UNIVERSITI TUNKU ABDUL RAHMAN (UTAR)

DEPARTMENT OF CHEMICAL SCIENCE

UDEC 2232 CHEMISTRY LABORATORY IV

Marks

EXPERIMENT No. : COMPONENT A, EXPERIMENT 1. TITLES; PART I: THIN LAYER CHROMATOGRAPHY PART II: THE SEPARATION OF PYRENE AND P-NITROANILINE BY COLUMN CHROMATOGRAPHY

LECTURER

: DR. DR. SIM KOOI MOW

NAME OF STUDENT : NICOLE ANN FRANCIS FERNANDEZ ID NUMBER GROUP GROUP MEMBERS : 1003561 :1 : TAN JUVEIN GOH WEI JIAN

Objectives :

( a ) Part I :

( i ) To learn the technique of TLC and the visualization of colorless components. ( ii ) To identify an unknown drug by a TLC comparison with standard compounds.

( b ) Part II :

( i ) To learn the technique of Column Chromatography ( ii ) To separate the mixture of pyrene and p-nitroaniline by Column Chromatography.

Introduction: Chromatography is the science which is studies the separation of molecules based on differences in their structure and/or composition. In general, chromatography involves moving a preparation of the materials to be separated - the "test preparation" - over a stationary support. The molecules in the test preparation will have different interactions with the stationary support leading to separation of similar molecules. Test molecules which display tighter interactions with the support will tend to move more slowly through the support than those molecules with weaker interactions. In this way, different types of molecules can be separated from each other as they move over the support material. Chromatographic separations can be carried out using a variety of supports, including immobilized silica on glass plates (thin layer chromatography), volatile gases (gas chromatography), paper (paper chromatography), and liquids which may incorporate hydrophilic, insoluble molecules (liquid chromatography). The separation of the components of a mixture depends in the phase each component remains in and the rate at which each travels. The stationary phase does not move, while the other mobile phase travels past the fixed phase. Due to various functional groups, each compound travels past the stationary phase at a different speed. The strength of the following types of interactions : ion-dipole, dipole-dipole, hydrogen bonding, dipole induced dipole, and Van der waals forces.

Different types of chromatography use a various types of stationary and mobile phases. In this experiment, the solid phase is silica gel while the mobile phase is an organic solvent ( may be a single type or a mixture of solvents ). This means that the compounds to be separated must choose between being absorbed to the solid silica gel or moving along in the organic solvent. The silica gel us either packed into a column or adhered to a sheet of glass, plastic or aluminium, depending on the type of chromatography.

When a column is used, the compound mixture is placed on top and the solvents are run down the column separating the mixture along the way. With the silica gel on a plate, the compounds are placed close to the bottom and the mixture is separated as the solvent travels up by means of capillary action. Since silica gel is a porous form of SiO2, the surface of gel contains Si-OH and Si-O-Si functional groups. With silica gel, the dominant interactive force between the adsorbent and the materials to be separated are of the dipole-dipole type. Highly polar molecules interact fairly strongly with the polar Si-O bonds of these adsorbents and will tend to stick or adsorb onto the fine particles of the adsorbent while weakly polar molecules are held less tightly. Weakly polar molecules thus generally tend to move through the adsorbent more rapidly than the polar species.

Another factor that established the rate at which a compound travels past silica gel is the polarity of the solvent. A polar solvent will compete for silica absorption sites, disallowing the compounds to do so. This promotes a faster rate at which all compounds travel. The order in which the compounds move remains the same, while moving faster as the polarity of the eluent ( solvent system ) increases.

Column chromatography is advantageous over most other chromatographic techniques because it can be used in both analytical and preparative applications. Not only can column chromatography be used to determine the number of components of a mixture, but it can also be used to separate and purify substantial quantities of those components for subsequent analysis. This is in contrast to paper chromatography, which is solely an analytical method. For example, while paper chromatography is easily applied to see whether a purple coloured beverage contains a mixture of dyes, it is not practical to further analyze the separated dyes given the necessarily very small size of the initial sample. A preparative method like column

