BSc(Hons) Forensic Investigation

Honours Project

School of Engineering and Built EnvironmentGlasgow Caledonian University

Cowcaddens RoadGlasgow G4 0BA

Contents

Section PageAbstract iList of Figures

List of Tables

ii

Chapter 1 – Introduction

1.1 – Aims and Objectives

1.2 – The Chemistry of Aromatic Compounds

1.2.1 – The Structure of Benzene

1

1.2.2 – Substitution Effects 41.2.2 – Substitution Effects 5

1.4 – Chemistry and Applications of Benzocaine 8

1.5 – Multi-step synthesis of Benzocaine

1.5.1 – Acetylation

10

1.5.2 – Oxidation 111.5.3 - Hydrolysis 121.5.4 – Fischer Esterification 131.6 – Instrumental Analysis

1.6.1 – Fourier Transform Infra-Red Spectroscopy

14

1.6.2 – Chromatography 161.6.2.1 – Thin Layer Chromatography 171.6.2.2 – High Performance Liquid Chromatography (HPLC) 18Chapter 2 – Experimental

2.1 – Equipment and Chemicals

22

2.2 – Acetylation of p-toluidine to p-methylacetanilide 242.2 – Oxidation of p-methylacetanilide to p-acetamidobenzoic acid

2.3 – Hydrolysis of p-acetamidobenzoic acid to PABA

25

2.4 – Fischer Esterification of PABA to Benzocaine 26Chapter 3 – Results

3.1 Acetylation

28

3.2 – Oxidation 333.3 – Hydrolysis 363.4 – Fischer Esterification 40Chapter 4 – Conclusion

4.1 – Discussion

46

4.2 – Further Work 48Appendix 1 – Table of mobile phases used for TLC

Appendix 2 – Table of solvent polarity parameters (P’)

50

Acknowledgments 51Bibliography 52

Abstract

The multi-step synthesis of benzocaine was carried out starting with p-toluidine in a

four step reaction. The reactions were acetylation, oxidation, hydrolysis and fischer

esterification. The product of each reaction was carried over to the next to complete

the synthesis of benzocaine. The product of each reaction was analysed using

melting point (Mp), thin layer chromatography (TLC), and fourier transform infra-red

spectroscopy (FT-IR) and the results were compared to data obtained for the

standard compounds to monitor the efficiency and progress of the reaction. The final

benzocaine product was also analysed using reversed phase high performance liquid

chromatography (HPLC) using a SphereClone 5U 005 (S) clone column, 250 x 4.60

mm x 5 microns dimensions and a 50:50 methanol:water mobile phase. The Mp,

TLC, IR and HPLC results confirmed that benzocaine had been successfully

produced using the multi-step synthesis. The HPLC chromatogram also showed that

the final product was contaminated with large amounts of p-aminobenzoic acid

(PABA) which had not been fully converted during the final (fischer esterification)

reaction.

i

List of Figures

Figure 1: Bromobenzene (C6H5BR) – note 6 membered ring of benzene illustrated using Kekule’s line

representationFigure 2: Top – Kekule’s resonance structures of benzene, Bottom –New benzene model displaying delocalised

electron cloud in between both resonance forms Figure 3: General structure of a local anaestheticFigure 4: Benzocaine (C9H11NO2)Figure 5: Acetylation of p-Toluidine to p-Methylacetanilide [7]

Figure 6: Oxidation of p-Mehtylacetanilide to p-Acetamidobenzoic Acid [7]

Figure 7: Hydrolysis of p-Acetamidobenzoic Acid to p-Aminobenzoic Acid (PABA) [7]

Figure 8: Esterification of p-Acetamidobenzoic Acid to Benzocaine [7]

Figure 9: HPLC schematicFigure 10: Top left going clockwise: Jasco 6100 FT-IR spectrometer, std. mp apparatus, UV lamp stand 9607-00,

Jasco HPLC instrumentFigure 11: p-Methylacetanilide ProductFigure12: TLC Plate 1 UV 254 nm: A) p-toluidine, B) p-methylacetanilide control, C) p-methylacetanilide productFigure13: p-Toluidine IR spectrumFigure 14: p-Methylacetanilide IR spectrum: top – control, bottom – productFigure 15: TLC Plate 2 UV 254 nm: A) p-methylacetamidobenzoic acid control, B) p-methylacetamidobenzoic acid

productFigure 16: p-Methylacetamidobenzoic acid IR spectrum: top – control, bottom – productFigure 17: Top – PABA 1 product, Bottom – PABA 2 productFigure 18: TLC plates 3 and 4 UV 254 nm: left – PABA 1, right – PABA 2Figure 19: PABA control IR spectrumFigure 20: Top – PABA 1 IR spectrum, bottom – PABA 2 IR spectrumFigure 21: Benzocaine productFigure 22: TLC plate 5 UV 254 nm – A) benzocaine control, B) benzocaine productFigure 23: Benzocaine IR spectrum: top – control, bottom – productFigure 24: Benzocaine HPLC Chromatogram 50:50 MeOH:H2O: top – control, bottom – product

List of Tables

Table 1: Hydrolysis reaction 1 and 2Table 2: Product results

ii

Chapter 1 – Introduction

1.1 – Aims and Objectives

• To successfully carry out the multi-step synthesis of benzocaine using the self-

designed method.

• To determine the efficiency of the process using Mp, TLC, IR and reversed

phase HPLC.

1.2 – The Chemistry of Aromatic Compounds

1.2.1 – The Structure of Benzene

An aromatic chemical is a substance which exhibits a fragrant smell, such as those

arising from fruits and spices. Common examples of aromatic compounds include

benzaldehyde, which is found in fruits (cherries, peaches, almonds); toluene found in

balsam resins, and benzene, the main petrochemical found in coal distillate [1].

In the early years, spices were important international commodities, however there

was an element of danger surrounding the spice trade as most of the products were

either pirated or destroyed in conflict. Eventually there was a growing need to extract

these chemicals from their plant sources and determine their structure so they could

be effectively synthesised at home. Large industrial quantities could then be

produced at a lower cost without the added risks associated with overseas trading [2].

1

In 1825, Michael Faraday was the first person to isolate benzene, the most common

and simple of the aromatics. Nowadays, benzene is isolated from petroleum by

cracking gasoline fractions and is used in the production of important industrial

chemicals such as styrene, phenol, acetone and cyclohexane. Aromatic compounds

are commonly found in many drugs, especially those used as painkillers and

anaesthetics. They can be found bounded to various side chains and functional

groups. Benzocaine in particular contains an aromatic ring attached to an ester group

(naturally occurring, fragrant compounds obtained from fruits and flowers). Other

important biological compounds are also aromatic in parts of their molecular structure

e.g. estrone hormone [2], [1].

