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A. Title of Experiment : Compound Extraction, Separation, and
Purification Technique
B. Date of Experiment : Wednesday, 13rd March 2013
C. Finish of Experiment : Wednesday, 13rd March 2013
D. Objective of Experiment :
1) Selecting the tools that is needed correspond to the experiment
2) Selecting materials that is needed correspond to the experiment
3) Doing the isolation technique correctly
4) Selecting the appropriate solvent to conduct the separation
5) Doing the separation technique correctly
6) Purifying the compound and doing recristalyzing technique
7) Operating IR Instrument correctly
8) Identifying compound through functional group interpretation using
IR spectra
E. Basic Theory :
Basic separation of the TLC is the difference in migration velocity
between stationary phases is a solid and mobile phase is a mixture
of solvent (eluen), which is also known as the mixed solvent developer.
Type of eluent used depends on the type of sample to be separated.
Eluent causes the entire stain spots on the plate rises to the upper limit
of the plate without any separation, is said to be too polar. On the
contrary, if the stain spots did not move, meaning the less polar eluent.
The way that used to determine the exact type of eluent is
concentrated ring method. Results obtained compared with the following
image.
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Chromatographic separations are accomplished by continuously
passing one sample-free phase, called a mobile phase, over a second
sample-free phase that remains fixed, or stationary. The sample is injected,
or placed, into the mobile phase. As it moves with the mobile phase, the
samples components partition themselves between the mobile and
stationary phases. Those components whose distribution ratio favors the
stationary phase require a longer time to pass through the system. Given
sufficient time, and sufficient stationary and mobile phase, solutes with
similar distribution ratios can be separated. The history of modern
chromatography can be traced to the turn of the century when the Russian
botanist Mikhail Tswett (18721919) used a column packed with a
stationary phase of calcium carbonate to separate colored pigments from
plant extracts. The sample was placed at the top of the column and carried
through the stationary phase using a mobile phase of petroleum ether. As
the sample moved through the column, the pigments in the plant extract
separated into individual colored bands. Once the pigments were
adequately separated, the calcium carbonate was removed from the
column, sectioned, and the pigments recovered by extraction. Tswett
named the technique chromatography, combining the Greek words for
color and to write. There was little interest in Tswetts technique until
1931 when chromatography was reintroduced as an analytical technique
for biochemical separations. Pioneering work by Martin and Synge in
19412 established the importance of liquidliquid partition
chromatography and led to the development of a theory for
chromatographic separations; they were awarded the 1952 Nobel Prize inchemistry for this work. Since then, chromatography in its many forms has
become the most important and widely used separation technique. Other
separation methods, such as electrophoresis, effect a separation without the
use of a stationary phase.
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Thin Layer Chromatography
Thin layer chromatography is done exactly as it says - using a thin,
uniform layer of silica gel or alumina coated onto a piece of glass, metal or
rigid plastic. The silica gel (or the alumina) is the stationary phase. The
stationary phase for thin layer chromatography also often contains a
substance which fluoresces in UV light - for reasons you will see later. The
mobile phase is a suitable liquid solvent or mixture of solvents.
A pencil line is drawn near the bottom of the plate and a small drop
of a solution of the dye mixture is placed on it. Any labelling on the plate
to show the original position of the drop must also be in pencil. If any of
this was done in ink, dyes from the ink would also move as the
chromatogram developed.
When the spot of mixture is dry, the plate is stood in a shallow
layer of solvent in a covered beaker. It is important that the solvent level is
below the line with the spot on it.The reason for covering the beaker is to make sure that the
atmosphere in the beaker is saturated with solvent vapour. To help this, the
beaker is often lined with some filter paper soaked in solvent. Saturating
the atmosphere in the beaker with vapour stops the solvent from
evaporating as it rises up the plate.
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As the solvent slowly travels up the plate, the different components
of the dye mixture travel at different rates and the mixture is separated into
different coloured spots. The solvent is allowed to rise until it almost
reaches the top of the plate. That will give the maximum separation of the
dye components for this particular combination of solvent and stationary
phase.
