Methylxanthines

The Determination of Methylxanthines in Cocoa, Coffee
and Tea using HPLC
FACULTY OF APPLIED SCIENCE
LIMERICK INSTITUE OF TECHNOLOGY
MOYLISH PARK
LIMERICK.
Bachelor of Science (Honours) in Chemical Instrumentation & Analysis
(Level 8)
Submitted by: Iqimat Oloko
Supervisor: Dr. Michael Monaghan

DECLARATION
I certify that this thesis which I now submit for my project for the part of my final year in
Chemical Instrumentation and Analysis , is entirely my own work and has not been taken from
the work of others, save and to the extent that such work has been cited and acknowledged
within the text of my work.
This thesis was prepared according to the regulations provided by Applied Science Department
The Institute has permission to keep, lend or copy this thesis in whole or in part, on condition
that any such use of the material of the thesis is duly acknowledged.

Acknowledgements.
I would like to thank Dr. Michael Monaghan for his help and knowledge in
delivered to this project. I would like to thank all the LIT technicians for all their
support over the laboratory period.

Abstract
The determination of Methylxanthines (Caffeine, Theobromine and Theophylline) was
analysed using HPLC. The standards were used as a calibration curve to quantify the samples
carried out. The samples carried out are Cocoa, Coffee and Tea bags.
Methods were developed for the analysing of methylxanthine and was validated. SPE
extraction Pre-treatment was utilised on the sample and it was characterised using HPLC.
Another method were developed in determining the caffeine in sample by GC- Mass
spectrometry
The HPLC was used to analysed amount of methylxanthine are in each samples which result
to that Theobromine is mainly made from Cocoa observing from the HPLC chromatogram. It
was also observed that the Coffee has a lot of Caffeine which is about 1.2g of caffeine in
Richman Blend Coffee. Theophylline was hardly found in the three samples, just a little bit of
trace of theophylline.

Abbreviations
HPLC- High Pressure liquid Chromatography
RP- HPLC- Reversed Phase –High Pressure liquid Chromatography
GC-MASS spectrometry – Gas Chromatography –Mass Spectrometry
SPE- Solid Phase Extraction
FDA- Food and Drug Administration

Aims and Objective
The main aims is to determine the amount of methylxanthine in Cocoa powder, Coffee and Tea
using HPLC.
What are the objective to achieve in this project?
The first objective to achieve is
1. Method validation
Method development has to be validated in purpose of the analysis of methylxanthines. The
method development has to go under what type of mobile phase, flow rate and column to use.
2. How to extract the samples
The steps of SPE extraction of methylxanthine from samples has to well understand if reversed
phase or normal phase will be use.
3. Optimization of separation
The method validated has to sure it can separating the three components of methyxlanthine
with good theoretical plate number, resolution factor and tailing factor.
4. Quantitative
To quantify the amount of caffeine, theobromine and theophylline in each samples.
5. Using another method to qualify methylxanthine
To use GC-Mass spectrometry to identify the methylxanthine and observe if the fragment of
the three methylxanthine will be known.

Table of Contents
1 Introduction ..................................................................................................................................11
Coffee....................................................................................................................................11
Tea.........................................................................................................................................12
Cocoa.....................................................................................................................................13
Methylxanthine Chemistry and general Information ...........................................................14
1.4.1 The physiochemical properties.....................................................................................16
Clinical Use of Methyxlanthine .............................................................................................17
1.5.1 Methylxanthine and CNS stimulation ...........................................................................17
1.5.2 Methyxanthine and Diuretic.........................................................................................18
1.5.3 Methyxlanthines and Smooth muscles.........................................................................18
World Consumption of Caffeine ...........................................................................................19
1.6.1 Caffeine Content in Beverages......................................................................................19
1.6.2 Theobromine content in Cocoa ....................................................................................19
SPE Apparatus.......................................................................................................................20
High Pressure liquid Chromatography..................................................................................21
1.8.1 HPLC Instrumentation...................................................................................................21
HPLC Methods Development................................................................................................23
Gas Chromatography -Mass spectrometry...........................................................................25
Methods of Analysis Methylxanthines in food .....................................................................26
2 METHOD........................................................................................................................................29
Materials ...............................................................................................................................30
Preparation of mobile phase ................................................................................................30
Preparation of Caffeine.........................................................................................................30
Preparation of Theobromine ................................................................................................30
Sample Preparation ..............................................................................................................30
Instrument ............................................................................................................................31
2.6.1 HPLC ..............................................................................................................................31
2.6.2 GC-Mass spectrometry .................................................................................................31
3 Method Validation........................................................................................................................32
Validation of the Chromatographic Method ........................................................................33
3.1.1 Linearity and Range.......................................................................................................33