chromatography allows you to do just that. Separating the purple food dye on an appropriately set up column with good technique will leave you with cleanly separated blue and red dyes in large enough amounts for further investigation. Thus, column chromatography should be used any time you want to separate a mixture of liquids or solutes into its components, and work with these components individually. In fact, it is the most frequently used method of purifying mixtures of products in research laboratories. Thin-Layer Chromatography (TLC) is a simple and inexpensive technique that is often used to judge the purity of a synthesized compound or to indicate the extent of progress of a chemical reaction. In this technique, a small quantity of a solution of the mixture to be analyzed is deposited as a small spot on a TLC plate, which consists of a thin layer of silica gel (SiO2) or alumina (Al2O3) coated on a glass or plastic sheet. The plate constitutes the stationary phase. The sheet is then placed in a chamber containing a small amount of solvent, which is the mobile phase. The solvent gradually moves up the plate via capillary action, and it carries the deposited substances along with it at different rates. The desired result is that each component of the deposited mixture is moved a different distance up the plate by the solvent. The components then appear as a series of spots at different locations up the plate. Substances can be identified from their so-called Rf values. The Rf value for a substance is the ratio of the distance that the substance travels to the distance that the solvent travels up the plate. There are several factors that affect the resolution of separation on the TLC plate. The first is the presence of adsorbents. The weight ratio of adsorbent to sample is important to obtain accurate separation. If too much sample is applied, the active adsorbing sites will be saturated and the column will be flooded, resulting in poor separation. In most cases, a ratio of 20 to 1 is satisfactory but sometimes up to 100 to 1 is necessary. Even the ratio of column height to diameter is important; about 8 to 1 is considered optimal. Next, the solvent is important to the compound separation, keeping in mind that the more polar the solvent, the faster the compounds move. In some cases, a solvent system may increase in polarity by gradually changing the composition of the solvent mixture. The type of functional groups present in the compound can also affect the resolution of separation on the plate. Compounds with highly polar groups are strongly adsorbed and eluted less readily than less polar compounds. The strength of adsorption for compounds having the following types of polar functional groups decreases in the order listed below.

However, variations may occur depending on the overall structure of each specific compound.

Part I: TLC Analysis of Analgesic Drugs In this experiment, TLC will be used to examine the composition of various analgesic (pain relieving)drugs. Among these are aspirin, and acetaminophen. Caffeine is sometimes added to these formulations to overcome drowsiness.

Apparatus: UV lamp, capillary tube, 250 ml beaker

Materials: Aspirin, acetaminophen, caffeine, unknown A, unknown B, TLC plates, ethyl acetate, hexane, iodine.

Procedure: A: Spotting of the TLC plates 1. A tLC plate was obtained andset down on a clean and dry surface. Care was taken not to bend the plate excessively since the silica adsorbent might flake off. 2. A line was drawn across the plate gently at about 1.0cm from the bottom of the plate using a 2B pencil. 3. Then, five 2-3mm lines were drawn perpendicular to the line across the bottom of the TLC plate, about 0.8cm apart from one another. The first and the last lines were checked to be 0.8cm from the edge of the palte to mark the points where the samples will be spotted or applied to the TLC plate. 4. Using the 5 different analgesics, the plate was spotted using a capillary action tube for each sample. 5. First, the acetaminophen was spotted, then the caffeine, then the unknown A, then aspirin and finally the unknown B. The spots were made as small as possible to avoid tailing once the plate is developed. The plate was examined under the ultraviolet light to see that enough of each compound had been applied, and more was added if not.

B: Developing the TLC plate 1. A developing chamber was prepared as indicated by using a 250mL beaker as the chamber, a half-piece of filter paper inside, and aluminium foil to cover. 2. The eluent (1:3, mixture of ethyl acetate:hexane) was poured into the beaker to a depth of under 1cm (about15mL). The prepared TLC plate was placed in the developing chamber, ensuring that the solvent level is below the pencil line. 3. After the solvent had risen to near the top of the plate (about 0.5cm from the top), the plate was removed and the solvent front marked with a pencil. The solvent was allowed to evaporate from the plate in the fumehood. C: Visualisation 1. The colourless compounds were visualised by illumination of the plate with an ultraviolet lamp. Usually, aromatic compounds will show a bright fluorescence, which may have a characteristic colour. 2. The spots were outlines with a 2B pencil. The spots may also have been visualised by putting the plate in an iodine chamber for a couple of minutes.