Stationary phase on a thin layer plate often has a substance added
to it which will fluoresce when exposed to UV light. That means that if it
shined UV light on it, it will glow. That glow is masked at the position
where the spots are on the final chromatogram - even if those spots are
invisible to the eye. That means that if you shine UV light on the plate, it
will all glow apart from where the spots are. The spots show up as darker
patches.
While the UV is still shining on the plate, you obviously have to
mark the positions of the spots by drawing a pencil circle around them. As
soon as you switch off the UV source, the spots will disappear again.
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As the solvent begins to soak up the plate, it first dissolves the
compounds in the spot that you have put on the base line. The compounds
present will then tend to get carried up the chromatography plate as the
solvent continues to move upwards.
How fast the compounds get carried up the plate depends on two
things:
How soluble the compound is in the solvent. This will depend on
how much attraction there is between the molecules of the
compound and those of the solvent.
How much the compound sticks to the stationary phase - the silica
get, for example. This will depend on how much attraction there is
between the molecules of the compound and the silica gel.
Infrared spectroscopy is a technique based on the vibrations of the
atoms of a molecule. An infrared spectrum is commonly obtained by
passing infrared radiation through a sample and determining what fraction
of the incident radiation is absorbed at a particular energy. The energy at
which any peak in an absorption spectrum appears corresponds to the
frequency of a vibration of a part of a sample molecule. In this
introductory chapter, the basic ideas and definitions associated with
infrared spectroscopy will be described. The vibrations of molecules will
be looked at here, as these are crucial to the interpretation of infrared
spectra.
The visible part of the electromagnetic spectrum is, by definition,
radiation visible to the human eye. Other detection systems reveal
radiation beyond the visible regions of the spectrum and these areclassified as radiowave, microwave,
The infrared portion of the electromagnetic spectrum is usually
divided into three regions; the near-, mid- and far- infrared, named for
their relation to the visible spectrum. The higher-energy near-IR,
approximately 140004000 cm1 (0.82.5 m wavelength) can excite
overtone or harmonic vibrations. The mid-infrared, approximately 4000
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400 cm1 (2.525 m) may be used to study the fundamental vibrations
and associated rotational-vibrational structure. The far-infrared,
approximately 40010 cm1 (251000 m), lying adjacent to the
microwave region, has low energy and may be used for rotational
spectroscopy. The names and classifications of these subregions are
conventions, and are only loosely based on the relative molecular or
electromagnetic properties.
The role and scheme of IR
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Alkanes contain only CH and CC bonds, but there is plenty of
information to be obtained from the infrared spectra of these molecules.
The most useful are those arising from CH stretching and CH bending.
CH stretching bands in aliphatic hydrocarbons appear in the 30002800
cm1 range and the CH stretching bands of methyl groups and methylene
groups are readily differentiated. For methyl groups, asymmetric CH
stretching occurs at 2870 cm1, while symmetric CH stretching occurs at
2960 cm1. By comparison, methylene groups show asymmetric
stretching at 2930 cm1 and symmetric stretching at 2850 cm1. CH
bending gives rise to bands in the region below 1500 cm1. Methyl groups
produce two bending bands, i.e. a symmetrical band at 1380 cm1 and an
asymmetrical band at 1475 cm1. Methylene groups give rise to four
bending vibrations: scissoring (1465 cm1), rocking (720 cm1), wagging
(1305 cm1) and twisting (1300 cm1). The intensity of the methylene
CH2 rocking band is useful as four or more CH2 groups are required in a
chain to produce a distinct band near 720 cm1. Shorter chains show a
more variable band, for instance, the CH2 rocking band for C4H10 is near
734 cm1. Although these are the main characteristic bands associated
with aliphatic hydrocarbons, there are a number of bands that appear in the
spectra of such compounds as there is a wide range of structures possible.
