Grignard Synthesis of Triphenylmethanol

Objective:

1. To synthesis triphenylmethanol from Grignard reaction

2. To study the method to produce Grignard reagent

Introduction:

Grignard reagents are organomagnesium halides (RMgX), and are one of the most synthetically useful and versatile classes of reagents available to the organic chemist. An alkyl, benzyl, or aromatic halide is reacted with a magnesium metal by using an anhydrous solvent in order to produce Grignard reagent. Ether or tetrahydrofuran are usually can be used as the anhydrous solvent in producing the particular reagent. Tetrahydrofuran is a strong base and it has a better solvating ability, it may used when Grignard reagent does not readily form in diethyl ether. This is considered as an organometallic compound which consists of the combination of a metal and organic molecule. Figure 1 in below shows the general reaction mechanism for the formation of Grignard reagent.

The chemical reaction between an organic halide and a magnesium metal can produce an alkyl or aryl free radical and magnesium free radical. The formation of Grignard reagent has been occurs. The bonding between carbon and magnesium is a covalent bond but it is highly polarized because the magnesium is bonded to halide which is an electron withdrawing group. This causes the formation of partial positively charge and partial negatively charge on the magnesium atom and alkyl or aryl group respectively. Hence, the carbanion has both characteristics of a good nucleophile and a strong base. Its basicity allows it to react with the electrophile carbon in a carbonyl group. Besides, Grignard reagent also works with acidic compound such as carboxylic acid, phenol, thiol, alcohol, and even water. One of the most important reactions is the addition of Grignard reagent to the carbonyl compound like aldehyde, ketone, and ester in order to produce the corresponding secondary alcohol and tertiary alcohol.

Figure 2

The most challenging part of this experiment is to avoid the Grignard reagent contact with water. The partial negative charge on the carbon atom that bonded to magnesium exhibits a very basic property. The water molecule will destroy the nucleophilic property of Grignard reagent. So, several precaution steps must be taken in the procedures to avoid the Grignard reagent reacts with water: the reaction flask is dried in the oven before use; iodine is vaporized in the flask tie up traces of water and to activate the surface of magnesium; the anhydrous diethyl ether should be used.

Figure 3

The figure 3 above shows the Grignard reagent and water reaction. The metal hydroxide (alkoxide) formed in the above reaction is appears as insoluble white solid (HO-MgBr or RO-MgBr) in the diethyl ether solvent. Thus, the process of producing Grignard reagent must be start over again in a dry glass if the insoluble white solid is observable during the formation of Grignard reagent.

For a variety of reasons, anhydrous diethyl ether is the solvent choice for carrying out a Grignard synthesis. One of the reasons is the vapors of the highly volatile diethyl ether helps to prevent the oxygen in the atmosphere to reach the reaction solution. Grignard reagent will reacts with oxygen which hydroperoxides is produced. This compound is highly unstable if exposed to the air so that the compound is usually not isolated from the solvent. Other than that, the basic oxygen atoms in ether molecules are actually coordinate with and help to stabilize the Grignard reagent. The figure 4 below shows how the anhydrous diethyl ether protects Grignard reagent from oxidation:

Figure 4

There is a layer of oxide coated on the surface of magnesium which used to synthesis Grignard reagent. The oxide layer works to prevent it to react with alkyl bromide. The formation of Grignard reagent is highly exothermic which will produce a lot of heat energy from the system. Once the reaction has been initiated, the system will reflux itself in the bottom flask without any external heat source. The adding of a drying tube that contains calcium chloride to the reflux apparatus is used to protect the reaction from atmospheric moisture.

In this experiment, bromobenzene is the alkyl halide used to generate Grignard reagent.

Figure 5

Once the Grignard reagent is readily formed, the carbonyl compound has been introduced into the reagent in order to synthesis the expected product. The methyl benzoate (ester) acts as the carbonyl containing compound in the experiment. The reaction between methyl benzoate and Grignard reagent is showing in the following figure 6:

Figure 6

Dissociation of magnesium alkoxide produces a ketone which tends to react further with more Grignard reagent. The final step of the synthesis is involving hydrolysis of the magnesium alkoxide by using a mineral acid. As the result, the reaction synthesizes an alcohol, triphenylmethanol and magnesium salt (water soluble).

A side reaction may take place in the reaction between phenylmagnesium bromide and bromobenzene. The side product has been produced is biphenyl which consists of two phenyl rings. The biphenyl is known as impurity in the experiment. The impurity can be removed from the product through a method of recrystallization since biphenyl is much more soluble in ligroin compared to triphenylmethanol.

Apparatus: dropping funnel, two neck round bottomed flask, drying tube, condenser, sonicator, magnetic stirrer, hot plate, separating funnel, beaker, Buchner funnel, glass funnel, melting point apparatus

Materials: magnesium turning, anhydrous diethyl ether, bromobenzene, calcium chloride, methyl benzoate, iodine crystal, 10% H₂SO₄, ice, sodium bisulfate, sodium sulfate anhydrous, petroleum ether, methyl spirit

Procedure:

Part A – Preparation of phenylmagnesium bromide (phenyl Grignard reagent)

1. A condenser and a 50ml dropping funnel were set up to a 250ml two neck round bottom flask.

2. A calcium chloride drying tube was inserted into the top of the condenser.

3. 1.4g of magnesium turning was weighed and placed into the two neck round bottom flask with a stir bar. 10ml of anhydrous diethyl ether was added immediately into the two neck bottom flask.

