SCITAMA
EXPERIMENT 1
Isolation of Casein from Milk,
Acid/Base Hydrolysis and
Neutralization
MILK
- It is a food substance for
persons of all ages.
- Many specialized products
like yogurt, cheese, butter
and ice cream are staples of
our diet.
- It is the most nutritionally
complete food especially for
the young mammals.
(including babies)
- Milk is required for the
growth of the newborn since
the main function of it is
to supply nutrients such as
essential amino acids needed
by the body.
- It is a good source of
calcium and phosphorus but
lacks important elements
like iron and vitamin C.
- Milk contains 3.4% protein,
9 essential amino acids
found in milk protein:
M,I,L,K,W,V,P,T
- Different kinds of milk:
Non-fat, skim milk. Low fat,
pure fat milk.
PROTEINS
- Are naturally unbranched
polymer, with monomer unit
called amino acids.
- Amino acid chains are linked
by peptide bonds.
- At least 50 amino acid
residues are found in the
peptide.
- Can be categorized in two
types namely: Fibrous and
Globular protein.
- pH.
CASEIN
- Main protein in milk
- Globular in shape
- Function as a storage
protein
PHOSPHOPROTEIN
- Has phosphate groups
attached to the hydroxyl
groups of some amino acid
side-chains.
CALCIUM CASEINATE
- Calcium salt, insoluble in
solutions with pH less than
4.6
- Calcium caseinate has an
isoelectric point of 4.6 pH
- High number of Proline
residues, which do not
interact; little tertiary
structure
- Cannot denature without
disulfide bridges;
hydrophobic
- Mixture of 4 milk (casein):
1-casein, 2-casein, -
casein, and K-casein;
forming a micelle
- 1-casein, 2-casein, and -
casein are both insoluble in
water; solubilized by the
micelle
- K-casein has a hydrophilic
portion; has the ability to
solubilize the other two
caseins by the formation and
stabilization of the
micelles.
-
ISOELECTRIC PRECIPITATION
- Precipitation formation in a
liquid or solid reaction.
- Solid formed during a
reaction is a precipitate.
- Precipitation occurs when
casein is in its isoelectric
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ISOELECTRIC pH
- Casein protein is
isoelectric at 4.6
- Minimized intermolecular
repulsions
- Displays a minimum water
solubility
- non-fat milk is more simpler
medium compared to whole
milk
- Fats are stabilizers
- Heating casein to unfold the
globular structure
- 55 ºC and not higher
- pH of milk = 6.6
- isoelectric pH of casein =
4.6 (point at which protein
is least soluble)
- 10% acetic acid (not too
strong)
- causes the casein micelles
to form curds by decreasing
the pH
- aggregation occurs as a
result of entropically
driven hydrophobic
interactions
- precipitates casein by
coagulation
- increases solubility of
organic calcium and
phosphorus in the micelle
HYDROLYSIS
- The cleavage of chemical
bonds by the addition of
water
- Used to break peptide bonds
ACID HYDROLYSIS
- The likely used method in
the analysis of proteins and
polypeptides.
- Acid is used as a catalyst
- Proceeds without
racemization and with less
destruction of certain amino
acids (S,T,C,R)than alkaline
treatment
- W (tryptophan) is completely
destroyed.
- N --> D, Q --> E
BASE HYDROLYSIS
- Destroys more amino acids
compared with acid
hydrolysis. (S, T, C, and R
are destroyed in base
hydrolysis)
- Ba(OH)2 – for easy
separation
- The base itself also acts as
a catalyst
Why autoclave the hydrolyzate?
- Frees amino acid in high
pressure as the acid/base
speeds up the hydrolysis
- For the heat catalyzed
reaction
NEUTRALIZATION
- A chemical reaction in which
an acid and a base interact
with the formation of a salt
ACID HYDROLYSIS
- Ba(OH)2 – neutralizing agent
BASE HYDROLYSIS
- H2SO4 – neutralizing agent
ACID + BASE  SALT = WATER
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EXPERIMENT 2
Color Reactions of Intact Protein
Casein and Acid/Base Hydrolysis
Casein
- Main protein in milk
- Exists as the Ca salt
- Phosphoprotein
- Mixture of min of 3 similar
proteins (, - & x-casein)
- 80% of protein present in
milk
- contains the essential AAs
(V,P,H,M,A,T,I,L)
- isolated at isoelectric pH
(pI), least soluble
(isoelectric precipitation)
- accomplished by addition of
dilute acid
- net charge at pI=O
Hydrolysis
- bond cleavage of labile
bonds simultaneous with the
addition of water.
- Needed to break amide bonds
in intact proteins to
produce amino acids
Acid Hydrolysis
- Catalyzed by strong acids
such as H2SO4, HCl, HNO3,
etc. (15 psi/5hrs)
 Total hydrolysis
 Does NOT promote
racemization of a-C
configuration
 Trp is destroyed and
converted to humin (black
pigment)
 Thr and Ser are destroyed
 Asn and Gln are converted to
Asp and Glu
Base/Alkaline Hydrolysis
- Uses strong bases such as
Ba(OH)2, NaOH, KOH, etc.
(15psi/5hrs)
 Trp is NOT destroyed
 Promotes racemization
 Thr and Cys are lost
 Arg is destroyed & converted
to urea & ornithine
Biuret Test
- General test for intact
proteins and protein
hydrolyzates (at least a
tripeptide)
 Named after a compound
biuret
 Reagents:
CuSO4 soln & dilute NaOh
 Positive result: formation
of pink to violet to blue
color
 Principle: complexation of
Cu+2 with amide N atoms
 No reaction with dipeptides,
urea, coagulated proteins
and amino acids (except Ser
& Thr)
 Mechanism:
Ninhydrin Test
- General test for compounds
with a free -amino groups
 one of the most sensitive
color reactions known
 reagents:
ninhydrin
(triketohydrindene hydrate
in ethanol)
 positive result: blue to
blue violet color
 principle: oxidative
deamination and
decarboxylation; reduction
of ninhydrin
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 proline, hydroxyproline, and
2-, 3-, and 4-aminobenzoic
acids fail to give a blue
color but produce a yellow
color instead
 ammonium salts give a
positive test.
 Some amines such as aniline,
yield orange to red colors
which is negative test.
Xanthoproteic Test
- General test for aromatic
amino acids such as
tryptophan, phenylalanine,
histidine, and tyrosine (H,
F, W, Y)
 Presence of electron
donating substituents
enhances reaction rate
 Reagents: conc. HNO3 and
conc. NaOH (neutralize
excess acid)
 Positive results: formation
of yellow ppt and after
addition of excess NaOH
(alkaline), an orange ppt
forms
 Principle: nitration of
aromatic rings (i.e. indole
in tryptophan) via
electrophilic aromatic
substitution
Hopkins-Cole Test
- Detects the presence of
indole group in tryptophan
 Reagents: magnesium, oxalic
acid, and conc. H2SO4
 Positive result: pink to
violet interphase
 Principle: reduction of
oxalic acid to glyoxilic
acid & acid catalyzed
condensation of 2
tryptophans with glyoxilic
acid
 Mechanism:
Sakaguchi Test
- Specific for arginine
(guanido group)
 Reagents: -napthol, NaOH
and NaOBr (and urea to
stabilize color and destroy
excess OBr-anions)
 Positive result: red to red-
orange color
 Principle: base-catalyzed
condensation of -napthol
with the guanido group of
arginine
Bradford Assay
- Simple, fast, inexpensive,
highly sensitive
 Uses the Coomassie Brilliant
Blue G-250 dye reagent
(binds electrostatically
with arginine residues in
anionic form and by pi-
stacking interactions with
aromatic AA’s)
 Read at 595 nm (UV
spectrophotometer)
 Intensity of color (measured
by absorbance) is directly
proportional to the
concentration of protein
(Beer’s Law) A = abc
 Unknown concentration is
measured using linear
regression analysis
 Y = mx + b
Where: y = measured
absorbance
m = slope
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x = concentration of
unknown
b = y-intercept
 For standard protein
preparations, use C1V1=C2V2
when dilutions are done on
standard solutions

