazine Phenylosazone Ammonia Aniline
The reaction proceed in three steps:-
1- In the first step the condensation of one phenylhydrazine molecule with

the corresponding sugar , yield phenylhydrazone compound.
30
H
H-C = O
NH - NH2 H-C=N-N
H OH NH - NH2
C
H C OH
HO C H
HO C H
H C OH
Heat H C OH
CH3COOH
H C OH H2O
H C OH
CH 2OH
CH 2OH
D-glucose glucose phenylhydrozone
H
H-C=N-N
H C O
HO C H NH 2
H C OH + CH3COONH4 +
H C OH
CH 2OH
ketophenylhydrazone
2- In the second step, oxidation takes place resulting in a free carbonyl group.
3- In the third step , condensation of anther phenylhydrazine molecule can
form insoluble osazones.
Note:- only reducing sugars react with phenylhydrazine in slightly acidic
medium to give osazones.
31
H
H C N - NH
H-C=N-N
H C O NH- NH2
C N - NH -
HO C H
H C OH
HO C H
H C OH
H C OH
CH 2OH
H C OH
CH 2OH
ketophenylhydrazone
Osazone
The Osazone crystals of reducing sugars have characteristic shapes which enable
us to identify different sugars. The oreticaily , glucose , mannose and fructose
form the same Osazone crystals since the difference in structure and
configuration about carbon atoms number 1 & 2 are abolished in Osazone
formation and since the configuration on carbon atoms number 3 , 4 , 5 and 6 is
the same for all three sugars.
CHO CHO
CHO
C - OH C=O
HO C
HO C HO C
HO C
C - OH C - OH
C - OH
C - OH C - OH
C - OH
CH 2 - OH CH2 - OH
CH2 - OH
Glucose Mannose Fructose
However, still we can use this test for identification due to the fact that the time
of formation of crystals varies depending on the sugar. Furthermore, some
Osazones are precipitated only from hot solution , others only upon cooling.
32
Exp. 13
1- Mix thoroughly in a morter two parts of phenylhydrazine hydrochloride and
three parts of anhydrous sodium acetate ( by weight ).
2- Introduce 3 ml of 1% solution of each sugar ( glucose , galactose , lactose ,
maltose , fructose , xylose and mannose ) into labeled tubes respectively .
3- Add about 0.7 to 1 gm of freshly prepared phenylhydrazine sodium acetate
reagent mixture . mix well and stopper the tubes with cotton or marbles.
4- Place the tubes immediately in a boiling water bath, and observe for 15
minutes. A yellow crystalline precipitate will soon appear in test tubes
containing mannose , glucose and fructose.
5- Remove these test tubes from water bath and cool them under tap water.
6- Continue heating the rest of the tubes in which no Osazone has
precipitated , for 40 minutes. Add 4 ml of water to each tube and continue
boiling for a few minutes.
7- Place few crystals di the precipitated Osazone on a microscopic slides and
slip cover glass on top.
8- Examine the Osazone crystals of all sugars tested under microscope ,
preferably with in one hour of the time of Osazone formation . compare the
shapes of the crystals for the sugars with those shown in the following
figures.
Note :- each student should form Osazone crystals with one sugar.
33
Part 3
Determination of Glucose Concentration by
Nelson’s Method
Objective:-
To determine glucose concentration , in solution , spectrophotometry.
Introduction:-
In Nelson’s method glucose first reduces cupric ion to cuprous ion. This is the
same reaction which occurs in Benedict’s Fehling’s and Barfooed’s tests for
reducing sugars. As you remember, red cuprous oxide (Cu2O) is insoluble and
precipitates out from the solution. It cannot be measured spectrophotometrically
since one of the requirements for accurate spectrophotometric determinations is
that the colored molecules should be randomly and evenly distributed in the
solvent. However cuprous ion is capable of reducing a solution of arsenomolybdic
acid to arsenomolybdous acid. This compound has blue color ( molybdene blue),
and absorbs light at a wavelength of 680 nm.
Biological fluids such as plasma are normaly deprotenized prior to glucose

determination, this is done for several reasons:-
1- Polypeptide chains may form a blue complex with the cupric ion through
the free electron pairs at the amino groups in the chain. This complex will
affect the spectrophotometric measurements since it is colored
H O R O R O R
H H
N-C-C-N-C - C- N- C-C
H H H
Cu+2
34
2- Biological fluids may contain glycoproteins which have a carbohydrate

potion capable of reducing Cu+2 to Cu+2 and forming a colored complex
resulting in erroneous readings.
3- Proteins may also cause turbidity of solution which is bound to affect the
spectrophotometric reading.
Exp. 14
Notes: * carry out all tests in duplicate.
* A blank and a series of standards must be carried through with each
Series of unknown samples.
* The stock standard solution of glucose is ready preparedand it’s
Concentration is 100 µg / ml .
1- Prepare a seriec of glucose standard solution concentrations : 20 , 40 , 60 ,

80 , 100 µg/ ml by diluting with 0.2 % benzoic acid
2- Place 0.5 ml of each glucose solution prepared above into 5 labelled test
tubes respectively. Place 0.5 ml of D.W. to tube number 6 (blank ) and
finally place 0.5 ml of unknown sample in tube number 7 .
3- Add 1 ml of the alkaline copper sulfate solution to each test tube, mix well
and cover with a marble or cotton.
4- Heat the test tubes in a boiling water bath for exactly 20 minutes.
5- Remove the tubes simultaneously and cool them for 5 minutes under
running water.
6- Add 1 ml of arsenomolybdate reagent to each tube and mix well to dissolve
the cuprous oxide ( using a vortex).
7- Dilute the content of each tube to 10 ml with D.W. by adding 7.5 ml of D.W.
35
8- Now the color is very stable. By using the spectorophotometer instrument.

Read absorbance at 680 nm for each test tube against the blank.
Notes :-
 The blank does not contain glucose, but it contains the solvent in
which glucose is dissolved and all other reagents.
 Before any measurements are taken, the instrument must be
calibrated. This is explained under the “operation of
spectrophotometer “discussed later on in this experiment.
 Read the absorbance of your samples in the order of increasing
concentration using the same tube. Rinse the tube between each
sample with the sample to be read next.
9- Prepare a standard curve, on a graph paper, by plotting absorbance (A)
versus glucose concentrations.
10- Place the absorbance of the unknown sample on this graph to find the
corresponding concentration.
Notes:-
 Your unknown sample must fall in the range from 0 to x moles / liter
to be valid [see the figure below]. If your sample gives an
absorbance greater than x moles / L than you must dilute the
solution and repeat the procedure.
 You should obtain a straight line graph. The corresponding
concentration. Sometimes the graph is not linear. If your decrease,
use only the straight line portion of the graph.
36
A 680
x
x
xl
l
l
x l
l
l
l
x l
x l
l
X Cconcentration, moles / l
How to dilute the stock standard solution ?

1- In the first step you would have to decide how much you are going to need
of each dilution. According to the procedure, you need 0.5 ml / test.
Since you are going to do duplicate, so you need 1 ml of each dilution. To
be on the safe side, prepare 5 ml of each dilution.
2- The second step is to figure out , how many micrograms (µg) of glucose you
are going to have in 5 ml if one ml contains 20 µg , 40 µg , …….. etc. based
on that informations you should count, how many milliliters of the stock
standard solution is needed since 1 ml of that standard contains 100 µg
glucose.
3- The final step is to count, how many milliliters of 0.2 % benzoic acid needs
to be added to make a total of 5 ml.
Example:- how you can prepare 40 µg / ml glucose solution?
40 µg of glucose is present in 1 ml so, the amount of glucose in 5 ml of
solution will be 40 * 5/1 = 200 µg .
But in the standard solution, the concentration is 100 µg / 1 ml, so we must
take 2 ml of the stock solution of glucose and complete the volume to 5m
by adding 3 ml of 0.2 % benzoic acid.
37
tube Final µg glucose/ 5 ml of ml of benzoic acid added

conc. ml stock to make a total of 5 ml
µg/ml 100
µg/ml
Blank ------- ------- 0 5
1 20 100 1 4
2 40 200 2 3
3 60 300 3 2
4 80 400 4 1
5 100 500 5 0
Reagents:-
 Stock standard solution of glucose:-
100 g/ml of glucose in 0.2 % benzoic acid.
 Alkaline copper sulfate reagent:-
4 ml Nelson’s solution B to 100 ml with Nelson’s solution A.
 Nelson’s solution A:-
Dissolve 25 g anhydrous sodium carbonate, Na2CO3, 25 g of
potassium sodium tartarate, 20 g sodium bicarbonate, NaHCO3, and
2oo g anhydrous sodium sulfate, Na2SO4, in 700 ml D.W. and dilute
to 1000 ml. mix well and filter if not clear.
 Nelson’s solution B:-
Dissolve 75 g copper sulfate, CuSO4 .5H2O in 500 ml of D.W. add
0.25 ml concentrated sulfuric acid, H2SO4, and mix well.
 Arsenomolybdate reagent :-
Dissolve 50 g ammonium molybdte (NH4)6Mo7O24.4H2O, in 900 ml
D.W. add 42 ml concentrated sulfuric acid while stirring. Dissolve 6
gm disodium orthoarsenate, Na2HAsO4.7H2O, in 50 ml D.W. and add
it to the above acid molybdate. Mix well and store the reagent in a
glass stopper brown bottle for 24 to 48 hours in an incubator at 37
0
C. The reagent should be yellow in color with no green tint.
38
Operation of spectronic laboratory spectrophotometer:-

1- Turn on the instrument.
2- Allow the instrument to warm up about 5 minutes.
3- Adjust the wave length knob to the desired wavelength and select the
lamp, [deuterium below 325 nm].
4- Select absorbance or transmittance range.
5- Insert a tube filled with blank (reference sample) in the instrument.
a- Always place the tubes in the instrument, so the etched line on the cell
is aligned with the mark on the edge of the cell receptacle.
b- Always set the cell firmly in the receptacle and close the cover before
taking any reading.
6- Set absorbance to zero ( or transmittance to 100% )
7- Remove the blank and insert sample and read the result from the display.
8- Repeat steps 3, 4, 5 and 6 each time the wavelength is changed.
Rules for using cuvettes and spectrophotometric tubes:-

Cuvettes when inserted in a spectrophotometer or colorimeter are an essential
part of the optical system. They should receive careful treatment. A photometer
does not differentiate between a light impediment caused by a scratched cuvette,
lint, air, bubbles, finger marks, or the optical density of the solution. Minor optical
faults in cuvettes can introduce substantial and quite unnecessary analytical
errors.
The following rules should therefore be observed:-

1- Protect the tubes or cuvettes from scratches by ;
a- Not allowing them to rub against one another or against hard surfaces.
b- Avoiding the use of brushes and abrasive cleaning agents.
2- Clean the tubes or cuvettes in cleaning solution ( cuvettes can be left in
absolute ethanol overnight ) and rinse thoroughly .
39
3- Rinse at least once with the sample solution and fill the tube or cuvette
about ¾ full with the sampl;e .
4- Wipe the lower half of the tube or cuvette free from moisture, lint and
finger marks before placing it in the instrument, using a soft cloth or soft
paper tissue. After wiping, handle the tube or cuvette by the top edge.
5- Make certain that no bubbles cling to the inner surface of the cuvette.
6- Round tubes have an index line which should be aligned with a mark on the
instrument in which they are used.
7- Find two tubes or cuvettes which give the same reading when filled with a
colored solution (= a matching pair). Use one for the blank and the other
one for all measurements. By doing this you minimize the difference
between tubes (or cuvettes).
Quantitative spectrophotometric work:-