D: Comparison of the Unknown with Reference Standards 1. The plate was sketched in the notebook and the Rf values for each spot were calculated. 2. The Unknown drug was determined based on the Rf value.

Part II: The separation of pyrene and p-nitroaniline by column chromatography. Apparatus: Glass column, UV lamp, capillary tube, 250ml beaker, test tubes, glass funnel. Materials: Pyrene, p-nitroaniline, TLC plates, ethyl acetate, hexane, iodine.

Procedure: A: Column preparation 1. A 40cm chromatography column, 15g of deactivated silica gel and 120ml of the developing solvent mixture (ethyl acetate : hexane) were obtained. 2. A slurry of the adsorbent (silica gel) with 50ml of solvent in a 250ml Erlenmeyer flask was prepared. 3. A small plug of cotton wool was pushed into the constriction at the bottom of the column using a piece of glass tubing. The column was clamped in a vertical position and a 0.5cm layer of sodium sulphate anhydrous was added on top of the cotton wool. 4. After ensuring that the stopcock was closed, 15ml of solvent was poured in and allowed to set. Then, all the slurry was quickly decanted through a funnel into the column. 5. The stopcock was opened and the solvent allowed to drain while tapping the walls of the column with the ends of a folded piece of rubber tubing. 6. The majority of packing was known to occur in the first 15 minutes and regular tapping was over the next 20-30 minutes as the solvent drained out, and additional setting occurred which was monitored by marking with a felt tip pen or masking tape. 7. Once the solvent level was within 6cm of the top of the adsorbent, the packing was considered to be complete. A 0.5cm levellayer of sodium sulphate anhydrous was carefully added on the adsorbent. 8. Excess solvent was drained off until its level was precisely on top of the sodium sulphate anhydrous and the stopcock was closed.

B: Separation and collection of Pyrene and p-nitroaniline. 1. 0.02g of the pyrene and 0.02g of p-nitroaniline were mixed and a few drops of ethyl acetate was used to dissolve as much of the mixture as possible. 2. The solution was carefully transferred directly to the top of the sodium sulphate anhydrous layer with a dropper. 3. The solvent was drained off until the mixture solution was just below the top of the sodium sulphate anhydrous.

4. The walls of the column were rinsed with about 1ml of fresh solvent (ethyl acetate:hexane, 1:3) and drained until the level was once again below the top of the sodium sulphate anhydrous. The rinsing of the walls was repeated until the solvent above the silica gel was colourless. 5. The column was very carefully filled with fresh solvent (ethyl acetate:hexane, 1:3) and allowed to drain. 6. The separation of the bands was observed as the column developed. The colourless band of pyrene was collected into 3 test tubes. 7. When the edge of the yellow band (p-nitroaniline) reached the lower part of the column, a new test tube was used to replace the old one and the yellow band was collected into three fractions. 8. Each fraction was concentrated to a small volume by evaporation (in a hot water bath) for analysis by TLC.

C: Analysis of the fractions. 1. The fractions were spotted on a TLC silica gel plate along with the reference pyrene and p-nitroaniline. 2. The TLC plate was developed in a developing chamber containing a 1:3 mixture of etyl acetate/hexane. 3. The TLC plate was visualised with the ultraviolet lamp (or iodine chamber) to determine which fractions were pure pyrene and which were pure p-nitroaniline. 4. The chromatogram was drawn in the laboratory notebook while identifying and labelling the spots in the chromatogram. 5. The Rf values for pyrene and p-nitroaniline were calculated. 6. The pure fractions of pyrene and p-nitroaniline were combined in pre-weighed test tubes respectively. The solvent of both flasks were evaporated on a steam bath. 7. Once the solvent had evaporated, the weights of pure pyrene and p-nitroaniline were calculated.

