KLT Analysis

7/28/2019 KLT Analysis

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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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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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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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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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KLT Analysis

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