Spectrophotometer is an instrument used for the measurement of light absorbing
compounds. You are accustomed to judging the concentration of tea by the
intensity of its color. This principle can also be used in the laboratory for
quantitative determinations of compounds. Every substance absorbs light of one
wavelength or another. Substances may or may not appear colored to the eye
depending on whether they absorb light in the visible region between 400 and
700 nanometers or in the UV range below 400 nm. Most chemicals which do not
have color can be converted completely too colored compounds by proper
chemical reaction:-
Colorless compound + color forming -------------→ color proportional to the
To be assayed reagents amount of colorless compound
The first is Lambert's low, which states that each successive unit layer of a
solution containing a colored compound absorbs an equal fraction of the light
passing through it. The other important law, Beer's law deals with concentration.
Generalized Beer's law states that the light absorption is proportional to the
40
number of molecules of absorbing substance in the solution through which the

light passes.
These two relationships are combined into Lambert's – Beer law:-
The laws of light absorption:-

When light passes through a solution some may be absorbed the two
fundamental laws describing such absorption are those of Lambert & Beer, which
gives rise to the equation:-
lE/ l0 = e - KCt
In which
lE = intensity of emerging light
l0 = intensity of incident light
K = A constant
C = Concentration of the absorbing substance
t = path length of the light passing through the solution
e = 2.718 the base of natural logarithms.
This equation indicates that when monochromatic light passes through a solution
the intensity of the light transmitted decreases exponentially
a- With increasing path length

b- With the increasing concentration of the absorbing substance.
The first of these follow from Lambert's law, the second from Beer's law.
From Lambert's law the proportion of light absorbed (lo –l E) / l0 or transmitted
(lE / l0 ) is independent of the intensity of light entering the solution ( l0 ). Thus a

constant proportion of light entering each successive layer of the solution is
absorbed; end absolute amount absorbed in each layer diminishes progressively.
41
The ratio of (lE / l0) is known as the transmission (T), transmittance or

transmittancy. Transmission * 100 is the percentage transmission (% T):-
Hence T = e -Kct
In T = -Kct
- Log T = Kct where (K) is another constant.
The quantity (- log T) or log (l/ T) is termed the extinction (E), optical density or
absorbance.
Thus E = log (l /T) = log 100 / T%
So that E = 2 – log (T %)
Several terms for the extinction measured under defined conditions are
recognized. The extinction coefficient is the extinction a path length of ( 1 cm ),
and the specific extinction coefficient, E ( l% / l cm ) or abs or absorptivity index is
that at a path length of ( 1 cm ) & a concentration of 1 % ( 10 g./L). The molecular
coefficient (E) refers a path length of (1 cm) & a concentration of (1 mol /L).
Transmission Extinction % Transmission % Extinction %

%
99 0.44 49 30.98
98 0.88 48 31.88
97 1.32 47 32.79
96 1.77 46 33.72
95 2.23 45 34.68
94 2.69 44 35.65
93 3.15 43 36.65
92 3.62 42 37.68
91 4.1 41 38.72
90 4.58 40 39.79
89 5.06 39 40.89
88 5.55 38 42.02
87 6.05 37 43.18
86 6.55 36 44.37
42
85 7.06 35 45.59
84 7.57 34 46.85
83 8.098 33 48.15
82 8.62 32 49.49
81 9.15 31 50.86
80 9.69 30 52.29
79 10.24 29 53.76
78 10.79 28 55.28
77 11.35 27 56.86
76 11.92 26 58.5
75 12.40 25 60.12
74 13.08 24 61.98
73 13.67 23 63.83
72 14.27 22 65.76
71 14.87 21 67.78
70 15.49 20 69.9
69 16.12 19 72.12
68 16.75 18 74.47
67 17.39 17 79.96
66 18.05 16 79.59
65 18.71 15 82.39
64 19.38 14 83.39
63 20.07 13 88.61
62 20.76 12 92.08
61 21.47 11 95.86
60 22.18 10 104.58
58 23.66 8 109.69
57 24.01 7 115.49
56 25.18 6 122.18
55 25.96 5 130.1
54 26.76 4 139.79
53 27.59 3 152.29
52 28.4 2 169.9
51 29.24 1 200.0
50 30.1
43
In practice the instrument is usually set for 100 percent, transmission or

extinction with water or a blank in position. The test solution is than introduced
Since E= - log (T) E = Kct.
So if the thickness remains the same E is proportional to the concentration, as a

result, provided Beer's law is obeyed. When extinction is plotted against
concentration a straight line is obtained. On the other hand, if percent,
transmission is read, as it is in some instruments, and is plotted against
concentration.
Extinction % transmission
1.0 100
0.8 80
0.6 60
0.4 40
0.2 20
Concentration Concentration
Graphs showing the relationship between concentration & extinction, and

between concentration & % transmission.
For transmission we do not get a straight line but a curve as shown in fig. to
convert this into a straight, semi logarithmic paper must be used with the
concentration axis liner & the percent transmission logarithmic.
So we see that if a suitable standard is prepared & the extinctions of this & the
unknown solution are read, we have
Conc. Of unknown extinction of unknown
----------------------------- = ---------------------------------------
Conc. Of standard extinction of standard

44
So concentration of unknown
Extinction of unknown
------------------------------------- x concentration of standard
Extinction of standard
Throughout this book this has been expressed:-
Reading of unknown
Concentration of unknown = ----------------------------- x concentration of standard
Reading of standard
Since this formula only holds when beer's law is obeyed, this should be verified by
preparing a standard curve for the range of concentrations covered by the
determination.
Satisfactory results are only obtained with extinction rang from (0.2- 0.7).
Exp. 15
((Identification of unknown carbohydrates))
Making use of the tests for carbohydrates outlined in experiments 1 & 2, design
your own scheme for the identification of a sample suspected of containing
carbohydrates. Perform the Molisch's test to be certain that the substance is a
carbohydrate. Then your scheme may include tests for the different classes of
carbohydrates [ e.g .mono, di and polysaccharides] . the presence of class of
carbohydrates does not rule out the presence of other classes.
45
Following the scheme for the identification of samples suspected of containing

carbohydrate and names the unknown carbohydrate.
1- Molisch's test must be done firstly before you start application of scheme
to be sure that you have carbohydrate solution.
2- Pipet 5 ml of unknown into dry and clean test tube and apply all test
according to the scheme.
46
starch, sucrose, galactose, glucose, fructose, pentoses, lactose, maltose
2 Benedict's test
(-) non reducing (+) reducing
clear blue brick red Cu2O

starch , sucrose galactose,glucose,fructose,pentoses,lactose,maltose
3 iodine test
(+) blue (-) colorless
(+)brick red Cu2O (-) reaction(5 min)
starch sucrose
lactose,maltose
galactose,glucose,fructose,pentoses
(-) (+)
5 Bial's test
no gas CO2 gas
(-)brown (+) blue
lactose
maltose
galactose, glucose, fructose pentoses
6 Seliwanoff's test
(-)no color (1 min) (+) red color (1 min)
galactose, glucose fructose
galactose glucose
scheme for identification of more important carbohydrates, also

each compound can be identify by osazone test
47
Part (4)
EXP 16
Studies on some amino acids ionic properties
All amino acid contain ionizable groups that act as weak acids or bases, giving off
or taking on protons when PH is altered :-
H3N+-CH2-COO- + H+ ↔ H3N+- CH2-COOH
H3N+-CH2-COO- + OH- ↔ H2N-CH2-COO- + H2O
Unprotonated from (base)
PH = PKa + log ----------------------------------------
Protonated from (acid)
The PKa is the negative log of the ionization constant of the ionizable group.
Examination of this equation leads to a further understanding of the term PKa.
This useful equation is applied in the titration of glycine with acid and base.
Glycine has two ionizable groups; a carboxyl group and an amino group,with PKa
values of 2.4 and 9.6 respectively. In water, at PH 6 glycine exists as adipolar ion ,
or zwitterions, in which the carboxyl group is unprotonated (-COO- ) and the
portonated ti give the substituted ammonium ion (-NH3+). (verify this using the
Handerson – Hasselbalch equation,PH6, and the respective PKa values).
Addition of acid to the solution will lower the PH rapidly at first and then more
slowly as the buffering action of the carboxyl is exerted.
Titration of amino acid with H2SO4

1- Measure 20 ml of a neutral (moncamino, monocarboxylic) amino acid such
as glycine.
48
2- Add 20 ml D.W. using a burette and PH – meter ( or universal indicator

paper or solution ).
3- Titrate the amino acid with 2NH2SO4, two drops at time for the first ten
drops, and then 4 drops at a time until the solution reaches PH1.0.
4- After the the addition of each increment of acid, mix the solution, and
record the PH (from burette) of acid consumed.
Titration of amino acid with NaOH

1- Measure a second 20 ml of the same amino acid, and then add 20 ml D.W.
2- Titrate with 2N NaOH in the same manner as for the acid titration until the
solution reaches PH 12.
Results:-
To determine the true titration curve of any substance. It is necessary to
determine how much acid or base is consumed in titrating the solvent (water) to
each PH and then subtract this amount from the total amount of acid or base
consumed in reaching that PH. The following example with the acid side the
titration of an amino acid illustrates one of several available methods of
correcting for such acid or base dilution.
1- For both the sample and the water blank, plot the volume of the acid added
versus the PH reached.
2- From the graph or the original data, prepare a table, recording the amount of
acid required to reach PH. Then substract the volume of acid required to bring
the water blank to any given PH from the volume of acid, required to bring the
sample to the same PH. This difference represents the mount of acid
consumed in the titration of the sample.
3- Using the data from your table, plot the PH versus the number of equivalents
of acid needed to titrate the amino acid sample to a given PH.
Similar method of correcting for dilution can also be applied to the base side of
the curve. Prepare complete corrected titration curve for the amino acid tested.
The number of equivalents of acid or base consumed in passing through one full
49
inflaction of a curve ( in the zone of a single ionization represents the quantity of