Results: The Rf values for the distances can be calculated using the formula;

Part I: TLC Analysis of Analgesic Drugs Distance of solvent Distance of compounds; Acetaminophen (Ac) = 0.9cm Caffeine (Ca) Unknown A (A) Aspirin (As) Unknown B (B) = 0.2cm = 0.3cm = 0.3cm = 0.7cm = 8.0 cm

Rf of acetaminophen = 0.9 / 8.0 = 0.1125 Rf of caffeine = 0.2 / 8.0 = 0.025 Rf of unknown A = 0.3 / 8.0 = 0.0375 Rf of aspirin = 0.3 / 8.0 = 0.0375

Rf of unknown B

= 0.7 / 8.0 = 0.0875

Based on the Rf values, unknown A has the same Rf value as aspirin, and the Rf value of unknown B is closest to the Rf value of acetaminophen. Therefore, unknown A is determined to be aspirin and unknown B is acetaminophen.

Part II: The separation of Pyrene and p-Nitroaniline by column chromatography Distance of solvent = 8.0 cm

Distance of spots from bottom of TLC plate; 1 2 3 4 5 6 7 8 = 6.3cm = 6.3cm = 6.3cm = 6.3cm = 1.8cm = 1.9cm = 1.8cm =1.8cm

Average distance of spots 1, 2, 3 and 4

= (6.3 + 6.3 + 6.3 + 6.3) / 4 = 6.3cm

Rf value of pyrene

= 6.3 / 8.0 = 0.7875

Average distance travelled by spots 5, 6, 7 and 8: = (1.8 + 1.9 + 1.8 + 1.8) / 4 = 1.825 Rf value of p-nitroaniline = 1.825 / 8.0 = 0.228 During this column chromatography, pyrene was collected as the colourless solution that was collected from the column. Four fractions were collected to make the spots on the TLC plate for observation. P-nitroaniline was the yellow solution that was collected from the column. Another four fractions were collected for observation on the TLC plate. Based on Rf values, pyrene with larger Rf is less polar compare to p-nitroaniline which has smaller Rf value.

Discussion:

In this experiment, thin layer chromatography was used to identify the unknown A and B. But in part II, TLC was used in order to check components purity. As the solvent moves past the spot that was applied in experiment, equilibrium was established for each component of the mixture between the molecules of that component which were adsorbed on the solid and the molecules which were in solution. But, before developing the TLC plate into the chamber, a filter paper was dipped in the chamber and covered by aluminum foil in order to let the atmosphere in the chamber saturated with solvent. In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others. This was due to the interaction of the component and adsorbent. The component bind to adsorbent depend on the strength of the types of interaction, such as ion-dipole, dipole-dipole, hydrogen bonding, dipole induced dipole, and van der Waals forces. With silica gel, the dominant interactive forces between the adsorbent and the components to be separated were of the dipole-dipole type. Highly polar molecules interact fairly strongly with the polar SiO bonds of these adsorbents and will tend to stick onto the fine particles of the adsorbent while

weakly polar molecules were held less tightly. Weakly polar molecules thus generally tend to move through the adsorbent more rapidly than the polar species. From the experiment, noticed that some of the spots on the TLC plate were colorless. In order to visualize them, the TLC plate need exposed under ultraviolet lamp. This was because the adsorbent of TLC plate was impregnated with a fluor and zinc sulfide. The fluor enabled most organic compounds to be visualized when the plate was held under a UV lamp and it will become glow. That glow was masked at the position where the spots were on the final chromatogram - even if those spots were invisible to the eye. That means that if shine with UV light on the plate, it will all glow apart from where the spots were. Under UV lamp, the first and second spot will not appeared, this was due to both of the spots were solvent molecules. Rf value is referred to the distance traveled by the compound divided by the distance traveled by the solvent. The larger an Rf of a compound, the further the distance it travels on the TLC plate. The compound with a larger Rf value is less polar because it interacts less strongly with the polar adsorbent. Plus, the compound with a small Rf value is more polar because when it has more polarity, it can break the bond. Therefore, we can know the structures of the compounds when comparing two different compounds run under identical chromatography conditions, we can predict that a compound of low polarity will have a larger R f value than a polar compound run on the same plate. For part I of the experiment, the Rf value of unknown A is 0.0375. Based on this Rf value, unknown A has the same Rf value as aspirin. The Rf value of unknown B is 0.0875 while the Rf value of acetaminophen is 0.1125. Unknown B is closest to the Rf value of acetaminophen. Therefore, unknown A is determined to be aspirin and unknown B is acetaminophen. For part II, we conducted a column chromatography to separate pyrene and p-nitroaniline. This time the silica gel is used in this experiment as it is adsorbent. This experiment has different particles sizes and the particles sizes affect how the solvents flow down the column. Sodium sulphate anhydrous was added to the adsorbent in order to absorb excess water. The polarity of this solvent in the column chromatography can affect the rate at which the compounds move through the column. More polar the solvent, compounds can be remove faster.