4. 6.2ml of bromobenzene and 30ml of anhydrous diethyl ether were added into the 50ml dropping funnel.

5. 5ml of the mixture in dropping funnel was added to the ether/magnesium mixture. The mixture is stirred with a magnetic stirrer.

6. If the reaction does not begin immediately, both palms were placed around the bottom of the flask to keep it warm.

7. A small amount of iodine crystal was added directly into the magnesium surface if the reaction does not take place after 5-10minutes.

8. Once the reaction was initiated and the formation of Grignard reagent became steady, the ether refluxed itself. The remaining mixture in dropping funnel was added dropwise into the round bottom flask.

9. The solution was allowed to reflux for 10 minutes.

Part B – Preparation of triphenylmethanol from Grignard reagent

1. After reflux, the round bottom flask was cooled down in ice bath and a solution of 3.2 ml of benzoate dissolved in 15ml anhydrous diethyl ether was placed in the dropping funnel.

2. The solution was added over 5 minutes to avoid the solution to get too exothermic.

3. Once the reaction was completed, the solution was heated to reflux for 10 minutes to complete the reaction.

4. After reflux, the round bottom flask was cooled with ice bath and the mixture was poured into a 600ml beaker with contains 75g of ice and 30ml of 10% H₂SO₄.

5. The mixture was stirred until all white solid was dissolved.

6. The mixture was poured into the separating funnel and separated the two layers. The ether layer was washed with 15ml of H₂SO₄, followed by 15ml of water and then with a solution of 1g of sodium bisulfite dissolved in 12ml of water.

7. The ether layer was dried over sodium sulfate anhydrous and filtered with cotton wool. 15ml of petroleum ether was added to the ether layer. The ether layer was concentrated over a steam bath at 60-80 °C until the triphenylmethanol was formed. The product was cooled in an ice bath.

8. The product was recrystallized from methyl spirit.

9. The weight, yield and melting point of triphenylmethanol were determined.

Results and calculation

Weight of magnesium = 1.4009g

Volume of bromobenzene = 6.2ml

Volume of methyl benzoate = 3.2ml

Weight of watch glass = 14.8091g

Weight of watch glass + weight of product = 13.7410

Weight of product = 1.0681g

Atomic weight of magnesium = 24.31g/mol

Number of mole of magnesium = 1.4009 g / 24.31 g mol^-1

= 0.0576mol

Molecular weight of bromobenzene = 157g/mol

Density of bromobenzene = 1.495 g/cm³

Weight of bromobenzene = density x volume

= 1.495 g/cm³ x 6.2cm³

= 9.269g

Number of mole of bromobenzene = 9.269g/ 157 g mol^-1

= 0.0590mol

Since the magnesium is limiting reagent, so the number of mole of Grignard reagent produced is limited by number of mole of magnesium.

Number of mole of Grignard reagent produced = 0.0576mol

Density of methyl benzoate = 1.0837 g/cm³

Weight of methyl benzoate = density x volume

= 1.0837 g/cm³ x 3.2cm³

= 3.468 g

Molecular weight of methyl benzoate = 136.144g/mol

Number of mole of methyl benzoate = 3.468 g / 136.144g mol^-1

= 0.0255 mol

Since the Grignard reagent is excess, the product is limited by methyl benzoate so that it is limiting reagent in the reaction.

Thus, the number of mole of triphenylmethanol = 0.0255 mol.

Molecular weight of triphenylmethanol = 260.318g/mol

Theoretical weight of triphenylmethanol = 0.0255mol x 260.318g/mol

= 6.6381 g

Actual weight of triphenylmethanol =

Percentage yield = actual yield / theoretical yield x 100%

= 1.0681g / 6.6381g x 100%

= 16.09%

Melting point of triphenylmethanol = 153°C ~ 156°C

Discussion:

The purpose of this experiment is to synthesis triphenylmethanol by using Grignard reagent. In order to synthesis triphenylmethanol, Grignard reagent is playing an important role because Grignard reagent is the key reagent in this experiment. The presence of water in the process of generating Grignard reagent will causes the particular reagent to be decomposed. So, the solvent used in the experiment must not contain any water such as diethyl ether since it is a water free solvent. In order to make sure the water in air can be eliminated, a small amount of calcium chloride has been placed with the drying tube on the top the condenser. The calcium chloride acts as a drying agent which to absorb all the water from the air in the reflux apparatus and it prevent the atmospheric moisture. The purpose of using magnetic bar is to increase the rate of reaction for Grignard reagent.