acid required to titration one ionizable group in the quantity of amino acid you
have assayed. Using this relationship and nothing the number of full inflections
on your curve, calculate an equivalent weight for your amino acid. Compare this
with the known molleculer weight of the compound further, compare the
observed PKa values for the amino acid in question.
1.0
0.8
0.6 x PK2
Base
0.4
Equivalents loaded
0.2
0.0
x
0.2 PI
Acid
0.4
0.6 x
PK1
0.8
A
1.0
2 4 6 8 10 12
PH
Titration curve of monoamino monocarboxylic acid such as glycine with PK1= 2.3
and PK2= 9.6 . in acid solution A the substance present is glycine hydrochloride,in
B it is sodium glycine. The isoelectric point P1= 6 .
50
EXP ( 17 )
Color Reaction of Proteins
Objective:-
To identify the different proteins and amino acid by color reaction tests.
Introduction:-
The chemistry of living things involves of large and complex molecules. It is based
on the chemistry of the carbon atom and the fact that carbon atoms can be
connected to form long chains or rings. This results in a vast array of molecules.
The structure of each molecule is related to its function. Changes in the structure
may result in abnormal function which we call disease. Some of the most
common types of organic molecules found in living things are proteins,
carbohydrates, lipids and nucleic acids.
Proteins are polyamides and have molecular weights above 5000. Polyamides of
molecular weight below 5000 are usually referred to as polypeptides. Amino acids
are the building blocks of proteins. An amino acid consists of an amino group, a
carboxyl group, a hydrogen atom, and a distinctive R group bonded to a carbon
atom. Color producing tests such as the Biuret, Ninhydrin, Millon's, Hopkins –
Cole, and unoxidized sulfur tests are used to detect proteins biological mixtures.
Some of these reactions [Biuret and Ninhydrin] are general tests that give positive
results with all proteins and amino acids.
1- Biuret test:-
The Biuret test depends upon the reaction of cupric ions [ Cu+2] in an
alkaline solution with peptide linkages of the protein to produce a purple
color. This color is atoms of two peptide chains as shown below:-
51
O=C
C=O
NH HN H
H
C C
R R
Cu+2
O=C C=O
NH HN
C C
A Biuret compound is formed when urea heated. If alkaline cupric ions are added
to biuret solution, a violet color is produced. This is a characteristic color of not
only biuret but also proteins and peptides which contain a structure similar to
biuret. The biuret test requires the presence of at least two peptide linkages per
molecule to be positive. The biuret reaction can be said to be polypeptide –
specific, since proteins are about the only compounds found in. nature that have
the poly peptide character required for biuret assay, therefore, the biuret test is
remarkably specific for proteins.
O
C
H2N NH2 NH3 + H2N-CO -NH-CO - NH2
Urea biuret
1– into three separate test tubes, add 2 ml of the following solution: 2% agg
albumin, 1% gelatin and 0.5 % alanine one solution per tube.
2- into a fourth test tube add 20 mg of powdered casein.
3 – add 2 ml of 10 % NaOH into each of the four tubes and mix the
contents.
4 – add 5 to 10 drops of 0.5 % CuSO4 into each tube.
52
5 – report your observations.
2- Ninhydrin test:-
Ninhydrine reacts with α- amino acids to yield a characteristic blue violet
products [ decarboxylation ]. The overall reaction is shown below, for the
sequence of steps involved:
O
O O
H3C H CH3
OH
2
OH
+ NH2 C N C
+ O + CO2 + 3H2O
HO
O O O
O
Ninhydrine amino acid blue- colored adduct acetaldehyde
Most amino acids give almost the same color, except proline makes a pale- yellow
product with ninhydrine. This is due to the fact that proline is an amino acid
instead of having traditional α- amino acid structure.
1- Into three clean test tubes pipet 5 ml of each the following solutions: 2%
egg albumin, 0.5% glycine and water < one solution per tube.
2- Add 1 ml of 0.1 % ninhydrine solution.
3- Mix the contents of each tube and heat in a boiling water bath.
4- Record your observation with each solution.
3&4- Millon’s and Hopkins – Cole tests:-

These tests are actually specific for certain functional groups of amino- acids and
not for the amino acids themselves. Millon’s test is specific for the phenolic
hydroxyl group of tyrosine and the Hopkin’s – Cole test is specific for the indole
53
group of tryptophan. A colored comlex is formed between tryptophan and

aldehyde group of glyoxylic acid.
O O
OH OH
NH2 NH2
HO NH
Tyrosine (Tyr) Tryptophan (Trp)
Amino acid are not the only chemical components in the sample, which contain
the particular functional group which gives the positive reaction. Millon’s test will
be conducted with one compound which is not an amino acid nor a protein. This
compounds are tested with Millon’s reagent. Red color is produced due to a
mercury salt of nitrated tyrosine.
O O COOH
H3C OH
OH OH
NH2 NH2
Alanine (Ala) Phenylalanine (Phe)

Salicylic acid
Test the solutions lsted below:
2% egg albumin, 0.5% tyrosine, 0.5 % alanine, 0.5% salicylic acid.
1- Into four clean test tubes pipet 4 ml of each solution per tube.
2- Add 5 to 10 drops of frech Millon’s reagent into each tube.
54
3- Mix the contents of each tube and place them in a boiling water bath.
4- Note any changes in the test tubes, and record your observations.
1- Add 2 ml of 2% ovalbumin, 1% tryptophan , 0.5 % tyrosine solutions in
separate test tubes. Add 3 ml of Hopkins- cole reagent to each tube and
mix. [ sol. A]
2- Add 3 ml of conc.H2SO4 into a separate test tube [ sol. B].
3- Very carefully and cautiously pour solution B down the side of solution A.
Note:- work so carfully that conc.H2SO4 and solution Ado not become
mixed.
4- Note any color that develops at the interface of the two liquids and record
your observations.
5 – Unoxidized sulfur test:-

This test is positive only in the presence of amino acids containing sulfhydryl
[ SH ] or disulfide group [- S- S-]. Methionine is the third amino acid which
contains sulfur but not in the form of SH or S-S groups.
O
O O
HS OH S
HS OH NH2 H3C OH
NH2 NH2
O
HS OH
NH2
Cysteine (Cys) cystine Methionine (Met)
When proteins boiled in strong alkali, the -SH and –SS groups are converted to inorganic
sulfide. If we add lead acetate solution a black precipitate of lead sulfide [ PbS] is form D:-
55
O
NaOH Pb(CH3COOH)2
HS OH inorganic sulfide PbS
0
NH2 100 C
Black ppt.
1- Add 2 ml of each of the following solutions[ 2% ovalbumin, 0.5 % ysteine,
0.5 % methionine] into three separate test tubes.
2- Add 5 ml of 10% NaOH and 2 drops of 5% lead acetate, Pb(CH3COO)2, into
all tubes.
3- Mix the content of each tube and place them in a boiling water bath for 10
minutes.
4- Record your observations.
Qualitative color tests of proteins are summarized in the following table:-
Name of Reagent used Specific for Sample material

test
1 Biuret Alkaline CuSO4 in Poly Egg albumin,
sod.pot.tartrate solution. piptide gelatin,alanine, casein
2 Ninhyderin Ninhydrine solution Amino acid Egg albumin,
glycine,aspartic acid.
3 Millon’s Mercuric & mercurous Phenolic Egg albumin,
nitrates in HNO3&HNO2 hydroxyl tyrosine,alanine,ph.alani
group of ne, salicylic acid.
tyrosine
4 Hopkins- Glyoxylic acid in H2SO4. Indole Egg albumin.
cole group of
tryptopha
n.
5 unoxidized Strong NaOH & acetate. SH- & SS- Egg albumin, cystine,
groups in methionine.
56
cystine &
cysteine
respectivel
y.
6 sakaguchi Alkaline- naphthol & Guanidine Egg albumin, arginine.
hypochlorite group of
argininr.
7 Nitroprussi Dil.ammoniacal Free Egg albumin, cysteine
de Na2(NO)Fe(CN)5 sulphhdril
8 Ehrlich P- indol Egg albumin, tryptophan
dimethyaminobenzaldeh
yde in H2SO4
9 Pauly Alkaline diazotized Histidine Egg albumin,histidine ,
sulphanilic acid &tyrosine tyrosine
Exp.t (18 )
Precipitation of proteins
Introduction:-
Protein in solution show profound changes in solubility as a function of four
parameters.
1- Salts of heavy metals.

2- PH
3- Ionic strength.
4- The dielectric constant of the solvent.
Each of the four parameter will be tested by adding salts of heavy metals, acids,
neutral salts, or organic solvents such as ethanol into protein solutions.
Figure ( 1) illustrates how the solubility of most globular proteins changes. When
PH of the protein solution is changed.
57
Solubility
4.8 5.2 5.6 PH

Figure (1):the effect of PH on the solubility of B- lactoglobulin at 25 0C
The solubility is at minimum around PH 5.3 on either side of PH 5.3 the solubility
rises very sharply. The PH at which it occurs varies slightly from one protein to
another. And is near to the iso electric point. The ability of neutral salts to
influence the solubility of proteins is related to the ionic strength of the salt
solution. The ionic strength of a solution, µ, is given by equation:-
µ = ∑ Ci Z2 i
Where c is the concentration and z is the electric charge of each ion contributed
by the salt. The sign sigma means that ionic strength is the sum of the
concentration multiplied by the charge of each ion present in the solution . as the
equation shows, ionic strength is directly proportional to the electric charge to
the second power. This means that salts of divalent ions such as (NH4)2SO4 are
more effective at increasing the ionic strength than salts of monovalent ions such
as NaCI or NH4CI.
The effect of salt in increasing the solubility of protein is called” salting in “. The
solubility is a function of the ionic strength, which is calculated from the molar
concentration of the ions and their charge
Where µ is the ionic strength, m the molarities and the charge of the ion. the
summation sign ∑ denotes that ( mZ2) terms are added for each of the ions.
58
e.g. :- 0.1 M solution of NaCI
The ionic strength µ = ½ ( 0.1 x l2 + 0.1 x l2 )
= 0.1
Thus for a salt of univalent ions the ionic strength is equal to the molarity.
For 0.1 N solution of Na2SO4
µ = ½( 0.2 x l2 + 0.1 x22 ) = 0.3
A second variable is added:- the effect of ionic strength is tested simultaneously

with PH by adding NaCI (neutral salt)and repeating the test at different PH values.
Figure (2) shows that the protein least soluble at 5.3 regardless of the
concentration of NaCI present.
Solubility
20 mM
10 mM
5mM
4.8 5.2 5.6 PH

Figure (2): the effect of PH and salt concentration on the solubility of protein at
25 0C .
Ions of neutral salts have the ability to increase the solubility of many globular
proteins but only in very low concentration. This phenomenon is called salting- in.
59
By adding more salt the ionic strength is increased further and the solubility of a
protein begins to decrease as shown in figure (3)
solubility
conc. of salt
Figure(3): the effect of a neutral salt on the solubility of protein.
The high concentration of salt removes water of hydration surrounding the

charged protein molecules. This causes the protein to precipitate. A protein can
be almost completely precipitated from the solution, this is known as salting- out.
The PH at which a protein is least soluble is called isoelectric PH of that protein[ in

figure 1 I PH=5.3]. at the isoelectric PH the protein molecule has no net electric
charge, therefore there is no electrostatic repulsion between neighbouring-
protein moluecules and they tend to precipitate out.
At PH values above and below the isoelectric point all protein molecules will have
a net charge. When the solution is more acidic the net charge in all protein
molecules will be positive.above the isoelectric PH the net charge in all protein
molecules will be negative.
60
1- Precipitation of proteins by salts of heavy metals:-

A positive molecule will repel anther with the same charge. Similarly, a negatively
charged molecule will repel other negative molecules and no precipitation will
occur on either side of the isoelectric PH.
R-CHNH2-COO- + Ag+ --------→ R-CHNH2- COOAg ↓
Protein silver proteinate
Heavy metal ions [Pd+2, Hg+2, Ag+ , etc ] are very dangerous if swallowed because
they inactivate certain key enzymes which all are protein by nature. Egg white and
milk contain protein which form insoluble salts with heavy metals converting the
toxic metal to nontoxic to human body. If a metallic poison has been ingested
accidentally, it is rendered nontoxic when it is precipitated in the form of a
proteinate and vomiting is induced.
Salts of heavy metals such as AgNO3 and HgCI2 are used as germicides because
they have the ability to disrupt disulfide linkages thus causing denaturation of
protein by definition , a germicide is an agent that destroys microorganisms,
especially pathogenic organisms. The term is associated with the death of all
diseased microorganisms but it does not necessarily include the capacity of
destroying bacterial spores.
a- Separate egg yolk from egg white. Egg white contains 11% solids of which
10 % is made up of numerous globular protein , mainly ovalbumin.
b- Add about 300 ml of distilled water to 30 g of egg white.
c- Swirl the beaker carefully but do not shake vigorously because ovalbumin is
readily denatured and coagulated by exposure to new surfaces upon
shaking.
61
d- Filter through filter paper, use this protein solution in the following four
experiments.
e- Prepare four test tubes as indicated in the table below:-
1 2 3 4
1% ovalbumin, ml 7 7 7 7
5% Pb(CH3COO)2 5-10 drops - - -
5% HgCI2 - 5-10 drops - -
5% AgNO3 - - 5-10 drops -
D. W - - - 5-10 drops
0.05 N NaOH 5-10 drops 5-10 drops 5-10 drops 5-10 drops
Make sure that you add an equal number of drops D.W. to tube 4 as reagents to
other tubes in order to keep the volume constant in each tube.
2- Precipitation of protein by alkaloidal reagents:-

