Acid hydrolysis of

proteins for
chromatographic
analysis of amino
acids.

Members:

 are

largebiomolecules
ormacromolecules consisting of one or
more long chains of amino acid residues.
Are complex compounds that contains
amino acids.
They are hydrolized by acids, alkalies
and enzymes, hereby they are broken
down to simpler molecules and finally to
their individual amino acid components.
Proteins perform a vast array of functions
within living organisms, including
catalyzing metabolic reactions, DNA
replication, responding to stimuli, and
transporting molecules from one location
to another

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One technique in following the course
s
of the hydrolysis of protein is the use
of paper chromatography to separate
and identify the amino acid contents.

Chromatography
is

a common technique for

separating chemical substances.
is partially characterized by the
medium on which the separation
occurs.
This medium is commonly identified
as the stationary phase.
Stationary phases that are typically
used include paper (as in this
experiment), thin plates coated with
silica gel or alumina, or columns
packed with the same substances.
The mobile phase is the medium
that accompanies the analyzed
substance as it moves through the
stationary phase. B

What is paper
Chromatography?

Paper Chromatography
is

ananalytical method that is used to separate

coloured chemicals or substances. This can also be
used in secondary or primary colours in ink
experiments.
Theretention factor (R ) may be defined as the ratio of
the distance travelled by the substance to the
distance travelled by the solvent. R values are usually
expressed as a fraction of two decimal places. If
Rvalue of a solution is zero, the solute remains in the
stationary phase and thus it is immobile. If R value =
1 then the solute has no affinity for the stationary
phase and travels with the solvent front.

Protein Hydrolysis:
Acid Hydrolysis

a process in which a
protic acid is used to
catalyze the cleavage
of a chemical bond via
a nucleophilic
substitution reaction,
with the addition of the
elements of water
(H2O).

Protein Hydrolysis:
Alkaline Hydrolysis
the

process thus
destroys all of these
classes of compounds,
reducing them to their
building blocks and, in
some cases, degrading
them even further into
smaller molecules

Protein Hydrolysis:
Enzymatic Hydrolysis
a

process in which enzymes

facilitate the cleavage of bonds
in molecules with the addition of
the elements of water.

It

plays an important role in the

digestion of food.

It

may be used to help provide

renewable energy, as with
Cellulosic ethanol.

Color Reaction of Hydrolyzate :

Biuret Test

Color Reaction of Hydrolyzate :

Xanthoproteic Test

Color Reaction of Hydrolyzate :

Ninhydrin Test

Color Reaction of Hydrolyzate :

Reduced Sulfur Test

Color Reaction of Hydrolyzate :

Hopkins Cole Test
is

specific for tryptophan, the only

amino acid containing an indole
group. The indole ring reacts with
glyoxylic acid in the presence of a
strong acid to form a violet cyclic
product

only

reacts with proteins containing

tryptophan. The protein solution is
hydrolyzed by the concentrated
sulfuric acid at the solution interface.
Once the tryptophan is free, it reacts
with the glyoxylic acid to form the
violet product

Color Reaction of Hydrolyzate :

Sakaguchi Test
a

chemical test
used for detecting
the presence of
arginine in
proteins

PROCEDURE # 1
Protein Hydrolysis
1.1 Acid Hydrolysis (Casein)
1.2Alkaline Hydrolysis (Gluten)
1.3 Enzymatic Hydrolysis (Bean
Protein

TOPIC
1.1 Acid
Hydrolysis
(Casein)

1.2 Alkaline
Hydrolysis(
Gluten)

TEST

APPARATU
S
Erlenmeye
r Flask
Cotton
Plug
Autoclave
Filter Paper
Funnel

CHEMICALS

0.5 g
Casein
5 ml 6N
H2So4
Solid
Ba(OH)2
Sat.
Ba(OH)2
Erlenmeye 0.5 gluten
r Flask
6.4 g
Ba(OH)2
Cotton
Plug
3 ml 6N
H2So4
Test tube
Autoclave 8N H2So4
Filter
paper

TOPIC

1.3
Enzymatic
Hydrolysis

TEST

APPARATUS

CHEMICALS

Beaker
Water Bath
Microscope

10 g Protein
(Bean
protein
Powdered
Albumin
Gluten
50 mL 0.05N
Na2CO3
10 mL 2%
Pancreatic
Solution