Aromatic compounds can be identified from their structure. Benzene is the most

basic aromatic compound on the planet. The benzene molecule takes the form of a

six-membered-ring. The ring is effectively a hydrocarbon molecule composed of six

carbon atoms bonded together in a flat, planar, hexagonal shape. Each carbon is

bonded to one hydrogen atom giving the molecular formula C6H6. According to the

structure proposed by the famous German chemist, Friedrich August Kekule, the six-

membered ring formation of benzene also contains three alternating double bonds.

The presence of these double bonds would suggest that benzene is an unsaturated

highly reactive compound. Later, it was found this was not entirely true [1].

By the mid 1800s it was found that the double bonds in benzene did not break as

easily and it was unable to undergo the basic electrophilic addition reactions

observed in alkenes. It was found that benzene reacts with bromine in the presence

of iron to produce the substituted product, bromobenzene (C6H5Br), rather than the

addition product, C6H6Br2 (ref to “Figure 1”). In bromobenzene one of the hydrogen

atoms was replaced by one bromine atom. This was unusual as it was expected that

one of the carbon to carbon double bonds would break and each carbon atom would

bond to one of the bromine atoms. This was due to the stability of the benzene

molecule.

2

To account for this stability, it was assumed that the double bonds did not remain

fixed in three distinct positions rather the pi-electrons (associated with the double

bonds) were free roaming and circulated the entire molecule.

Figure 1: Bromobenzene (C6H5BR) – note 6 membered ring of benzene illustrated

using Kekule’s line representation

[http://wtt-pro.nist.gov/wtt-pro/index.html?cmp=bromobenzene]

In the progressive world of science, it was inevitable that scientists would eventually

find flaws in Kekule’s original benzene model [1].

Later on, it was discovered that all of the C-C bonds in benzene were equivalent. All

six bonds were found to be 139 pm long (between 134 pm for C=C bonds and 154

pm for common C-C bonds). This discovery confirmed that Kekule’s theory was not

entirely correct and benzene did not contain three distinct double bonds [1].

The most recent model addresses some of the short-comings of Kekule’s model.

First of all it is known that each carbon atom is sp2 hybridised and each of these have

p-orbitals which are perpendicular to the ring as well as sitting above and below the

plane of the ring. Each carbon has one p-orbital which can overlap equally well with

the p-orbitals of both of its neighbouring carbon atoms to form a double bond. As this

bonding is not restricted to one site, the pi-electrons found in the p-orbitals are

referred to as de-localised electrons.

3

The de-localised electrons are shared around the ring in a cloud form making it

impossible to pinpoint three distinct localised pi-bonds. This is the main reason as to

why the new benzene model is widely accepted over the Kekule proposal [1].

Kekule’s lined representation of benzene can still be used to illustrate the resonance

stability of the molecule (ref to “Figure 2”). It is claimed that benzene is a resonance

hybrid of two forms. This means that benzene does not switch between one and the

other as its true structure is found to be somewhere in between the two forms [1].

Figure 2: Top – Kekule’s resonance structures of benzene, Bottom –New benzene

model displaying delocalised electron cloud in between both resonance forms

[http://commons.wikimedia.org/wiki/File:Benzene_resonance_structures.png]

1.2.2 – Substitution Effects

When benzene undergoes a substitution reaction with another compound or

molecule, the substituent can have various affects on the reactivity of the ring. These

new substituted compounds will react in different ways depending on whether the

substituent is an activating group (donates electrons to the ring) or a deactivating

group (withdraws electrons from the ring) [1].

4

For example, -OH reacts with benzene to produce phenol (C6H5OH). The hydroxyl

group is an activating group which makes phenol one thousand times more reactive

towards another substance by providing an electron rich environment. On the other

end, the nitration of benzene produces nitrobenzene (C6H5NO2). The NO2 group is a

deactivating group which makes nitrobenzene less reactive towards another

substance (electrons are removed from the ring). These phenomena can arise from

either the difference in electronegativity between the ring and the substituent

(inductive effect) or from the overlap of the ring orbital with the substituent orbital

(resonance effect) [1].

These groups are also classified according to how they orientate the ring structure

towards the reagent. They can be termed either ortho, para or meta. Common ortho

and para activators include -OCH3, -NH2, -CH3 and -OH. Common ortho and para

deactivators include the halides. At present there are no known meta activators.

Common meta deactivators include -NO2, -COOH, -COCH3 and -CN [1].

1.3 – History of Local Anaesthetics

In the present day, the ‘Class A’ drug known as cocaine is associated with negative

connotations. It is abused by drug addicts for its stimulant effects and is an important

commodity utilised by the criminal underworld. Common household/medical

substances such a baking soda and lidocaine can also be added to pure cocaine to

produce synthetic derivatives commonly known as “crack” cocaine however, pure

cocaine is still a naturally occurring chemical which, like many illegal drugs, is

isolated from plants.

The purest form of cocaine exists as an alkaloid substance derived from the leaves of

the coca shrub (Erythoxylon coca). This plant grows in the Andes Mountains of Peru

at about 1500 - 1600 feet above sea level. Excavations of pre-Inca burial urns found

that the leaves of the coca shrub were placed inside.

5

Many believe that the ancient Peruvian natives would release the cocaine by

smearing the leaves with lime. The natives would then roll the leaves and chew them

to experience the stimulation provided by the chemicals [3].

In the world of science and medicine cocaine was first isolated in 1860 by German

chemist Albert Niemann who noted its unpleasant and bitter taste. He also found that

cocaine led to a numbing sensation on the tongue. In 1880, Vassily Von Anrep found

that subcutaneous injection of cocaine produced a similar numbing sensation on the

skin. He found that the user was unable to feel any pain at the site of injection [3].

By 1884 the famous neurologist, Sigmund Freud, utilised cocaine for his studies on

addiction. He attempted to wean patients off of their morphine addictions but found

that cocaine had greater addictive properties and those who stopped using the

former became addicted to latter. Freud’s assistant, Karl Koller, later found that

adding a few drops of cocaine to the eyeball would stop involuntary movements

during surgery. This test concluded that cocaine could deaden the reflexes and block

signal to nerve conduction. Koller’s tests led to the widespread use of cocaine as a

local anaesthetic to numb specific nerves during operations. During 1884 -1885 it

was commonly used as an anaesthetic in the fields of surgery and dentistry [3].