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F. Tools and Materials :
Tools
Capillary Pipe
Filter Paper
Vial bottle 5 mL
Pipette
Chamber
Spatula
Graduated Cylinder 10 mL
Beaker Glass 50 mL & 100 mL
UV lamp
Chemicals
Sample
Methanol
Hexana
Chloroform
TLC Plat
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IR instrument
Pencil
Funnel
TLC Plat
Stirrer Rod
UV lamp
G. Procedure :
Sample Preparation
Eluent Preparation
sample
Is diluted with 2
mL of methanol
Solution of sample
14 mL of hexane + 4 mL of chloroform + 2 mL of
methanol
Is Mixed into a
chamber
Is Closed
Eluent for sample
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TLC Plat Preparation
Sample Separation
Plat of TLC (4cm x 20cm)
Is drawn a line 0.3cm from top
of plat and 1.0cm from bottom
of plat
Is spotted in bottom line with
0.5cm from each spot
Plat of TLC after being drawn
Is spotted with sample
until it is run out
Plat of TLC after being spotted by sample
Is put into a chamber containing eluent
Is let until eluent reaches limit of topline of
TLC plat
Is taken slowly
Is let until dry in 1 minutePlat of TLC after being dried
Is put on UV lamp to observe spots
Is dredged by using spatula
Is washed by using 2mL of methanolFiltrate
Is tested with
IR
spectroscopyIR Data of sample
Is hot plated
Crystal of sample
Plat of TLC after being drawn
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H. Observation
Procedure Result Hypothesis/Reaction Conclusion
1.
2.
Sample: white crystal
Methanol: colorless
Sample + methanol:
colorless solution
Hexane + chloroform
+ methanol: colorless
Sample + CH3OH
sample (aq)
Functional group that may
be contained:
O-H
C=C
NO2
C-O
C-N
C-H
NH2
C-Cl
The compound (sample) is
a polar compound because
the solvent (eluent) that is
used is methanol and
Sample can be
separated by using
eluent that is mixture
of 10 mL of hexane, 8
mL of chloroform, 2
mL of methanol
Sample can be
recrystallized after
being filtrated to the
same crystal.
Sample can be
predicted by using IR
test.
10mg of sample
Is diluted with
2mL of
methanol
Solution of sample
10mL of hexane + 8mL of chloroform +
2mL of methanol
Is mixed into a
chamber
Is closed
Effluent for sample
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Procedure Result Hypothesis/Reaction Conclusion
3.
Sample on TLC plat in
UV light: purple
Sample after dredging:
white powder
Filtrate: colorless
Crystal of sample after
recrystallization: white
crystal
chloroform.
Plat of TLC (4cm x 20cm)
Is drawn a line 0.3cm from top of
plat and 1.0cm from bottom ofplat
Is spotted in bottom line with
0.5cm from each spotPlat of TLC after being drawn
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Procedure Result Hypothesis/Reaction Conclusion
4.
Is spotted with
sample until it is
run out
Plat of TLC after being spotted by
sample
Is put into a chamber containing
effluent
Is let until effluent reaches limit of
topline of TLC plat
Is taken slowly by using pinset
Is let until dry in 1 minutePlat of TLC after being
dried
Plat of TLC after being
drawn
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Procedure Result Hypothesis/Reaction Conclusion
Is tested with IR
spectroscopy
Is
recrystallliz
ed
Crystal of sample
Is put on UV lamp to
observe spots
Is dredged by using
spatula
Is washed by using 2mL
of methanol
Filtrate
IR Data of
sample
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I. Analysis
In this experiment of extraction technique, separation and
purification with thin layer chromatography (TLC) technique. Thin layer
Chromatography is a method of qualitative and quantitative that involve 2
changing, those are properties of stationary phase and properties of mobile
phase.
Thin layer chromatography is performed on a sheet of glass, plastic
or aluminium foil which is coated with thin layer of adsorbent material,
usually silica gel, aluminium oxide or cellulose (blotter paper). Silica gel
or aluminium oxide is a stationary phase, and eluent is mobile phase that
has important act in elusion process for feed solution to pass stationary
phase (adsorbent). Interaction between adsorbent with eluent determine the
occurrence of component separation.
Preparing sample
An unknown sample is white crystal in the solid state is diluted
with 2 ml of methanol until the mixture perfectly dissolve. After that is
preparing TLC plate that will spotted with the sample solution. The size of
TLC plate is 4cm x 20 cm, then make the side line above and below, the
side line in the above is 0.3 cm and below is 1 cm. Then making spot in
the TLC with distance 0.5 cm of each spot. It is done by using pencil, if we
make it with pen, the dye of the pen will move with chromatogram that is
formed. So will make accumulation of spot that make sample spot not
detected. The purpose of making side line in TLC plat is to show the initial
position of eluent and the final position of eluent that movement.And then preparing the eluent from mixture of n-hexane, CHCl3
(chlorofom) and methanol. The comparison that used is 10:8:2. After that
entered the eluent in to chamber. The chamber must be closed is to make
sure that the condition in chamber is saturated with the vapour of solvent.