TOPIC

TEST
Biuret Test

Color
Reaction of
Proteins

APPARATUS

CHEMICALS

-Test tube

-1 mL 6M NaoH
-0.1% CuS04

Xanthoproteic Test

-Test tube
-Water bath

-1 mL conc. HNO3
-1 mL 3M NaOH

Ninhydrin Test

-Test tube
-Water bath

-1 mL 0.2% Ninhydrin

Hopkins Cole Test

-Test tube

-Hopkins Cole reagent

-1 mL conc. H2S04

Sakaguchi Test

-Test tube

-1 mL 10% NaOH
-1 mL 0.02% Naphthol
soln.
-1mL 2% Sodium
Hypobromite

-Test tube
-Water Bath

-5 mL 5% NaOH
-Crystals of Pb(Ac)2

(Casein, Gluten)

Lead Acetate Test

PROCEDURE #2
Chromatographic
Analysis

TOPIC

Chromatog
raphic
Analysis

TEST

APPARATUS

CHEMICALS

Beaker
Aluminum
Foil
Whatman
Filter Paper
Capillary
tubings
Dropper
Chamber

Ethanol
Ammonia
Ninhydrin Spray
Reagent
1mL 1% Argine
1mL 1%
Phenylalanine
1mL 1% Alanine
1mL 1% Lysine
1mL 1%
Tryptophan
1mL 1%
Glutamic Acid
1mL 1% Glycine

Acid hydrolysis:
Casein
Procedures

To 0.5 g of casein in a 50 ml E.R Flask, add 5 ml

6N H2So4. Cover the flask with cotton plug and
label flask properly.
Add 30 ml of distilled water and neutralize the
solution by adding solid Ba(OH)2 little by little.
(pH paper indicator maybe used to determine
complete neutralization). Use sat. Ba(OH)2
Solution if pH is near 7.
Filter the resulting milky solution. Dilute the
filtrate to 50 ml with distilled water.
Keep in a covered container & store in
refrigerator for Chromatographic analysis and
test for specific color reaction.
Perform test for specific amino acid. Request
chemical for Color reaction of Protein.

Alkaline
Hydrolysis
(Gluten)
Procedures

Place 0.5 g gluten in a 50 ml E.F flask, add 10 ml

boiling water & 6.4 g Ba(OH)2.
Plug with cotton and warm gently while mixing to
dissolve most of the hydroxides.
Label the test tube with tag.
Autoclave at 15 psi for 3 hours. Note the appearance of
the sample after autoclaving.
Dilute hydrolyzate with 320 ml distilled water and
neutralize by adding 3 ml of 6N H2SO4. Check the pH
after each adding the acid. When the pH has dropped
to 10, use 8N H2So4 until the pH reached a value of 7.
Filter off precipitate and wash it twice with 5 mL
portions of boiling water.
Make the combine filtrate up to 25 mL with water and
store hydrolyzate in a refrigerator for further analysis.
Perform test for specific amino acid. Request
chemical for Color reaction of Protein.

Enzymatic
Hydrolysis (Bean
Protein)
Procedures

 Suspend

10 g of protein (Bean protein),

(powdered albumin or gluten if
sufficient sample is taken from Expt. 4
cut into small pieces) in 50 ml distilled
water in a beaker.
Add 50 ml of 0.05N Na2Co3 as a
buffer.
Add 210 mL of 2% pancreatic soln. Mix
well.
Add 1 mL of 10% thymol in ethanol as
a preservative. Shake very thoroughly
and place in a water bath for 30
minutes at 37 degree Celsius.
Examine some crystals that may
separate under the microscope,
identify by doing test for specific
amino acids.

Chromatographic
Analysis
Procedures:

Prepare 30 mL of the solvent,

ethanol-water-ammonia (80+10+10)
ratio mixture in a 1 litter beaker.
Cover it with aluminum foil for about
15 minutes in order to reach
equilibrium.
Meanwhile, measure and cut 13x22
cm sheet of Whatman filter paper.
With a pencil, draw a line
lengthwise, 10 cm at the bottom of
the paper. Labeel it as the origin (0)
zero. Mark 8-10 spots at equal
intervals on the line with a pencil.
Using capillary tubings, apply 5
drops of amino acid standards with
drying in between spotting. (Use a
different capillary tube for each
amino acid to prevent
contamination)

Spot the protein hydrolyzate(in protein hydrolysis) to another

point in the same manner. (Step 5)
Staple the paper ro form a cylinder, with the applied amino
acids and the sample hydrolyzate on the outer space making
sure that the edges do not overlapped.
Develop the paper in a liter beaker containing the solvent.
Cover the beaker with aluminum foil.
Watch until the solvent reaches the uppermost part of the paper
about 1 cm. from the upper edge.
Remove from the chamber and put in an oven for 5 minutes to
dry. Spray the dry paper with Ninhydrin spray reagent. The
amino acids will appear ass blue or purple or yellow spots.
Encircle all spots with pencil
Calculate the Rf value of each amino acid
What are the Rf values of the unknown amino acids present in
hydrolyzate.
What could be the amino acid constituents of the protein
hydrolyzate based on the Rf values.