There were major disadvantages to the use of cocaine in the medicinal sector. It was

found that overindulgence of cocaine could easily lead to mental and physical

deterioration which could eventually result in acute death. The reason for this was

because cocaine was highly toxic as well as being highly addictive. The lethal dose of

cocaine was also too close to that of the therapeutic dose meaning that the risks

associated with cocaine use were far greater than the reward. This led to a wide

scale production of cocaine derivatives which were less toxic in order to find

something more suitable for everyday use [3], [4].

6

The first cocaine substitute was eucaine. Eucaine was not habit forming and did not

produce mydriasis (pupil dilation). Eucaine however, was still very toxic when

compared to piperocaine which was found to be one third less toxic than cocaine.

The most successful of these substances was procaine which was also known by its

trade name ‘novocaine’. ‘Novocaine’ was used for many years. It was the fourth less

toxic derivative and non-habit forming. The toxic dose of novocaine was and still is

almost ten times the effective amount. The difference between the therapeutic and

toxic dose increases the margin of safety for its use as an anaesthetic [3].

The reason to how these drugs act on the nervous system to relieve pain is not fully

understood. It is speculated that their main site of action is at the nerve membrane,

where their molecules appear to compete with calcium at an undefined receptor site.

Somehow the drugs can alter the permeability of the membrane so it stops

responding to electrical impulses – reduces signal conduction [3].

The general structure of a local anaesthetic includes a substituted aromatic ring

bonded to an ester group which in turn is bonded to a central carbon chain, the end

of which contains either a secondary or tertiary amine group (ref to “Figure 3”) . It is

claimed that the tertiary amine groups are important in order to enhance solubility of

the molecule in the injection solvent. Most of these compounds can react with

hydrochloric acid (HCl) to produce their hydrochloride salt forms – leads to greater

polarity, easily dissolved in water, suitable for injections. The ester groups are

important for detoxification of the drugs once they have entered the system. This is

achieved through hydrolysis of the ester linkage in the blood stream. Compounds

without the ester group are known to be longer lasting and are generally more toxic [3].

7

Figure 3: General structure of a local anaesthetic

[http://www.gpattutor.com/SampleContent/SampleContent2.aspx]

Hundreds of new anaesthetics have been synthesised and tested however, there is

still the need to find that “one” compound which provides the lowest risks and the

greatest benefits from its use in medicine.

1.4 – Chemistry and Applications of Benzocaine

Benzocaine (C9H11NO2) is a local anaesthetic also known by its trade names,

‘anaesthesin’ and ‘americaine’. Its can be named chemically as ethyl-4-amino

benzoate or 4-amino benzoic acid-ethyl ester and has a molecular weight of

165.19 g [5], [6], [7].

Unlike the drugs mentioned in “section 1.3” benzocaine is not used as an intravenous

anaesthetic. This is because benzocaine lacks the tertiary amino group at the end of

the molecule (ref to “Figure 4”). This means that it remains insoluble in water and

therefore cannot be administered via syringe. Benzocaine is still used in ointments for

topical pain relief e.g. sunscreen formulations. It can also be found in throat lozenges,

teething gels and sprays for pharyngeal and dermal anaesthesia and rectal

suppositories. Benzocaine abuse can induce methemglobinemia resulting in weak

oxygen delivery to the tissues. The decrease in oxygen carrying capacity may

eventually lead to atherosclerotic vascular disease, restrictive/obstructive pulmonary

disease, sepsis and trauma [3], [4], [8].

8

This investigation looked at carrying out four different reactions for the multi-step

synthesis of benzocaine, using p-toluidine as the starting material. The reactions

were acetylation, oxidation, hydrolysis and fischer esterification.

Figure 4: Benzocaine (C9H11NO2)

[http://www.sigmaaldrich.com/catalog/product/sigma/e1501?lang=en&region=GB]

9

1.5 – Multi-step synthesis of Benzocaine

1.5.1 – Acetylation

Figure 5: Acetylation of p-Toluidine to p-Methylacetanilide [7]

Acetylation is carried out using acetic anhydride in a nucleophilic addition reaction to

add an acetyl group (C2H3O) on to the amine group (NH2). This in turn produces the

acetanilide group (C2H4NO).

Reaction Mechanism:

Nucleophilic nitrogen attacks the first electrophilic carbonyl (C=O) on acetic

anhydride. The C=O bond is broken [9].

The same C=O bond reforms and the next C-O bond breaks expelling the acetoxy

group [9].

The loss of proton from the product to the acetoxy group produces p-

methylacetanilide and acetic acid [9].

p-ToluidineC7H9N

Acetic AnhydrideC4H6O3

p-MethylacetanilideC9H11NO

Acetic Acid

Acetic AcidC2H4O2

10

1.5.2 – Oxidation

Figure 6: Oxidation of p-Mehtylacetanilide to p-Acetamidobenzoic Acid [7]

The oxidation of p-methylacetanilide using potassium permanganate as (KMnO4) as

the oxidising agent removes two hydrogens from the methyl group and replaces them

with two oxygens to produce a carboxylic acid (COOH). The potassium

permanganate is reduced to manganese dioxide (MnO2) [10].

p-MethylacetanilideC9H11NO

KMnO4

HEAT

p-Acetamidobenzoic AcidC9H9NO3

11

1.5.3 - Hydrolysis

Figure 7: Hydrolysis of p-Acetamidobenzoic Acid to p-Aminobenzoic Acid (PABA) [7]

Hydrolysis is carried out in the presence of HCl to split the acetanilide group

(C2H4NO) into the constituent acetyl group (C2H3O) and reform the amine group (NH2)

giving p-aminobenzoic acid. The acetyl group bonds to the hydroxyl (-OH) group from

the broken water molecule to give acetic acid [10].

p-Acetamidobenzoic AcidC9H9NO3

p-Aminobenzoic Acid (PABA)C7H7NO2

Acetic AcidC2H4O2

HCl

12

1.5.4 – Fischer Esterification

Figure 8: Esterification of p-Acetamidobenzoic Acid to Benzocaine [7]

This reaction was named after its founder, Emil Fischer, who developed the acid

catalysed esterification with an alcohol. The reaction utilises nucleophilic

substitution.

The water molecule is formed from the hydroxyl group of the acid and the hydrogen

from the alcohol (in this case ethanol). The catalyst in this case is sulfuric acid

(H2SO4) [2].

Reaction Mechanism:

H2SO4 causes protonation of the acid group (COOH) on PABA enhancing the

reactivity towards the nucleophilic ethanol (CH3CH2OH) [2].

H2SO4 is deprotonated to hydrogen sulphate (HSO4-) [2].

The acid group now has a delocalised positive charge which exists in three

resonance forms amongst the carbon and two oxygens (+COOH) [2].