Saturated condition in chamber with the vapour can prevent the
vaporization of solvent. Because of solvent move slowly in TLC plat, the
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different component in the mixture will move with different speed and will
show the differences of spot colour in the plate. The eluent let to move
until the above side line and the TCL plat must be taken from the chamber.
In the plat that have spot with sample give the similar colour of the spot
with the colour of TLC plat so to see the spot that formed is using UV
light. The light of UV light is purple. After put below the UV light, the
spot movement is clearly to see and then the spot that formed give sign by
using pencil. After that the area that signed is dredging using metal ruler.
The result of dredging on TLC plat collected in filter paper that
have been arranged on funnel, then dissolve with 2 ml of methanol and
filtered. The filtrate is collected in the vial glass.
Recrystallization
After that to get crystal from the filtrate that has collected in vial
glass we do recrystallization. The result that taken from the spot in TLC
plat dissolve using methanol as solute. In this experiment, the
recrystallization do from sample. To get the crystal from sample, filtrate
heated by using hot plate, the solution evaporate and the crystal is formed.
The colour of crystal is white, After that the crystal that we got is process
by using IR to identified the compound from sample by interpretation of
functional group.
Identification the Functional group by Using IR Spectrometry
Crystal that we got before analyze using IR spectrometry. Sample
that will test firstly start with make pellet, we make pellet with mix the
crystal with KBr. KBr as alkyl halide that used as window materials that
has transparent character until 385 cm
-1
and will not reat in wavelength IR.The sample refined with KBr use mortar until homogenous then the
sample pressing so that form pellet. Pellet that formed cant broke because
the influence cluster tops that read by IR instrument. Pellet putted into IR
instrument to analyze the cluster function.
From the spectrum that is got from IR instrument, the wavenumber
range of the sample is between 4000 600 cm-1. This range include in the
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mid-infrared region. The mid infrared region is 4000 - 400 cm-1. The mid-
infrared region can be approximately divided into four regions and the
nature of a group frequency may generally be determined by the region in
which it is located. The regions are generalized as follows: the XH
stretching region (40002500 cm1), the triple-bond region (25002000
cm1), the double-bond region (20001500 cm)and the fingerprint region
(1500600 cm1). The sample that is tested include four regions, they are
stretching region, the triple-bond region, double region, and fingerprint
region.
From the analysis, we got peaks with frequency that is:
Sample frequency
(cm-1)
Theoretical frequency
(cm-1)Molecular Motion
3792.2 ~3650 or 3400-3300 O-H stretch (alcohol)
3095.1
2008.1
1993.4
1633.6 1690-1630 C=C stretch (alkenes)
1633.3
1453.4
1390.3 1600-1530&1390-1300 -NO2 (aliphatic)
1389.71251
1243.5 1320-1210 C-O stretch (carboxylic acids)
1149.8 1200-1025 C-N Stretch (amines)
1149.8
1007.4
898.7 950850 Third overtone CH stretching
801.2
797.5 750-850 NH2 wagging and twisting
681.3 785-540 C-Cl stretch (alkyl halides)
674.9
From the frequency that we got, we estimate the sample include in
carboxylic acid group. This estimation strength with founded frequency of
O-H stretch in region 3400-3300 cm-1; C=C stretch in alkenes in region
1690-1630 cm-1; -NO2 (aliphatic) in region 1600-1530&1390-1300 cm-1;
C-O stretch in carboxylic acids in region 1320-1210 cm-1; C-N stretch in
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amines in region 1200-1025 cm-1; Third overtone CH stretching in region
950850 cm-1; NH2 wagging and twisting in region 750-850 cm-1; and C-Cl
stretch in region 785-540 cm-1. In our spectrum we cant found the
molecular motion in frequency 1993.4 cm-1, it may be a pengotor. In our
spectrum there is so many pollutant.