EXPERIMENT 4:
Rate of Enzymatic Activity of
Salivary - amylase Based on
Changes in pH and Temperature

Enzymes
- Biological molecules that
catalyze many important
chemical reactions inside
the body.
- Most enzymes are proteins.
- Enzymes are biological
catalysts.

- Catalysts are substances

that increases the rate of
chemical reactions taking
place.
- They do this by lowering
the activation energy
needed to start a
reaction.

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- This increases the
production of products,
and thus faster reactions.
Lock and Key
- Like a lock and key, each
are specific and unique to
one another.
- This means that both - amylase
components, enzyme and
substance, fit each other
to a very high degree.
- The shape of the substance
fits into the binding site
of the enzyme.
- All enzymes are catalyst,
but not all catalyst are
enzymes.
Induced fit
- On the other hand, the
induced fit model, aka the
glove and hand model.
- The flexibility of an
enzyme to conform.
- Thus, the binding of the
substance to the enzyme is
due to the ability of the
enzyme to adjust and be Salivary Amylase
flexible.
Amylase
- It is an enzyme that
breaks down starch into
sugars.
- It can be found in human
saliva where it begins the
chemical process of
digestion.
- Foods that contain much
starch but little sugar,
taste slightly sweet as
they are chewed because
amylase turns some of
their starch into sugar in
the mouth.
- also called
glucanohydrolase or
glycogenase.
- are calcium
metalloenzymes, completely
unable to function in the
absence of calcium.
-amylase breaks down
carbohydrates, yielding:
1. from amylose: glucose and
maltose
2. from amylopectin:
maltose, glucose and
dextrin
- Both the salivary and
pancreatic amylases are -
amylases.
- A digestive enzyme
secreted by the salivary
glands.
- -amylase, utilizes starch
as a substrate and
produces reducing sugars
as products:
1. maltose (disaccharide)
2. dextrin (oligosaccharide)
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enzymatic hydrolysis of starch
breaks large, insoluble starch
molecules into soluble starches:
1. amylodextrin
2. erythrodextrin
3. achrodextrin
 Amylodextrin: an
intermediate product of the
hydrolysis od starch that is
soluble in water and gives a
blue color with iodine.
 Erythrodextrin: a dextrin
that gives red color with
iodine.
 Achrodextrin: dextrins
formed, which give no color
when tested with iodine:
thus salivary amylase is
active.
Starch
- A white, tasteless, solid
carbohydrate.
- A mixture of two
polysaccharides, Linear
amylose and branched
amylopectin.
- Chemical formula of:
- Converted to glucose as a
source of energy for both
plants and animals.
- The presence of starch can
be detected by the blue-
black color when iodine
solution is added to the
sample.
Hydrolysis
- Chemical decomposition in
which a compound is split
into other compounds by
reacting with water.
- starch hydrolysis is the
conversion of starch to
dextrin, maltotriose,
maltose and glucose.
Factors affecting enzyme activity:
1. Temperature & pH
2. Enzyme & substrate
concentration
3. Inhibitors & Activators
Achromic point
- The rate of biochemical
reaction of salivary -
amylase is based on the
disappearance of reactant
(starch)
- The experiment is also
based on the determination
of achromic point, which
is the time taken by
salivary amylase to
hydrolyze starch to
achrodextrin at optimum
conditions.
Starch-Iodine Complex
- Digestion of starch is
monitored by noting the
disappearance of the blue-
black color given by the
starch-iodine complex.
- The complex is formed by
the complexation of
amylose with an iodide
polymer where triiodide
(I3-) forms by reacting
iodine (I2) with iodide
(I-)
Allosteric regulation
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- is the regulation of an
enzyme by binding an
effector molecule at the
protein’s allosteric site,
a site other than the
protein’s active site.
- An effector molecule is a
molecule that selectively
binds to a protein and
regulates its biological
activity; such that it
acts as a ligand that can
increase or decrease
enzyme activity.
- -amylase, chloride ion is
the allosteric effector.
*-amylases have a binding
site for chloride ion,
which contains a
positively charged amino
acid residue that assists
in the binding of the
negatively charged
chloride ion, and the size
of the binding pocket
excludes larger negative
ions.
- The binding chloride
causes a conformational
change to -amylase
switching it to the more
active state.
Effect of temperature
- Rate of enzyme catalyzed
reactions increase
proportionally with the
rise in temperature.
- This is due to the
increase in kinetic energy
that consequently increase
the frequency of collision
between enzymes and
substrates.
- Thus, the increase in
temperature increases the
amount of energy available
for the reactants to reach
its transition state, the
ES complex.
- The rate of most enzyme
reactions approximately
double for every 10C rise
in temperature (Q10=2)
- Optimum Temperature is the
temperature at which the
enzyme has maximum
activity.
- When the optimal
temperature is reached,
the increase in reaction
rate due to increase in
temperature will suddenly
drop.
- Above the maximum velocity
the enzyme denatures.
- When an enzyme denatures,
its conformation or
structure alters
irreversibly.
- The increased thermal
agitation breaks down the
secondary, and tertiary
structures of the enzyme
that rely on weak hydrogen
bonds, van der Waals
forces and ionic bonds.
- This alters the shape of
the active site such that
the substrate no longer
has a perfect fit.
 According to research, the
optimum temperature of
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salivary amylase (- will pick up a hydrogen
amylase) is 37C. ion (protonation)
- This results in the
 At low temperature, there
inability to form ionic
is minimal to no enzymatic
bonds between the
activity.
substrate and the enzyme.
At high temperature, the
enzyme denatures, and - Those bonds were necessary
to attach the substrate
ceases to function for the
and activate, thus at
substrate no longer has a
lower pH, the enzyme is
perfect fit to the active
inactive.
site of the enzyme.
At extreme pH, the enzyme
denatures.
- The tertiary structure of
the enzyme held together
by ionic bonds can be
disrupted, and it can lose
Effect of pH its shape.
- This change in the pH will  Consequently, this
affect the polar and non alters the shape of the
polar intramolecular active site such that
attractive and repulsive the substrate no longer
forces and alter the has a perfect fit.
interaction between enzyme
and substrate.
- At optimum pH, the
substrate attaches itself
to the enzyme via two
ionic bonds.
- The group allowing ionic
bonding are caused by the
transfer of a hydrogen ion
from a -COOH group in the
side chain of one amino
acid residue to an –NH2
group in the side chain of
EXPERIMENT 5:
another.
Characterization and
- At lower pH, or acidic
Identification of Carbohydrates
conditions, the –NH3+
Using General and Specific
group will not be
Tests
affected, but the –COO
Carbohydrates