CH3CH2OH attacks the carbon of the acid to form the new C-O ester bond and

breaks the C=O bond [2].

H2SO4

BenzocaineC9H11NO2

p-Aminobenzoic Acid (PABA)C7H7NO2

H2O

13

The oxygen of the broken C=O bond is now protonated to a second -OH. The acid

now possesses two hydroxyl groups [2].

The H+ from the -OH on the alcohol is expelled from the molecule. This H+ is free-

flowing and moves to bond with the first -OH on the acid forming H2O. The H2O is

expelled from the molecule [2].

HSO4- from the deprotonated catalyst regenerates into H2SO4 by binding to the final

H+ on the second -OH. The C=O bond reforms and the reaction is complete.

Benzocaine and water is formed and the catalyst has been regenerated [2].

1.6 – Instrumental Analysis

1.6.1 – Fourier Transform Infra-Red Spectroscopy

Fourier transform infra-red spectroscopy (FT-IR) is a technique used to identify the

functional groups present within a molecule. This can give structural information

about the compound present within a sample. The technique is based on the

interaction of molecules with IR radiation. Different organic compounds can absorb

energy at specific wavelengths of light while transmitting others [1].

The visible region of the electromagnetic spectrum ranges from 3.8 x 10-7 m – 7.8 x

10-7 m (380-780 nm). The IR region of the spectrum runs from 7.8 x 10-7 m to

approximately 7.8 x 10-4 m. In IR spectroscopy, we are only interested in the midpoint

of the IR region. This region covers 2.5 x 10-5 – 2.5 x 10-6 cm [1].

All organic molecules possess a certain amount of energy from the transitions

between vibrational levels which causes their bonds to stretch and contract in a

spring-like connection. These bonds are constantly vibrating (stretching/compressing)

at specific frequencies.

14

When a molecule is irradiated, it will absorb the incoming energy if the frequency of

the incident wave matches the frequency of the bond vibration. The bonds of the

known functional groups absorb light at characteristic frequencies [1], [11].

The absorption of light by the functional groups is processed into an IR spectrum.

The spectrum displays a series of downward spikes which indicate an absorption

band at a specific frequency. Frequency runs across the x-axis and is given in

wavenumber (1/λ cm-1) ranging from 400 – 4000 cm-1. Transmittance (%) runs along

the y-axis and generally ranges from 0 – 100 [1].

In modern spectrometers, a single beam of light is passed through the sample. The

spectrum for the sample is obtained and stored in a digital form. In a single run, the

spectrum for the background signal is obtained then the spectrum for the sample

coupled with the background signal is obtained and stored. The instrument software

then subtracts the background signal to give the spectrum for the sample reading

using the fourier transform mathematical process [11].

Sample preparation is dependant on the state. Liquid samples can be examined as

films formed when one drop of sample is placed between two sodium chloride plates

to hold the sample in place. The plates are transparent to the IR region of use. Solid

samples can be examined as solutions, nujol mulls and potassium bromide discs

(KBr). Solutions are examined in a sodium chloride cell, 1mm thick, which is

transparent to the IR region of use. Samples in nujol mulls are prepared by adding

one drop of nujol (liquid hydrocarbon) to around 1 mg powdered sample. This method

suffers from unwanted C-H absorption peaks arising from the nujol as well as some

compounds being insoluble in nujol. One way to avoid the presence of interfering

peaks is by adding ~1 mg powdered sample to dry KBr and pressing it down into a

thin disc via hydraulic press. A vacuum pump is also connected to the system to

remove all of the water from the sample to greatly reduce background signal. This

method is the most time consuming but can also provide some good clear spectrums

[11].

15

Functional groups have characteristic peaks which occur within specific wavenumber

ranges. For example a peak representing absorption by the carbon to carbon triple

bond can be seen in the wavenumber range of 2500 – 2000 cm-1. The carbon to

hydrogen absorption can be seen in the 3100 – 3000 cm-1 range and the peak

representative of the C=O ester bond can be observed at a wavenumber of

1735 cm-1. The section below 1500 cm-1 is termed the fingerprint region and the

peaks in this region are considered unique to the molecule in question [1].

By analysing the IR spectrum and comparing the distribution of peaks to an IR

correlation chart, the analyst can identify what functional groups are present in the

molecule, what type of bond is causing the absorption and what is the molecular

motion of the bond. The pattern analysis helps to identify the structures of the

molecules. In an unknown sample, the use of IR can help to identify the types of

compounds present in the material [1].

It is also sometimes helpful to identify the peaks which are not present as this would

give an indication to molecules that are not present in the sample. The absence of

peaks indicates 100% transmittance and alludes to the absence of a particular

compound. This type of analysis may be especially suited to check the progress of a

reaction where the expected spectrum for the product is already known [1].

1.6.2 – Chromatography

Chromatography is a wide-spread laboratory technique that can be utilised for drug

analysis. The principles of any chromatographic technique are based on the theory

that a mixture of different compounds can be separated out into its individual

components. The separation occurs due to the difference in polarities between the

molecules of each individual compound which make up the mixture [12].

16

Chromatography is carried out by dissolving the sample in an organic solvent which

has a certain level of polarity. This solvent is termed the mobile phase. The mobile

phase is passed through a solid support (usually a column) which is bonded to a

chemical substance which has the opposing polarity of the mobile phase. This

chemical is termed the stationary phase. As the mobile phase passes through the

column, the molecules will interact in specific ways with both the mobile phase and

stationary phase. If the mobile phase is highly polar, then the components which are

the most polar will remain in the solution and be the first to reach the end of the

column. The less polar components will have a higher affinity for the stationary phase

than the mobile phase and find it harder to dissolve back into solution. The least polar

components will therefore be eluted last [12].

1.6.2.1 – Thin Layer Chromatography

Thin layer chromatography (TLC) is a simple separation method that can be used for

qualitative and semi-quantitative analysis. It can also be used to optimise HPLC

conditions and is used in many cases as a presumptive test before HPLC is carried

out [13].

The technique is based on the same separation theory that underpins

chromatography. TLC uses a thin solid support in the form of glass, metal or plastic

coated with a sorbent material e.g. silica or alumina which is the stationary phase. A

pencil is used to draw a line across the plate, ~1 cm from the bottom. A small aliquot

of sample is transferred on to the line and then left to dry. The marked end of the

plate is then placed into a shallow bath of mobile phase inside a closed chamber to

stop the solvent from evaporating. The internal atmosphere of the chamber is left to

saturate with the solvent vapour. The mobile phase rises up the plate and separation

of the sample mixture is achieved due to the varying degrees of affinity of each

component with the mobile phase and stationary phase [13].