In order to produce Grignard reagent, the magnesium turning was added with anhydrous diethyl ether. Magnesium turning (thin shaving with high surface area) is usually used in preparation of Grignard reagent due to its large surface area that can increase the reaction rate. The diethyl ether functions as the medium for the Grignard reaction to take place and stabilize the reagent. This is because the solvent (diethyl ether) is highly volatile solvent which can prevent the water from atmosphere approaching to the Grignard reagent after the Grignard reagent is formed. Besides, diethyl ether is easily removed from the reaction mixture since it has a low boiling point of 36°C. A mixture of bromobenzene and diethyl ether was prepared in the dropping funnel. The adding of diethyl ether in the mixture is works for the similar function which make sure the solvent is free from water. The addition of ether and bromobenzene mixture into the magnesium surface might not allow the reaction to occur due to lack of heat energy.

The iodine crystal was added into the magnesium surface because the heats from water bath or palm were not enough to initiate the reaction. Alternatively, the iodine crystal was added instead of increasing the temperature that supplied to the system in order to prevent the explosion since diethyl ether is highly flammable. The iodine crystal facilitate the reaction either activating the magnesium through removal of its oxide coating or by oxidizing the bromide in organic compound to form negatively charged bromide which is more reactive towards magnesium. A second alternative is place the flask containing reaction mixture over a sonicator to start the Grignard reaction. The sonicator is used to produce ultrasonic wave in which helps to remove the oxide coating physically.

When the reaction has been initiated, the appearance of bubbles on the solvent surface indicated that the formation of phenylmagnesium bromide start to occurr. The bromobenzene is reacted with magnesium metal to form phenylmagnesium bromide which is known as Grignard reagent. The chemical reaction between magnesium and bromobenzene is shows in below:

The formation of Grignard reagent is solvated by diethyl ether which protects the reagent from attacked by water molecules. If the water reacts with Grignard reagent, the decomposition of the particular reagent will occur. The mixture of ether and bromobenzene was added slowly to make sure that the Grignard reagent form steadily in the reaction. The side product may be generated in high yield if the mixture is added in a large volume at the same time. The formation of Grignard reagent is an exothermic process. Thus, the system can refluxed itself without any heat supply to it.

After reflux, the Grignard reagent produced was cooled down in an ice bath in order to reduce its temperature. This is to prevent the immediate addition of solvent from evaporating quickly due to high temperature of Grignard reagent if cooling down process is not taken. The methyl benzoate is the subsequent reactant which was used to react with Grignard reagent in this experiment. In order to avoid the reaction between Grignard reagent and methyl benzoate get too exothermic, the methyl benzoate in anhydrous diethyl ether must be added in a small amount. The system was refluxed itself to produce (Ph)₃CO-Mg-Br as product.

The Grignard reagent can be dissociated to form negatively charged carbanion which attacked the carbonyl carbon with partial positively charged. The carbonyl carbon of methyl benzoate was attacked by the nucleophilic carbanion during reflux. The nucleophilic addition of Grignard reagent to methyl benzoate caused the methoxide became the leaving group from the intermediate and the formation of benzophenone. Since the benzophenone consists of a carbonyl carbon as functional group, this favored the second nucleophilic attack of Grignard reagent and (Ph)₃CO^- anion with three benzene ring has been produced in the solution through reflux. The solution was treated by using sulphuric acid to protonate the (Ph)₃CO^- anion to generate the triphenylmethanol, (Ph)₃COH as product. Triphenylmethanol has a synonym which is known as triphenylcarbinol. The formation of triphenylmethanol is highly exothermic, so ice bath was used to reduce the temperature and heat energy produced from the system. In this stage, some white solid were precipitated out in the cold solution, the white solid is the desired product. The mechanism of formation of triphenylmethanol by using Grignard reagent via nucleophilic addition is shown in the following Diagram 1:

Diagram 1

In order to remove the impurities and side product, the washing process is necessary. The ether layer was washed with sulphuric acid and followed by water. The aqueous solution was used to remove the water-soluble impurities in the mixture. Then, sodium bisulfide was a base which was used to neutralize the acid added before. Sodium sulfate anhydrous has been introduced to remove all the water in the mixture since it is a drying agent and the clump of solid sodium sulfate was filtered. In the process of producing triphenylmethanol, some side products have been produced at the same time such as biphenyl.

Therefore, petroleum ether was used in the experiment in order to let the biphenyl dissolved in it so that this side product can be removed via recrystallization. This might be due to the higher polarity of the triphenylmethanol compared to biphenyl which enables the triphenylmethanol to dissolve easily in the polar methyl spirit.

Recrystallization of triphenylmethanol has been carried out to purify the product. Methyl spirit (denatured alcohol) is a mixture of methanol and ethanol which was used as the dissolve medium in recrystallization. After recrystallization, the product is still not pure enough since its melting point of 153°C ~ 156°C is lower than the theoretical melting point which is 162°C. This deviation may be due to the product is not completely dry so that it affects the melting point of the product. The yield of the product in the experiment is 1.0681g which contributes to the percentage yield of 16.09%. The percentage yield is very low may be due to there are many impurities were formed in the reaction since the impurities compete the material which required for the formation of desired product.