Plants produce nitrogen – containing substances which are moderately basic and
are therefore called alkaloids ( alkali – like). These alkaloids are most often
heterocyclic rings. Some familiar examples include caffeine , nicotine , and
cocaine. Tannic acid and picric acid are called alkaloidal reagents since they are
used in precipitating alkaloids. Tannic acid is a very complex acid. The commercial
tannic acid has a formula C76H52O46. Picric acid is 2,4,6, trinitrophenyl.
Tannic and picric acids are used in solution for burns treatment because they are
safe. They prevent the growth of microorganisms by precipitating bacterial
protein as shown below. When PH of a protein solution is below the isoelectric
point, the net charge of the protein is positive. The protein will now act as a base
and can form salts with added acids:-
62
H H
-
R C COOH + Picrat R C COOH
NH3+ NH3- picrate
Protein Protein picrate
1- Place 5 ml of 1% ovalbumin solution into two test tubes.
2- Acidify the contain of bath tubes by adding 1 ml of 0.5 N acetic acid.
3- Into tube 1 add 2ml of saturated picric acid solution.
4- Into tube 2 add 2 ml of 10 % tannic acid solution.
3- Precipitation of protein by ethanol:-
The addition of organic solvents which are miscible to water decreases the
solubility of most globular proteins in water to such as extent that they
precipitate out of solution. By adding ethanol the dielectric constant than water [
25 and 78 respectively ]. Its addition to an aqueous protein solution increases the
attractive fprce between opposite charges, thus decreasing the degree of
ionization of the R- groups of the protein. As a result, the protein molecules tend
to aggregate and precipitate.
e
1 . e2
F= ---------------
D.r2
F= the attractive force between two ions of opposite charge, e1 and e2 are the
charges of the ions and r is the distance between the ions.
D = the dielectric constant.
63
From the equation above one can conclude that the bigger the dielectric
constant, the smaller is the attractive force between two ions of opposite charge.
Alcohol are used as disinfectants and cleansers. By definition ,a disinfectant, is an
agent that destroys harmful micro – organisms or inactivates viruses. Alcohols
denature proteins in bacteria thereby decreasing the number of viable bacteria
remaining on the skin without damaging the skin itself.
1- Make two dilutions of 95% ethanol as shown below:-
A B
95% ethanol , ml 1 1
D.W. ml 1 4
mix mix
Ethanol conc. 84% 19%
2- Into three separate test tubes pipet 1 ml of 95% , 48% , and 19% ethanol.
3- Into all three tubes add 4 drops of the fresh ovalbumin solution prepared in
the experiment.
4- Add 5 ml of D.W. into those tubes in which a precipitate had formed.
Report what effect addition of water had on the precipitate.
4- Precipitation of proteins by high concentrations of neutral salts:-

When high concentration of inorganic salts present in protein solution, the
solubility of the protein is reduced leading to its precipitation. This happens due
to the ability of the ions of salts to become hydrated ( to bind water) and
therefore to compete with the protein molecules for water.
64
Note:- work with 1 % solution of casein and ovalbumin prepared from fresh milk
and egg white respectively.
1- The ovalbumin solution which was prepared in this experiment needs to be

diluted first with D.W. in the ratio 1: 4 .
2- Preparation of casein :- into fresh skim milk add slowly 0.5 N HCI until PH
reaches 4.8 use a magntic stirrer and a PH meter. Let the precipitate settle
down for a while. Filter it through filter paper. The precipitate remaining on
the filter paper is casein. Assume that it contains 60 % water. Weigh 5 gm
of the precipitate and dissolve it into 200 ml of 0.1 % NaOH in order to
obtain 1% casein solution. If the solution remains very turbid , filter it
through filter paper.
3- For both protein solution prepare three tubes as follows:-
A B C
Protein solution , ml 5 5 5
D.W. ml 5 - -
Saturated (NH4)2SO4 , ml - 5 5
Solid (NH4)2SO4 - - Till 100 % saturation
5-Determination of optimum conditions for precipitation of proteins:-
Note:- work with 1% ovalbumin solution prepared from fresh egg white as
explained in the experiment.
1- Dilute the ovalbumin solution with D.W in the ratio of 1:4 [ e.g. 5ml of
1% ovalbumin solution + 20 ml water].
2- Divide the diluted protein solution into seven test tubes , 2ml into each
tube.
3- Add the following solutions :-
65
Tube Reagent
1 1 drop of 0.05 N NaOH
2 1 drop of 0.05 N HCI
3 3 drops of 0.05 N HCI
4 1 drop of 0.05 N CH3COOH
5 1 drop of 0.05 N CH3COOH +3 drops of 10% NaCI
6 Nothing
7 3 drops of 10 % NaCI
4- Place all seven tubes in a beaker of warm water and heat slowly under
constant observation. You may be able to observe precipitation in some
of the tubes even before exposing them to heat.
Exp. No (19)
Proteins solubility and paper chromatography
Objectives:-
1- To test the solubility of different proteins in different solvents.
2- To separate and identify amino acids by paper chromatography technique.
Introduction:-
Proteins are polyamides and have molecular weight above 5,000. Those
polyamides of molecular weight below 5,000 are usually referred to as
polypeptides. A mino acids are the building blocks of proteins.
1- Solubility of proteins:-
Albumins such as egg white albumin are soluble in distilled water. Ovalbumin
is classified as a conjugated protein , a phosphoglycoprotein, since phosphate
and carbohydrates molecules are attached to the polypeptide chain. Simple
66
proteins contain only amino acids. Albumins are coagulated by heat. Complete
and irreversible denaturation of ovalbumin is induced by heating to 560C .
some other proteins are not coagulated by heat such as casein of milk. Casein
is only weekly soluble in water but it is soluble in aqueous solutions of
alkalies.derived proteins are defined as molecules derived from a physical or
chemical treatment of a native protein such as gelatin. Gelatin originates from
collagen which is the fibrous protein in higher animals. The collagen of
connective tissues is the most abundant of all proteins in higher animals. The
collagen of connective tissues is the most abundant of all proteins in higher
vertebrates making up one third of the total body protein, and noting that
tendons and bones are held together by connective tissues. In boiling water,
collagen is solubilized , it’s structure unfolds resulting in a mixture of individual
soluble protein strands. Therefore boiling in water converts collagen into
gelatin which is a hertrogenous mixture of polypeptides. Gelatin is soluble in
hot water and exist in colloidal suspenension and a semisolid known as gel
when cooled.
Each individual protein under normal conditions of temperature and PH will

have a specific shape [ i.e. native conformation]. If this native conformation of
a protein is altered or destroyed the protein loses its ability to function
properly.
Proteins fall into two categories; fibrous proteins and globular proteins. In –
fibrous proteins many long polypeptide strands are interwind with each other
while globular proteins are individual polypeptide strands which bend and turn
to form an overall spherical type of structure.
The kinds of amino acids which are present in fibrous protein are different from
those in globular proteins. Fibrous proteins are (predominantly ) from amino acids
possessing small and non polar R groups. Therefore they have much hydrophobic
character and are insoluble in water. Globular proteins are formed predominantly
from amino acids having polar ionic R groups. Because of their hydrophilic
character, globular proteins are soluble in water.
67
The structure of proteins molecules are primary, secondary, and tertiary. The
primary structure is the actual sequence of amino acids [ e.g : gly- ser-asp-phe-
ala]. The alphahelix is the prominent secondary structure in proteins. The
tertiay structure applies to the globular protein molecules which bend in
definite ways due to attractive forces between various R groups. These forces
are :-
1- Ionic bonds between the different R group of a aspartic acid and lysine.
2- Hydrogen bonds between polar R groups such as serine and threonine ( OH
group).
3- Disulfide links can be formed between cysteine residues.
4- Hydrophobic R group are attracted towards, each other, away from an-
aquaeous environment.
68
Test the solubility of the three proteins [ albumin, casein, and gelatin] in
cold water, in hot water and in 0.1 %NaOH.
1-Place a small amount of each protein into each tube.
2-heat the tubes in a water bath and record which of the four proteins
were soluble in hot water.
3-repeat the test using cold 0.1 NaOH as a solvent.
Note:-
do not mix ovalbumin solution with a vortex mixer because this protein is
readily denatured and coagulated by exposure to new surfaces which
happens during mixing . after heating ,let the tube containing ovalbumin in
water to cool to room temperature.
2- Acid hydrolysis of proteins:-
Proteins can be broken down stepwise by hydrolysis using either acid, alkai,
or enzymes. Cleavage of proteins by acid hydrolysis is the most widely used
method. A solution of L- amino acids is obtained by complete hydrolysis of
protein which requires 20% HCI and boiling for up 48 hrs. tryptophan is
susceptible to decomposition by mineral acids and is lost diring acid
hydrolysis of proteins.
1- Weigh about 100 mg of a protein into an ampule.
2- Add 2 ml of conc. H2SO4 (8N).
3- Seal the ampule in a flame . place this ampule inside a beaker in an oven
and keep it at 100 0C for 20 hours.
4- Allow the ampoule to cool at room temp. and open it using a file.
5- Neutralize the contents with barium hydroxide by adding crystalline
Ba(OH)2 until PH is 3-4 then saturated solution Ba(OH)2 to reach PH 7 (
use indicator – paper to determine the PH)
6- The white ppt of BaSO4 is removed by centrifugation.
7- Obtain about 10 ml of clear yellowish supernatant.
69
3- Paper chromatography of amino acids:

Paper chromatography is relatively rapid qualitative analysis of mixtures of
amino acids. Paper chromatography is a type of liquid partition chromatography
in which amino acids are portioned between the water ( the stationary phase)
which is tightly bound within the cellulose fibers of paper, and as eluting organic
solvent ( the mobile phase) migration upward across the paper by capillary
cations. The individual amino acids move with the solvent at varying rates
depending on the partition coefficient of each amino acids between the water
phase and the organic solvent (the mobile phase).
Those amino acids having the highest solubility in the organic solvent
relative to their solubility in water will have the greatest mobility and will migrate
upward on the paper most rapidly. The PH – may also be important in a particular
separation. Many solvent systems contain acetic acid or ammonia to create a
strongly acidic of basic environment.
Some amino acids will separate into individual steps while others will
remain unresolved in overlapping spots. The two dimensional paper
chromatography is standard practice to overcome the problems of overlapping.
When the two dimensional procedure is used, the paper is first spotted with the
sample and developed with solvent 1, it is removed from the developing chamber
and allowed to dry , the paper is then turned 90 degrees in orientation to the
original direction of solvent flow and developed with solvent 2, which will
separate the amino acids that did not separate with the first solvent. The end
result is that all amino acids in the sample will be resolved by the two dimensional
paper chromatography.
Notice that only one sample can be run at the same time when the two
dimensional paper chromatography is used. It is desirable to run samples of
known amino acids simultaneously with the unknown sample. When standard
amino acids are run on the same paper, the unknown spots can be identified
70
reliably, much more reliably than by comparing the obtained experimental Rf [rate
of flow]values with the standard Rf value.
There is always some variation in the quality and type of paper used , in the
purity and exact composition of the solvent, also the temperature may vary.
Therefore the best practice is to obtain Rf values for standard amino acids
simultaneously with the unknown under identical experimental conditions on the
same paper.
supplies needed:-
1- Ovalbumin: use fresh egg white since 54% of all protein in egg white is
ovalbumin.
2- Casein : use fresh skimmed milk; add 1N HCI until PH of milk is 4.6
casein precipitates out. Filter through whatman no 1 . the ppt which
remains on the filter paper is casein.
3- Commercial gelatin.
4- 0.1% NaOH.
5- Conc.H2SO4.
6- Ba(OH)2.8H2O
7- Standard amino acids:- phenyl alanine, alanine, aspartic acid, serine,
tryptophan.
8- The detection reagent :0.5% solution of ninhydrine in 95% ethnol or
acetone.
9- The solvent system:- 1- butanol : glacial acetic acid : water [ 4:1: 5].
Shake this mixture in a separatory funnel. The upper layer is butanol
saturated with water. The upper layer is used as solvent.
1- Pour the solvent into the developing chamber which is lined with filter
paper so that the atmosphere is maintained saturated with the solvent
vapors, cover the beaker.[ make sure that the lining becomes totally
welled with the solvent].
71
2- Draw a pencil line along the side 1.5 cm from the edge.
3- Place eight or nine light dots along this line about 2 cm apart. Label each
of the dots. [ keep your fingers off the paper except along the edges
because finger prints contain significant amount of aminoacds].
4- Place spots of amino acids and of the hydrolysate on the paper [ or a
mixture of amino acids]
Notes:-
1- Be sure to use separate capillary tubes for each standard amino acid to
avoid contamination.
2- In spotting a small drops (2-3 mm) in diameter is placed on the paper and
are allowed to dry.
5- Repeat this several times ( 5 – 10 ). The spot should be allowed to dry
between each additional application of sample.
6- Coil the paper into a cylinder and fasten it with staples and place it in
the developing chamber. The cylinder should not touch the sides of the
beaker.
Note:-
the level of solution in the beaker should be below the spots of the paper.
7- When the solvent front has migrated up close to the upper edge of the
paper remove the paper from the developing chamber and put a light
pencil mark to indicate the position of the solvent front ( developing
time is 50 min).
8- Let the paper cylinder ti dry in the hood (you can speed up drying using
the hot air blower).
Notes:-
1- Spraying with ninhydrine should be done in a hood behind an appropriate
shield.
72
2- Be cautions to neither breathe the aerosol and must not allow contact with
your skin. If ninhydrine does come in contact with the skin wash the
affected area at once with soap and warm water.
9- After spraying with ninhydrine , the paper should be dried the oven at
110 0C because heat is ne4cessary to the color forming reaction.
10- Mark the center of each spot.
11- Calculate the Rf ( rate of flow) values for each known amino acids and
each of the spots resolved in the hydrolysate.
Distance traveled by sample in cm

