amino acid and protein
DESCRIPTION
This is my complete report of Organic Chemistry 2TRANSCRIPT
HERMIN HARDYANTI UTAMI (111 304 0202)
CHEMISTRY DEPARTMENT
MATHEMATIC AND SCIENCE
FACULTY
STATE UNIVERSITY OF MAKASSAR
2012
ORGANIC CHEMISTRY 2
COMPLETE REPORT
RATIFICATION PAGE
The complete report of Organic Chemistry II with the title of “Caffeine
Extraction” which made by :
Name : Hermin Hardyanti Utami
ID : 111 304 0202
Group : VII
Class : ICP B
Have been checked by assistant and assistant coordinator. So, this report is
accepted.
Makkassar, December 2012
Assistant Coordinator
Fandi Ahmad, S.Pd.
Assistant
Muh. Jufri
Known By,
Responsibility Lecturer
Dra. Hj. Ramdani, M.Si.
A. Title of experiment
Amino acid and Protein
B. Purpose of experiment
1. Can prove the existence of a peptide bond
2. Can understand xanthoproteat reaction and biuret test against a wide - range
of protein content.
3. Can understand the solubility and amphoteric properties of amino acids.
C. Preview of Literature
Amino acids. 2,4-Dinitropheny deriVatsves. Thereuctron betwen 1 Fluoro-2,4-
dinitrobenzene and amino acids leade to 2,4-dinitrophenyl derivatsves. These are
oftenerystalline and process relatively sharp melting points.
2,4-(NOL)2 C6H3F + NH2 –CHR- CO2H 3
)( NaHCOi
HClii )(
2,4-(NO2)2. C6H3-NH-CHR-CO2H
To a solution or suspension of 0,25 gr of the amino acid in 5 ml of water
and 0,5 gr of sodiumhydrogen carbunale, add a solution and exrtact with ethet
(2x5 ml) to remove unchanged reagents. Pour the aqueous layer into 12 ml off
cold 15 percent hydrochloric acid mith virgorous a gitatron. This mixture should
be distinctly acid to congo red indicator paper. If the product separates as an oil
pour try to incude crystallisation by scratching or strring. Collect the derivative
bysuction filtration and recrystallise it from so percent ethanol.
(Vogel.1989:1279).
You are certain to be familiar with there common, pure proteins: albumin in
eggs, casenin milk and gelatin. Albumin egg while contains around 13% albumin,
from latin albus, white when you shake up egg white with water, you get what
looks like on almost cler solution. But this not true solution such as you get when
dissolve sall or sugar. It is another kind of “solution” called a “colloidal
dispersion”. For more about collloidal dispersions.
(Bren, Robert. 1960:99).
Mechanisme for reaction amino acid ninhydrinto from folored:
Paper chormatography onceplayed an imprortantrole in biochemical
analysis because it privided a method for separating, amino acids using very
simple equipment. Although more modern techniques are now more commonly
used, we’ll describe the principles are employed in modern separation techiques.
( Bruice.2003:969).
The technique of paper chromatography separales amino acid on the basic
of polarity.A few drops of a solution of a solution of an amino acid micture are
applied to the bottom of strip of filter paper the edge of the paper is then placed in
a solvent (typically a mixture of water acetic acid and butanol). The solvent
moves up the paper by capsllaryaction, carrying the amino acids wiht it.
Depending on their polaritres, the amino acids have different of finitres for mobile
(solvent) and stationary( paper) phases and there fore travel up the paper at
different rates.
Fedding prefermences of nectarivorous ants for sugarsand amino acids were
studied in an australian tropical rainforest usning artifical nectar solution .fipty
one ant species were recorded feeding on the solution. Preferences among
carbuhydrates were principally concordant betwen ant species in parred tests
sucrose was often preferred over fuctose, glucosa, maltose, melezitose, raffinose
and xylose respectively. Attractiveness of sucrose baits in creased with
concentation. (Blushgen, N.2004:155)
The main determinansof anathelete’s protein needs are their training regime
and habitual nutrisent intake. Most athletes ingest sufficient protein in their
habitual diet. Additional proteins will confer only a minimal, albert arguably
important, additional advantage. Given sufficient energy intake, lean body mass
can be maintained when a wide range of range of protein intake
(Tipton, K.O.2004:65).