Precaution steps:

1. Avoid the diethyl ether from any heat source since it is extremely flammable.

2. Carry out the reaction away from any heat source.

3. Place all the glassware in a 110°C in order to make sure all the glassware is totally dry.

4. Use solvent to wash the glassware instead of distilled water.

Synthesis of Dibenzalacetone by Aldol Condensation

Objective:

1. To carry out a mixed aldol condensation reaction

2. To study the mechanism of aldol condensation reaction

Introduction:

The reaction of an aldehyde with a ketone employing sodium hydroxide as the base is an example of a mixed aldol condensation reaction, the Claisen-Schmidt reaction. The double mixed-aldol condensation reaction between acetone and benzaldehyde was carried out. Acetone has α-hydrogens (on both sides) and thus can be deprotonated to give a nucleophilic enolate anion. The alkoxide produced is protonated by solvent, giving a β-hydroxyketone, which undergoes base-catalyzed dehydration. The elimination process is particularly fast in this case because the alkene is stabilized by conjugation to not only the carbonyl but also the benzene. In this experiment, excess benzaldehyde such that the aldol condensation can occur on both sides of the ketone.

Dibenzalacetone is readily prepared by condensation of acetone with two equivalent of benzaldehyde. The aldehyde carbonyl is more reactive than that of the ketone and therefore reacts rapidly with the anion of the the ketone to give a β-hydroxyketone, which easily undergoes base catalyzed dehydration. Depending on the relative quantities of the reactants, the reaction can give either mono- or dibenzalacetone.

Dibenzalacetone is a fairly innocuous substance in which its spectral properties indicate why it is used in sun-protection preparations. In the present experiment, sufficient ethanol is present as solvent to readily dissolve the starting material, benzaldehyde and also the intermediate, benzalacetone. The benzalacetone once formed, can then easily to react with another mole of benzaldehyde to give the desired product in this experiment, dibenzalacetone.

Apparatus: Erlenmeyer flask, Buchner funnel, glass funnel, melting point apparatus, UV/Vis spectrometer, FTIR spectrometer

Materials: Benzaladehyde, acetone, sodium hydroxide, 95% ethanol, ethyl acetate, ice

Procedure:

1. 5g of NaOH was added to 25ml of H­₂O in an Erlenmeyer flask and the solution was swirled.

2. 25ml of 95% ethanol was added and the solution was allowed to come nearly to room temperature.

3. 2.9g of acetone and 10.5ml of benzaladehyde were added.

4. After 15 minutes of occasional swirling, the products was filtered on a Buchner funnel.

5. The product was washed with cold ethanol and was allowed to suck dry.

6. The yellowish product was recrystallized from ethyl acetate.

7. After recrystallization, a yellow crystalline was obtained.

8. The weight, yield, and melting point of the product were determined.

9. The UV and IR spectra of dibenzalacetone were ontained.

Results:

Weight of watch glass = 36.1291 g

Weight of watch glass + products = 45.6878 g

Weight of products = 9.5587

Melting point of products = 109 °C

Moles of benzaldehyde used = 0.1 mol

Moles of acetone used = 0.05 mol

Moles of dibenzalacetone produced = Moles of acetone used

= 0.05 mol

Theoretical weight of dibenzalacetone produced = 0.05 mol * 234.29 g mol^-1

= 11.7145 g

Percentage yield of dibenzalacetone = 9.5587 g / 11.7145 g × 100 %

= 81.60 %

Discussion:

Condensation is a process which joins two or more molecules usually with the loss of a small molecule such as water or an alcohol. Aldol condensation (Claisen-Schmidt reaction) definitely is a process which join two carbonyl groups with a loss of water molecule in order to form β-hydroxyketone. The product is also known as adol because it containing two functional groups which includes aldehyde (or ketone) group and alcohol group. The product dibenzalacetone was formed from the reaction between an acetone molecule and two benzaldehyde molecules. Generally, the aldol condensation is carried out under a base condition.

Sodium hydroxide was mixed with distilled water then was used to react with sufficient ethanol as the first step. The particular reaction is an exothermic reaction which released the heat energy to the surrounding from the reaction. The sodium hydroxide was functioned as a catalyst in the reaction. The ethanol acts as a solvent which allows the acetone and benzaldehyde to dissolve and react with each other. After that, acetone and benzaldehyde were mixed in the solvent which turns to yellow colour quickly. Eventually, the product was formed with a yellow precipitate appear in the reaction after a few seconds. However, there are some impurities and side products were formed in the yellow precipitate. So, recrystallization was carried out by using ethyl acetate as solvent in order to purify the product and hence a pure product could be obtained for the ultraviolet (UV) and IR spectra analysis. In the recrystallization process, the yellow precipitate in ethyl acetate was immersed into an ice-bath in order to obtain a higher yield of product. This is because the heat energy in the precipitate easily to be released since the precipitation formation is an exothermic reaction and hence it maximizes the formation rate of the product.

Acetone is considered as a stable and unreactive compound, so it should be converted into anionic form to increase its nucleophile properties to initiate the reaction. The sodium hydroxide dissolves in water to produce hydroxide ion and it tends to attack the α-hydrogen in acetone and to form water molecule. The deprotonation of acetone caused the enolate ion was produced as nucleophile which will be used in the synthesis of dibenzalacetone. An enolate ion was formed which it exists as resonance-stabilized structure which shown in the following diagram:

Diagram 1

The acetaldehyde enolate ion attack to the benzylic carbon of benzaldehyde via nucleophilic addition to form the intermediate as shown in below:

Diagram 2

The oxygen attached to the benzylic position of carbon tends to attract one proton from water molecule to form hydroxide group in the intermediate. This is the formation of an aldol since the molecule consists of a carbonyl group and an alcohol group. In the basic condition, the hydroxide ion tends to remove one proton from the α-carbon resulting the formation of C=C double bond at the α and β carbon. At the same time, the hydroxide group attached to the β carbon forms a leaving group. After the condensation, benzalacetone was formed after two water molecules leaved as shown:

Diagram 3

The benzalacetone tends to form benzalacetone enolate ion after the hydroxide group from the surrounding attack the proton which attached to the carbon at benzylic position.