Rf = --------------------------------------------------------
Distance traveled by solvent in cm
12- Identify the amino acids of the hydrolysate (mivture) as far as

possible with the Rf value of standard amino acids
Paper chromatography of amino acids; Rf values various solvents

Solvent systems
Amino acid 1 2 3 4 5 6
Alnine 0.58 0.30 0.11 0.09 0.45 0.22
Arginine 0.56 0.138 0.03 005 0.22 0.07
Asparagines 0.44 0.172 0.02 - - -
Aspartic acid 0.18 0.106 0.03 0.10 0.27 0.09
Cystine - - - 0.01 0.04 0.11
Glutamic acid 0.31 0.12 0.05 0.01 0.37 0.11
Glutamine 0.60 0.21 0.04 - - -
Glycine 0.39 0.22 0.06 0.05 0.32 0.19
Histidine 0.64 0.25 0.03 0.09 0.22 0.22
Isoleucine 0.84 0.55 0.36 0.40 0.62 0.47
Leucine 0.84 0.55 0.34 0.46 0.64 0.52
Lysine 0.48 0.08 0.02 0.02 0.11 0.16
Methionine - - - 0.05 0.48 0.40
Phenylalanine 0.84 0.58 0.29 0.38 0.55 0.58
73
Proline 0.86 0.31 0.14 - 0.47 0.29

Serine 0.36 0.23 0.05 0.02 0.32 0.29
Threonine 0.49 030 0.09 0.04 0.45 0.51
Tryptophan 0.80 0.66 0.22 0.30 0.35 0.41
Tyrosine 0.67 0.64 0.16 0.19 0.41 0.34
valine 0.78 0.44 0.24 0.15 0.63 0.37
Solvent system:-
1- Phenol saturared with water ( PH 5.0- 5.5 )
2- Collidine – lutidine ( 1:3 ) saturated with water
3- 1- butanol – acetic acid ( 9 :1) saturated with water
4- 1 – butanol saturated with 2N NH4OH
5- Methanol – water – pyridine ( 80 :20 :4)
6- 1 – butanol - methylethyl ketone – water diethylamine ( 40 – 40 – 20 – 4 )
Even if the spots you obtain from the casein hydrolysate would not be distinct
clear after doing the paper chromatography you should be able to detect one of
the disadvantages of acid hydrolysis ; the spot corresponding to tryptophan is
missing which means that tryptophan was decomposed during acid hydrolysis.
74
Exp. 20
Determination of proteins
Objectives:-
1- To prepare standard curves using different methods for protein
determination.
2- To determine the concentration of unknown sample protein.
Introduction:-
The level of total plasma protein in healthy adults is 6.0 to 8.2 gm/ 100 ml , of
which about 55.2% is albumin and the rest is the different globulins. The total
amount of plasma proteins tend to increase in dehydration that causes
hemoconcentration. It is also increased in cases of abnormal proteins formation
in the plasma ;as in the Bence-Jones proteins, and the abnormal macroglobulins.
Plasma proteins may be decreased as a result of dietary deficiencies, after
hemorrhage, and in some kidney diseases due to the loss of albumin. It is also
decreased in some liver diseases that leads to decrease albumin synthesis by the
liver.
1- Biuret Method:-
Substances that contain two or more peptide bounds give a blue violet
color with alkaline copper sulfate solution. The intensity of the color
obtained is a measure of the number of peptide bounds present in the
protein sample. The reaction is not absolutely specific for peptide bounds,
since any compound containing two carbonyl groups linked together
through atom N or C atom will give a positive test. The Biuret reaction was
named after the compound " Biuret " that gives a positive test, forming a
copper Biuret complex. The Biuret compound can be formed by heating
solid urea.
75
1- Dilute the albumin solution provided ( 10 mg / ml ) to prepare five solutions
[ standard solution ] containing ; 2mg/ ml , 4mg/ml ,6 mg/ml , 8 mg/ ml ,
and 10 mg/ ml ]. This can be done by using the following quantities of the
albumin solution 2ml ,4ml ,6ml ,8ml , 10 ml, and completing the solution in
each case by saline to 10 ml volume.
2- In a series of test tubes labeled to denote the concentration of albumin
used, add 1 ml of each of the dilute standard solution prepared above . in
another two test tubes, add 1 ml of saline in the first tube and label it as
blank and 1 ml of unknown sample in the second test tube.
3- To each of the tubes prepared , add 4 ml of the biuret reagent . mix well ,
and allow to stand at room temperature for 30 minutes. Read the
developed color in the colorimeter at 540 nm, against the blank tube.
4- On a squared paper , draw the curve representing the relationship between
the albumin concentration and the optical density . show that the curve is a
straight line which proves that the Beer’s is obeyed by this reaction.
76
NH2 NH2 O = C-NH2

- NH3
C= O + C = O
Heat H-N
NH2 NH2
O = C - NH2
Urea Biuret
OH OH
O = C-NH2
O = C-NH2 Cu H2N - C = O
H-N Cu(OH)2
H-N N-H
2NaOH
O = C - NH2
O = C - NH2 H2N - C = O
OH OH
Biuret Copper biuret complex ( violet)
2- Lowery method[ it's called folin – ciocalteu method]

Lowery method is at least ten times more sensitive than the Biuret method.
The procedure employs two color – forming reactions. First, cupric ions
(Cu+2) in an alkaline solution yield a purple color in the presence of peptide
bounds just like in the low – sensitivity Biuret test. Second, And more
important , Lowry method employs a complex inorganic salt mixture [
phosphomolybdate- phosphotungstate] that yields an intense bluish –
green color in the presence of tyrosine and tryptophan. Because the
content of these two amino acids varies substantially within proteins, the
color yield per mg of protein is not constant. It may differ from that of a
77
protein which is used as a standard BSA [ bovine serum albumin] is not the
best possible standard protein for lowery method because it happens to be
deficient in these two amino acids. One important disadvantage with the
lowery test is that it is altered by the presence of high amount of NH4+ ions
from (NH4)2SO4 . the ammonium sulfate concentration should be kept
below 0.15% in the test solution in order to minimize the interference. The
lowery’s method is more sensitive than the biuret because it can measure
the concentration of proteins in more dilute solutions, within the
acceptable accuracy. The relative sensitivities of the two methods
measuring the same compounds can be expressed as the ratio between the
optical density of the more sensitive method and the optical density of the
less sensitive method when both are measuring a certain concentration of
the compound.
Protein concentration is measured spectrophotometrically using
spectrophotometer or colorimeter. Colorimetry is based on measuring the
intensity of the color produced by a certain compound , to determine its
concentration. The concentration is calculated by comparing the intensity
of the color produced by the compound, with the color intensity produced
by a certain concentration of the pure compound” standard”. The intensity
of the color is determined by the “colorimeter” using the wave length at
which the color absorbs light maximally [ the maximum wave length]. In
most cases , the reagent alone may produce some color. This amount can
be subtracted from by reading the color against the “blank” that contains
only the color of the reagents. The Beer’s Law states that the increase in
the optical density is directly proportional to the increase of the standard
concentration, and the relationship should be represented by a straight
line. However , due to certain considerations, the law is not obeyed by
some reactions when the concentration of the standard is increased
beyond certain limits.
The “standard curve” is done to satisfy the following purposes; to
determine the range of the sample concentration within which the Beer’s
law is obeyed, to determine the sensitivity of the test [ the least amount
78
that can be determined with agreeable accuracy ] , and to test the precision
of the operator. The standard curve can also be used to convert the optical
densities to the corresponding concentrations. However, in some cases it is
advised to run a standard sample with each set of determinations to avoid
the effect that arises from the variations in the experimental procedure.
1- Dilute the albumin solution provided ( 1mg/ml ) to prepare 5 solutions
containing ; 0.02 mg / ml, 0.04 mg / ml , 0.06 mg /ml , 0.08 mg /ml , and 1
mg / ml . this can be done by diluting each of the following amounts of the
albumin solution provided to 10 ml by distilled water; 0.2 ml , 0.4 ml , 0.6
ml , 0.8 ml , and 1.0 ml.
2- In a series of test tubes labeled to show the concentration of the albumin
solution used, add 1 ml of the corresponding diluted standard solution
prepared above. In another two test tubes add 1 ml of water in the first and
label it as blank and add one ml of unknown sample in the second test
tube.
3- To each of these tubes add 5 ml of the alkaline solution.
4- Mix thoroughly and allow to stand at room temperature for ten minutes or
longer.
5- Add to each tube 0.5 ml of the diluted Folin reagent with immediate mixing.
6- Wait for 30 minutes , then read the optical densities against the blank at
750 nm.
7- Draw the standard curve to prove that Bee’s law is obeyed by this reaction
at these concentration. Use the standard curves of both the biuret method
and the Lowry’s method to compare between their sensitivities. This can be
done by comparing the optical densities measured by both method for a
certain concentration of the protein solution.
79
Reagents:-
1- Biuret reagent:-
dissolve 9 gm of Na, K –tartarate in 500 ml of 0.2 N NaOH . add 3gm of
copper sulfate ( CuSO4. 5 H2O) and dissolve by stirring ,then add 5 gm o
potassium iodide and make the volume to one liter with 0.2 N NaOH.
The Biuret reagent stores indefinitely, it is only discarded whenever a
black or reddish precipitate develops which will indicate contamination.
2- Alkaline solution:-
Prepared fresh on the day of use by mixing 50 ml of the alkaline sodium
carbonate solution with 1 ml of the CuSO4 – Cu tartarate solution.
 Alkaline sodium carbonate :- 2% of sodium carbonate in 0.1 N
sodium hydroxide.
 Copper sulfate – copper tartarate solution :- 0.5 % of CuSO4.5H2O in
1% sodium potassium tartarate.
3- Folin’s reagent:-
Dilute one part of the commercial folin reagent with two parts of
distilled water. The reagent is a solution of Na tingstate and Na
molybdate in phosphoric and hydrochloric acid.
Exp 21
Electrophoresis
Electrophoresis is the name given to the movement of charged particles through
an electrolyte subjected to an electric field, if these are differently charged they
will move in opposite directions, the positively charged migrating to the cathode
and the negatively charged to the anode. The rate of migration of particles of a
like charge will depend among other things on the number of charges each
carries.
80
As a result of different rates of migration , a complex mixture such as plasma

proteins can be separated into a number of fractions of alike mobility, the
sharpness of separation depending upon the extent to which each fraction is
homogeneous in its mobility.
Factors influencing the rate of migration:-

