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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
Eluen distance = 3,8
Rf = Spo t distanceeluen distance
= 0,83,8
= 0,21
V. Spot distance = 0,9
Eluen distance = 3,8
Rf = Spot distanceeluen distance
= 0,93,8
= 0,24
VI. Spot distance = 0,8
Eluen distance = 3,8
Rf = Spot distanceeluen distance
= 0,83,8
= 0,21
VII. Spot distance = 0,8
Eluen distance = 3,8
Rf = Spot distanceeluen distance
= 0,83,8
= 0,21
VIII. Spot distance = 0,8
Eluen distance = 3,8
Rf = Spot distanceeluen distance
= 0,83,8
= 0,21
IX. Spot distance = 0,9
Eluen distance = 3,9
Rf = Spot distanceeluen distance
= 0,93,9
= 0,23
X. Spot distance = 0,8
Eluen distance = 3,9
Rf = Spot distanceeluen distance
= 0,83,9
= 0,20
XI. Spot distance = 0,8
Eluen distance = 3,9
Rf = Spot distanceeluen distance
= 0,83,9
= 0,20
XII. Spot distance = 0,8
Eluen distance = 3,9
Rf = Spot distanceeluen distance
= 0,83,9
= 0,20
XIII. Spot distance = 0,8
Eluen distance = 3,9
Rf = Spot distanceeluen distance
= 0,83,9
= 0,20
XIV. Spot distance = 0,9
Eluen distance = 3,9
Rf = Spot distanceeluen distance
= 0,93,9
= 0,23
XV. Spot distance = 0,9
Eluen distance = 3,9
Rf = Spot distanceeluen distance
= 0,93,9
= 0,23
XVI. Spot distance = 0,9
Eluen distance = 3,9
Rf = Spot distanceeluen distance
= 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.
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.