3.1.2 Precision – percentage RSD value.................................................................................34
3.1.3 Accuracy........................................................................................................................34
3.1.4 Limit of Detection .........................................................................................................36
3.1.5 Limit of Quantification ..................................................................................................37
3.1.6 Specificity......................................................................................................................38
4 Discussion......................................................................................................................................39
Method development and validation of Caffeine.................................................................40
Sample...................................................................................................................................46
Gas Chromatography ............................................................................................................48
5 Conclusion.....................................................................................................................................51
6 Future work...................................................................................................................................51
7 Calculations...................................................................................................................................52
Theobromine.........................................................................................................................52
Theophylline..........................................................................................................................57
Caffeine.................................................................................................................................59
8 References ....................................................................................................................................65
9 Appendices....................................................................................................................................68

1 Introduction
Coffee
Methylxanthines;caffeine(1,3,7-trimethyxanthine),theobromine(3,7-dimethylxanthine),and
theophylline(1,3-dimethylxanthine) can be normally found in coffee beans, tea leaves, cocoa
beans and any other kinds of the plant for drugs (Paradkar, M.M, and Irudayaraj, 2002). The
historical origins of the use of methylxanthine are unknown and dressed in myth. Coffee, tea
are the most recent common use. Coffee became widespread in the fifteenth and sixteenth
centuries and in Europe it occurred in the eighteenth and nineteenth centuries. The word Coffee
derived from qahva which means denoting a drink from plants. From the initial cultures of
Ethiopia, cultivation of coffee bushes soon came to be dominated by Yemen, the city became
a centre to denote drink. Venetian traders introduced coffee to Europe. In London, the first
coffee shop opened in 1652 and was located in St. Michael’s Alley, Cornwall. (B.Fredholm,
2011, p. 3) Coffee drinking has been introduced into France nine years earlier and by 1690,
250 coffee houses were registered in France; by 1782, the number had risen to 1800. Now
coffee is grown in 50 different countries around the world. (B.Fredholm, 2011, p. 4)
.
Figure 1: showing an Arabica coffee see (vine, 2015)

Tea
Tea became popular use during the Ming Dynasty in China and during the eighteenth century
in Britain. Tea use increased in popularity under the Chinese Ming Dynasty (1368-1644),
which represented a return to power of the Han People, it was at this period tea began to be
brewed by steeping cured loose leaves in boiling water, and this was tried by Europeans, it was
this method of making tea that became popular in the world (B.Fredholm, 2011, p. 6). Tea
arrived in Europe about the same time as Coffee. The first green tea leaves were brought from
China to Amsterdam by the Dutch East India Company by 1636. From its early history, Tea
has become one of the world's ubiquitous drinks. Tea drinking in one form or another is part
of the diverse cultures of many lands. Recent years have seen considerable interest in tea’s
therapeutically properties, which is one of the factors leading to an increase in tea consumption.
It was esteemed for its ability to dispel tiredness, to stimulate the mind and to raise energy
levels, even helps to banish fevers and cure head and stomach problem. (Saberi, 2010).
Figure 2 : showing a Tea leaves (Camellia sinensis) from Köhler's Medicinal Plants, 1897 (WikiTea, 2016)