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 Most abundant class of
organic compounds found
in living organisms.
 Has a general formula
of
 N>3
Simple sugars
- Carbohydrates that are
quickly absorbed by the
body to produce energy.
Glycosidic bond
- A bond that connects the
anomeric carbon of a sugar
to alcohol oxygen.
- Classified as either an
alpha or beta linkage
Reducing sugars
- Saccharides bearing
anomeric carbons that have
not formed glycosidic
bonds.
 Classification
- Number of carbon atoms
(e.g. triose = 3C; pentose
= 5C)
- Nature of reactive
carbonyl group (aldehyde
or ketone)
- pH = 6.7
Major source of metabolic
energy for plants and animals
that depend on plants for food.
Serve as structural material
One of three essential
components of DNA (deoxyribose)
and RNA (ribose)
Basic carbohydrate unit
2 Types:
1. aldoses – contain an
aldehyde group
2. ketoses – contain a ketone
group
Cannot be broken down into
smaller units by hydrolysis
Name Description
Xylose Aldopentose: Reducing
sugar; isolated from
wood; not metabolized
by humans
Glucose Aldohexose; reducing
sugar; also called
blood sugar
Galactose Aldohexose; reducing
sugar
Fructose Ketohexose; reducing
sugar; commonly found
with glucose in fruit
juices
If it is composed of
monosaccharide units, it can be
hydrolyzed
Name Description
Lactose Milk sugar; GLU+GAL;
glycosidic bond is 𝛽(1-6)
Maltose Malt sugar; GLU+GLU;
glycosidic bond is (1-4)
Sucrose Table sugar; GLU+FRU;
glycosidic bond is  (1-
2) or  (1-4)
Composed of monosaccharide
units linked together by
glycosidic bonds.
Homopolysaccharides
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- consist of one type of
monosaccharide
Heteropolysaccharides
- consist of more than one
type of monosaccharide
POLYSACCHARIDE
NAME DESCRIPTION
Amylose Linear; composed of
glucose residues; (1-
4) glycosidic bonds,
component of starch
Cellulose Unbranched; composed of
glucose units; (1-4)
glycosidic bonds;
component of cell walls
Glycogen Branched; composed of
glucose units; (1-4)
glycosidic bonds, (1-
6) bonds at branching;
storage polysaccharide
in animals
Molish test
- General test for the
presence of carbohydrates
- This test is useful for
identifying any compound
which can be dehydrated to
furfural or
hydroxymethylfurfural in
the presence of H2SO4
- Furfural – is derived from
the dehydration of
pentoses and pentosans,
while
hydroxymethylfurfural is
produced from hexoses and
hexosans.
- Reagents: conc. H2SO4 –
used to dehydrate
carbohydrates to form a
furfural or 5-
hydroxymethylfurfural
- -naphtol in 95% ethanol –
reacts with the cyclic
aldehydes to form purple
colored condensation
products.
1. Dehydration
2. Condensation with -
naphthol
The furfurals further react
with -naphthol present in
the test reagent to produce
a purple product.
Anthrone test
- Calorimetric test that
causes blue-green solution
to appear when sugar is
present in a sample.
- This test determines how
much sugar concentration
is in a sample of any
substance, including
carbohydrates.
- Reagents: conc. H2SO4;
anthrone reagent
- Principle: hydrolysis of
carbohydrates, dehydration
forming either a furfural
or a 5-
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hydroxymethylfurfural; - down or hydrolyzed into
condensation of anthrone smaller carbohydrate
via anthranol units. The blue-black
intermediate. color is not produced.
Therefore, it can indicate
Carbohydrates are completion of hydrolysis
dehydrated by using conc. when a color change does
Sulfuric acid to form not occur.
furfural, which in turn - Iodine atoms can then fit
condenses with anthrone to into the helices to form a
form a bluish-green starch-iodine or glycogen-
complex. iodine complex. The starch
in the form of amylose and
amylopectin has less
Iodine test branches than glycogen.
- Test for helical Helices of starch are
carbohydrates such as longer than glycogen
amylose. therefore more iodine
- Polysaccharides can trap atoms bind.
iodine molecules and - The result is that the
produce a deep blue-black color produced by a
product. starch-iodine complex is
- Lugol’s iodine reagent more intense than that
yields a blue-black color obtained with glycogen-
in the presence of starch. iodine complex.
- Performed on
monosaccharides and
disaccharides that does SPECIFIC TEST FOR CARBOHYDRATES
not yield deeply colored
products. PART I. Mucic acid test for
- A positive test is a blue- galactose and lactose.
black color in starch PART II. Tests based on the
while red in glycogen. reducing properties of sugars.
- The reaction is due to the PART III. Tests for the
formation of polyiodide production of furfural or a
chains from the reaction furfural derivative.
of amylose and iodine.
- Principle: The amylose in PART I
starch forms helices where
iodine molecules assemble, MUCIC ACID TEST
forming a dark blue or - For galactose and lactose
black color. When starch - Another name for galactric
is broken acid

