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Complete report of Organic Chemistry I with the title “Column

Chromatography and Thin Layer Chromatography” which made by:

Name : Sri Agustiani Basir

Reg. Number : 101304159

Class : ICP Chemistry

Group : IV

Department : Chemistry

After checked by assistant and assistant coordinator. So, this report accepted.

Makassar, June 2011

Assistant coordinator Assistant

(Ahmad Fudhail Majid, S.Pd ) ( Humaerah Azahra )

Known by,

Lecturer of Responsibility

( Iwan Dini, S.Si, M.Si )

A. Title of experiment

The title of experiment is Column Chromatography and Thin Layer

Chromatography.

B. Purpose of experiment

In the end of experiment, the student excepted understands about:

1. The student to know basic techniques column chromatography and thin layer

chromatography.

2. The student to know basic principles of chromatography.

3. The student to know basic principles from the influence of substituent to the

electrofil in aromatic compound.

C. Preview of literature

Chromatography is an extremely sensitive technique for purification and

separation of substances.

Chromatography possesses two important features:

a. Only a very small sample is required for analysis, and

b. It is capable of effectively separating very complex mixtures (Henry: 1981).

Chromatography was first introduced by Michael Tswet (1906), a botanist

from Rusia. In this experiments he managed to separate the chlorophyll and other

color pigments in plant extracts using calcium carbonate powder is filled into

glass columns and petroleum ether as solvent.

Understanding chromatography involves separation method based on the

differential distribution of sample components between two phases. According to

this definition always involves two – phases chromatography, the stationary phase

and mobile phase. Stationary phase can be either solids or liquids that are bound

to the solid surface (paper or an absorbent). Whereas the mobile phase to a liquid

called the eluent or solvent, or an inert carrier gas. The movement of this mobile

phase resulted in differential migration of components in the sample.

Based on the separation mechanism known as four different types of

chromatography, namely: a. adsorption chromatography, b. partition

chromatography, c. ion exchange chromatography, and d. exclusion

chromatography. In adsorption chromatography, a solid stationary phase and

phase motion can be either liquid or gas. Solute adsorbed by the surface of solid

particles. An example of this type of chromatography is thin layer

chromatography (TLC).

Basically, thin layer chromatography is very similar to paper

chromatography, particularly on how to do it. The apparent difference in the

separation media, which used a thin layer of fine adsorbent on board glass,

aluminum or plastic instead of paper. A thin layer of this adsorbent in the

separation process serves at the stationary phase.

Adsorbent material can be used as stationary phase silica gel, alumina and

cellulose powder. Silica gel particles containing hydroxyl groups on its surface

which will form a hydrogen bond with polar molecules. Water is absorbed in the

gel to prevent the polar molecules. From reaching the surface. To fix the gel is

activated by heating so that the absorbed water can be issued. Alumina can

separated weak polar compounds (Subagyo:2000).

Organic compounds analytic interaction model with silica gel:

All types of chromatography involves a process of equilibrium dynamic

molecules and rapidly between 2 phase (stationary and mobile). Equilibrium

between the two phases is dependent on 3 factors:

1. Polarity and molecular size tube separated

2. Polarity stationary phases

3. Polarity mobile phase

Molecular polarity is determined by its structure.

Therefore, the more polar molecules will be separated, the stronger

interaction with the stationary phase, resulting molecule it stays longer in the

stationary phase. In contrast, non polar molecules are smaller affinity of the

stationary phase will tend to be in the mobile phase longer and will be eluted first.

The more polar compounds will be separated, so if you use a polar

stationary phase such us silica gel, the compound will be bound strongly to the

stationary phase and will be separated in order last.

In general, if the chromatography used a polar stationary phase, first

choose non – polar solvents as the mobile phase to elute the components in the

mixture (Anonym:2011).

Factory equipment maker to chromatography from year to year race

improve accuracy, sensitivity an ease of operations, because it is not surprising

when a device is often quickly become outdated. Similarly, in the field of

purification of a product such as chemicals and pharmaceuticals, preparative

chromatography plays a fairly important. Fields that use services such as

preparative chromatography preparation enzymes, purification of drugs results

biosynthesis, the isolation of bioactive materials from natural materials, the

manufacture of standard compounds in the petroleum industry, purification of

drinking water and water for laboratory, industrial waste water cleaning/

household etc other (Chairil:1994).

Phenol also known as carbolic acid, is an organic compound with the

chemical formula C6H5OH.

Phenol is appreciably soluble in water, with about 8,3 g dissolving in 100 mL

(0,88 M). Phenol is highly reactive toward electrophilic aromatic substituent as

the oxygen atom’s π electrons donate electron density into the ring (Rasyid:2009).