Diagram 4

The same process has been take place as in the Diagram 2 but with the more bulky benzalacetone enolate ion as the material. The benzalacetone enolate ion acts as a nucleophile which attacks another benzaldehyde. The protonation of the aldol took place followed by the hydroxide groups have been eliminated as leaving groups. As a result, the nucleophilic addition and base-catalyzed dehydration lead to the formation of the desired product which is dibenzalacetone. The mechanism of dibenzalacetone formation was shown in the Diagram 5:

 

 

 

 

 

 

 

 

Diagram 5

The overall mechanism of the dibenzalacetone was summarized in the Diagram 7 as shown in below:

Diagram 7

The percentage yield of dibenzalacetone in this experiment is 83.27%. Some of the product has been lost during the process of recrystallization. In recrystallization, some of the product dissolved in the ethyl acetate. The melting point of the product is lower than the actual melting point (110 °C ~ 111 °C). This is because there is some impurities exist in the particular compound which will tend to lower the melting point of the dibenzalacetone.

Precaution steps:

1. Avoid to carry out the experiment near the fire since the organic solvent are mostly flammable.

2. Avoid to smell benzaldehyde directly.

3. Handle carefully with sodium hydroxide since it is corrosive.

Thin Layer Chromatography and Column Chromatography

Objective:

Part I:

1. To learn the technique of TLC and the visualization of colourless components.

2. To identify an unknown drug by a TLC comparison with standard compounds.

Part II:

1. To learn the technique of column chromatography.

2. To separate the mixture of pyrene and p-nitoraniline by column chromatography.

Introduction:

Chromatography is a common laboratory technique to separate and analyze two or more analytes in the mixture by distribution of two phases: a stationary phase and a mobile phase. The stationary phase is a phase which allows the mobile phase to travel along. These two phases can be solid-liquid, liquid-liquid or gas liquid. This method works on the principle that different compounds with different solubilities and adsorptions to the two phases which they are to be partitioned. The compounds to be retained on the stationary phase are more interacted with it while the compounds to be moving carried along by the mobile phase. The rates of migration for each component on the system is depends on the degree of the compounds of mixture are adsorbed by the stationary phase and their degree of solubilities on the mobile phase. The stronger the adsorption by stationary phase, the slower the compounds travels along the mobile phase. As a result, the distance of separation for each compound in the mixture will be different. The types of chromatography is divided into few types which include gas chromatography(GC), high performance liquid chromatography(HPLC), thin layer chromatography(TLC), and column chromatography(CC). However, only TLC and CC are applied in this experiment.

Thin layer chromatography (TLC) is a solid-liquid form of chromatography where the stationary phase is usually a polar adsorbent while the mobile phase can be one single solvent or a combination of solvents. TLC is a quick and inexpensive technique that can be used to 1) determine the number of compounds in a mixture, 2) identify the compounds, 3) monitor the progress of a reaction, 4) determine the effectiveness of a purification, 5) determine the appropriate conditions for column chromatography separation, and 6) analyze the fractions obtained from column chromatography. In thin layer chromatography, the stationary phase is refers to polar adsorbent, usually is silica gel or aluminium oxide which is coated on an aluminium plate. Generally, polar solvent is used as the mobile phase in the system to carry the analytes by passing through the stationary phase. The stationary phase in TLC chromatography is typically silica gel, (SiO₂.xH₂O)_n which has been shown in the diagram 1 below:

Diagram 1

Different compounds are spotted on the silica gel plate (stationary phase), the prepared solvent (mobile phase) will carry the compounds goes up along the plate through capillary action which the solvent travels from the bottom to the solvent front. Once the dilute solutions of compounds are spotted on the plate, then development of solvent is the next. After that, the position of each compounds can be visualized under the presence of ultraviolet (UV) light. The compounds in the mobile phase will have different interaction with the polar stationary phase. The factors are mainly depends on the polarity of adsorbent (silica gel in this experiment), solvent polarities, and functional groups of the compounds. The polar adsorbent will more strongly attract the polar molecules of compounds and it will have lower affinity to the non-polar compounds. Hence, the movement of compounds with different polarities could be different. In addition, the polarity of solvent is very important to the compound separations, a solvent system may increase in its polarity by changing the composition of the solvent mixture. The more polar the solvent, the faster the compounds can be drawn up, which means the further the compounds move. The comparison of the polarities of solvent are listed down in the diagram 2.

