PAPER
CHROMATOGRAPHY
GROUP 5 (A2)
OBJECTIVES
1.EXPLAIN THE PRINCIPLE OF
SEPARATING AMINO ACIDS BASE ON
THEIR SOLUBILITY/PH OF
CONCENTRATION
2.COMPUTATION FOR THE RF VALUE OF
EACH AMINO ACID USED IN THE
EXPERIMENT
QUESTION #1
EXPLAIN THE PRINCIPLE OF
PAPER CHROMATOGRAPHY.
PROCEDURES
1. USING WHATMAN #3 PAPER DRAW A PESO
COIN-SIZED CIRCLE IN MIDDLE WITH
PENCIL
2. FOLD PAPER INTO QUADRANTS AND LINE
WITH PENCIL
3. MAKE A SMALL HOLE JUST BIG ENOUGH FOR
WICK TO
BE INSERTED IN
IN EACH QUADRANT WRITE ONE OF THE
FOLLOWING IN PENCIL : ALANINE, GLUTAMIC
ACID, LYSINE AND UNKNOWN
PROCEDURES
4. DROP 1 DROP (0.02ML) OF 1 SOLUTION (AMINO ACID) ON DRAWN
INNER CIRCLE
OF PAPER.
5. REPEAT FOR EACH QUADRANT.
6. DRY WITH A BLOWER.
7. PASS PAPER THROUGH AN OPEN BOTTLE OF CONCENTRATED NH4OH
TO
NEUTRALIZE THE AMINO ACIDS.
8. CUT WICK TO 2 INCHES LONG, AND INSERT INTO MIDDLE HOLE.
PROCEDURES
9. POUR 1CM HIGH DEVELOPING SOLVENT INTO PETRI
DISH AND PLACE PAPER ON TOP
10. COVER PAPER WITH LID OF DISH
11. WAIT FOR SOLVENT TO REACH EDGES OF PETRI
DISH
12. REMOVE PAPER FROM DISH AND REMOVE THE
WICK
13. ALLOW TO DRY IN THE HOOD
PROCEDURES
14. SPRAY NINHYDRIN SOLUTION ONTO
PAPER
15. DRY WITH BLOWER UNTIL VIOLET
LINES APPEAR
16. CALCULATE RF-VALUES AND ATTACH
PAPER
DISC TO MANUAL
UNKNOWNS:
UNKNOWN 1 : ALANINE
UNKNOWN 2 : LYSINE
RESULT OF THE EXPERIMENT
EXPECTED RESULT
QUESTION #2
IDENTIFY THE AMINO ACIDS USED IN
THE EXPERIMENT ACCORDING TO
POLARITY. ASSOCIATE THEM WITH THE
POLARITY OF THE FILTER PAPER AND
THE SOLVENT. EXPLAIN THE
ASSOCIATION.
A
AMINO ACIDS
BUILDING
BLOCKS OF
PROTEIN
FUNCTIONS
1. BUILDING BLOCKS OF PROTEINS
2. PRECURSOR OF VARIOUS SUBSTANCES
• HEME, PURINE, CREATINE, GLUTATHIONE, HIPPURIC ACID
• EPINEPHRINE, SEROTONIN,MELATONIN
3. SOURCE OF ENERGY
4. SPECIAL AMINO ACIDS AS COMPONENTS OF CERTAIN TYPES
OF PROTEINS
• COLLAGEN
• PROTHROMBIN
5. SOME AMINO ACIDS OR THEIR DERIVATIVES ACT AS
CHEMICAL MESSENGERS
CLASSIFICATION OF
AMINO ACIDS BASED ON POLARITY
THE R- GROUP DETERMINES
WHETHER THE AMINO
ACID IS POLAR, NON-POLAR OR
NEUTRAL.
AMINO ACIDS USED IN
THE EXPERIMENT
Glutamic Acid Lysine Alanine
FILTER PAPER
(STATIONARY PHASE)
PAPER IS MADE OF CELLULOSE
FIBRES, AND CELLULOSE IS A
POLYMER OF THE SIMPLE SUGAR,
GLUCOSE.
