Organic Chemistry 3d3rx

EXPERIMENT 5

Title: Separation of Compound Using Chromatographic Techniques Objective: To separate various components in a mixture by using column chromatography and to test the purity of the compounds separated by using thin layer chromatography Introduction : To separate components in mixture usually chromatography are used. By put a mixture in a stationary phase and a mobile phase is the ed through the stationary phase . The stationary phase can be of porous solids or a layer of liquid covering the surface of a suitable solid while the mobile phase can be either a liquid or a gas. The adsorbents that are normally used as stationary phase are sucrose, cellulose, starch and inorganic carbonates but most separations are carried out using silica gel or alumina. In this experiment silica gel was used . The solvent used as the mobile phase usually has low boiling point and low viscosity, such as petroleum ether, ligroin, diethyl ether, dichloromethane, ethyl acetate, acetone, ethanol and methanol. The different components moved with mobile phase due to the fact that the different components in the starting mixture are adsorbed by the stationary phase and desorbed back into the mobile phase to different degrees. The difference in the adsorbsion-desorbsion properties result in each compound ing through a given amount of stationary phase (often a column) at a different time (retention time). Thus, the components of the mixture are separated. In a column chromatography, the stationary phase (typically alumina or silica) is held in a column (usually glass) and the mobile phase is ed down this column by aid of gravity. A column used is typically 5-50cm long and 5-50mm wide and usually has a stopcock (tap) on the bottom to halt the flow of the mobile phase. The fraction that are collected from the column and analysed so as to locate and identify the individual components. In this experiment column chromatography is used to separate various components in a mixture and thin layer chromatography (TLC) is used to test the purity of the compounds separated. The rate at which a compound moves in respect to the solvent front, Rf, is characteristic of that compound under standard conditions. The Rf values can be calculate by using the following equation: Rf = Distance of Compound / Distance of Solvent

Apparatus : Column , Chromatographic plate , Test tube, capillary tube ,Chromatographic tank ,Glass wool , Beaker Conical flask (50 mL)/test tubes Chemicals : Mixture of m-nitroaniline and pyrene Silica gel Hexane, Ethyl acetate Anhydrous sodium sulphate

Procedure : COLUMN CHROMATOGRAPHY Packing the column : The Slurry Method 1. The silica gel was mixed with hexane in a conical flask. The mixture was stir. 2. The column was packed by full it with hexane .The a cotton wool was placed at the bottom of the column by using a glass rod. All entrapped has been forced out. 3. A small amount of anhydrous sodium sulphate (or fine sand) was poured into the column so that a small clean layer is formed on top of the glass wool. 4. Tap the column to level the surface of the sodium sulphate. Wash down any sodium sulphate that adheres to the side of the column with a minimum volume of solvent was wash . The sodium sulphate layer forms a base that s the column adsorbent and prevents it from washing through the stopcock. 5. The slurry in portions was poured into a beaker .The column was tap constantly and gently at the side with a pencil fitted with a rubber stopper or with a rubber tube until all the material has settled. 6. The solvent was drain until it is just level with the top of the absorbent. The column not let to run dry. Anhydrous sodium sulphate was added to the surface of the adsorbent to protect the surface of the adsorbent from been disturbed.

Applying the sample to the column 1. The mixture of m-nitroaniline and pyrene was applied carefully around the circumference of the adsorbent using a capillary pipette so that a layer of the mixture is formed evenly on to top of the sodium sulphate covering the absorbent. 2. The solvent was drain out until to bring the liquid level to the top of the absorbent again. A little eluent was pipette around the inside of the column to rinse down any inherent

sample and once again, drain out the solvent until the liquid level to the top of the absorbent again. 3. The eluent was added and the eluate begin collected. Added the eluent continuously to ensure that the liquid level is nearly constant throughout the elution. 4. The eluate was collected in a clean, dry flask.

THIN LAYER CHROMATOGRAPHY Spotting the plate 1. The pencil was used to draw a straight line on the thin layer chromatography plate and mark two spots on the plate . 2. These spots had high enough (a point about 1 cm from the bottom) to ensure that the compounds placed on it will not dissolve in the developing solvent. 3. A capillary tube was filled with the solution to be examined by dipping one end of the tube into the solution. Capillary action fills the tube. 4. The tube was empty by touching it lightly to the thin layer plate on the spot marked. When the tube touches the plate, the solution is transferred to the plate as a small spot.