1- Support media:-
Paper was the original support medium but has been largely abandoned
and several others are now in use, these fall into two main groups
according to whether they act only as support or whether their properties
affect the separation in some other manner. These groups are (1) paper,
cellulose acetate and agar gel, (2) starch and acrylamide gels.
2- Composition and concentration of buffer:-
Many buffer of differing composition and PH have been used , in the case
or protein the rate of migration increase as the PH of the buffer is removed
from the isoelectric point . at PH 8.6 , all protein are anions and good
separations are obtained . the rate of migration also increase with decrease
in ionic strength of the buffer but the bands become more diffuse at low
ionic strength, probably due to impaired buffering capacity.
3- Voltage ,current, and heating effect:-
The mobility is proportional to the potential gradient along the strip. This is
not the same as the voltage across the electrodes on the voltmeter of the
power supply, variations in design of tanks and ionic strength greatly
modify the potential drop between electrodes and the ends of the paper
strips . the strips the greater the current passed and the greater the
potential drop across the fixed resistance of the buffer compartments.
Types of electrpphoresis:-
1- Paper electrophoresis.
a- Low voltage paper electrophoersis.
b- High voltage paper electrophoersis.
81
2- Thin layer electrophoresis.

3- Cellulose acetate membrane electrophoresis.
4- A ger gel electrophoreses
5- Starch gel electrophoresis.
6- Acrylamide gel electrophoresis.
7- Acrylamide gel disc electrophoresis.
8- Isoelectric focusing ( electro – focusing).
Theory:-
Many biological molecules carry an electrical charge, the mangnitude of which
depends on the particular molecule and also the PH and composition of the
suspend ing medium. These charged molecules migrate in solution to the
electrodes of opposite polarity when an electric field is applied, and this principle
is used in electrophoresis to separate molecules of differing charges.
The mobility of the molecules thus depends on the viscosity of the medium (n)
and the size, shape (r), and charge on the molecule (q). the electrophoretic
mobility depends mainly on the ionizable groups present on the surface of the
particle, and the sign and magnitude of the charge carried by the ionizing groups
varies according to the ionic strength and PH of the medium in a characteristic
manner. Separation of molecules can therefore be effected by selecting the
appropriate medium.
Electrophoretic separation of serum proteins:-

The total serum proteins include many different fractions which have a wide
range of isoelectric points; thus at a given PH they will carry different net charge
and so move at different rates in an electric field. Six zones are recognized at PH
8.6 albumin, α1, α2 ,β1, β2 , and ϒ- globulin.
82
Paper electrophoresis
The general procedure for an electrophorectic run on paper can be presented in
six steps:-
1- Placing the paper strip:-

The strip is wetted with buffer before setting it in place in the apparatus .
the paper should not be too wet since this results in smearing the pattern.
The strip is then placed in taut suspension or between glass or plastic plates
depending on the type of apparatus.
2- Equilibration period :-
A period of about 0.5 – 1.5 hour with the potential applied is allowed for
attaining equilibrium , during which time the paper may gain or lose buffer.
83
3- Application of sample :-
The current is turned off, and the sample is applied to the paper as a spot
by a micropipette or as a strip across the full width of the paper. The
Beckman applicator, which holds 10 µ L sample between two closely spaced
parallel wires, is very convenient for applying the sample. The sample
should not contain particulate matter since this may foul the run. The
quantity and concentration of sample to be applied is dependent on width
and thickness of the paper strip and the nature of the components. The
quantities usually employed, range frome about 5 to 100 µL.
4- Electrophoresis:-
V Q
Femf  EQ 
d
Fdrag  6r
when Femf  Fdrag , velocity is constant
84
 Large, highly charged proteins may actually migrate toward the like-charged electrode.
5- Identification of components:-
As soon as the strip have been removed from the cell they should be
dried quickly in the horizontal position , or the bands will shift and diffuse.
The components on the paper can then be identified by coloring the
bands by dye absorption, chemical reaction , ultraviolet light absorption,
fluorescence, or radioautography.
6- Quantitation:-
The colored components can be quantitated by two methods: (a) the
colored bands are cut out , and the color is eluted and then read
photometrically, (b) the strip with colored bands of the absolute
quantites of the respective components. It is essential that the
densitometer is standardized for each component to be scanned. If
densitometer values are to be used in quantitation the values must first
be converted to some function giving linear correlation with component
concentrations. The strip may be scanned dry , or the transparency of the
paper can be increased with mineral oil methysalicylate, benzyl alcohol,
or 1:1 mixtures of mineral oil and bromonaphthalene ( refractive index
85
1.55 ) profoundly different results can be obtained between the dry and
oiled strips.
Cellulose Acetate Electrophoresis (CAE)

The principle of paper electrophoresis apply also to cellulose acetate
electrophoresis. Cellulose acetate , however , has the following advantages over
paper :
(a) cellulose acetate striops can be used directly without any special preparation ;
(b) wet cellulose acetate is stronger than any other supporting medium and,
therefore , easy to handle;
(c) very small quantities of sample can be conveniently applied and are sufficient;
(d) only a short time for electrophoresis is required;
(e) better resolution of bands of components is achieved;
(f) cellulose acetate strips can be easily cleared after staining;
(g) good transparency of the strips facilitates quantitation by densitometry.
Cellulose acetate strips:-

Cellulose acetate strips are made by acetylation of cellulose , and the membrans
generally do not vary much in pore size , density , thickness, and other physical
properties. These membranes are stable in methanol, ethanol, propanol,
butanol,benzene, toluene, and petroleum ether, but will dissolve in glacial acetic
acid, chloroform, acetone, ethyl acetate, and methylene chloride. The thickness of
the membranes is approximately 120 µ. And the pores have a diameter of
approximately 0.4 µ. Cellulose acetate is also available with a mylar backing from
several commercial sources. The various types and brands of cellulose acetate are
as follows:-
86
Titan Helena Laboratories
Celagram Shandon Scientific Company, Incorporated
Sepraphore lll Gelman Instrument Company
Sartorius Membrane Pilters Brinkmann Instrumente
Celotate Millipore Corporation
Oxoid Oxo , Limited
Sand S 2500 Cellulose Schieicher and schuell
Membrane
These membranes vary from each other by the degree of acetylation , thickness,
and pore size. The decision as to which strip to use will depend on the procedure,
the components to be separated, and the electrophoretic chambers to be used.
The selection of strip is empirical, but it must be determined in such a manner
that optimum results are obtained.
Application of sample:-
Before applying the sample to acellulose acetate strip, the position of application
should be marked , marking should be performed when the strip is dry with a
marker that does not diffuse in either acid or alkaline solution, adheres to
cellulose acetate, and does not migrate in an electrical field. Grease pencils , ball,
point pens, and visipoint pens can be used for this purpose.
The strips should be impregnated,with buffer after which, visual inspection of the
strip ensures, that imperfection such as spots and ridges are not present. If these
faults are observed the strip should not be used since they will affect the
electrophoresis of the components and the resolution of the bands. For
impregnation with buffer the membranes should be allowed, to float on the
surface of the buffer in order to absorb the buffer from underneath. Then the
87
strips are immersed completely in the buffer. After impregnation, strips are
inspected for trapped air, which appears as white spots. The strips are then
blotted between filter paper to remove excess buffer, but they should be kept
moist and not allowed to develop dry areas.
The positioning of the strips in the electrophoresis cell or chamber depends on

the type of chamber. In some chambers, the strips are held in a horizontal
position by magnetic bars ; in other; the moist ends are pressed against the edges
of the supporting rack. The membranes with Mylar backing are positioned with
the active surface on top and the Mylar backing on the bottoms. After the strip is
positioned the sample is applied to the strip in the form of a fine, uniform streak
along the mark line. This can be carried out ‘by using a capillary tube,
micropipette or a wire applicator with parallel wires that are very close to each
other.
The sample size is generally 1-2 µ for proteins, 5- 10 µ L for lipoproteins and
approximately 10 µ L. for lactic acid dehydrogenase isoenzymes. The samples are
applied at the anodal or cathodal end of the strip depending on the charges of the
substances to be separated and the PH of the buffer.
When samples are applied as strips, “edge effects” are commonly seen. This is a
convexity of the component bands in the direction of migration possibly due to
increased evaporation of solvent at the edge, which results in increased
electrolyte concentration, cooler strip temperature and increased capillary flow
from the reservoir, each of which would cause a slowing down of migration. This
phenomenon becomes less pronounced as strip width is decreased. Every effort
must be made to minimize this distortion, for it makes it difficult to cut out the
bands for elution and introduces” stray radiant energy” in densitimetry.
Buffer:-
The buffer used in cellulose acetate electrophoresis are generally more dilute
than those used with paper. The ionic strength of the buffer will determine the
migration distance; the lower the ionic strength the greater the distance. It is also
88
true that diffusion of separated components is inversely related to the ionic

strength of the buffer. The resolution of bands, however , gets poorer with
increasing migration distance since the width of the bands will increase because
of diffusion. Some of the commonly employed buffers are the barbital buffers
with concentration that vary between 0.05 and 0.1 M and the PH 8.6 tris – EDTA –
borate buffer at PH 8.9 and 9.6 buffer solution should be changed frequently,
especially at high ambient temperatures since the buffer will concentrate during
use due to evaporation. Since thew number of electrophoretic determinations
will vary from laboratory to laboratory, it is recommended that the buffer be
changed after acertain number of runs depending on the procedure.
Instruments:-
Many electrophoretic chambers are available for the electrophorotic separation
of proteins. The ones most commonly used are the RIOI Microzone, Shandon,
Photovolt, and Gelman chambers. These are all horizontal chambers with the
exception of the photovolt chamber, in which the strips with Mylar backing are
not placed horizontally but rather from a bow type bridge. The densitometers
used in most laboratories are the Beckman Analytrol. Photovolt DENSICORD, AND
THE Microzone Densitometer. The main requirement for a power supply is that it
can maintain either a constant voltage or a constant current. Since the resistance
will change in an electrophoresis run, the current will change accordingly and a
constant power stabilizer should therefore be used.
Electrophoresis :-
The optimum conditions for an electrophoresis run have to be determined for
each technic. The factors affecting the electrophoretic seoaration of components
are :-
(a) the type of supporting media

(b) composition and ionic strength of buffer,
(c) temperature of buffer
(d) current oe voltage applied
89
(e) time of electrophoresis .
generally a protein electrophoresis will be completed in approximately 30 min.