17

When the mobile phase has reached the appropriate distance the plate is removed

and analysed for the presence and number of coloured spots. The point at which the

mobile phase has stopped is marked and is termed the solvent front. The presence of

a row of individual spots running along the plate indicates that separation of the

mixture has been achieved [13].

TLC can also be used to compare a number of different samples on the one plate. In

the case of synthesis experiments, a control substance can be added along with the

product of the reaction. Comparison of the resolution and migration distances of both

spots can provide an indication of whether or not the synthesis has been successful

[13].

Semi-quantitative analysis can be carried out by measuring the retention factor (Rf)

of the sample. The Rf is a measure of the migration distances of the sample from the

starting point and is calculated as a ratio:

Rf = distance travelled by component/distance travelled by solvent

The Rf value lies between 0 to 1 with 0 indicating that the component has not moved

at all and 1 indicating that the component has run off the edge of the plate with the

solvent i.e. polarity needs to be reduced [13].

Most commercial TLC plates also contain fluorescence indicators which can be used

to observe spots under UV illumination [13].

1.6.2.2 – High Performance Liquid Chromatography (HPLC)

HPLC is a separation technique used for high molecular weight, thermally unstable

compounds. In HPLC the sample is dissolved in an organic solvent and run through a

long narrow column.

18

The column feeds into a detector system which analyses the separation and, using

the appropriate software, processes this information into a chromatogram which

displays distinct peaks for each component (ref to “Figure 9”). There are several

HPLC modes which include normal phase, reversed phase, ion exchange and size

exclusion separation. This investigation used reversed phase HPLC for sample

analysis [12].

The stationary phase can be in a solid or liquid form which is immobilised on the

inside of the column. In normal phase HPLC the stationary phase is polar and is

based on silica gel which contains free hydroxyl groups (-OH). In reversed phase

(most common) the silica gel is bonded to an organochlorosilane to produce a non-

polar stationary phase. The most common stationary phases include octadecylsilane

(ODS, C18) and octylsilane (C8). Both of these molecules contain long hydrocarbon

chains which are aligned perpendicular to the support particle to give a bristle like

structure. The functional groups of the stationary phase can also be varied to include

phenyl, C6, C4, C2, NH2 and NO2, each of which can be chosen to vary the polarity of

the stationary phase to suit a specific application [12].

The mobile phase for reversed phase HPLC is a highly polar organic solvent as

oppose to normal phase where the solvent can range from being non-polar to

moderately polar. In normal phase the least polar component of a mixture is eluted

first while the most polar component is eluted last due to stronger intermolecular

forces occurring between the polar component and the polar stationary phase. In

reversed phase the most polar component is eluted first while the least polar

component is eluted last due to the interaction between polar component and polar

mobile phase i.e. component remains in liquid and travels further [12].

The selection of mobile phase composition is important in controlling the separation.

Comparing solvent strength using the solvent polarity parameter (P’) is a good

indication of mobile phase polarity. The lower the P’ value the longer it will take the

solvent to elute.

19

Water is generally used as the base solvent and is mixed with the appropriate solvent

(organic modifier) to increase polarity. Reducing the proportion of water in the

composition will decrease the polarity [12].

The set-up of a HPLC instrument includes one or more reservoir bottles which house

the mobile phase which can also be fixed to degassing system to remove dissolved

gases. The reservoir is attached to a pump for delivery of high output pressures and

constant pulse free flow rates [12].

An injection valve is placed before the column for sample introduction via syringe.

The column itself is usually made from stainless steel and is between 2.5 - 25 cm

long with an internal diameter of around 5 mm and 2 mm for small bore columns [12].

Guard columns can also be used to protect and prolong the lifetime of the analytical

column. The guard columns are packed with the same stationary phase and remove

and retain substances from the sample which can stick to the analytical column

decreasing separation efficiency [12].

Figure 9: HPLC schematic

[http://arycho.wordpress.com/tag/hplc/]

20

A variety of detectors can be used with HPLC. These include Refractive Index (RI)

detectors which can detect almost all analytes. The most commonly used is the UV

detector. These detectors are used to identify organic molecules that can absorb light

in the UV range of the spectrum (190 – 880 nm). Absorption of light is proportional to

chemical concentration according to Beer’s Law:

Beer’s Law: A= έCl

A is the absorbance, έ is the molar absorptivity, C is concentration (mol/L) and l is the

path length (cm) [12].

The UV detector works by shining a light from a tungsten lamp through a

monochromator to select the appropriate wavelength. The light then goes through a

beam splitter which passes half of the incident beam through a sample flow cell

which is measured for transmitted radiation by the photodetector. The other half

passes through a reference detector which senses variation in the beam from the

source. Signal processors then ratio the two signals and the output displayed in the

form a chromatogram [12].

The chromatogram displays a series of peaks and is plotted as signal vs. time (min).

Each of the eluted components will form discrete bands or peaks when processed

through the appropriate software. Each of these peaks will be associated with a

specific retention time (the time taken for the analyte to pass through the column).

The number of different peaks should be indicative of the number of compounds

found in the mixture. The height of each peak corresponds to the concentration of

each compound in that mixture [12].

21

Chapter 2 – Experimental

2.1 – Equipment and Chemicals

• Standard chemicals for each reaction supplied by Sigma Aldrich™ Co. LLC.

(used as controls)

• Standard melting point apparatus

• Scales

• Reverse Phase HPLC – ‘SphereClone’ 5U 005 (S) clone column, 250 x 4.60

mm x 5 microns dimensions with UV Detector and 50:50 MeOH:H2O mobile

phase

• Pre-coated TLC sheets ‘Alugram’, Silica, G/UV254 with Fluorescence/UV

indicator

• UV light (254 nm)

• ‘Jasco 6100’ FT-IR Spectrometer

• Celite filter agent

This experiment was initially designed as a 6-step synthesis. There were two

previous steps that were to be performed before the acetylation reaction. Using

toluene as the starting material, a nitration reaction was to be carried out to produce

p-nitrotoluene. The p-nitrotoluene was then to be reduced using tin as the reducing

agent into p-toluidine.

It was found that nitration of toluene could produce three isomers in varying ratios.

These were 2-nitrotoluene, 3-nitrotoluene and 4-nitrotoluene (aka p-nitrotoluene).

There is also a possibility that di-nitrotoluene and tri-nitrotoluene (TNT) could also

have been produced [14].

22

Due to the volatile and explosive nature of trinitrotoluene, the nitration and reduction

steps were abandoned and the experiment began with the acetylation step using p-

toluidine as the starting material.