Results

for Protein Hydrolysis

(CASEIN)

Observation

Acid

Alkaline

When H2So4
Brown Solution Milky white
was added &
and
Solution
after it was
precipitate
hydrolyzed,
the solution
became acidic;
turned neutral
in addition of
Ba(OH02

Enzymatic

White
precipitate

Results
for Protein Hydrolysis
(Gluten)

Observation

When added with

10 mL boiling
water and 6.4g
Ba(OH)2 it was
hydrolysed. the
solution became
basic upon adding
3mL of 6N H2SO4 ;
turned neutral in
addition of 8N
H2SO4.

Acid

Alkaline

Enzymatic

Results
for Protein Hydrolysis
(Bean Protein)

Bean Protein
Observation

Acid

10g (protein) +
50ml H2O=
some of the
ppt rise
+ 50 mL Na2CO3yellow green
color
+ pancreanin=
dissolved

After place ment

on H2O, both for
30 mins. At 37
degree celcius =
the ppt
suspended at
the bottom of
the flask
Crystals
separate at the
top of the liquid
portion

Hopskin cole
Formation of
Bubbles
Formation of
white layer of
ppt
Pink color

Evolution of gas
Formation of
bubbles
Formation of
ppt

Alkaline

Enzymatic

Biuret test=
orange-pale
orange
Xantropoteic test
=formation of
yellow pptand
bubbles

Ninhydrin TestReddish Brown

Results
for Color Reaction
of Hydrolyzate

Color reaction of Hydrolyzate:

Test

Acid
Hydrolysis

Alk.
Hydrolysis

Enzymatic

1. Biuret

-light blue to
deep purple

- blue-violet
soln

- Orange to pale
orange

- blue-violet
soln

- reddish-brown
soln

2. Ninhydrin
3.
Xanthoproteic

Intense yellow
color

- Clear yellow
soln

-Evolution of
gas
-Formation of
bubbles

4. Hopkins Cole

Clear brown
Solution (-)

- Clear violet
ring

-Formation of
bubbles
-Formation of
white layer

5. Sakaguchi

Darker red color - Clear yellow

soln

Results
for Chromatographic
Analysis

Chromatographic Analysis
Distance travelled by:
Std. Amino
Acid

Spot

Solvent
Front

Rf Value

Phenylalanine

3.4 cm

5 cm

0.68

Tryptophan

2.97 cm

4.5 cm

0.66

Arginine

0.9 cm

4.5 cm

0.20

Lysine

0.84 cm

6 cm

0.14

Glutamic Acid

1.95 cm

6.5 cm

0.30

Glycine

1.69 cm

6.5 cm

0.26

Alanine

2.39 cm

6.3 cm

0.38

Rf= Distance travelled by spot

Distance travelled by the solvent front.

Distance traveled by:

Amino Acid
Hydrolyzate

Spot

Solvent
Front

Rf Value

Guide Questions:

What are the advantages

and disadvantages of
enzymatic hydrolysis and
acid hydrolysis?
Enzymatic hydrolysis advantages
include being a mild process
compared to other chemical
conversions, it requires less energy,
and produces less by-product. The
disadvantages include low reaction
rates and a high yield of sugar
monomers.

W
th rite
po e h an
a c ly p y d e q
id ep rol u
so tid ys ati
lu e is on
tio in of
fo
n
r
st
ro
ng

What are the

limitations in the
use of paper
chromatography?
Semi-quantitative in nature.
Overlapping of spots of components
having close Rf values.
Higher concentration of components
often leads to streaking instead of
well-defined spots.
Errors in Rf calculations can result
from uneven flow of solvent front.
This can be caused by running out of
solvent at the bottom of the
chamber, uneven cutting of the filter
paper or unevenness of the bottom
of the development chamber.

What are the

limitations in the
use of paper
chromatography?
Improper sample spotting, spotting
below the marked line resulting in
dipping into the solvent or
accidental dipping of spot into
solvent while inserting the paper
into the solvent chamber.
Paper chromatographic techniques
cannot be used in separation of
volatilesubstances such as
hydrocarbons and volatile fatty
acids.
The lower limit for the detection of
most compounds is 1-5 microgram

Classification - Amino acid

classification according to reactive
group or R group
Aliphatic - Glycine, Alanine, Valine,
Leucine, Isoleucine
Aromatic - Phenylalanine, Tyrosine,
Tryptophan
OH- - Serine, Threonine
Acidic - Aspartic Acid, Glutamic
Acid
Acid amide - Aspargine, Glutamine
Basic - Arginine, Lysine, Histidine
Sulphur - Cystine, Methionine
Cyclic - Proline

Classify

the
Amino Acids
according to R
Group.

Thank You for Listening