Diagram 2

The second factor that affects the interaction between stationary phase and compounds is functional group of each compound. The highly polar groups in compound will cause the stronger adsorption and eluted less readily to the stationary phase compared to less polar compounds. Hence, the highly polar compounds will tend to interact strongly with the polar adsorbents and absorb onto the fine particles of the absorbent, hence it cannot travel further. The adsorption strengths of each compound having the following types of functional groups in the order of increasing group polarities.

However, the variation may take place which depends on the overall structure of each compound.

Column chromatography is one of the most useful methods for the separation and purification of both solids and liquids when carrying out small-scale experiments. Like TLC, the silica gel is used as a stationary phase while an organic solvent is used as the mobile phase which its polarity should be lower than silica gel. Column chromatography is carried out in a glass tube that is clamped vertically with the mixture of samples at the top. The samples are dissolved in a small quantity of solvent which is used to apply on the top of the vertical column. In this case, the solvent (mobile phase) will tend to flow down through stationary phase (silica gel) instead of the capillary action. The compound with less polar characteristics will elute faster through the column due to the silica gel has the strong affinity towards the more polar compounds. Eventually, the compounds start to be separated as the solvent is allowed to flow through the stationary phase. Due to the difference in their polarities, the solvent acts as the mobile phase will carry the less polar compounds further down from the top in the system. Below the column, several flasks are used to collect the solvent with compound in various fractions. The solvent is continually added to the top of the column until each band resolves and is carefully collected. As a result, the experiment is end up with the separation of two compounds from the mixture into two different portions. With coloured substances, the bands might be visible and easily to be collected as they run off the column. However, colourless compounds can be observed directly. So, the particular compound in eluting solvent is collected in many small fractions and testing each of them by using TLC. A fresh solvent (mixture of solvent similar to the eluent in column chromatography) is being used in TLC for the next step of identifying the compounds.

The total distance traveled by the compounds on silica gel plate are measured and is being compared to each other. The migration rate of each compound is compared by using the retardation factor, R_f. Retardation factor is the ratio of distance traveled of the compound to the distance of the solvent traveled. Retardation factor, R_f is the distance of compound traveled divided by the total distance of solvent travelled in TLC plate. If two spots in the TLC plate travel the same distance or have the same value of R_f, then both compounds might be concluded as the same compounds.

Part I: TLC analysis on analgesics drugs

Apparatus: UV lamp, capillary tube, 250ml beaker

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

Procedure:

PartA: Spotting the TLC plate

1. A TLC plate was obtained from instructor. Holding the edges of the plate carefully.

2. Set the plate down on a clean and dry surface, then a line was drawn across the plate about 1.0cm from the bottom of the plate by using a 2B pencil.

3. Five lines of 2-3mm were drawn, spaced about 0.6cm apart and running perpendicularly through the lines across the bottom of the TLC plate. 0.5cm must be spaced from each side of the edges.

4. 5 different analgesics were spotted on each 2-3mm lines. Firstly, acetaminophen was spotted on the plate, followed by caffeine, unknown A, aspirin and lastly the unknown B. The plate was examined under the UV light to check whether enough each solution has been applied.

Part B: Developing the TLC plate

1. A developing chamber was prepared by using a 250ml beaker, a half-piece of filter paper inside and aluminium foil to cover.

2. Mixture of 1:3 of ethyl acetate : hexane was poured into the beaker to the depth of about 1cm. The TLC plate was placed in the developing chamber.

3. After the solvent has risen to near the top of the plate, the plate was removed.

Part C: Visualization

1. The colourless compounds were visualized by illumination of the plate with UV lamp.

2. The spots were outlined by using a 2B pencil. The spots may be visualized by putting the plate in an iodine chamber for a couples of minutes.

Part D: Comparison of the unknown with reference standards

1. The plate was sketched in notebook and the R_f value was calculated for each spot.

2. The unknown drug was determined based on R_f 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:

Part A: Column preparation

1. A 49cm chromatography column, 15g of deactivated Silica gel and 110ml of developing solvent mixture (ethyl acetate:hexane; 1:3) were obtained.

2. A slurry of the adsorbent( silica gel) was prepared with a solvent in a 250ml Erlenmeyer flask.

3. A small plug of cotton was pushed into the constriction at the bottom of the column. The column was clamped in a vertical position and 0.5cm layer of the sodium sulfate anhydrous was added on top of the cotton.

4. Ensuring the stopcock of the column is closed, 15ml of solvent was poured in. After setting, all the slurry was quickly decanted through a funnel into the column.

5. The stopcock was opened and allowed the solvent to drain while tapping the walls of the column with the ends of a folded price of rubber tubing.

6. Once the solvent level is within 6cm of the top of the adsorbent, the packing should be essentially complete. 0.5cm level layer of sodium sulfate anhydrous was added on the adsorbent.

7. Excess solvent was drain off until its level is precisely on top of the sodium sulfate anhydrous and the stopcock was closed.

Part B: Separation and collection of pyrene and p-nitroaniline

1. The mixture of pyrene and p-nitroaniline was took and a few drops of ethyl acetate was added to dissolve as much as possible.

2. The solution was transferred directly to the top of the sulfate anhydrous layer with a dropper.

3. The solvent was drain off until the mixture solution is just below the top of the sodium sulfate anhydrous.

4. The wall was rinse with 1ml of fresh solvent(ethyl acetate/hexane 1:3) and was drain until the level was once again below the top of sodium sulfate anhydrous. The rinsing of the walls was repeated until the solvent above the silica gel is virtually colourless.