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Reaction: oxidation of most
monosaccharides by nitric acid
yields soluble dicarboxylic
acid. However, oxidation of
galactose yields an insoluble
mucid acid. Lactose will also
yield a mucic acid due to
hydrolysis of the glycosidic
linkage between the glucose and
galactose subunits.
Reagent: conc. HNO3
Positive result: Rhombic
crystals (broken glass)
Principle involved: 1,6-
oxidation of sugars whereby
galactose-containing
carbohydrates form a meso
compound which upon standing
yields crystals.
PART II. Tests based on the
reducing property of a
carbohydrate (sugar)
A. Benedict’s test –
performed under mildly
basic conditions
Reagents: CuSO4, Na2CO3,
Na3C6H5O7, Sodium citrate (pH
10.5) in water
Sodium carbonate is required to
make the solution alkaline.
Benedict’s reagent incorporates
sodium citrate to keep the
cupric salts in solution by
forming complex ions with them,
preventing precipitation of
CuCO3.
Positive result: formation of
bricked ppt (Copper I oxide)
*a greenish color indicates
only a little reducing sugar,
yellow, a bit more, and red, a
lot.
Reactions: Monosaccharides and
these disaccharides that have a
potential aldehyde group will
reduce benedict’s reagent to
produce a red ppt of copper (I)
oxide. Ketoses also act as
reducing sugars because the
ketone group on carbon 2
isomerizes to give an aldehyde
group on carbon 1.
Principle: oxidation of
carbohydrates (reducing sugars)
by copper ions to form a
carboxylate ion group
Sodium gluconate is the sodium
salt of gluconic acid (aldonic
acid-only aldehyde is
oxidized).
Why is sucrose the only
nonreducing agent while maltose
and sucrose are?
- Maltose and lactose both
has reducing ends to them
due to a free anomeric
carbon. Sucrose’s carbon
is not free.
This is because sucrose is a
disaccharide composed of an
aldose and a ketose, the latter
of which forms a five membered
ring (instead of 6) with the
sixth carbon hanging off the
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end of the ring, attached to
what would be considered a
“free anomeric” carbon.
B. Barfoed’s test
- Distinguishes reducing
monosaccharides and
reducing disaccharides by
a difference in the rate
of reaction.
Reagents: Cu(Ac)2 copper
acetate
Dilute CH3COOH acetic acid
(pH 4.6)
Positive result: formation
of brick red ppt (Cu2O)
Principle: Oxidation of a
reducing monosaccharide in
an acidic condition is
faster than a disaccharide.
Reactions: Reducing
monosaccharides are oxidized
by the copper ion in
solution, forming carboxylic
acid and red ppt of copper
(I) oxide within 3 mins.
Reducing disaccharides
undergo the reaction in a
much slower rate.
PART III. Test for production
of furfural or a furfural
derivative.
Aldopentoses and ketopentoses
rapidly undergo dehydration to
give furfural under acidic
conditions.
Ketohexoses rapidly yields 5-
hydroxymethylfurfural under
acidic conditions.
Aldohexoses are slowly
dehydrated to 5-
hydroxymethylfurfural under
acidic conditions.
A. Bial’s Orcinol Test for
Pentoses (xylose)
- Used to differentiate
pentoses from hexoses.
- Dilute sugar solutions
(0.02M) are necessary in
this test to reduce the
potential interference as
the color for ketose
yields similar to orcinol.
Reagents: Orcinol (5-
methylresorcinol) –
condensation reagent
FeCl3 – catalyst
HCL
Positive result: formation of a
blue green condensation product
Principle: dehydration forming
a furfural & condensation with
orcinol
Reactions:
- Pentose sugars yield
furfural on dehydration in
acidic solution. Furfural
reacts wit orcinol and
ferric chloride to give a
blue green condensation
product
- xylose is positive
- hexose sugars give 5-
hydroxymethylfurfural
which reacts with the
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reagent to yield colors
such as green, brown, and
reddish brown.
B. Seliwanoff’s test for
Ketohexose (fructose &
sucrose)
- Depends on the relative
rates of dehydration of
carbohydrates.
- Used to distinguish
aldohexoses (glucose &
galactose) from
ketohexoses
- Concentration of HCl must
not me more than 12%.
Reagents:
Specific tests
Resorcinol – condensation
reagent
HCl- dehydrating acid
Positive result: Formation
of cherry red condensation
product
*sucrose hydrolyzes to give
fructose, which eventually
reacts to produce cherry red
colored product.
Reaction:
Ketohexose reacts rapidly to
give 5-
hydroxymethylfurfural, while
an aldohexose reacts more
slowly. 5-
hyroxymethylfurfural reacts
the with resorcinol to give
cherry red condensation
product within 2 mins. An
aldohexose will show a light
pink color that takes a
longer time to develop when
reacted with Seliwanoff’s
reagent.
SUMMARY TO TEST RESULTS
General tests
 Molisch test – positive
results for all three
polysaccharides; glycogen,
cellulose, and amylose.
 Anthrone test – positive
for all three
polysaccharides; glycogen,
cellulose, and amylose.
 Iodine test – Both
glycogen and amylose
yielded positive results.
 Mucic acid test – positive
– galactose & lactose
 Benedict’s test – positive
– reducing sugars; a
negative result for
sucrose
 Barfoed’s test – positive
for monosaccharides;
negative for disaccharides
 Bial’s Orcinol test –
positive – pentoses;
negative –hexoses
 Seliwanoff’s test –
positive- ketohexoses;
negative – aldohexoses
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EXPERIMENT 6:
CHARACTERIZATION OF SAPONIFIABLE
LIPIDS

