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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
n
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P
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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
What is paper
Chromatography?
Paper Chromatography
is
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
It
It
only
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
-Test tube
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)
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
Alkaline
Hydrolysis
(Gluten)
Procedures
Enzymatic
Hydrolysis (Bean
Protein)
Procedures
Suspend
Chromatographic
Analysis
Procedures:
Results
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
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
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
Results
for Color Reaction
of Hydrolyzate
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
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
Spot
Solvent
Front
Rf Value
Guide Questions:
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
Classify
the
Amino Acids
according to R
Group.