Amino acid contain two funotional group are amine group (-NH2) and
carboxil group (-COOH). Amino acids nature contain amine group that bond in
carbum atom - to carboxil.
R-CH-COOH R-CH-COO-
NH3 +NH4
Amino acid amino acid in dipolar
(Tim Dosen.2012:17)
D. Apparatus and Chemical
1. Appararus
a. Test tube and rack 6 pieces/1 piece
b. Measuring cup 10 ml 1 piece
c. Drop pipette
d. 500 ml beaker 1 piece
e. wooden clamps
f. stir
g. spirit lamp
h. Kasa asbestos and leg three
i. Wash bottle
j. Funnel
k. Reflux
l. Condensor
m.rough and smooth cloth
2. Chemicals
a. Glisin
b. L- tirosin
c. L- aspartat
d. Aquadest
e. Sodium hydroxide (NaOH) 10 %
f. Litmus papers
g. Indicator universal
h. Chloride acid (HCl) 10 % dan 20 %
i. Kasein
j. Ice cubes
k. Sodium nitric (NaNO2) 5 %
l. Urea
m. Matches
n. Filtering Paper
o. Cupper(II)Sulphate CuSO4 2 %
p. Nitric acid concentrated (HNO3)
E. Work Procedure
1. Solubility and amphoteric properties
a. 1) Insert the test tube into the glycine
2) Add 2 ml of distilled water / aqudest into a test tube
3) Test the acidity of the resulting solution with litmus paper
4) Repeat the experiment with L-aspartic acid and L-tyrosine
b. a) Add 1 mL of 10% NaOH solution to the suspension of 0.1 grams of L
tyrosine in 2 mL of water, and record the results.
b) Insert a piece of litmus paper into the solution and menambahkansetetes by
drop until the solution is acidic started.
c) Stir for 1 minute and then observed and recorded the results.
d) Add 10 drops of the acid solution again, then observed.
c. a) Enter 0.1 grams of casein (milk protein) into a test tube.
b) Add 5 ml of distilled water and 2 mL of 10% NaOH laruta.
c) Close the test tube and shake until a colloidal solution.
2. Reaction with nitrous acid
1. a) Enter 0.1 grams of glycine into a test tube
b) Add 5 ml of 10% HCl solution
c) In another test tube, add 5 ml of 10% HCl solution for comparison.
d) Cooling the second test tube to ice water.
e) Adding carefully 1 ml of 5% NaNO2 solution. Into each test tube and
recorded the results.
2. Cooling the casein solution was prepared in ice water, then add 1 ml
solution of NaNO2.
3. Biuret test
1. a) Include 0.5 g urea into a dry test tube
b) Heat slowly until the urea melt and gas is formed.
c) Mecatat gas odor is tested with litmus paper wet mouth tube.
d) Continue heating until the formation of gas stops and the rest from solid.
e) Cool the solution, and then dissolving the solid in hot distilled water.
f) Filter the solution and add to the filtrate 2 ml of 10% NaOH solution, then
2 -3 drops of 2% CuSO4 solution.
g) Stir the solution and observe the color.
h) For comparison, dissolve 0.5 grams of urea in 3 ml of water.
i) Add 2 ml of 10% NaOH solution
j) Then add 2 -3 drops of 2% CuSO4 solution, and then compare the results
with the above observations.
2. a) Include 2 ml of distilled water in a 2 ml casein solution provided (1.c)
b) Then add 2 drops of 2% CuSO4 solution Stirring and observe.