when a current of 1.5 mA per strip is applied. If the ammeter reads offscale in the
direction of high amperage, a “shorting” of the circuit as a result of spillage of
buffer is the most likely cause. The direction of the current should be changed
after each run to prevent the gradual change in buffer composition between the
two reservoirs that would occur over many successive runs. Reference dyes are
often used to monitor the rate of migration. For example, albumin stained with
bromphenol blue is frequently used internal control in electrophoresis.
Staining:-
The staining solution used for cellulose acetate electrophoresis are generally less
concentrated than those used for paper electrophoresis . aqueous as well as
alcoholic staining solution are employed by different workers, but the former
ones are generally preferred. If alcoholic stains are used, additional washings
through an aqueous buffer are required to prevent shrinkage of the strips.
Proteins stain with the anionic dyes Ponceau S or nigrosin by chemical reaction
with the amino groups. Other chemical mechanisms that occur in staining include
the oxidation or reduction of the dye. Sometimes the separated components are
identified after their chemical conversion to fluorophors.
Strips stained with aqueous staining solution are commonly washed with a 5?%
acetic acid solution until the background is white or until the washing solution is
no longer colored with the dye. This rinse solution does not affect cellulose
acetate. The stained strips are then dried by draining off excess rinse solution
followed by air drying. Then they are placed in methanol, followed by a clearing
solution containing glacial acetic acid or dioxin to make them transparent and
suitable for densitometry. Cellulose acetate strips can also be cleared by oil
impregnation. This procedure of clearing is not permanent, and the strips can be
returned to their original state by washing them in a fat solvent such as ether.
90
Quantition:-
Quantitative determinations can be carried out by cutting out the stained bands
and eluting the dye that is then measured photo metricallynmtry. The dye in the
cut – up band can also be dissolved completely in organic solvents prior to
reading the color in a photometer or spectrophotometer.
A more convenient and rapid procedure for quantitation of components is

continuous staining densitometry or fluoronetry. The advantage of quantitation in
this manner is that the cleared strip with the stained hands can be used without
any further preparation. The responses of the photometer can be recorded and
the resultant curves can be evaluated by a variety of manual or automated
techniques. An automatic calculation of densitometry scans of electrophoretic
strips utilizing a disk integrator, automatic digital readout, and programmable
desktop computer can be used.
Thin – layer electrophoresis

Thin – layer electrophoresis requires smaller amounts of sample than is needed
for paper electrophoresis. Furthermore, the procedures are simpler and require
less elapsed time for analysis.
Thin layer supports Kieselguhr, Silica gel, cellulose, and alumina are usually used
as supports. If the surface is cellulose the plates cannot be sprayed with strong
reagents, and if alumina is employed, tailing of the dyes can be expected. In
general, the best results have been obtained with Kieselguhr and silica gel.
Prepared plates with. Most of the common supports are now available from
commercial sources.
91
Buffer:-
Buffers containing voltatile solvents such as butanol, acetone , pyridine, etc , are
recommended since they can be eliminated easily when the plates are being dried
and therefore do not interfere with the spraying reagent.
Application of sample and electrophoresis:-

The plates must be dry when the sample is applied in the form of a small spot
with a micropipet. When the plate is sprayed with buffer care should be taken not
to use too much. The plates are then subjected to plates are dried at
approximately 100 0C and colors from the separated compounds are developed
with a suitable reagent.
Cellulose acetate electrophoresis of proteins

(method of Kohn)
Principle:-
Serum proteins are separated by elelctrophoresis on cellulose acetate using a
Tris- barbital buffer, PH 8.8 . the electrophoretograms are stained with ponceiou S
and quantified by transmission densitometry.
Procedure:-
The procedure presented is for the photovolt electrophoresis system which
utilizes 12 x75 mm cellulose acetate strips with Mylar backing and integrating
densitometer for quantification. This procdure is similar to those of several other
commercially available systems. The manufactuer’s instruction manuals should be
consulted for technical details.
92
1- With a wax pencil label strips at one end of the Mylar side. It is important
not to touch the membrane surface with bare fingers in this and all
subsequent steps.
2- Place strips on the surface of the buffer. Slowly immerse into a staining dish
containing sufficient ( approximately 250 ml) Tris – barbital buffer to cover
strips. Soak for at least 2 min.
3- Add Tris- barbital buffer to appropriate levels in the electrophoresis
reservoirs and tilt to equalize fluid levels.
4- Remove the strips from buffer solution and individually blot each strip
between two blotters.
5- Place strips in electrophoresis chamber, so that both ends are dipped in the
buffer.
6- Apply samles to cellulose acetate strips at the cathodic side of the chamber
using the sample, applicator. Excess sample should be avoided by carefully
blotting the underside of the applicatortip.
7- Place cover on electrophoresis chamber and allow electrophoresis to
proceed for 20 min at a constant voltage of 220 volts.
8- Turn electrical current off remove strips.
9- Immerse in Ponceau S staining solution in staining dish for 3 min.
10- Drain for a few seconds and rinse for 3 min each of three staining
dishes containing 5% acetic acid.
11- Drain on absorbent paper for 10 sec. immerse in absolute methanol
for exactly 2 min.
12- Drain and immerse in clearing solution for 1 min.
13- Scan strips on an integrating densitometer using a 570 mm filter and
a 0.1 x 6 mm slit (note 1 )
93
Notes:-
1- Reuse of solution:-
The buffer solution in the staining dish as well as in the chamber should be
changed daily. The polarity of the electrophoresis chamber should be
reversed after each electrophoresis run with the system described, this is
conveniently achieved by a toggle switch. The dye solution is suitable for
100 strips or 1 week. Rinse solutions should be serially replaced when
coloration becomes excessive – i.e., discard the first rinse solution, move
the second solution to the first position, the third to the second position,
and place fresh solution in the third position. The absolute methanol and
clearing solutions should be replaced daily.
2- Sample stability:-
It has been reported that á2 – globulin decreases and Ɣ – globulin increases
after 1 day at 20 0C . on further storage at either 4 0C or 20 0C the β-
lipoproteins tend to migrate more toward the α 2 – globulins. In contrast,
we have not found significant changes in the electro-phoretic pattern of
serum stored at 30 0Cfor 4 days , 4 0C for 2 weeks or – 20 0C for months.
3- Plasma vs serum:- fibrinogen is represented by a narrow band in the β2 –
globulin region, which may be mistaken for an M- component. It is
therefore customary to use serum instead of plasma.
Normal values:-
There are two methods of expressing the quantity of individual fractions in an
electrophoretogram :
(a)- in relative terms as percentage of total protein
(b)- in absolute terms of concentration, e.g., g/100 ml.
There are arguments in favor of each method of expression . a disadvantage of

expressing individual components as percentages of the total is that on increase
or decrease in any one of the major fractions results in a corresponding decrease
94
or increase in the percentages of all other fractions even though their absolute
concentration may be normal. On the other hand, in dehydration or water excess
syndromes, the absolute concentrations may be increased or decreased,
respectively, simply as a result of contraction or expansion of the intravascular
fluid volume. An obvious solution would be to report the results in both ways.
The reference range as g./ 100 ml is total protein 6 – 8 ,
albumin 3.5 – 5
α 1 globulin 0.1 – 0.4
α2 globulin 0.5 – 1.1
β globulin 0.6 – 1.2 and Ɣ globulin 0.5 – 1.5 .
expressed as percentage of total the reference range is :-
albumin 52 – 97
α 1 globulin 2.4 -4.6
α2 globulin 6.6 – 13.6
β globulin 9.1 – 14.7
Ɣ globulin 9.0 – 20.6
The effect of various factors is as follows:-

Sex :-
There is no sex difference ,pregnant females at term have significantly decreased
albumin and increased β globulin and slightly increased. α 1 globulin ; α2 globulin
and Ɣ- globulins are not significantly altered during pregnancy.
95
Age:-
Relative to adults, cord blood has decreased concentrations of total protein,
albumin, and α 2 and β globulins. α 1 globulin is slightly increased , and Ɣ globulin
is normal or increased. Adult levels are reached for all fractions except Ɣ globulin
by 3 months of age. Ɣ globulin in cord blood is largely of maternal origin.
Catabolism of the passively transferred protein results in a marked decrease in Ɣ-
globulin.
Reagents:-
1- Tris – barbital buffer:-
PH 8.8 , dissolve 5.75 g Tris ( hydroxymethyl ) aminomethane, 2.45 g
barbital, and 9.81 g sodium barbital in approximately 250 ml warm water.
Mix , allow to cool , and dilute to 1 liter. Adjust PH to 8.8 if necessary .
buffer is 0.0475 M with respect to both Tris and sodium barbital.
2- Pouceau S solution:-
Dissolve 5 g in 1 liter 7.5 % ( w/v) trichloroacetic acid
3- Acetic acid 5%
4- Methanol anhydrous
5- Clearing solution :-
Mix 200 ml glacial acetic acid , 770 ml anhydrous methanol , and 30 ml
ethylene glycol.
96
Exp. 22
Enzymes (1)
Objectives :-
1- To prepare enzyme rich solutions by simple extraction procedures.
2- To confirm the chemical nature of enzymes.
3- To demonstrate enzyme activity.
Introduction:-
Enzymes are proteins that catalyze biological reactions, and they influence the
rate at which equilibrium is attained, but do not affect the overall equilibrium of
the reaction.
How could one prove that a solution contains an active enzyme ?
E + S ------------------→ ES ---------------→ P + S
When a substrate is added into an enzyme rich solution , the enzyme , if active,
would react with the substrate and form an enzyme – substrate complex which is
eventually converted to products and to free enzyme. Notice that the enzyme is
not consumed in this reaction. Therefore, a very small quantity of enzyme keeps
on catalyzing this reaction until all substrate has been converted to products.
Measuring very small quantities of enzyme is tedious and mostly impossible.
However, to obtain qualitative proof than an enzyme catalyzed reaction in fact
takes place, one can do several things:-
1- To demonstrate the formation of the product (s)

2- To demonstrate the disappearance of subatrate.
In the following experiments, the action of catalase, acid phosphatase and urease
is demonstrated on the basis of the appearance of products.
97
1- Catalase :- (an oxido – reductase):-

Catalase is found in both animals and plants. In this experiment a solution rich
in catalase is prepared from fresh potatoes. Catalase is the trivial name of the
enzyme. It is not very descriptive. The systematic name is hydrogen peroxide –
hydrogen peroxide oxido – reductase . it catalyzes a reaction in which one
molecules of H2O2 acts as a donor of hydrogen atoms and a second molecule of
H2O2 acts as an acceptor of hydrogen atoms. The products of the reaction are
water and molecular oxygen, which is released as gas.
Catalase
2H2O2 ------------------------------→ 2H2O + O2 -------- (1)
The above reaction is called the catalectic type of reaction.however, the same
enzyme is able tocatalyze anther type of reaction [peroxidatic type]. In the
peroxidatic type of reaction one H2O2 acts as an acceptor of hydrogen atoms
but another molecule [not H2O2 ] acts as a donor of hydrogen atoms.
catalase
H2O2 + 2AH2 ------------------→ 2H2O + HAAH -------------- (2)
Polymerized product
The other molecule which serve as hydrogen donors have certain aromatic
compounds such as pyrogallol ( 1,2,3 – benzyne triol) , P – cresol ( 4 – methyl
phenol) or guaiacol ( O – methoxy phenol).
98
CH3 OH
OH
OCH3
OH
OH
OH
Pyrogallol Guaiacol
P - cresol
Reaction number 1 and 2 are summarized below:-
Type of reaction Hydrogen acceptor Hydrogen donor

Catalatic type H2O2 H2O2
Peroxidatic H2O2 Aromatic compounds
Notice that while being able to catalyze peroxidatic reaction, catalase is not a very
efficient catalyst for these types of reactions:-
2- Acid phosphatase:-
Phosphatase are enzymes that catalyze the hydrolysis of phosphate
monoesters:-
O
ll phosphatase
R - O - P - O - + H2O R - OH + P i
I
O-
Acid phosphatase is a nonspecific enzyme which means that it works on

several substrates . one can take advantage of the broad substrate specificity
by using an artificial, synthetic substrate. The presence of enzyme activity is
going to be demonstrated by using disodium phenylphosphate as substrate:-
99
OH
O OH
acid phosphatase
O - P - ONa + H2O + HO - P - OH
O O
Disodium phenylphosphate phenol phosphoric acid
As shown above, when acid phosphatase enzyme reacts with disodium

phenylphosphate the end products are phenol and phosphoric acid. The
presence of phenol can be easily detected by a color forming reaction . folin
phenol reagent yields color due to reduction of the phosphomolybdate –
phosphotungstate salts in the reagent in the presence of phenol.
The other product, phosphoric acid , can also undergo a sequence of reaction
to produce color. The principle behind the detection of phosphoric acid is as
follows:- first a yellow phosphomolybdic acid complex is formed by the
reaction of H3PO4 with ammonium molybdate. This complex is then reduced
quantitatively to a heteropply – blue by addition of crystalline ascorbic acid
followed by short boiling .
100
O=C
I O=C
HO-C O Areduced complex I
A yellow phosphomolybdic O=C
acid complex II of blue color
HO-C I O
I O=C
H-C I
I H-C
HO -CH I
I HO -CH
CH2OH I
CH2OH
Ascorbic acid dehydro ascorbic acid
3- Urease :-
Urea is the main end – product of nitrogen metabolism in mammals. Urease
catalyzes thwe hydrolysis of urea as follow :-
NH2
I
C= O + H2O 2NH3 + CO2
I
NH2
The experiment is carried out using commercially prepared urease in crystalline

form. This is done for two reasons :-
1- The commercial urease enzyme does not dissolve easily in water.