Cocoa
The cocoa tree belongs to the genus Theobroma cacao tree, a group of small trees which occurs
in the wild in the Amazon basin and other tropical areas of south and Central America. Over
different species in the genus but a cocoa tree is only cultivated widely. The tree forms flowers
from small cushions on its trunk and large branches. The flowers polluted produced pods that
vary from 100 to 350mm in length and width. In 1502, cocoa seeds were bought by Columbus
to King Ferdinand on his fourth and final voyage (Tannenbanum, 1941). The first plant
products containing methylxanthines. The Spaniards promptly discovered the great value of
cocoa, which is highly used beverages and currency. The first documented arrival of cocoa in
Spain was 1544 and first official shipment reached Seville from Veracruz (Tannenbanum,
1941). Cocoa contains the amines and alkaloids theobromine about 0.5% to2.7%, caffeine
approximately 0.25% in cocoa. A standard chocolate bar (40 to 50g) contains theobromine (86
to 240mg) and caffeine (9 to 31mg). The characteristic bitter taste of cocoa is generated by the
reaction of diketopiperazines with theobromine during roasting. Theobromine is produced
commercially from cocoa husks (Health, 2009).
Figure 3: showing a tree of Cocoa in Papu Vaai in Asau and Saena Penaia’s plantation at Lafi (Tavita, 2012)

Methylxanthine Chemistry and general Information
Methylxanthines are secondary plant metabolites which are derived from purine compounds
nucleoids. These purine base are found in human body, tissues and organism. The types of
methylanthien are Caffeine(1,3,7-trimethylxanthine),Theophylline(1,3-dimethylxanthines)
and Theobromine(3,7-dimethylxanthine), these are found in tea, coffee and cocoa.
Caffeine Theophylline Theobromine
Figure 4: Showing the structure of methylxanthines
Physical and chemical properties of Methylxanthines
Properties Caffeine Theophylline Theobromine
Molecular Formula C8H10N4O2 C7H8N4O2 C8H10N4O2
Molecular Weight 194.19g/mol 180.16 180.16
Melting Point 235-238 271-273 357°C
Log P -0.55 -0.77 -0.78
pKa 10.4 8.81 9.28
Water Solubility 11.0mg/mL 9.74mg/mL
Boiling Point 178 454 357
Table 1:showing the physical properties of methylxanthines (Group, 2007)
CH3
H3C
CH3
CH3
H3C
H
CH3
CH3

Caffeine is a chemical compound which occurs naturally from plant sources. Caffeine belong
to family of heterocyclic groups of compounds which are called purines.Caffiene
IUPAC(International Units of Pure and Applied Chemistry) name is 3,7-dihydro-1,3,7-
trimethyl-1H-purine-2,6-dione and it has a common name called 3,7-trimethylxanthines. From
the structure in figure 4, caffeine is classified as alkaloids since it occurs as a metabolism of
nitrogen metabolism. (Group, 2007)
Theobromine is an alkaloid generally found in cocoa beans and hence chocolate. It is the least
active as a central nervous stimulant of the three naturally occurring methylxanthines.
Theobromine IUPAC (International Units of Pure and Applied Chemistry) name is 3, 7-
Dimethyl-3, 7-dihydro-1H-purine-2,6-dione and has a common name called 3, 7-
dimethylxanthine. Theobromine is white in colour and slightly soluble in water. Theobromine
has weakly acidic properties, combining with bases to forms salts. Theobromine also has even
weaker basic properties, combining with acids to form salts which are decomposed in aqueous
solution. (Group, 2007)
Theophylline is an alkaloid found in plants such as the leaves of the tea brush. It is primarily
used as a drug for the treatment of asthma. The major pharmacological actions of theophylline
include stimulation of the cardiac muscle causing a complete emptying of the heart; relaxation
of the bronchial muscle; acting as a diuretic increasing urine output and stimulation of the
central nervous system. (Group, 2007)