4. Xantoproteat test
a. Placing 0.1 grams of casein into a test tube.
b. Add 2 ml of concentrated Nitric Acid
c. Heats slowly
d. Observing the color that occurs
e. Cooling the reaction mixture
f. Neutralize carefully with 10% NaOH solution, then add a bit of excess base.
g. Take note if the color of the solution changes.
5. Hydrolisis Protein
a. Entering 0.5 grams of casein into a test tube.
b. Adding 2 ml of 20% HCl solution, then heated above the spirit lamp.
c. Cooling the reaction mixture to room temperature.
d. Cooling the partial hydrolysis results in ice water.
e. Comparing the results with the experiment above
f. on the other, neutralizing with 10% NaOH.
g. Add 3 ml of 10% NaOH solution and 2 drops of 2% CuSO4 solution.
h. Preheat over spirit lamp, and then compare the results with the experiment
above.
F . Observation result
1. Solubility and amphoteric properties
a. Solubility test
1) 2 ml water (transparant) + glisin (white)→netral and soluble
2) 2 ml water (transparant) + tirosin (white)→acid pH=5 and not soluble
3) 2 ml water (transparant) + aspartic acid (white)→acid pH=3 and less soluble
b. Amphoteric test
1. 2mL aquades + 0,1 g L-Tirosin (white)+ 1ml NaOH 10%→yellow
transparant→base→HCl 10% 50 drops→yellow→stir→10 drops HCl
10%→acid solution pH=2.
2. 0,1 g kasein + 5mL aquades→transparant solution + 2mL NaOH
10%→transparant solution
2. Test with nitrous acid
a.1) 0,1 g glisin + 5mL HCl 10% transparent solution cooling 0ºC
transparent
solution+ 1mL NaNO2 5% transparent solution and bubbles
2) 5mL HCl 10% cooling 0ºC
transparent solution + 1mL NaNO2 5%
- transparent solution and not bubbles
b. 0,1 g kasein + 5mL aquades transparent solution and nit bubbles gas.
3. Biuret test
a. 0,5 g urea heat
transparent solution→litmus(base)→transparant solution+hot
water→ white precipitate → filter → filtrate + NaOH 10%→transparant
solution+CuSO4 2% 2 drops (blue) → purple solution
Compare :
0,5 g urea + 3mL aquades → transparent + NaOH 10% 2ml → transparent +
CuSO4 2% 2 drops (blue) blue solution
b. 0,1 g kasein + 2mL aquades transparent solution+ 2 drops CuSO4 2%
→blue solution.
4. Xantoproteat test
0,1 g kasein + 2mL HNO3 concentrated (transparent) → heated → transparent +
NaOH 10% until netral + indicator universal → netral → base solution (hot)
5.Hydrolysis protein
0,5 g kasein + 20 mL HCl 20% concentrated → transparant → reflux 40
minutes→18 ml (turbid)→divide 2 part
1st part → 9 ml +NaOH 3ml → yellow + 2 drops CuSO4 2% → purple solution.
2nd
part → cool down + NaOH 20 % → yellow solution + CuSO4 2% 2 drops
→purple solution
G. Discussion
1. Solubility and amphoteric properties
Amino acid that used are glysin, L-aspartic acid and L-tyrosine in water
solubility. Water easy solute in water because not have alkil group or aril group
only hydrogen because free group amin bigger than carboxyl then both of amin
group and carboxyl in amino acid will be act each other result zwitteren ion.
Because of that, dipolar structure then amino acid soluble in water.
H CH COOH + H2O H CH COO-
NH2 NH3+
glysin
Aspartic acid get the pH is 3 and difficult soluble in water. L-aspartic acid is acid
because contain two carboxyl group. Reaction:
HOOC CH2 CH2 COOH + H2O
-OOC CH2 CH COO-
+NH3 L tyrosine is acid with pH is 5 and not soluble in water because R in amino acid is
amphoteric. Amino acid is non esensial amino acid that have benzene
ring(aromatic). L-tyrosine is acid because ion H+ that disosiation in solution from
carboxyl group. Reaction :
COOH COO-
H2N C H + H2O H3N+ C H + H+
CH2 CH2
OH OH
Amphoteric test used L tyrosin and casein as sample. L-tyrosine added
with water and NaOH 10% produce yellow solution. The function of NaOH is to
base the solution, then add HCl to acid the solution. It is same with theory that
amino acid react with acid and base condition (amphoteric properties).