2- In solution the enzyme tends to lose its activity.
Ammonia is a weak base. When ammonia is produced , the solution becomes

more and more alkaline. If an acid – base indicator is added into the solution, one
can be easily demonstrate the formation of ammonia. Acid – base indicators have
101
the ability to change color depending on the pH of the solution. Phenol – red
indicator has a usefil pH range from 6.8 to 8.2 .
Below pH range from 68 phenol red has a yellow color and above pH 8.2 it is red
to purple.
A.1 Extraction of catalase from fresh potato:-
1- peel half of a potato and grate it into fine pieces which are placed in a beaker
and add 100 ml of D.W.
3- Mix with a glass rod and let stand for 10 min.

4- After through whatman No. 1 filter paper and collect about 20 ml of potato
extract.
A.2 Biuret test :-

1- take 3 ml of potato extract.
3- Add an equal volume of 10 % NaOH and 5 – 10 drops of 0.5 % CuSO4.

Report your observations.
A .3 Demonstration of catalatic type of reaction :-

1- Take 5 ml of potato extract into a test tube.
2- Add 1ml of 3% hydrogen peroxide solution. Record your observations.
A .4 Demonstration of peroxidatic type of reaction:-

1- Place 4 ml of potato extract into two test tube.
2- Add 1 ml of D.W. in the first tube and add 1 ml of 2% pyrogallol solution
which contains some H2O2 in the second tube
Notice that the acceptor of hydrogen atoms is still going to be H2O2 although the
donor now is an aromatic compound. You should be able to observe a change of
color to take place in tube two because pyrogallol is oxidized to a polymerized
102
product, HAAH, which has a characteristic color. Catalase reaction does not take
place in tube one since one substrate was added into it . compare the color of
tube one to that of tube two. Report your observations.
Lactate dehydrogenase
Demonstration of activity:-
Place 5 ml of yeast suspension in a test tube add an equal volume of 0.02%
solution of methydne blue and 10 drops of 5 % sodium lactate solution. Prepare a
second tube containing water instead of lactate. Cover with a layer of mineral oil.
Incubate both tubes at 37 0C and record the rate of decolonization of the dye in
each tube. The rate will be greater in the tube containing lactate. Yeast
suspension itself contain little lactate.
Extraction of phosphatase acid from wheat germs:-

The seeds of plants are a particularly rich source of acid phosphatase. The amount
of phosphatase activity in seeds usually increases sharply upon germination. In
this experiment wheat germs are used as a convenient source of acid
phosphatase.
1- Mix 5 gm of wheat germs with 100 ml of D.W. in beaker. Let the mixture
stand for 20 min in an ice – bath . stir it every now and then with a glass
rod.
2- Filter the extract through Whatman No. 1 filter paper. Keep this enzyme
preparation in an ice – bath to avoid inactivation of acid phosphatase.
103
Demonstration of acid phosphatase activity:-

Demonstrate the presence of bith products after acid phosphatase has acted
upon the synthetic substrate, disodium phenylphosphate, for 30 min under
optimum conditions. One essential point about enzyme assays in general is that
one must perform control experiments to show that the assay is in fact due to
enzyme action.
1- Take part of the filtered extract ( e.g.,10 ml ) into a small beaker and boil it
gently for 5 – 10 minutes.
2- Conduct the test as shown in the table and answer the following
questions:-
A B C D
Fresh enzyme solution , ml - 1 - 1
Bolled enzyme solution , ml 1 - 1 -
Disodium 5 5 5 5
phenylphosphate, ml
Incubate all four tubes for 30 min. at 40 0C
10 % Na2CD3, ml 5 5
Folin phenol reagent, ml 1 1
Mix & let stand for 5
min.
Record colors in tube
A&D
3
Ammonium molybdate, ml 2 2
Crystal line ascorbic acid 10 mg 10 mg
Mix & boil in flame
Record color in tubes C& D
Biuret test :-
1- Take about 10 mg of crystalline urease extracted from melonseeds.
2- Add 1 ml of distilled water and mix well using the vortex mixer.
104
3- Add1 ml of 10 % NaOH.
4- Add a few drops of 0.5 % CuSO4 solution. Do not shake.
Exp 23
Objective:- to study the different factors that affect enzyme activity.
Introduction:-
a. Subcellular location of enzymes:-
Each cell contains subcellular organelles such as lysosome, mitochondria ,
nucleus, cell membranes etc. it is important to remember that there is no uniform
concentration of enzymes throughout the cell. Some enzymes are located in the
cytoplasm whereas othersare bound to cell membranes. Still others are inside the
nucleus, in the mitochondria or within other cellular organelles. When cell walls
are disruoted, those enzymes which are free in the cytoplasm can be separated
from others by simple centrifugation methods.
Enzymes which are located inside various cellular organelles or bound to cell
membranes would settle down with the cell debris upon centrifugation. Working
with any biological material is complicated because of the presence of numerous
other components in the same sample. For example other active enzymes in a
solution can produce false results by decomposing the products of the enzymes
under study. In some cases it is essential to work with fractions of the studied
material which have proven to be fairly free from interfering substances.
c- Influence of enzyme concentration on reaction velocity:

In order to demonstrate how the concentration of an enzyme affects the reaction
velocity, it is important and essential to keep the other variables constant.
Therefore, the substrate concentration, PH and temperature is maintained the
same while the only thing which is varied is the amount of enzyme added to each
tube. In this experiment we work with amylase enzyme.the substrate for amylase
105
is starch. It is broken down to smaller and smaller units, first to different dextrins,
then to the disaccharide maltose, and eventually to glucose.
Enzymatic hydrolysis of starch is shown in the following sequence:-
Amylase
Starch ------------→amylodextrin -----→erythrodextrin ---→ maltose ------→glucose
Blue color blue brownish colorless with
With iodine With iodine
Since pure enzymes are difficult to obtain and would be very expensive to use,
diluted saliva [reagent A] or serum obtained from the blood of an animal is used
as the source of amylase.
The principle of this enzyme assay is as follows:-
A measured amount of diluted saliva A (serum or any other materials to be

analyzed) is placed in a solution containing the substrate for the enzyme under
study (i.e. starch for amylase). The solution is monitored over a period of time for
the disappearance of substrate. The rate at which blue color disappears indicating
hydrolysis of starch, is a direct measurement of amylase activity in salive or
serum.
d- The influence of temperature on enzyme reaction:-
Extremes of temperature can denature proteins and destroy their biological

activity. Enzymes are no exception in this respect; boiling an enzyme solution
usually destroys its catalytic activity, as does exposure to very acidic or basic
solution.
For each enzyme there is a temperature at which it is most active and its activity
diminishes, when the temperature is changed in either direction awwy from this
optimum [ see the figure below].
106
Most enzymes have their greatest activity somewhere between 25 & 37 0C,
corresponding to the temperature of most cellular environments.
Enzymes are sensitive to temperature due to the fact that they are globular
proteins. temperature changes cause changes in the three – dimensional shape of
proteins molecules which may become less compact. Even small changes in the
dimensions of its active site can make an enzyme less efficient as a catalyst.
e- The influence of PH on enzyme reaction :-
Many enzymes work most efficiently at a PH near 7 which is the condition found
inside living cells. Their catalytic activity decreases as the PH is shifted a way from
this optimum. The following figure shows the hypothetical PH dependence of the
catalytic activity of an enzyme.
107
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Book · January 2015

Abdulhussien Aljebory And Tamadhur J. Alsalman

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groups important in biochemistry

The acid may be organic or inorganic . Esters of H3PO4 ( phosphorylated sugars

R-CH2-SH R-C – S – R/ R-S-R/

Thioalcohols thioesters thioether

In addition , the disulfides ( left) and peroxides ( right)

Play an important role in protein structure and in prostaglandins biosyntheses

Aldehydes and ketones:-

Sugars , being polyhydric alcohols are also either aldehydes or ketones.

RR/ C = O --------------------------------→ RR/ CH - OH

3- Hemi acetal and acetal formation:-

β – hydroxyacids derived from these are of importance in fatty acid

CH3CH = O + CH3CH = O -------- [ OH- ]-----------→ CH3CHOH – CH2 – CH = O

R – COOH ------ [ 4H+ ] ---------→ R – CH2OH

R – CO – O – H + HO – CO – R/ ------- - H2O ------→ R – CO – O – CO –R/

When both acids are the same , a symmetric anhydride is produced .

CH3 – CO – O – PO (OH)2 Acetyl phosphate

CH3 – CO – OH + H- NH2 ------- - H2O -----→CH3 – CO – NH2

Acetic acid acetamide

General reactions of carbohydrates

Carbohydrates may be defined as polyhydroxy aldehydes or ketones, or

1- Monosaccharides(simple sugar molecules)

They lack the free aldehyde or ketone groups , an example is sucrose

1- Heating with strong acids causes the hydrolysis of disaccharides and

3- These furfurals are colorless compounds which undergo condensation

Phenol a - naphthol Anthrone

To prepare Molisch’s reagent :-

Dissolve 2 gm of anthrone in 1 liter conc. H2SO4

3- Bial’s Orcinol test:-

1- Add 3 ml of bial’s reagent into three separate test tubes.

4-Seliwanoff’s Resorcinol test:-

HOCH2 C C CHO + Red condensation prpduct

Test Phenolic Acid Sugar Color

5-Mucic aci test:-

2- effects of alkaline on carbohydrates

R- C-H + R-OH R-C-H

Similarly, an aldehyde group reacts with an alcohol group forming a hemiacetal.

Glucose Internal hemiacetal structure

α- form ↔ open chain form ↔ β- form

Oxidation product ( leaves the equilibrium)

RCHO [ or α- hydroxyl ketone ] + 2 Cu+2 -----------→RCOOH + Cu2O + 3H2O

The experimental conditions of these three tests vary :-

distinguish between monosaccharides and disaccharides by the speed of

Procedure of Benedict’s test:-

Procedure of Fehling’s test:-

Iodine test is used to distinguish polysaccharides from other carbohydrates and

Carbohydrates -------------------------------------→ monosaccharides

From previous experiments recall what happened to carbohydrates in

Carbohydrates ----------------------------------→ hydrolysis & dehydration

Disaccharides may be hydrolyzed to yield two monosaccharide molecules. So

Sucrose --------------------------------→ fructose + glucose

Starch ---------------------------------------------→ glucose

With dilute acid