LIPIDS
- Insoluble in water, soluble
in non-polar solvents
FATS
- Solid/semisolid
- Obtained from animals
OIL
- Liquid
- Obtained from plants

GLYCEROL
- simple polyol (alcohol
containing multiple hydroxyl
groups) compound
- Soluble in H2O
FREE FATTY ACID
- carboxylic acid with a long
aliphatic (non aromatic)
tail
LECITHIN
- generic term for fatty
substances in animal and
plant tissues (available
from sources such as soy
beans, eggs, milk, marine
sources, rapeseed,
cottonseed, and sunflower)
- low solubility in H2O
DICHROLOMETHANE (CH2CL2)
- organic compound
- immiscible with water,
miscible with organic
solvents

FATTY ACIDS

Myristic acid (14:0)

Palmitic acid (15:0)

Palmitoleic acid (15:1)

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Stearic acid (18:0)
Oleic acid (18:1)
Linoleic acid (18:2)
Linolenic acid (18:3)
Arachidonic acid (20:4)
Fatty acids absent in animals:
Puntadecanoic acid (15:0)
Heptadecanoic acid (17:0)
Nonadecanoic acid (19:0)
COCONUT OIL
- extracted from the kernel or
meat of matured coconuts
- composed mainly of lauric
acid
- high saturated fat content
- slow to oxidize, resistant
to rancidification (chemical
decomposition of fats, oils,
and other lipids)
- lasts up to 2 years without
spoiling
CANOLA OIL
- extracted from rapeseed
- low in saturated fat
OLIVE OIL
- obtained from olives
- composed mainly of oleic
acid and palmitic acid and
of other fatty acids
PALM OIL
- derived from mesocarp of the
fruit of the oil palms
- has 30 phenolic compounds
- has high saturated vegetable
fats
SESAME OIL
- derived from sesame seeds
- high proportion of
polyunsaturated linoleic
fatty acids
CORN OIL
- extracted from the germ of
corn
- high smoke point
 the number that indicates
the degree of unsaturation
of an oil
 defined as the amount of
bromine in grams accepted by
100g of test material
 the higher the bromine
number, the more unsaturated
the oil
 similar to the iodine number
 because of the different
reactivity of bromine and
iodine, the bromine and
iodine number cannot be
compared or converted to the
other (Br is more than
reactive than I)
 DCM was used in order to
dissolve the oils and fats
and to make the sample
easily mix with the 5% BR-
DCM
 Use of DCM (dichloromethane)
with Bromine:
 Good solvent for
bromine
 Doesn’t react
with bromine
SAPONIFICATION NUMBER
- Defined as the number of
milligrams of potassium or
sodium hydroxide required to
neutralize the fatty acids
in 1g of fat.
- Also known as Koettstorfer
number.
- It gives information
concerning the character of
the fatty acids of the fat.
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- Inversely proportional to Corn 188-193
the molecular weight of the Palm 196-205
fat/oil. Coconut 246-260

Formation of Soap
- After the addition of NaOH  All vegetable oil samples
and shaking of the oil/fat, are positive for grease-spot
presence of bubbles and test
precipitate are on top of
 The least unsaturated oil
the solution.
sample is coconut oil while
- This indicates that the
the most unsaturated is the
formation of fatty acid
sesame oil
salts which is also known as
“soaps”
- The test for the presence of
glycerol.
- It is done through heating
the oil sample with KHSO4
- Upon heating with potassium
bisulfate (dehydration),
that would yield “acrolein”
(unsaturated aldehyde)
Reagents used: KHSO4 (dehydrating
reagent)
Principle: Oxidation; dehydration
with the presence of heat
 Further heating would result
to the polymerization of the
acrolein, which is indicated
by the blackening of the
reaction mixture.
 Both the pungent smell and
black color indicate the
presence of glycerol.
 Positive results for
phosphoglycerin (lecithin)
The saponification test is
positive for lipids that can
undergo base hydrolysis and has
ester linkages like Triglycerides
Waxes.

oil Saponification
no.
Rapeseed/canola 170-179
Olive 185-196

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- Phosphatidyl serine
- Sphingomyelin