1.4.1 The physiochemical properties
How can structure properties of compounds be described?
Lipinski rules are the guidelines for structural properties of drug like compounds. These rules
are used to describe the physiochemical properties of compounds.
The most common five rules set for the physiochemical properties of compounds are:
1. Not more than 5 hydrogen bond donors
2. Not more than 10 hydrogen bond acceptors
3. A molecular weight under 500g/mol
4. The partition coefficient lop P less than 5
Lipophilicity is the ability of a compound to dissolve in fats, oils, lipids and non –polar solvent.
It is also to partition the compound between immiscible non-polar and polar liquid phase. The
partitioned values are measured and termed ad Log P and Log D.
Log P is the partition coefficient of the compound between an organic phases an aqueous phase
at a pH whole all the compound molecules are in neutral form.
Log D is the distribution coefficient of the compound between an organic phase and aqueous
phase at a specified pH.
How does Log P affect Reversed Phase HPLC?
Reversed phase HPLC is the instrument used in when a mobile phase is polar and the stationary
phase is non-polar. The instrument will be explained better in this section.
This RP-HPLC will be used to explain how to separate ionisable compounds. In this process,
the higher the log P, the more hydrophobic the molecule. In RP-HPLC polar analyte interact
with silica surface silanol groups which undergo an adsorption type interaction with their
partitioning behaviour. This lead to bad peak shape along with increasing in retention times.
The structure of the standard molecule gives clues to their elution order and this is based on
the water solubility of the molecule. (Scientific, n.d.)
There are some observations governing the compounds elution. Which are:
1. The less water soluble, the more the retention
2. The retention time increases as the number of atoms increase
3. Branched-chain compounds elute more rapidly than normal isomers
4. Unsaturation decreases retention
5. Neutral polar and charged species shows the least retention time followed by the acid
then basic compounds elute rapidly. (Scientific, n.d.)

Clinical Use of Methyxlanthine
Methylxanthine have four most common pharmacological actions which are;
 CNS stimulation
 Diuresis
 Stimulation of Cardiac muscle
 Relaxation of smooth muscle; especially the bronchial muscle. (Rang, Dale, Ritter, &
Flower, 2004)
1.5.1 Methylxanthine and CNS stimulation
Of the methyxlamthine, caffeine is the most effective used to improve mental alertness. This
is usually achieved by blocking adenosine receptors in the brain. Adenosine signals the brain
that it is time for the body to sleep. Caffeine increases energy metabolism throughout the brain.
This stimulating effect of increased alertness can lead to Insomnia. The caffeine contained in
beverages 100mg in a cup is sufficient to cause nervousness and insomnia in sensitive
individual. (Rang, Dale, Ritter, & Flower, 2004) A very high dose of caffeine can cause
medullary stimulation and convulsions and may lead to death. Generally, caffeine has a
stimulant can be used as a potent killer; caffeine has found relevance in the preparation of pain-
relieving drugs especially for headache originating from eye strain by exerting a peripheral
action which is the site of an injury, this acts on muscles tissue by repairing tissue damage and
also reducing the inflammation. (Bennett Alan Weinberg and Bonnie K. Bealer, 2016)
( Clark & Landolt, 2015) designed a study associated with caffeine, coffee and sleep. The
method was conducted using a computerised literature search on web of science. The surveys
associated caffeine with variable of sleep quality in adolescent are estimated that 30% of
America adolescents consume caffeinated beverages on daily basis, soda(drinks) appears to be
the beverages of choice. Data from a nationally representative survey with 15,686 respondents
revealed that more than two-thirds drank soda once a day or more. Respondents drank coffee
less frequently, with over one half not drinking coffee at all and two thirds drinking it once a
week or less. Adolescents reporting high caffeine intake (consumption n=4,243) were 1.9times
likely to experience difficultly in sleeping and 1.8 times more likely to experience sleepiness
in the morning compared to adolescent with low caffeine, beside with shortness of sleep,
daytime sleepiness has been associated with high a moderate caffeine intake aiming adolescent.
( Clark & Landolt, 2015)

1.5.2 Methyxanthine and Diuretic
Theobromine has diuretic, stimulant properties, but unlike caffeine theobromine does not affect
the central nervous system. Diuretics work by causing the kidney to excrete increased amounts
of salts and water from the body. (Ruben Vardanyan, 2016). Diuretics is mostly common in
sports, which has been banned by most sports organizations because the athletes intend to cheat
in sports by excreting all the doping used for performance and most especially diuretics make
athletes lose water wright quickly (Ruben Vardanyan, 2016).
1.5.3 Methyxlanthines and Smooth muscles
Methylxanthines act as bronchodilators by relaxing
bronchial smooth muscle and help to narrow the
airways. Theophylline and Theobromine are the
primarily used for therapeutic drug for this process.
(Rang, et al., 2004)
Figure 5 :A diagram of the bronchus. The loosening of the muscles in the bronchus caused by theobromine helps alleviate
the symptoms of asthma. (Henry Vandyke Carter, 1918)

