amino acid and protein

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


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.


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


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


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.

amino acid and protein

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