EXPERIMENT 7: - Unlike triglycerides, which

Isolation and Characterization of have three fatty acids,
Complex Lipids in the Calf’s Brain phospholipids have two fatty
acids that help form a
Lipids diacylglycerol. The third
- Are a class of biological carbon of the glycerol
molecules defined by low solubility backbone is also occupied by
in water and high solubility in a modified phosphate group.
organic solvent. - Phosphatidylcholine and
- It is a broad class of phosphatidylserine are
biomolecule whose function ranges examples of two important
from being a membrane structural phospholipids that are found
material, energy storage and in plasma membranes
messenger molecules
- simple and complex
Cerebroside
NON-SAPONIFIABLE  Ceramide + monosaccharide
- Lipids without an ester  Galactocerebroside
Functional Group
E.g. Cholesterol Phosporylated Lipids
SAPONOFIABLE
 Have polar and nonpolar
- Lipids with an ester
groups (amphipathic)
Functional Group that can be
hydrolyzed under basic  Contain phosphoester groups
conditions  Lipids that contain other
groups of atoms other than
Simple Lipids carbon, hydrogen & oxygen
- Esters of fatty acids Lecithin: Phosphatidyl Choline
w/alcohols (Phosphatidycholine)
- Fats and waxes  Phosphatides of Choline
Complex Lipids  Glycerophospholipid
- Esters of fatty acids Non-Phosphorylated Lipids
containing groups in - No other constituents other
addition to an alcohol and a than C, H, O
fatty acid  Triglycerides
- Phospholipids (lecithin), - Composed of 3 fatty acyl
glycolipids groups esterified to
Phospholipids glycerol
- Major components of cell - Neutral
membrane - Non polar
 Contain phosphoric acid
H3PO4 CALF’S BRAIN
- Lecithin
- Cephalin