World Consumption of Caffeine
How much does the world take beverages daily?
Caffeine is used daily by millions of people to increase wakefulness, reduce fatigue and improve focus.
Up to 400 milligrams of caffeine appears to be safe for healthy adults. (Books, 2016)
Caffeine is present in number 0f dietary sources consumed worldwide for example tea, coffee, cocoa
beverages, bars, soft drinks.
Age Range Maximum recommended daily caffeine intake
4-6 45mg
7-9 62.5mg
10-12 85mg
Adult Up to 400mg
Table 2 showing the maximum intake of caffeine recommend in beverages (Books, 2016)
1.6.1 Caffeine Content in Beverages
Product Serving Size Caffeine per
serving(mg)
Caffeine mg/L
Cocoa Powder 1 bar(43g) 31 -
Milk Chocolate 1 bar(43g) 10 -
Coffee powder 207mL 80-135 386-652
Tea for 3 min 177 millilitres 22-74 124-418
Table 3 showing the caffeine content in beverages (Books, 2016)
1.6.2 Theobromine content in Cocoa
Cocoa types Mean theobromine content ratio(10-3
)
Cocoa 20.3
Cocoa cereals 0.695
Chocolate products 1.47
Cocoa beverages 2.66
Table 4 showing the amount of theobromine in cocoa.

SPE Apparatus
Solid phase extraction (SPE) involves running samples through a suitable sorbent bed. This
stationary phase is normally a silica based
sorbent which the functional groups are
bonded to. The sorbent is contained within a
polypropylene disposable syringe. The syringe
contains a packing material of sorbent ranging
from 100mg-5g that is held in place by
polyethylene frits. At the tip of the syringe is
called a leur tip which allow for ease fitting
onto the equipment (SPE manifold vacuum.)
The polypropylene syringes are available in
different sizes (1-10ml) capacities. (cutris,
2016)
Figure 6 showing Varian Vacuum SPE Manifold with Vacuum
pump,HyperSepTM VerifyTM-CX, HyperSepTMPhenyl and HyperSepTMPhenyl and HyperSepTMC18SPE Cartridges (E. M. Thurman,
2016)
The SPE Process generally consists of four stages which are:
Step 1. Wetting/Conditioning: First Methanol then followed by water (reversed phase).
 Wetting the sorbent allows the boned alkyl chains, which are twisted and collapsed on
the surface of the silica to be solvated so that they ‘spread open’ to form a ‘bristle’.
 Ensures good contact between the compound and the sorbent in the adsorption stage.
Step 2: Loading aqueous sample (Revered Phase)
 Sample is forced through the sorbent material by suction.
 With the choice of the sorbent, the compound of interest will be retained by the sorbent
in preference to extraneous material.
Step 3: Washing –interference elution
 This is the most important stage.
 Wash off unwanted materials using the same solution in which the sample was
dissolved, or another solution that will not remove the desired compound.
Step 4:
 Analyte elution and collect the elute
.