The products of each reaction were weighed, analysed by IR, TLC and melting point

and the data was compared with the control substances to check the progress and

efficiency of the reactions. The melting point standards were obtained from Sigma

Aldrich™ material safety data sheets. The PABA and benzocaine controls were

analysed using HPLC for standard chromatograms. The final benzocaine product

was analysed using HPLC and the chromatogram was compared to the control to

confirm if the synthesis had worked.

23

Figure 10: Top left going clockwise: ‘Jasco 6100’ FT-IR spectrometer, std. mp

apparatus, UV lamp stand 9607-00, ‘Jasco’ HPLC instrument

[http://www.coleparmer.com/buy/product/44830-uv-lamp-stand-18-0063-01.html] [http://mcf.nd.edu/instruments-and-

capabilities/] [http://www.coleparmer.com/buy/product/44830-uv-lamp-stand-18-0063-01.html]

2.2 – Acetylation of p-toluidine to p-methylacetanilide

Starting with p-toluidine, 5.3156 g was weighed out and added to a 250 ml

Erlenmeyer flask. A small amount of water was added and the solid flask was swirled

to dissolve the reactant. Using a Pasteur pipette, 5ml acetic anhydride was added

dropwise while swirling the liquid. The solution was left to react at room temperature

for ~5 min and then kept on a hot plate to dissolve any residual solid.

24

The solution was then cooled in an ice bath to recrystallise the product. The product

was collected using vacuum filtration. TLC was carried out using a 30:70

acetonitrile:water mobile phase.

2.2 – Oxidation of p-methylacetanilide to p-acetamidobenzoic acid

Potassium permanganate (KMnO4) solution was prepared by dissolving 2.9373 g

potassium permanganate in ~ 30 ml boiling water.

In a 250 ml Erlenmeyer flask, 1.1068 g p-methylacetanilide was added along with

2.9720 g magnesium sulphate heptahydrate (MgSO4 . 7H2O) and 70 ml water. The

solution was heated to about 85°C on a heating mantle. While swirling vigorously, the

hot permanganate solution was slowly added dropwise to avoid local build-up of the

oxidant. The solution was then left to stir for 5 min.

The solution was filtered through fluted filter paper packed with celite into a fresh

conical flask to remove the brown manganese dioxide (MnO2) that had formed.

Around 2 ml ethanol was added to the coloured filtrate to react with any excess

oxidant, and then the solution was boiled on a hot plate until the colour dissipated.

The solution was re-filtered using celite and the filtrate was left to cool in an ice bath.

The solution was acidified with 20% sulphuric acid until the pH was around 3-4. The

newly formed product was collected using vacuum filtration and the crystals were

rinsed with small amounts of cold water. TLC was carried out using a 30:70

acetonitrile:water mobile phase.

2.3 – Hydrolysis of p-acetamidobenzoic acid to PABA

Two separate reactions were carried out due to the low yield of p-acetamidobenzoic

acid that had been obtained from the oxidation. The reactions were practically the

same but differed in the fact that the compositions of the starting materials were

different. The volumes of hydrochloric acid (HCl) and concentrations of ammonia

25

solution (NH4) used in the reactions were also different to accommodate the reactant

yields (ref to “Table 1”).

Table 1: Hydrolysis reaction 1 and 2

Hydrochloric acid was added to a 250 ml round-bottom flask containing p-

acetamidobenzoic acid. The solution was set to reflux gently for 30 min until a yellow

solid had formed. The solution was left to cool at room temperature and 2.5 ml cold

water was added to the flask.

The solution was transferred to a fresh Erlenmeyer flask and aqueous ammonia

solution was added dropwise until the pH reached 7-8. A further 0.5 ml glacial acetic

acid was added and the solution was stirred vigorously. The solution was kept on ice

and crystallisation was induced using a seed crystal. The product was collected by

vacuum filtration. TLC analysis was carried out on both products using a 30:70

acetontrile:water mobile phase mixed with a few drops of acetic acid to improve

resolution of samples.

2.4 – Fischer Esterification of PABA to Benzocaine

The reaction used a PABA 1 and PABA 2 mixture as the starting material, the total

weight of which was 1.7885 g. A microscale esterification was carried out due to the

reduced yield. The volumes for all reagents were divided accordingly.

Reaction Starting material Product Vol of HCl

(ml)

[NH4]

1 1.0172 g p-Acetamidobenzoic Acid control PABA 1 5 4 M2 2.0267 g p-Acetamidobenzoic Acid control

and product mixed together

PABA 2 10 6 M

26

The PABA starting material was added to 50 ml round bottom flask and 20 ml

absolute ethanol (CH3CH2OH) was also added the flask. The mixture was swirled

gently and 3 boiling chips were added.

The mixture was heated until all solid PABA had dissolved. The new formed solution

was kept on ice and 2.5 ml sulfuric acid (H2SO4) was added dropwise. The solution

was left to reflux gently for 60-75 min - at approximately 15 min intervals, the solution

was removed from the condenser and swirled gently.

Neutralisation:

After reflux, the solution had turned clear and was transferred to a fresh Erlenmeyer

flask. A few drops of water were added and 10% sodium carbonate (Na2CO3) was

added dropwise until pH of the solution reached >9.

Isolation:

The solid product was dissolved in 35 ml ether. The solution was poured into a

separating funnel and the funnel was shaken until two layers were formed. The upper

ether layer was saved and the bottom layer was discarded. The ether layer was dried

with sodium sulfate (NaSO4) and gravity filtered into a clean flask to remove the

drying agent. The ether was removed from the product using a rotary evaporator until

residual ethanol and benzocaine product remained in the flask.

Purification:

A few drops of hot absolute ethanol were added to the flask and the mixture was

heated until all of the residual oil had dissolved. A few drops of water were added

until cloudiness just appeared.

A few more drops of ethanol were added and the mixture was kept on ice. A seed

crystal was added and the solution was left overnight.

The product was collected the next day by vacuum filtration (filtered 3 times).

Benzocaine was dried in a vacuum oven before being weighed. TLC analysis was

carried out using a 50:50 methanol:water mobile phase.

27

Chapter 3 – Results

Table 2: Product results

3.1 Acetylation

Figure 11: p-Methylacetanilide Product

Product Yield (g) Melting Point Range

Control(°C)

(sigma aldrich ™ MSDS)

Melting Point

Product (°C)

Rf Control Rf Product

p-Methylacetanilide 5.7010 149-151 151 0.84 0.82p-Methylacetamidobenzoic

acid

0.3034 259-262 >264 0.84 0.81

PABA 1 0.4010 187-189 >260 0.88 0.88PABA 2 1.3875 187-189 >260 0.88 0.88

Benzocaine 0.0739 88-90 81 1.62 1.62

28

Small off-white crystals were formed. They were shiny and hair-like in appearance.