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- Composed of galactolipids
and sphingolipids,
particularly sphingomyelin
Galactolipid
- is a glycolipid with
galactose as a sugar but
unlike a glycosphingolipid
it lacks nitrogen.
- Glycolipids are found in
high concentrations in
myelin sheaths and in the
CNS.
- These lipids comprise
cerebroside fatty acid
esters, monogalactosyl
diglycerides, and
alkylgalactolipids
Sphingomyelin
- is a type of sphingolipid
found in animal cell
membranes, especially in the
membranous myelin sheath
that surrounds some nerve
cell axons.
- It usually consists of
phosphocholine and ceramide,
or a phosphoethanolamine
head group
- Can also be classified as
sphingophospholipids
- In humans, it represents
~85% of all sphingolipids,
and typically
make up 10-20 mol % of
plasma membrane lipids.
- Sphingomyelin content in
mammals ranges from 2 to 15%
in most tissues, with higher
concentrations found in
nerve tissues, red blood
cells, and the ocular lenses
ISOLATION TECHNIQUES
§ EXTRACTION
- Process of separating a
desired substance when it is
mixed with others oHexane
§ TRITURATION
- a process used to purify
crude chemical compounds
containing soluble
impurities o acetone
§ RECRYSTALLIZATION
- technique used to purify
chemicals. By dissolving
both impurities and a
compound in an appropriate
solvent, either the desired
compound or impurities can
be coaxed out of solution,
leaving the other behind.
o95% ethanol
Chloroform: methanol (2:1 v/v)
- Organic mixture
- Chloroform denatures
proteins, eliminates
protein-lipid complexes in
egg yolk, volatile, and is
denser than water
- Methanol weakens hydrogen
bonds between lipids and
proteins unbinding them
- The proportion of chloroform
and methanol (2:1, v/v) make
the mixture more stable
EXTRACTION
1% NaCl Solution
- The different density causes
two layers to form; aqueous
(top) and organic (bottom)
- Makes the soln more polar
and separates the polar
compounds from the lipids
Kazel Mae Ramos, 2016
 SCITAMA
which are non-polar
compounds.
- Removes some water from the
lipid extract
DRYING
Anhydrous Sodium Sulfate Na2SO4
- An inorganic anhydrous salt
- A strong drying agent
- Is used because the
molecules in the lipid
extract have a high affinity
in water
Hydroquinone
- Functions as a powerful
antioxidant, preventing the
oxidation of the lipids
- The two hydroxyl groups are
easily oxidized
Acetone
- Separates the phosphorylated
lipids from non-
phosphorylated lipids
- Is easy to remove by
evaporation after being used
as a solvent
- Non-phosphorylated lipids
(e.g sterols) will dissolve
in the acetone solution
- The acetone can dissolve the
non-phosphorylated lipids
because they are both highly
non polar molecules.
EXPERIMENT 8:
Isolation and Characterization of
DNA from Allium cepa (Onion)
Why onion?
- It is used because it has a
low starch content which
allows the DNA to be seen
more clearly.
Why choose white over red onion?
- Composed of longer strands
of DNA compared to red
oinions
- The isolation of DNA from
onion has three basic steps:
a. Homogenization
- Break up the onion tissues
to separate and open cells
b. Deproteinization
- Purify the homogenate by
removing proteins that are
associated with cells and
DNA molecules
c. Precipitation of DNA
- Force DNA to come out of
solution, or precipitate, by
using very cold alcohol
Homogenizing Solution
- Separates the DNA from the
chromosomal protein through
its chemical components
which will cause the
proteins to precipitate out
of solution
1. Sodium dodecyl sulfate (SDS)
- Detergent used to break down
nuclear membrane
- Anionic detergent which
emulsify lipid and protein
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components of the cell by
disrupting the noncovalent
interactions that hold the
cell membrane together
- Detergent form complexes
with these lipids and
proteins causing them to
precipitate out of the
solution
- Chelating detergent
2. Ethylenediaminetetraacetic
acid (EDTA)
- Inactivates DNAse (breaks
down DNA)
3. Sodium chloride (NaCl salts)
and sodium citrate
- NaCl and sodium citrate
provide sodium ions that
neutralize the negative
charge of the DNA backbone
(phosphates)
Why heat in a water bath until the
solution reaches 60C?
- Heating at 60C softens the
phospholipid in the cell
membrane and denatures DNAse
Why should the temperature be kept
at 60C?
- To prevent the denaturation
of DNA that involves the
breaking of hydrogen bonds
between the bases in the
duplex