High Pressure liquid Chromatography
High pressure liquid chromatography (HPLC) is one technique widely used in an analytical
laboratory. HPLC is based on the analysis of organic molecules and ions with their
involvement of adsorption, partition and ion exchange, depending on the type of stationary
phase used. HPLC contains a solid stationary phase, normally packed stainless- steel 3-4mm
bore and 10-30cm long column, and a liquid mobile phase (P.J.Higson, 2004). The two basic
important question in HPLC focus on how particular compounds can be separated and why
particular compounds were separated by HPLC method. Separations of the component of a
solution results from the difference in the relative distribution ratio of the solutes between the
two phases. HPLC can be used as qualification and quantification analysis; these can determine
the purity and the content of many pharmaceutical substances. HPLC operates in two phases;
Reverse phase chromatography and normal phase. Reverse phase chromatography is usually
the most common process to use. A non- polar stationary phase and polar mobile phase was
used. The solvent for mobile phase for this chromatography is mixture of water and methanol
or acetonitrile. In normal phase chromatography the stationary phase is usually polar in nature
(silica gel) and the mobile phase is non polar (hexane) (Shu, Chem, n.d.). In order to maximize
the result or sensitive results, the analyte are usually dissolved with the mobile phase. The
mobile phase allows the efficient separation within a minimum of time.
1.8.1 HPLC Instrumentation
HPLC components are shown on the diagram below in figure 1. The different component of
HPLC are Injection, HPLC Column, Mobile phase, Pump and Detector. How does these
component works on HPLC? The solvent is the mobile phase. The mobile phase acts as a
transportation of sample into the system. The mobile phase are inertness onto the sample. For
normally phase the mobile phase are usually non-polar (hexane) and for reversed phase the
mobile are usually polar (water: methanol or acterntile). The mobile phase has to be free from
impurities. The pump (Eluent Delivery system) is the most important component of HPLC
which is used to generate pressure with a specific flow rate of the mobile phase. Variations of
flow rate of the mobile phase has impact on the elution time of the sample. For quantification,
a precise flow rate improves the reproducibility of gradient elution on a standard size columns
of stationary phase. Injectors supply a constant volume injection of sample into the mobile
phase and transport the sample into the HPLC column. HPLC column is packing material called
stationary phase. The stationary phase enhance the separation of the sample after run. A
detector gives precise response for the sample separated.

The most common detector use is Photodide array detector. Phototodide array detector provide
a fast low noise spectra; analysis, resolution determined by number of diodes deployed over
specific wavelength
range.
Figure 7 showing the
diagram of photo diode
array detector from
(Scientific, 2013)
HPLC photo diode
array is coupled to
the column to elute out the separation component by molecular weight, hydrophobicity
(reverse-phase) or ionic charge. This makes it very important for HPLC analysis. Specific
PDA detectors usually handle the over all of the spectrum from the UV at 190nm to near IR at
1micron where some others PDA detectors target to the UV, visual or near IR with up to 2048
elements provided for ultimate flexibility in resolution. These type of machine provide
sampling speeds of up 190Hz and provide thermoelectric temperature to minimize the noise
limit. (Labcompare, 2009). The mobile phase leaves the detector and later sent to the waste
reservoir. (Waters, 2006 )
Figure 8(a,b) : showing the diagram of HPLC from Waters technology (Waters, 2006 )and b from Limerick Institute of
Technology analytical Laboratory

HPLC Methods Development
Carrying out quantitative analysis in any set of samples, it is very important to develop a
specific preparation and separation. In developing a method for separation, problem must be
identified, and the most problem are based on the physicochemical property of the standard
compounds and the methods available for the separations. Chromatographic method
development and system suitability are the problems for separation. According to FDA,
analytical method validation characteristics is the new methodology to justify the acceptance
criteria, using a qualified instrumentation. The typical Validation characteristic are:
 Specificity: The ability of a test method to measure an analyte without interference from
other sample.
 Linearity: The measure of a test methods ability to obtain test results directly
proportional to the concentration of analyte in the sample. The acceptance criteria for
linearity is < 0.99
 Precision: The agreement of two or more measurement that are obtained under identical
condition using the same test method.
 Accuracy: The agreement between a measured value and accepted value
 Limit of Detection: The lowest concentration of analyte in a sample matrix that is
detected.
 Limit of Quantitation: The smallest amount of analyte in a sample matrix that can be
quantified with acceptable accuracy and precision.
 Range: Range is the magnitude for the analytical concentration where the precision,
accuracy and linearity all meet acceptable criteria.
System suitability tests are fundamental part of liquid chromatographic methods. They
confirmatory tests procedures carried out to ensure that the system will function correctly for
the intended use. The chromatographic test used to describe the performance of the column,
system and the separation are (Greay, 2016)