D. Apparatus and reagents

1. Apparatus

a. Thermometer 1100C 1 piece

b. Volumetric glass 10 mL 2 pieces

c. Erlenmeyer flask 50 mL 1 piece

d. Balance 1 piece

e. Stir bar 1 piece

f. Stopwatch 1 piece

g. Beaker glass 100 mL 4 pieces

h. Separating funnel 250 mL 1 piece

i. Spray bottle 1 piece

j. Column glass (contain the cotton) 1 piece

k. Erlenmeyer flask 250 mL 1 piece

l. Elution bottle 1 piece

m. Beaker glass 50 mL 7 pieces

n. Statif and clamp 1 piece

o. Spiritus, asbes, and tripod 1 piece

2. Reagents

a. Silica gel TLC

b. TLC plat

c. Concentrated Nitride acid (HNO3)

d. Phenol (C6H5OH)

e. Methylen Chloride (CHCl3)

f. H2O

g. Sodium Sulfate Inhidrate (Na2SO4)

h. Benzene (C6H6)

i. Iod Crystal

j. Ice

k. Tissue

l. Palm leaf rib

E. Work procedure

1. Phenol nitration

a. 1,5 ml of saturated HNO3 to mix 3,5 ml of water, to cool until 5oC

b. To add the mixture between HNO3 and water into erlenmeyer which is

contain 1,5 gram of phenol

c. To shake, and then to arrange the mixture temprature 20oC - 5oC for 15

minutes and then between 30o - 5oC for 15 minutesto cool it in water

d. To add 3,5 ml of cool water into the HNO3 solution and to extract for 2

timeswith 5 ml methylenchloride

e. To wish mixture with 5 ml of aquadest

f. To dry with sodium sulphate inhidrate , then vapor its solvent in water

evaporator, then to filtrate it.

2. Column Chromatography

a. To prepare glass column which contain cotton and fill into it Methylen

chloride

b. To add alumina into column

c. To add gel silica and put the mixture of phenol nitration and did fraction

for three fraction and to observe what happen

3. Making of thin layers chromatography (TLC)

a. To drop each fraction on plat of gel silica by using palm leaf rib

b. To set the plat on eluent bottle that contain of benzene

c. To vapor with iodium concentration

d. To calculate of Rf value

F. Observation result

a. Nitration of phenol

3,5 mL H2O + 1,5 mL HNO3 cooled 5 ° C→

+ 1,5 gr phenol shaked→

brown

solution cooled 20 °−5° C→

+ 3,5 mL ice water mixed→

brown oil + 5 mL

Methylen chloride extracted→

form two layers; up layer (brown) down layer

(black brownish) separated→

brown solution + 5 mL Methylen chloride → two

layers; up layer (brown) down layer (black brownish) separated→

black

brownish + H2O washed→

Nitrophenol + Na2SO4 anhydrate boiled→

black

brownish solution.

b. Column Chromatography

1 gram silica gel + 15 drops Nitrophenol green powder put into the column→

result 12 fractions.

c. Thin layer chromatography

The solutions from the fraction drop into the plat into the benzene and dry

with iodium solid. So, we get data:

Fraction I and II don’t show dot in TLC plat

Fraction III, Rf = Spot distanceeluen distance

= 0,83,8

= 0,21

Fraction IV, Rf = o ,83,8

= 0,21

Fraction V, Rf = o ,93,8

= 0,24

Fraction VI, Rf = o ,83,8

= 0,21

Fraction VII, Rf = o ,83,8

= 0,21

Fraction VIII, Rf = o ,83,8

= 0,21

Fraction IX, Rf = o ,93,9

= 0,23

Fraction X, Rf = o ,83,9

= 0,20

Fraction XI, Rf = o ,83,9

= 0,20

Fraction XII, Rf = o ,83,9

= 0,20

Fraction XIII, Rf = o ,83,9

= 0,20

Fraction XIV, Rf = o ,93,9

= 0,23

Fraction XV, Rf = o ,93,9

= 0,23

Fraction XVI, Rf = o ,93,9

= 0,23

Note: Rf 0,2 it is 2,4 – dinitrophenol

G. Analysis data

From observation result, we get data:

III. Spot distance = 0,8

Eluen distance = 3,8

Rf = Spot distanceeluen distance

= 0,83,8

= 0,21

IV. Spot distance = 0,8


Rf = Spo t distanceeluen distance

= 0,83,8

= 0,21

V. Spot distance = 0,9



= 0,93,8

= 0,24

VI. Spot distance = 0,8



= 0,83,8

= 0,21

VII. Spot distance = 0,8



= 0,83,8

= 0,21

VIII. Spot distance = 0,8



= 0,83,8

= 0,21

IX. Spot distance = 0,9



= 0,93,9

= 0,23

X. Spot distance = 0,8



= 0,83,9

= 0,20

XI. Spot distance = 0,8



= 0,83,9

= 0,20

XII. Spot distance = 0,8



= 0,83,9

= 0,20

XIII. Spot distance = 0,8



= 0,83,9

= 0,20

XIV. Spot distance = 0,9



= 0,93,9

= 0,23

XV. Spot distance = 0,9



= 0,93,9

= 0,23

XVI. Spot distance = 0,9



= 0,93,9

= 0,23

So, Rf is 0,2 it is 2,4 – dinitrophenol

H. Discussion

1. Nitration of phenol

This experiment concentrated HNO3 put into water produce colorless yellow

solution. The purpose of water added HNO3 so that not happen explosion or fully

because concentrated HNO3 very reactive and have a high concentration. When

the added HNO3 into water is cooled before, this purpose is concentrated HNO3

not occur spontaneous solution. And then a solution added phenol produce brown

solution. A solution arrange of temperature 200 – 50C function for the temperature