THE POLYMER CHAINS OF
CELLULOSE HAVE -OH GROUPS
STICKING OUT ALL AROUND THEM.
DEVELOPING SOLVENTS: n-butanol:
glacial acetic: water (4:1:5)
(MOBILE PHASE)
NON POLAR SOLVENT
NON-POLAR MOLECULES IN THE MIXTURE THAT YOU
ARE TRYING TO SEPARATE WILL HAVE LITTLE
ATTRACTION FOR THE WATER MOLECULES ATTACHED
TO THE CELLULOSE, AND SO WILL SPEND MOST OF
THEIR TIME DISSOLVED IN THE MOVING SOLVENT.
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QUESTION
BASED ON THE CHEMICAL#3STRUCTURE
OF THE AMINO ACIDS USED, RANK
THEM FROM MOST HYDROPHILIC TO
MOST HYDROPHOBIC. (HINT: THE MORE
CARBON AND HYDROGEN IN THE
STRUCTURE THE MORE HYDROPHOBIC,
POLAR GROUPS REDUCE
HYDROPHOBICITY AND IONIZABLE
GROUPS REDUCE HYDROPHOBICITY
MORE THAN POLAR GROUPS.)
CLASSIFICATION OF
AMINO ACIDS BASED ON POLARITY
Glutamic Acid Lysine Alanine
1. POLARS (Most soluble in water)
2. NONPOLARS with SHORT R-GROUP
3. NONPOLARS with LONG R-GROUPS (Least soluble in water)
Water Non-polar
solvents
Polar R- soluble Insoluble Will move but
groups for a short
distance
Non-polar R- Insoluble Soluble Move further
groups in the paper
QUESTION #5
HOW CAN PH CONCENTRATION
AFFECTS THE MIGRATION OF AMINO
ACIDS.
• The pH determines the degree of ionization of organic compounds;
it can also affect the rate of migration of these compounds. When
increase pH, increases ionization of organic acids. Decrease in pH,
increases ionization of organic bases.
• When pH increases, acidic amino acid tend to be more hydrophilic
than basic amino acid.
QUESTION #4
COMPUTE FOR THE RF VALUE OF EACH
OF THE AMINO ACIDS. COMPARE THE RF
VALUE OF ALANINE, GLUTAMIC ACID,
AND LYSINE, IS THIS CONSISTENT WITH
THEIR CHEMICAL STRUCTURE?
EXPLAIN.
•A bulky structure (branched isomer) has a weaker
bond compared to a straight chained isomer. This is
because the area of contact between molecules is
less compared to the straight chained isomer. Hence
it moves faster and have a higher Rf value.
ALANINE GLUTAMIC LYSINE UNKNOWN
ACID
1 0.87 0.93 0.91
COMPUTATION OF RF VALUE
Distance from the baseline
travelled by solute
RF value = ----------------------------------------
Distance from baseline
travelled by solvent
RF VALUE
48 cm
RF value of Alanine = ----------------
48 cm
=1
RF VALUE
45 cm
RF value of Lysine = ----------------
48 cm
= 0.93
RF VALUE
42 cm
RF value of Glutanine = ------------
48 cm
= 0.87
CONSISTENT WITH THEIR CHEMICAL
STRUCTURE
•Alanine is a non-polar travelled the farthest.
Followed by the polar Lysine. Lastly is the
Glutamic acid a polar amino acid and more
shorter chain than the lysine.
QUESTION #6
WHAT POSSIBLE ERRORS COULD
CONTRIBUTE TO THE NON-SUCCESSFUL
RESULTS?
REFERENCES
Paper chromatography. http://www.chemguide.co.uk/analysis/chromatography/paper.html.
https://www.chemguide.co.uk/organicprops/aminoacids/background.html
Retrieved September 2016.
General Veterinary Biochemistry.
http://ecoursesonline.iasri.res.in/mod/page/view.php?id=53406
THANK YOU!