Preparing the Developing Chamber 1. A piece of filter paper was used to linethe inside of the developing tank/ jar 2. The eluent solvent (hexane-ethyl acetate (2:1)) was poured into the developing tank/jar to a depth of a few millimeters and cap the developing tank/jar. 3. The filter paper inside the tank thoroughly moistened with the eluent solvent was confirm before the development.

Result : Total distance of solvent travelled from bottom line in TLC plate = 8.9cm Retardation factor, Rf = Distance of substances travelled from the bottom line / Total distance of solvent travelled from bottom line in TLC plate

Sample

Distance travelled by

Retardation factor

substances (cm)

(cm)

First fraction

0

0

Second fraction

4.3

0.483

Third fraction

4.6

0.519

Fourth Fraction

4.7

0.528

Discussion : For this experiment we used the mixture of m-nitroaniline and pyrene which have the different polarity. In the column chromatography the separated material was collected. The column chromatography allows to separate and collected the compounds individually. The used of silica as stationary phase in column chromatography. The most polar sample will be retained in the stationary phase longer which in m-nitroaniline . Thus the least polar compound will elute from the column first, followed by each compound in order of increasing polarity . The more polar has the ability to hydrogen bond The blue compound is obviously more polar than the yellow one - it perhaps even has the ability to hydrogen bond. That means that it must adsorb more strongly to the silica gel than the yellow one.The less polar yellow one spends more of its time in the solvent and therefore washes through the column much faster.

The structure of the pyrene show delocalized electron in the aromatic ring due to the lower polarity. The delocalizing π electrons are distributed evenly in the whole structure and hence it stabilize the pyrene.The pyrene also do not have substituent. The structure of aniline show the more substituent that gives more polarity. N and O atoms are the highly electronegative atoms which tend to withdraw the electrons from the benzene ring towards the atoms. This causes the arisen of partial positive charge and partial negative charge in the substituent and within the benzene ring. Consequently, the structure of p-nitroaniline induces the polar property of the compound and hence the polarity of p-nitroaniline is much more higher than pyrene. So the different in polarity effect the interaction with si

In thin layer chromatography , four fraction was spotted on the TLC. The separation of compounds is 1st to 3th spots are containing pyrene only while 4th is mixture of mnitroaniline and pyrene. By calculating the Rf value on TLC plate show higher polarity have lower Rf value . In TLC plate the more polar molecules will have lower Rf values .

There are precaution steps should be taken while doing this experiment. Firstly make sure the apparatus was clean to avoid contaminated. Next the beaker that contained TLC were not move. When plotting the spot on the plate make sure the diameter is not to bigger or smaller because the pyrene can easily interact with each other and mixed together and avoid using the pencil .

Conclusion : In conclusion the polarity of the substances effect in the column chromatography and Thin Layer Chromatography experiment . The most polar longer retained in stationary phase while in TLC plate the more polar have lower Rf value .

References http://en.wikipedia.org/wiki/Thin_layer_chromatography http://www.chromatography-online.org/Principles/Basic-ColumnChromatograph/Data-Acquisition.html

Organic Chemistry 1st Year Laboratory Report

Experiment No : 5 Title of Exp : SEPARATION OF COMPOUNDS USING CHROMATOGRAPHIC TECHNIQUES Date of Exp : 19 November 2013 Name

:Linda Aida bt Mohd Zaid (SED120005)

Group

:O

Subject

: Organic Chemistry 1st Year

Lecturer

: Dr. Thorsten Heidelberg

Tutor

:

Question 1. Explain the following in relation to chromatography. Mobile phase = The mobile phase is the part of the chromatographic system which carries the solutes through the stationary phase. Stationary phase = The stationary phase is the part of the chromatographic system though which the mobile phase flows where distribution of the solutes between the phases occurs Rf value = is the distance travelled by the substances over distance travelled by the solvent form 2. Calculate the Rf value for compounds X, Y and Z in TLC plate given : X = 6.8 cm / 8 cm = 0.85 cm Y = 2.6 cm/8 cm = 0.325 cm Z = 3.7 cm / 8 cm = 0.463 cm

3.Suggest one suitable chromatographic technique which can be used to separate a mixture of amino acids. Thin Layer Chromatography

4.What are the advantages of column chromatography over TLC? Can get the fraction of the mixture

5. Arrange the following compounds in the order of increasing polarity : ortho-nitroaniline, meta-nitroaniline ,para-nitroaniline