In sample we cant determine the compound that contain in the
sample with using IR spectrometry, because IR spectrometry only can
know the functional group that contain in the compound. To know exactly
the compound that contain in sample should identify with using other
spectrometry.
J. Conclusion
1. The sample also shown N-N stretching, the sample can be separated by
TLC method.
2. The sample is polar compound because the solve that is used is polar
solvent. It is methanol solution.
3. The sample can be purified again by using recrystalization
3. The sample can be identify by using IR spectrometry.4. The sample is identify as the compound that have boron group, ester
group, amine group, nitrate group and amide group.
K. Answer of Question
1. - Extraction is a technique often used when organic compounds
(mostly hydrophobic) dissolved or dispersed in water. Appropriate
solvent (enough for dissolved organic compounds; should not
hydrophobic) was added to the phase solution in water, the mixture
was stirred well so that the organic compound extracted well. The
organic compound layer and the water will be separated with a
separating funnel, and the organic compounds can be taken back
from the organic layer by removing the solvent.
- Separation and purification be done by purpose to get the substance
purely from a substance that has been contaminated or mixed. To
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obtain a substance pure, we must separate it from the mixture, is
done a system that can be separate pure substance and
contaminated substance in a mixture that is separating and
purifying. Separating and purifying compound can be done with
method like filtration, decantation, sublimation, crystallization,
distillation, adsorptions and extraction.
2. Chromatography is used to separate the mixture into its component
substances. All forms of chromatography works on this principle.
Chromatography is a separation technique based on a mixture of speed
difference propagation components in a particular medium. In
chromatography, components are separated between two phases,
namely the stationary phase and mobile phase. Stationary phase will
hold a blending component while phase will dissolve substance
mixture components. Components are easily retained on the stationary
phase will behind. While the component soluble in the mobile phase
will move faster. Used preparative TLC separation in order to obtain a
stable quality of organic compounds in the sample. It is appropriate
that the KLT-P is used absorbent (stationary phase) with a thickness
of 0.5 - 2 mm of silica gel or aluminum oxide and a large plate (size
20x20 cm and 20x40 cm
3. Eluent is the mobile phase plays an important role in the process of
elution for the feed solution to pass through the stationary phase
(adsorbent). Type of solvent used as eluent is hexane, chloroform,
methanol
4. Purification performed to separate pure substances with impurities orits contaminated substances. The basic principle of recrystallization:
a. The crystallization process starts by adding the compound to be
purified with hot solvent to the solubility of these compounds at the
level of super saturated. In these circumstances, if the solution is
cooled, the molecules soluble compounds will stick together, grow
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into crystals will settle to the bottom of the container. While the
dissolved dirt does not come settles.
b. The formation of the crystal itself consists of two stages. The first
stage is the nucleation primary or core formation, the stage in
which crystals begin to grow but not yet settled. This stage requires
a supersaturated state of the solute. When the solution is cooled, the
solvent can not "hold" all the substance dissolved, resulting
molecules are separated from the solvent sticking together, and
began to grow into the core crystal. The more cores are joined, the
sooner will the crystal growth.
c. The second phase after the primary nucleation is secondary
nucleation. At this stage the growth of crystal faster, which is
characterized by mutual attachment cores become solid crystals.
5. Equipment / IR spectrophotometer instrument is an instrument that
records the infrared spectrum traded and easy to use on a regular basis.
Infrared spectrophotometry very important in modern chemistry, a major
in the field of organic. Were instrumental in the discovery of functional
groups, the introduction of the compound analysis of mixtures.
6. These compounds can not be identified because the tool can only IR
instrument identify the functional groups only.
L. Refferences
Syarief, Sri Hidayati dkk. 2013 .Penuntun Praktikum Kimia Organik II.Surabaya : UNESA
Fessenden, Fessenden.1982.Kimia Organik Jilid 2.Edisi
Ketiga.Indonesia:Erlangga
Stuart, Barbara. Infrared Spectroscopy:Fundamentals and
Apllication._.Analytical Techniques in the Sciences
Anonymous.http://en.wikipedia.org/wiki/Thin_layer_chromatography.acce
ssed on 19 March 2013
Anonymous.http://www.erowid.org/archive/rhodium/chemistry/equipment
/recrystallization.html. accessed on 19 March 2013
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M. Attachment
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