5. The column was filled carefully with the fresh solvent(ethyl acetate/hexane 1:3) and allowed solvent to drain.

6. The separation of bands was observed as the column develops. 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 column, a new test tube was replaced and the yellow band was collected into three fractions.

8. Each fraction was concentrated to a small volume by evaporation for analysis by TLC.

Part C: Analysis of the fraction

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 mixture of ethyl acetate/hexane 1:3.

3. The TLC plate was visualized with the UV lamp to determine the fraction of pure pyrene and pure p-nitroaniline.

4. The chromatogram was drawn in the notebook.

5. The R_f value was calculated for pyrene and p-nitroaniline.

6. The pure fraction of pyrene was combined in a pre-weigh test tube and the pure fraction of p-nitroaniline in another test tube. Both solvents were evaporated on a stem bath.

7. Once the solvent has evaporated, the weight of the pure pyrene and p-nitroaniline were calculated.

Results:

Part I:

Total distance of solvent travelled from bottom line in TLC plate = 8.0cm

Retardation factor, R_f

= Distance of sample travelled from the bottom line / Total distance of solvent travelled from bottom line in TLC plate

 

Samples	Distance travelled from the bottom line (cm)	Retardation factor, Rf
Acetaminophen	0.50cm	0.0625
Caffeine	0.35cm	0.0438
Unknown A	0.50cm	0.0625
Aspirin	2.40cm	0.3344
Unknown B	2.20cm	0.2750

Diagram of acetaminophen, caffeine, unknown A, aspirin, and unknown B travelled on the TLC plate

Inference: unknown A is acetaminophen whereas the unknown B is aspirin.

Part II:

Total distance of solvent travelled from bottom line in TLC plate = 8.0cm

Retardation factor, R_f

= Distance of sample travelled from the bottom line / Total distance of solvent travelled from bottom line in TLC plate

 

Samples	Distance travelled from the bottom line (cm)	Retardation factor, Rf
1st fraction of pyrene	6.1cm	0.7625
2nd fraction of pyrene	6.1cm	0.7625
3rd fraction of pyrene	6.1cm	0.7625
4th fraction of pyrene	6.1cm	0.7625
1st fraction of p-notroaniline	1.6cm	0.2000
2nd fraction of p-notroaniline	1.7cm	0.2175
3rd fraction of p-notroaniline	1.7cm	0.2175
4th fraction of p-notroaniline	1.7cm	0.2175

Diagram of pyrene and p-nitoraniline travelled on the TLC plate

Inference: pyrene is present in the 1^st to 4^th spots while 5^th to 8^th spots containing p-nitroaniline.

Discussion:

In the thin layer chromatography, the eluent (solvent) is prepared by using a mixture of hexane and ethyl acetate in the ratio of 3:1. The polarity of the particular solvent cannot be too low because the polar compounds will not be able to carry by the eluent and will not be separated, so that the separation might not be observable. If the solvent of too high polarity is used, the polar compound will travel so fast that the separation between non-polar compound and polar compound to become so small and poor separation will be observed. The solvent mixture of ethyl acetate and hexane (1:3) is believed that it has the optimized solubility for the organic compounds to dissolve in the solvent. In another word, the compounds can be easily to be carried by the solvent in the TLC plate. A few drops of acetic acid were added into the particular solvent in order to protonate the organic compound on the TLC plate and prevent them from ionization. This is due to the deprotonation of organic compounds will cause the compound to form ions. So, the adding of acetic acid is used to maintain the structure of organic compound as they can travel up through the TLC plate. Before the TLC plate is placed into the solvent. A filter paper was dipped inside the solvent in a beaker which is covered by using an aluminium foil. This is to create a system that prevents the vapourization of organic solvent and hence the solvent is allowed to travel up along the plate faster. After the TLC plate was introduced into the solvent, the solvent is starting to migrate itself and the compounds on the TLC plate until the solvent front has been reached.

There are three components in the TLC which include the TLC plate with adsorbent, the development solvent and the organic compounds that to be analyzed. The adsorbent, silica gel consists of a three dimensional network of thousands of alternating silicon and oxygen bonds. It is a very polar and is capable of hydrogen bonding due to its partial positive charge in silicon and partial negative in oxygen. The silica gel with compete with the development solvent for the organic compounds as the solvent is traveling up through the TLC plate. The silica gel tends to bind the compounds (on stationary phase) while the development solvent tried to dissolve the compounds (on mobile phase) in order to carry the compounds along the plate as the solvent travels up. All the compounds are possible to be adsorbed into the stationary phase however the time of adsorption of compounds in the particular phase is depends on the polarity of each compound. The more polar the compound is, the longer the time taken that the compound adsorbed into the stationary phase so it eluting speed is slower (more time on stationary phase). Less polar compounds are weakly adsorbed, so the time taken for less polar compounds to be adsorbed on stationary phase is shorter. As a result, the less polar compounds can travel further along the plate compared to the more polar compounds.

