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A. Title of Experiment Purification Technique B. Date of Experiment C. Finish of Experiment D.

Objective of Experiment

: Compound Extraction, Separation, and : Wednesday, 13rd March 2013 : Wednesday, 13rd March 2013 :

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.

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 in chemistry 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.

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.

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.

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 are classified 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

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

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.

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

IR instrument Pencil Funnel TLC Plat Stirrer Rod UV lamp

G. Procedure : Sample Preparation sample

Is diluted with 2 mL of methanol

Solution of sample

Eluent Preparation 14 mL of hexane + 4 mL of chloroform + 2 mL of methanol Is Mixed into a chamber Is Closed Eluent for sample

TLC Plat Preparation 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 Sample Separation 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 Plat of TLC after being dried Is let until dry in 1 minute Is put on UV lamp to observe spots Is dredged by using spatula Filtrate Is washed by using 2mL of methanol

Is tested with IR spectroscopy IR Data of sample

Is hot plated

Crystal of sample

H. Observation Procedure 1. 10mg of sample Result Sample: white crystal Methanol: colorless Is diluted with 2mL of methanol Solution of sample Sample + methanol: colorless solution Hypothesis/Reaction Sample + CH3OH sample (aq) Functional group that may be contained: O-H C=C NO2 C-O 2. 10mL of hexane + 8mL of chloroform + 2mL of methanol Is mixed into a chamber Is closed Effluent for sample C-N Hexane + chloroform + methanol: colorless C-H NH2 C-Cl The compound (sample) is a polar compound because the solvent (eluent) that is used is methanol and Conclusion 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.

Procedure

Result Sample on TLC plat in UV light: purple

Hypothesis/Reaction chloroform.

Conclusion

3.

Plat of TLC (4cm x 20cm) Sample after dredging: white powder Is drawn a line 0.3cm from top of plat and 1.0cm from bottom of plat Filtrate: colorless Is spotted in bottom line with 0.5cm from each spot Crystal of sample after Plat of TLC after being drawn recrystallization: white crystal

Procedure Plat of TLC after being drawn 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 Plat of TLC after being Is let until dry in 1 minute dried

Result

Hypothesis/Reaction

Conclusion

Procedure

Result

Hypothesis/Reaction

Conclusion

Is put on UV lamp to observe spots Is dredged by using spatula Filtrate Is washed by using 2mL of methanol

Is tested with IR spectroscopy IR Data of sample Crystal of sample

Is recrystallliz ed

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

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

mid-infrared region. The mid infrared region is 4000 - 400 cm -1. The midinfrared 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) 3792.2 3095.1 2008.1 1993.4 1633.6 1633.3 1453.4 1390.3 1389.7 1251 1243.5 1149.8 1149.8 1007.4 898.7 801.2 797.5 681.3 674.9 Theoretical frequency (cm-1) ~3650 or 3400-3300 Molecular Motion O-H stretch (alcohol)

1690-1630 1600-1530&1390-1300 1320-1210 1200-1025 950850 750-850 785-540

C=C stretch (alkenes) -NO2 (aliphatic) C-O stretch (carboxylic acids) C-N Stretch (amines) Third overtone CH stretching NH2 wagging and twisting C-Cl stretch (alkyl halides)

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

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

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 methanol 4. Purification performed to separate pure substances with impurities or its 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 to pass through the stationary phase (adsorbent). Type of solvent used as eluent is hexane, chloroform,

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 Ketiga.Indonesia:Erlangga Stuart, Organik Jilid 2.Edisi and

Barbara. Infrared Spectroscopy:Fundamentals 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

M. Attachment