In this experiment, we had collected eight fractions from the column. The first four are transparent, which is pyrene, while the latter four are yellow colour which is p-nitroaniline. After we obtained the mixture, we added a few drops of ethyl acetate because it is to dissolve as much as possible of the mixture. We then carefully transferred the solution directly into the top of the sulphate anhydrous layer with a dropper. After that, we drained off the solvent until the mixture solution is just below the top of the sodium sulphate anhydrous. It is important to do so because we want to separate it well. We rinsed the walls with another 1mL of fresh solvent again which is ethyl acetate again and drained it until the level is once again below the top of the sodium sulphate anhydrous. We kept on repeating by rinsing the walls until the solvent is colourless. After that, we filled the column extra carefully with fresh ethyl acetate again and allowed the solvent to be drained out. We need to be observant of the separation of bands as the column developed. Then, once drained out, we need to collect the colourless band of pyrene into three test tubes. But once the edge of the yellow band which is pnitroaniline reached the lowest part of it, we must immediately replaced it with another new test tubes and collected all into three fractions too. Once everything had been collected, we had examined the silica gel plate under the ultraviolet light to see whether that is it enough for each compound had been applied. White portion that we took out is pyrene which is a colourless compound and is non-polar compounds while the yellow portion is p-nitroaniline which is more polar that took longer time to elute from the column. So, after examined, it is shown that pyrene is non-polar compound with a higher Rf value which means that it was not lag during migration and it collected within a shorter time. Therefore it is known that p-nitroaniline is a polar compound as it took a longer time to be eluted. So, from this experiment, we had learned a lot of things and realized that there are a few precautions steps that need to be followed and should not make any mistakes in the future. Firstly, if there are no spots seen on the plate, it is most probably that we did not have spot enough compound or maybe the solution of the compound is too dilute. Plus, by spotting it several times in one place, this allows the solvent to dry between applications. On the other hand, the solvent will dissolve the compounds into the solvent reservoir when the solvent level is progressing more than the original spot on the TLC plate. Therefore, we cant see the spots properly or it cant be seen at all. Besides that, it is crucial to have the suitable solvent for the compound separation. It is because that the more the solvent, the more polar it is, the faster the compound moves.

Conclusion: Thin Layer Chromatography (TLC) is a powerful technique to separate compounds based upon their polarity and interaction with silica and to assess the purity of a sample. Organic solvents act as a mobile phase and the stationary phase is alumina. Different polarity of the solvent will affect the rate of compounds moved in TLC plate. The higher the polarity, it can break the bond easily. The more polar the solvent, the faster the compounds move. Rf value refers to the distance traveled by the compound divided by the distance traveled by the solvent. The larger an Rf of a compound, the larger the distance it travels on the TLC plate. So, the results we get for Rf value for aspirin, acetaminophen and caffeine were 0.0375, 0.1125 and 0.025. Acetaminophen is known as non-polar compound, acetaminophen is known as moderately polar compound while caffeine is known as polar compound. Plus, unknown A is aspirin while unknown B is acetaminophen. Pyrene is non-polar compound and p-nitroaniline is polar compound. The Rf values are 0.788 and 0.228 respectively.

References: 1. P.W. Atkins & J.de Paula. (2006). Atkins Physical Chemistry, 8th edition, New York: Oxford. 2. Chromatography. Introdcutory theory. Retrieved 13th June 2011 from http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/chrom1.htm. 3. TLC of Analgesics. Retrieved 3rd July 2010. From http://facweb.northseattle.edu/amohamed/CHEM%20102N/Labs/Lab%205_TLC.pdf. 4. Douglas A.Skoog, Stanley R. Crouch and Douglas A. Skoog. Principle of instrumental analysis, 6th Edition, David Harris, page 762-768.