Removal of the first few layers
and mincing of the onion
- Cell lysis or cellular
disruption
- To extract biological
molecules including
organelles, proteins, DNA,
RNA and lipids from inside a
cell
Papain
- Proteolytic enzyme
- Degrades DNAses,
_____________ and proteins
- Denature the proteins
clinging to the DNA making
the molecule flexible and
easy to spoil
Why transfer the flask immediately
into an ice bath?
- Slows down DNA breakdown
- At room temperature, DNA
begins to denature by the
action of DNAse (present in
cell extracts)
Why is the onion tissue mixed in a
blender?
- To allow the release of DNA
with homogenization media,
which breaks down the cell
wall, cell membrane and
nucleus membrane
Why should it be blended for 45
seconds only?
- Exceeding to 45 seconds
exposes the DNA to sheer
forces that rapidly break
the DNA into shorter and
shorter length
Filtering of the homogenate
through a cheesecloth
- To remove the bigger debris,
such as cell walls,
membranes, or to remove any
solid material, resulting in
a clear cell homogenate
- More efficient compared to
filter paper
Why use an ice-cold 95% ethanol?
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- Ice-cold alcohol is added to
cause the DNA to precipitate
out of solution
- A 95% ethanol is recommended
since DNA is not soluble in
alcohol and the colder the
alcohol, the less soluble it
is
The DNA is precipitated out of
this solution using salt and
ethanol. Salt neutralizes the
charges on the phosphate groups in
the DNA backbone. The alcohol,
having a lower dielectric constant
than water, then allows the sodium
ions from the salt to interact
with the negatively charged
phosphate groups closely enough to
neutralize them and let the DNA
fall out of solution.
Since DNA strands are negative,
ethanol being an alcohol, which is
non-charged, acts to repel the
negative DNA.
UV/Vis Spectroscopy
- Determines the purity of the
isolated DNA
- The absorbance at 260 nm is
due to the DNA species,
while the absorbance at 280
nm is due to the protein
species
Absorbance ratio (A260/A280)
- Gives the relative
measurement of DNA and
protein content in the
isolated DNA
- A260/A280 – 1.8-1.9 – pure
DNA free of protein
contamination
- A260/A280 – 1.8-1.9 –
indicates protein
contamination
- A260/A280 – 1.8-1.9 –
indicates the presence of
RNA
B. Acid Hydrolysis
Reagents:
1.0M HCl
- causes the dissociation of DNA
into its components
- causes depurination
1.0M NaOH
- neutralizes the solution; needed
so that it wouldn’t affect the
tests
Why not use alkaline hydrolysis?
- Pentose of DNA does not have
an –OH group at the 2nd
carbon
- No formation of
monophosphate intermediate
- DNA is stable in alkaline
hydrolysis
Why is the DNA-HCl mixture heated
at 100C?
- To be able to destroy the
hydrogen, phosphodiester and
glycosidic bonds
Results to dissociation of DNA
into its components:
*phosphate group
*purine and pyrimidine
*deoxyribose
Result of hydrolysis: Breaking of
bonds associated with the DNA
molecule
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1. Hydrogen bonds
- Heating to temperature above
80C
2. Phosphodiester bonds
- Heating to temperatures
above 90C
- Acids, with pH less than
3. Glycosidic bonds
- Acids, with pH less than 2
Test for Deoxyribose (Dische
Reaction)
Standard: Deoxyribose standard
solution
Reagents: Diphenylamine reagent
(contains conc. H2SO4)
Positive result: blue colored
solution
Principle:
1. Dehydration of deoxyribose
by H2SO4 (formation of 5-
hydroxy-levulinaldehyde)
2. Complexation with
diphenylamine (blue color)
Test for Phosphate
Standard: Phosphate solution
Reagents: Conc. H2SO4, conc. HNO3,
2.5% ammonium molybdate solution
Positive result: yellow
precipitate (ammonium
phosphomolybdate)
Test for Purines (Murexide Test)
Standard: solid guanine
Reagents: Conc. HNO3, 10% KOH
Positive result: Red purple
residue
Principle:
1. Oxidation of purine by conc.
HNO3 (forming dialuric acid
and alloxan)
2. Condensation reaction of
alloxan to form alloxanthin
3. Neautralization, forming red
purple murexide (potassium
salt of purpurate)
Test for Pyrimidines (Wheeler-
Johnson)
Standard: Cytosine solution
Reagents: Bromine water, Ba(OH)2
Positive result: Purple
precipitate; negative for Thymine
because of methyl group
Principle:
1. Bromination of pyrimidine to
form dialuric acid
2. Neutralization of dialuric
acid by Ba(OH)2 to form
Barium salt of dialuric acid
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Kazel Mae Ramos, 2016