Reaction:
COOH COONa
H2N C H + NaOH H2N C H + H2O
CH2 CH2
OH OH
COONa COOH
H2N C H + HCl H2N C H + NaCl
CH2 CH2
OH OH
Cassein soluble in water get transparent solution then added NaOH 10% to
soluble casein and give acid condition, it is showed casein easy soluble in water.
Reaction :
H2N CH C NH CH C NH CH C OH + H2O
R O R O R O
H2N CH C NH CH C NH CH C O- + H+
R O R O R O
2.Reaction with nitrous acid
Glysin crystal reacted with HCl and NaNO2 produced transparent solution
and bubbles, it is showed that amino acid can reacted with NaNO2 produced N2
gases because free amino acid group. Reaction :
H2N-CH2-COOH+NaNO2→OH-CH2-COOH+H2O+N2↑
Compare solution without glysin crystal get transparent solution and not
bubble. It is because reaction HCl and NaNO2 not formed N2 gas. Reaction:
HCl+NaNO2→HNO2+NaCl
2HNO2→H2↑+NO2
The reaction of casein with NaNO2 solution produced clear solution and there is
no gas bubbles, it is proved that casein did not react with nitrous acid as amine-free casein
do not exist.
O O
|| ||
-NH-CH-C-NH-CH-C-O-
| |
CH2 CH2 +NaNO2 →
OH OH n
3. Uji Biuret
In this experiment, urea is main sample. Urea heated until formed
transparent solution and gases. Gases that produced is NH3 that stinky smell.
Reaction:
H2N C NH2 + H2N + H2N C N2H
O O
Urea
H2N C NH C NH2 + NH3 + CuSO4
O O
then added water that have done heated to soluble urea produce white precipitate
then filter and we get transparent solution, then added NaOH to give base
condition in solution to change the color and avoid precipitate CuSO4 and break
the peptide bond. Then added with CuSO4 produce blue prusi solution that sign
exsistence of peptide bond. Because of it, urea heated more than melting point
then urea will be change became biuret that reacted with CuSO4 produce purple
solution. Reaction:
H2N-C-NH-C-NH2 + CuSO4 →
|| || O O
Biuret
H3N NH3
C=O C=O complex ion
NH Cu2+ NH purple solution
C=O C=O
NH3 NH3
Comparing, urea solution (without heat) added water produce transparent solution
then added NaOH and CuSO4 produce blue solution, it is mean that not peptide
bond. It is because when adding CuSO4 not reacted with polypeptide that arrange
protein form complex that purple.
Casein that reacted with water and CuSO4 produce purple solution it is
mean that peptide bond because casein is protein that arrange from amino acid.
OH
+ CuSO4 →
CH2
HC-NH2
O=C-O- Cu- O-C=O
n
4. Xantoproteat test
The aim from this experiment to identify exixtence of benzene group in
protein. The principle is nitration benzene nuclear by nitric acid concentration and
produce orange solution. In this experiment, we get transparent solution casein, it
is not same with theory because mistaken of apperantice. The function HNO3 as
cause occur nitration reaction because the nuclear benzene from amino acid will
reacted with HNO3 and produce yellow solution. The function of NaOH as clearly
existence of benzene ring because in base condition.
Reaction:
COOH COOH
H2N C H + 2 HNO3 H2N C H + H2O
CH2 O2N CH2 NO2
OH n OH n
COOH COOH
H2N C H + NaOH H2N C H + H2O
O2N CH2 NO2 O2N CH2 NO2
OH OH
5. Protein hydrolisis
In this experiment, casein added with HCl 20% then reflux belong 40
minutes to break peptide bond of protein until decompetition become amino acid.