In this experiment, the analgesics drugs have been analyzed by using TLC are acetaminophen, caffeine, and aspirin. The structure of each compounds are shown as below:

The polarity of the compounds could be compared by looking at the structure of these compounds. The polarity of the compound is due to the effect of electronegativity in atoms and the asymmetrical structure of compound. The unequal sharing of electron within the bond causes the formation of an electric dipole which leads to partial positive and partial negative exist in the several atoms. The highly electronegative atoms present in these compounds are O, N, and F. These highly electronegative atoms tend to withdraw the electrons towards themselves from the aromatic ring and hence it polarizes the compounds. Nitrogen and oxygen atom present in these compounds have higher electronegativity compared to the carbon atom and hydrogen atom. Thus, nitrogen and oxygen atom acquire partial negative charge while the carbon and hydrogen atom acquire partial positive charge. Hence, caffeine has the highest polarity, followed by acetaminophen while aspirin possesses the lowest polarity. The sequence of increasing in polarity is arranged in the order below: 

Based on the retardation factor, R_f, the polarities of these compounds can be compared. The higher the R_f, the lower the polarity and hence the distance traveled by the compound would be longer. Aspirin has the highest R_f value. The interaction between aspirin and stationary phase (silica gel) is the weakest which allows the aspirin can travels up along the plate fastest. A stronger interaction in between the silica gel and acetaminophen causes the compound move slower when travels up the TLC plate. The polarity of the acetaminophen is considered lower than caffeine but the difference is quite small. This can be shown in the distance traveled by both compounds which corresponding to their R_f value. In this part, TLC method can be used to identify the two unknowns spotted on the TLC plate. The compounds with same polarities usually travel up through the plate in the same distance and possess the same R_f value provided the stationary phase and mobile phase are identical. Unknown A is predicted as acetaminophen while unknown B is forecasted as the aspirin. This is because the R_f value of unknown A and unknown B are same with the R_f value of acetaminophen and aspirin respectively.

Column chromatography is used to purify the individual organic compound from the mixture of compounds. In column chromatography, the stationary phase and mobile phase used are same as used in thin layer chromatography. The adsorbent (stationary phase) used is a solid which silica gel is usually being used. The eluent (mobile phase) used is the mixture of hexane and ethyl acetate in the ratio of 1:2. The compounds used were the pyrene and p-nitroaniline. The structure of each compound are shown as below: Based on the structure of the compound, the pyrene has the lower polarity due to its delocalized electron in the aromatic ring. The delocalizing π electrons are distributed evenly in the whole structure and hence it stabilize the pyrene. In addition, there is no other electronegative atom attach to the pyrene as substituent, so the electron are just delocalize within the four aromatic ring of pyrene. On the other hand, the p-nitroaniline has higher polarity due to its electronegative substituent in the structure. 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. Due to the difference in polarities in each compound, the interaction with the silica gel would be different. The polar compound would have the stronger interaction with the silica gel. This is because the polar silica gel tends to pull the polar p-nitroaniline toward the stationary site and causes the compound becomes more difficult carried by the solvent through the system. As a comparison, the less polar pyrene was weakly adsorbed into the stationary phase. The silica gel would not tend to attract the less polar compound into the stationary site and hence the particular compound can be carried by the solvent to reach the bottom of the column. The pyrene is said that it elute faster than the more polar p-nitroaniline.

The total number of fraction have been collected was 19 fractions after all the yellow bands (p-nitroaniline) transferred out from the column. After evaporated most of the solvent, the 19 fractions were concentrated into eight fractions which each of the fraction have been used to apply on a TLC plate by using TLC. The separation of compounds is considered as successful since the 1^st to 4^th spots are containing pyrene only. The 5^th to 8^th spots are only contain the compound of p-nitroaniline. These can be proved by checking the distance of compounds traveled on TLC plate of each fraction and the corresponding to the R_f­ value are the same. However, the colours of the 1^st and 4^th fractions of pyreme on the TLC plate were faded compared to the 2^nd and 3^rd fractions. This might be due to the concentration of pyrene in the former fractions were not the same with the latter fractions. The amount of pyrene collected in the 1^st fraction is less because it collected more solvent. The 4^th fraction also has less concentration of compound because the concentration left in the column after 2^nd and 3^rd fractions of compound have been collected. The four spotted compounds were noticed that they were connected to each other on the TLC plate. This might be due to the diameter of the spotted place was too big. The 1^st and 4^th fractions of p-nitroaniline also have the same condition which their colours on the TLC plate were faded. It was believed that the 1^st fraction and 4^th fraction of p-nitroaniline have the similar condition with the 1^st and 4^th fractions of pyrene. The concentrations of the p-nitronaniline were not enough so they appear lighter colours on the plate under the sources of ultraviolet light.

By using the iodine as the development solvent in TLC, the similar observation for the both pyrene and p-nitroaniline were observed. The distance traveled of compounds were almost similar compared to previous solvent. But, the difference was just the colour of the spots on the TLC plate.

Precaution steps:

1. Do not move the beaker after the TLC plate had introduced into the beaker.

2. Do not look directly to the ultraviolet lamp.

3. Avoid to use pencil on the TLC plate

4. Do not touch on the surface of silica gel of TLC plate by using finger.