The Capacity Factor
Capacity factor is a measurement of retention of an analyte of a chromatographic column.
K =
𝑡𝑅−𝑡𝑂
𝑡𝑂
tR is the retention time; time required for the peak
to elute
t0 is the time taken for the mobile phase to pass
through the column.
Figure 9 shwoing the diagram of capacity factor for HPLC
performance (Greay, 2016)
Resolution
The distance between two neighbouring peaks
Rs = (tR2 – tR1) / ((0.5 * (w1 + w2)) (1)
Figure 10 showing the resolution of 2 neighbouring peak
(Greay, 2016)
Efficiency Number plates: This is used to check how efficiency the column is. If the theoretical plate,
the retention time reduces and it is faster

Gas Chromatography -Mass spectrometry
Gas chromatography is a technique that involve the partition of analyte in the gaseous phase.
The mobile phase in GC is an inert gas (usually hydrogen, nitrogen or helium) and its functions
is to transport the analyte through the column. The stationary phase can be solid or liquid and
this is usually suspended unto the column housed in an oven. The purpose of the stationary
phase is to interact with the components of the mobile phase and this interaction results to
separation of analyte (University, n.d.). A schematic of a basic GC chromatograph is outlined
in figure 11.
Figure 11 A schematic diagram of GC chromatogram. (University, n.d.)
The introduction of a sample into the GC is done by a sample injection system which includes
an auto sampler, solvent and waste vials, a micro-syringe injector and a heated inlet. The
temperature of the inlet is usually 50°C above the boiling point of the least volatile component
of the sample. This ensures that the sample is introduced onto the column in a small narrow
volume and then goes to the detector for signals.
Mass spectrometry is usually coupled with GC and LC chromatography. The mass
spectrometer is an instrument which can measure the masses and relative concentrations of
atoms and molecules.
How does a mass spectrometer work?
1. Introduction
2. Sample introduction
3. Methods of sample ionisation
4. Analysis and separation of sample ions (Ashcroft, n.d.)
5. Detection and recording of sample ions

Methods of Analysis Methylxanthines in food
Many methods exist for determining the methylxanthines content of food and beverages. Some
of these methods include UV-Visible spectrophotometry, High-Performance Chromatography,
Gas chromatography, ion chromatography, capillary electrophoresis, micellar capillary
electrophoresis, gas chromatography, and solid-phase microextraction gas chromatography.
(Igelige Gerald, 2014) HPLC is the most commonly used analytical methods. This section
discusses highlight the uses of the different technique, methodology studies that involve the
analysis of methylxanthines in food products.
(Magalhães, Luis M; Machado, Sandia; Marcela, Segundo A; Lopes, Joao A; Pascoa, Ricardo
N.M.J, 2016)One study demonstrated using an FT-NIR spectroscopy method with an HPLC
coupled with diode-array detection to analyse 61 spent coffee ground samples.
Chromatographic separation of Ground coffee was ethanol/water (50/50, v/v) extract was
performed. The HPLC system used is (Jasco, Easton,USA)comprised of high-pressure
pump(PU-2089 PLUS0)equipped with reversed phase core-shell column to a photodiode array
detector. Within 30mins, the ground coffee is separated by an ethanol/water gradient. The main
of the experiment was to qualify major phenolic and methylxanthines present in the ground
coffee. The major phenolic found are (catechin, caffeic acid and chlorogenic) and
methylxanthine is (Caffeine and theophylline). Caffeine content was found the higher
concentration. Caffeine concentrations ranged from 740 to 12,400mg/kg of spent coffee
ground. Chlorogenic acid ranged from 157 to 3593mg/kg of spent coffee ground. The NIR
spectra of spent coffee ground samples were analysed using PCA model (Principal component
analysis). The near –Infrared spectroscopy was proposed as a rapid and none – destructive
technique to assess the content of three main phenolics and methylxanthine. PCA models using
NIR spectra samples were performed for the screening of possible outliers through squared