is not hot so the reaction can occur completely, if through of temperature 200 –

50C the solution form not nitration phenol but other solution. After that a solution

added ice water, function of the ice water for neutralization temperature and then

done the extraction with way filled HNO3 + H2O and phenol into separating

funnel and added methylen chloride, the function for separate organic compound

(phenol) with water. When extract formed two layers are up layer: brown solution

and down layer: brown like black solution. Form two layers caused different of

polarity and density of solution, water to have a characteristic polar and organic

compound have a characteristic non polar. Density of phenol bigger than water

(density of water = 1 gr/ mL, phenol = 1,07 gr/ mL) so can known up layer is

water and down layer is organic compound. Then organic compound layer

washed with water. The function of washed with water is to wash and bond of

acid for not residue so that produced Nitrophenol. Nitrophenol added Na2SO4 is to

bond of water which residue at Nitrophenol compound after that vaporized so that

gets pure Nitrophenol. Purpose of nitration of phenol for knows that phenol can

nitration or not, so that to know phenol can nitrate.

2. Column chromatography

Basic a principle of chromatography is separated mixture of compound that

the components base the different each speed of migration of component between

two phases is stationary and mobile phase.

At experiment, result from nitration into the column had contained silica gel,

function of the adding easy is elution. After nitration result out we must to wait

until yellow layer to appear and yellow drop out from column. The fraction which

contained there are 16 fraction. The function of column chromatography is to

separate of compounds which found in nitration phenol.

This column chromatography which function as stationary phase is silica gel,

silica gel is absorber which versatile and silica gel can bounded with hydrogen

with other solution and a solvent if there is water. And which function as mobile

phase is methylen chloride.

3. Thin layer chromatography

This experiment result from fraction of chromatography column tested which

use thin layer chromatography, purpose from tested is to identify compounds

which contain at fraction of column chromatography. Media which used in thin

layer chromatography is thin plate. Then to dip a palm leaf rib in each fraction

and put 1 drop result of fraction to each dot has been determine in layer plate.

After that put in elution bottle contained benzene, function of benzene is to

mediator in process absorbent of spot. Than vaporizing in iod solution which to

function is to show a spot can see so that to make easier identification process.

According to result of experiment obtained Rf is 0,2. Can concluded

according to Rf obtained is 2,4 – dinitrophenol. The mechanism reaction:

I. Closing

a. Conclusion

From the observation, we can get conclusion as follow:

1. – Basic techniques of column chromatography are applied

chromatography or adsorption base kind of phase which use.

- Basic techniques of thin layer chromatography are chromatography

which use TLC adsorbents (smooth) depend in glass of board.

2. Basic techniques of chromatography are separated mixture of compound

that the component bases the different each speed of migration of

component between two phases is stationary and mobile phase.

3. Result of experiment 2,4 – dinitrophenol.

b. Suggestion

1. For the practicant, we must have a great take care when observed the

change of color or reaction that was occur when done the experiment.

2. For the laborant, must attention for the reagent that need to done the

experiment.

BIBLIOGRAPHY

Anonyma. 2011. Penuntun Praktikum Kimia Organik (K12051) Farmasi.

http://diaharrazy.files.wordpress.com/2010/12/praktikumkoki2051fa2.pdf.

Online. Accessed on May 26th 2011.

Anwar, Chairil, dkk. 1994. Pengantar Praktikum Kimia Organik. Yogyakarta:

Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Gajah

Mada.

Henry,N.W. 1981. Matter Under Investigation. Hongkong: Jacaranda Kimia, FMIPA,

UNM.

Rasyid, Muhaidah. 2009. Kimia Organik I. Makassar: Badan penerbit Universitas

Negeri Makassar.

Subagyo. 2005. Kimia Analitik II. Malang: Penerbit Universitas Negeri Malang.

http://diaharrazy.files.wordpress.com/2010/12/praktikumkoki2051fa2.pdf

Answer The Question

1. With see the Rf value from derivate of phenol and known the thin layer

chromatography (TLC) principle is partition,how tol level or series the polarity

of phenol fraction ?

Answer : The levels or series the polarity of phenol fraction are :

a. 2,4,6-trinitriphenol

b. 2,4-dinitrophenol

c. p-nitrophenol

d. o-nitrophenol

2. Suggest a technique of thin layer chromatography used for getting pure

compound (preparatif)?

Answer: The method tthat we can use for getting pure compound in a thin layer

is preparation of nitrophenol because nitrophenol is first step to add

mixture which will be used in chromatography coulumn and will

continued thin layer chromatography.