The product had a soft handle.

Figure12: TLC Plate 1 UV 254 nm: A) p-toluidine, B) p-methylacetanilide control,

C) p-methylacetanilide product

Spot A was yellow (reactant) while spots B (control) and C (product) were pink. This

means that more than one compound was present on the plate. Spots B and C had

resolved at roughly the same distance and were found to have similar Rf values of

0.84 and 0.82 (ref to “Table 2”). This means that p-methylacetanilide may have been

successfully produced from p-toluidine by acetylation.

C

B

A

29

Figure13: p-Toluidine IR spectrum

Spectrum Analysis:

3500 – 3300 cm-1 – Two sharp peaks both, NH amine medium stretch

3100 – 3000 cm-1 – Small broad peaks, aromatic ring medium stretch

1700 – 1500 cm-1 – Two sharp peaks, C=C aromatic ring weak stretch

1300 – 1000 cm-1 – Small sharp peak, C-N amine medium-strong stretch

800 cm-1 – Small sharp peak, C-H aromatic strong out of plane bend

30

31

Figure 14: p-Methylacetanilide IR spectrum: top – control, bottom – product

Product spectrum from “Figure 14” displays a different pattern than the p-toluidine

spectrum. This confirms that both compounds are indeed different. Product spectrum

from “Figure 14” is similar to control spectrum. This means that: 1) Conversion of p-

toluidine had definitely taken place and 2) the acetylation reaction had worked and p-

methylacetanilide was produced as the product spectrum matched the control

spectrum.

Spectra Analysis:

3300 – 3200 cm-1 – Broad doublet peak, possibly N-H amine medium stretch

1700 – 1600 cm-1 – One sharp peak, C=O ketone strong stretch arising from

acetanilide group

1600 cm-1 – One sharp peak, C=C aromatic ring weak stretch

1400 – 1300 cm-1 – Three sharp peaks, possibly –CH3 medium bend arising from

methyl group at acetanilide end

800 – 700 cm-1 – Two sharp peaks, possible C-H aromatic out of plane strong bend

32

3.2 – Oxidation

Figure 15: TLC Plate 2 UV 254 nm: A) p-methylacetamidobenzoic acid control, B) p-

methylacetamidobenzoic acid product

Sports A and B had resolved with Rf values close to one another (ref to “Table 2”).

Spot A had an Rf value of 0.84 while spot B had an Rf value of 0.81. This indicated

that the reaction was progressing as expected and p-acetamidobenzoic acid had

been produced by oxidation however there was evidence of impurities.

B

A

B

Faded spots possible impurities

33

34

Figure 16: p-Methylacetamidobenzoic acid IR spectrum: top – control, bottom –

product

Product spectrum from “Figure 16” is similar to control spectrum. This means that: 1)

Conversion of p-methylacetanilide had definitely taken place and 2) the oxidation

reaction had worked and p-acetamidobenzoic acid had been produced as the product

spectrum matched the control spectrum.

Spectra Analysis:

3000 cm-1 – Sharp peak, NH amine medium stretch

3000 – 2500 cm-1 – Small broad peak, OH carboxylic acid broad stretch

1700 – 1600 cm-1 – Sharp peak, C=O ketone strong stretch arising from acetanilide

group

1600 cm-1 – One sharp peak, C=C aromatic ring weak stretch, present again at

around 1500 cm-1

1300 – 1200 cm-1 – Sharp triple peak, possibly C-O carboxylic acid strong stretch

900 – 700 cm-1 – Individual peak difficult to see, possible presence of C-H aromatic

strong out of plane bend

35

3.3 – Hydrolysis

Figure 17: Top – PABA 1 product, Bottom – PABA 2 product

PABA 1 was white and had a paper like appearance. It had a hard rough texture and

was extremely brittle.

36

PABA 2 was white, crumbled and fairly soft. PABA 2 would stick to the surface of the

watch glass and had an almost clay-like handle.

Figure 18: TLC plates 3 and 4 UV 254 nm: left – PABA 1, right – PABA 2

“Figure 18” shows TLC plates for PABA 1 and PABA2 products. PABA 1 used the

control p-acetamidobenzoic acid as the starting material. The spots for PABA 1 were

similar in size and had resolved with the same Rf values (ref to “Table 2”).

PABA 2 used a fusion of control and product p-acetamidobenzoic acid as the starting

material. The product spot on this plate looked to resolve further than that of the

control. There was a high chance that the product was contaminated as the p-

acetamidobenzoic acid product from the previous reaction also showed impurities (ref

to “Figure 15”).

Control

Product

Product

Control

37

Figure 19: PABA control IR spectrum

Spectrum analysis:

3500 - 3400 cm-1 – One sharp peak, NH amine medium stretch

3400 – 3300 cm-1 – One sharp peak, OH- carboxylic acid broad stretch

1700-1600 cm-1 – Small sharp peak, C=O carboxylic acid strong stretch

1600 cm-1 – One small sharp peak, C=C aromatic ring weak stretch

900 – 700 cm-1 – Two sharp peaks, both possibly indicative of C-H aromatic out of

plane bend

38

Figure 20: Top – PABA 1 IR spectrum, bottom – PABA 2 IR spectrum

39

PABA 1 and PABA 2 IR spectra were similar. There were slight differences between

the products and the control spectra. Most notable was the absence of the NH amine

absorption at 3500 – 3400 cm-1 range on both product spectra.

3.4 – Fischer Esterification

Figure 21: Benzocaine product

The benzocaine product was off-white as expected. The product had a powdered

appearance composed of some larger granules. The product could easily be broken

apart and it had a soft handle.

40

Figure 22: TLC plate 5 UV 254 nm – A) benzocaine control, B) benzocaine product

Spots A and B had resolved well. The Rf values were identical (refer to “Table 2”)

showing that the esterification had been accomplished and benzocaine had indeed

been produced.

B

A

41

Figure 23: Benzocaine IR spectrum: top – control, bottom – product

42

“Figure 23” shows the control and product spectra were similar. This, in theory,

meant that benzocaine had been produced from the multi-step synthesis and was

very pure. There was however a high chance that the sample contained impurities

stemming from the experimental set-up and other impurities found in the reactants,

specifically the PABA products.