The product of reflux is turbity solution. Residue solution divide two part, 1st part
cold and added NaOH 20% produced yellow solution then added CuSO4 and
produced purple solution. Others solution added with NaOH and CuSO4 and
produce purple solution. It is not same with theory because mistaken apperantice
when do the reflux and adding some reagent and because purple sign still contain
peptide bond and casein not hydrolysis.
Reaction is :
→ + H2O
tyrosin
casein
H. Closing
1. Conclusion
a. Glycine soluble in water and is acid, L-aspartic acid and is slightly soluble, L-
tyrosine is insoluble in water and acidic properties of casein and insoluble in water
and acidic.
b. In the biuret test, peptide bond is indicated by the color change to purple.
c. Xantoproteat Tests showed benzene groups on the protein and amino acids
(casein and L-tyrosine) are marked with orange color solution.
d. Amoni acid reaction with nitric acid to release N2 gas is characterized by the
presence of gas bubbles.
e. Hydrolysis of proteins can lead break peptide bonds that protein breaks down
into its constituent amino acids.
2.Suggestion
Practitioner should know the nature and structure of the amino acid used. It is
expected that the next practitioner to be more careful and meticulous in carrying
out experiments to minimize errors obtained more satisfactory results.
BIBLIOGRAPHY
Bluthgen, N. 2004. Preferences of Sugars and amino acids and their
conditionality in adiverse nectar-feeding antcommunity. Germany.
Journal of Animal Ecology
Brent, Robert. 1960. The Golden Book of Chemistry Experiments. New York.
Golden Press.
Bruice. 2003. Organic hemistry 4th
Edition. New York. Logman.
Tim Dosen Kimia Organik.2012. Penuntun Praktikum Kimia Organik II.
Makassar: Jurusan kimia FMIPA UNM.
Tipton, K.D. 2004. Protein and Amino acids for athletes. USA. Journal of Sports
Sciences.
Vogel. 1989. Practical Organic Chemstry 5th
Edition. New York. Longman
Group.
Answer Question
1. Give the molecular formula of glycine, aspartic acid, tyrosine, and describe the
acidity, alkalinity, and neutrality of the solution in water..
H2N CH COOH H2N CH COOH H2N CH COOH
HCH2 CH2
glysin COOH
aspartic acid
tyrosin
glycine has a dipolar structure in which both amine and carboxyl groups will interact
generate zwitter ion solubility in water so nearly neutral. Aspartic acid has R group
consisting of carbon atoms and has many carboxyl group to another so the solution is
acidic.
Tyrosine is an amino acid that has a dipolar structure in which both amine and carboxyl
groups interact with each other so that the zwitter ion produced nearly neutral solution in
water.
2.Write the equation that can explain what happens when the solution is slowly acidified.
COONa COOH
H2N C H + HCl H2N C H + NaCl
CH2 CH2
OH OH
3. Explain the differences in the nature of hydrolysis of casein with the results of
the nitric acid and the biuret test
a. The difference in the nature of casein by hydrolysis to yield nitric acid, formed
here N2 gas wave that signifies the hydrolysis of peptide bonds of protein
polymers.
b. The difference in the nature of the results of hydrolysis of casein with the biuret
test, still in the form of casein protein as peptide bond which is marked by a
purple discoloration of the solution, the color formed is caused by the formation
of complexes between Cu 2 + to the N atom of the peptide molecules.
4. Suggest an explanation of "aspartic acid will move more slowly than
phenylalanine", the paper chromatography experiment. On paper chromatography
experiment, aspartic acid will move faster than phenylalanine as aspartic acid has
a smaller molecular size compared to the size of molecules that aspartic acid
phenylalanine easily absorbed by the pores of the filter paper.