Spectra Analysis:

3500 – 3400 cm-1 – Medium sharp peak, characteristic N-H amine medium bend

absorption

1800 – 1700 cm-1 – Sharp peak, C=O ester strong stretch

1600 cm-1 – C=C aromatic ring weak stretch absorption, possible absorption at 1500

cm-1 as well

1300 – 1200 cm-1 – Possible C-N amine medium stretch absorption

1200 – 1000 cm-1 – Possible C-O ester strong stretch absorption

43

Figure 24: Benzocaine HPLC Chromatogram 50:50 MeOH:H2O: top – control, bottom

– product

PABABenzocaine

44

“Figure 24” displays the HPLC chromatogram for the benzocaine control and

benzocaine product. The control chromatogram showed that benzocaine had a

retention time of 7.310 min.

The product resolved at 7.273 min with a substantially smaller peak. As the retention

times were similar this product peak was taken to confirm that benzocaine had been

produced and the multi-step synthesis had worked to an extent.

The peak at 2.802 min on the product chromatogram was found to be PABA that had

not been fully converted during fischer esterification. This was confirmed by running a

standard PABA sample through the HPLC.

45

Chapter 4 – Conclusion

4.1 – Discussion

From “Table 2” it can be seen that the mp for the p-methylacetanilide product was in

the standard mp range obtained from the MSDS (151°C). The mp for p-acetamido

benzoic acid was over the standard range (>262°C) as were the values obtained for

the two PABA products (>189°C). These values alluded to the possibility that these

products were not 100% pure however confirmation of this could not have been

made on mp results alone as mp was known to be highly variable through out

different commercial and university laboratories. The mp results did however

corroborate later findings that did confirm the imperfections in the synthesis.

The Rf values were also similar for products and controls meaning that the reactions

had progressed and some form of reactant conversion had taken place at each step.

The TLC plate obtained for p-acetamidobenzoic acid (“Figure 15”) also showed extra

spots along with the control and product spots. They were noted as contamination

but it was unclear from where they had arisen from. The contamination could have

more than likely come from external factors when preparing the plates as one of

these spots resolved before the control spot, which was thought of as being a highly

pure compound.

The 30:70 acetonitrile water mobile phase was sufficient for adequate resolution of

products. Addition of two drops of acetic/HCl acid helped to aid resolution for the p-

acetamidobenzoic acid product and the two PABA products so the spots could be

easily visible along side the controls. A 50:50 methanol:water mobile phase provided

the best results for the TLC of benzocaine. The spots obtained from this mobile

46

phase were clear, regular shaped and streaking was greatly reduced (ref to “Figure

22”). The same mobile phase was chosen for the HPLC analysis due to these results.

The yield obtained after the oxidation reaction was substantially low however only

1.1068 g p-methylacetanilide from a possible 5.3156 g was carried over and used for

this reaction. Another reason for the low yield may have been because some of the

product may have adsorbed to manganese dioxide during the oxidation reaction.

Loss of product coupled with reduced amount of starting material may have resulted

in less PABA being produced.

The IR spectrum for the PABA 1 and PABA 2 products were also similar. These

results may have shown the limitations of IR as it failed to pick up any impurities

associated with PABA 2 (mixture of compounds).

The spectrum for the benzocaine product was similar to the control and all of the

relevant peaks were observed. There was however a high chance that the product

contained impurities due to the experimental set-up. Again they may not have been

detected due to limitations with the IR spectrometer. For confirmatory analysis the

final product was analysed using HPLC and it was found that benzocaine had been

produced with a retention time (7.273 min) similar to the control (7.310 min). Another

peak was found to appear at the 2 min mark (2.802). This peak was speculated as

being PABA which had not been fully converted during the esterification.

One reason as to the reduced conversion rate of PABA to benzocaine may have

been due to the reflux of the mixture during esterification. The heating mantle may

not have provided adequate heating of the mixture. Benzocaine yield may also have

been reduced due to loss of solvent as condensation during the reflux.

Multistep synthesis had been carried out for the production of benzocaine starting

with p-toluidine. The practical aspects of the method did work in that benzocaine had

been produced from a series of four different reactions starting with p-toluidine. This

showed that the conversion potential of reactants to products using the designed

process was indeed possible, and in this case the experiment was a success.

47

The final yield was low and the product was found to be contaminated with significant

amounts of PABA and other possible impurities.

4.2 – Further Work

Further work would look at optimising the reaction conditions to reduce the presence

of impurities and to increase the product yield. One way to increase product yield

would be to use more reactant. Loss of product during the experiment should also be

avoided.

HPLC analyses could be carried out after each stage in the synthesis to help monitor

the progress of the reaction and identify the quality of the products earlier on. Further

confirmatory analyses could employ the use of both IR spectroscopy followed up by

Nuclear Magnetic Resonance (NMR) to obtain high quality structural information. The

proton NMR spectrum of benzocaine would produce 5 distinct peaks. The NMR

peaks could be easily differentiated than compared with the apparent cluster of IR

peaks. This would also provide the added ability to highlight distinct compounds that

have contaminated the product. Both NMR and IR could still be used together in

order to provide reliable data.

Improvements to the IR analysis may include a sample preparation step using the

KBr discs. This method would ensure that all water has been removed from the

sample and greatly improve the signal to noise ratio. A clear spectrum should be

produced following this procedure.

The use of mass spectrometry (MS) coupled with HPLC would also provide

characteristic retention time data along with mass spectral fingerprint data of the

sample.

48

The fragmentation pattern of the sample could help to aid in the identification of any

impurities. LC-MS could be used after each reaction to help monitor the progress of

the reaction however, if impurities were found then this would increase the chances

of back-tracking and depending on time constraints, this could be a disadvantage.

The use of fluorescence and amperometric detectors may also be beneficial to the

analysis as these detectors have been found to be more sensitive than the commonly

used UV/Vis detectors in HPLC [12].

49

Appendix 1 – Table of mobile phases used for TLC

Solvent 1 Solvent 2 Acid RatioMethanol Water - 50:50Ethanol Water HCl 50:50

Acetonitrile Water Acetic Acid/HCl 30:70Acetonitrile - - 100

Cyclohexane - - 100Toluene - - 100Acetone - - 100

Appendix 2 – Table of solvent polarity parameters (P’)

Solvent P’Water 10.2

Acetonitrile 5.8Acetone 5.1Methanol 5.1Ethanol 4.3

[12]

Acknowledgments

Dr. Jim Neagle – Project Supervisor

Dr. Ray Ansell – Project Co-ordinator

50

School of Engineering and Built Environment

Forensics/Chemistry Academic Staff -

Provided guidance and information throughout the course

Colin Russell (Senior Chemistry Lab Technician) and the Laboratory Staff -

Provided the chemicals for the experiment, the instruments/glassware, and guidance

on methodology

Bibliography

51

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54