residuals statistics. All the compound was modelled in separate ways using PLS (Partial least
squares regression).For caffeine PLS model, the wavenumbers with the highest contribution
were 5945,4890,4500,4430 and 4145cm-1.
The first overtones region (N-H and C-H bond first
overtones) and could be associated with caffeine. The absorption at 4890cm-1
is related to the
C=O bond combined with N-H bond. CH3 bond absorption is 4500cm-1
and 4430cm-1
and the
CH bond combination occurs at 41545cm-1
. The Theophylline PLS models highest
wavenumbers are 4945, 4870, 4510, 4445, 4410 and 4380cm-1
. The N-H and C=O bond stretch
are 4945, 4870cm-1
and the CH3bond stretch are 4510, 4445, 4410, 4380cm-1
. (Magalhães, Luis
M; Machado, Sandia; Marcela, Segundo A; Lopes, Joao A; Pascoa, Ricardo N.M.J, 2016)
(João Rodrigo Santos, 2012) applied a chromatographic low-pressure flow injection system for
the of methylxanthines in coffee. In this work, the coupling of commercial monoliyhiccoulmn
to a traditional low-pressure flow rate injection system was proposed for the analysis of
theobromine, theophylline and caffeine in coffee brewed samples using UV detector set at
273nm. The flow system performance was studied at room temperature by increasing the
peristaltic pump for rotation using a mobile phase acetonitrile: water, 3:97, v/v up to maximum
value of 2.5mLmin-1
. Different compositions of mobile phase solution mixture of acetonitrile:
water were tested to achieve a good separation between theobromine, theophylline, and
caffeine. The different flow rate was tested at 0.65, 0.85 and 1.06mLmin-1
.A linear dependence
between peristaltic pump rotation speed and flow rate within the range. (João Rodrigo Santos,
2012)
The resolution values for theobromine and theophylline signal are within 1.88 and 2
(Mashkouri, Nahid Najafi; Hamid, Ahmadi Seyed; Afshin, Khorrami Rajabi, 2003) quantitated
the caffeine existing in black tea leaves based on the method of Fourier transform(FTIR)
spectroscopy using multiple linear regression. The caffeine was extracted using CHCl2 from

black tea leaves, wetted with an aqueous solution of NH3 solution. The spectroscopic data was
obtained at a wavenumber range of 1800-1300cm-1
with a micro flow cell with CaF windows
and path length of 0.518mm by accumulating 20 scans with the resolution of 4cm-1
. The method
had a detection limit of 35µg/mL, the sampling frequency of 6 h-1
and a coefficient of variation
of 0.8% for five independent measurements of tea samples of 3.68% w/w caffeine content.
Based on the results obtained by the authors, the caffeine content from FTIR (3.68±0.03% w/w)
and reference HPLC technique (3.60±0.07% w/w) provides similar results for caffeine. FTIR
has a better-developed method for determining caffeine in tea leaves through it precision,
accuracy, and quickness. (Mashkouri, Nahid Najafi; Hamid, Ahmadi Seyed; Afshin, Khorrami
Rajabi, 2003).
Another spectrometry based methodology (Solid phase Fourier transform- Raman
spectrometry, SP-FT-Raman) was developed for determination of caffeine in commercial
energy drinks. Raman spectrometer is equipped with C18bonded silica with average particle
size of 105 and 50-70um.A Gilson Minipuls P2 peristaltic pump and 1 mm i.d tygon pump tube
and PTFE 0.8 mm i.d. connecting tubes were used to introduce the sample. The caffeine content
of each sample was determined from Raman intensify between 573 and 542cm-1
with a
corrected baseline between 580-540cm-1
. The limit of detection of SP-FT-Raman method was
obtained at 18mg 1h-1
, with a repeatability of 3% as the relative standard deviation of five
analysis of 200mg 1-1
concentration of caffeine. FT-Raman coupled with solid-phase this
enhanced and increased the sensitivity of detecting caffeine by a factor of 31 times when
compared with Raman measurement alone. The SP-FT-Raman provides a sampling frequency
of 13.3 h−1
, higher than that of liquid chromatography (LC), which was 7.0 h−1
. (Sergio, et al.,
2005)

Materials
All chemicals used were of analytical grade; Caffeine, Theobromine and theophylline were
obtained from Sigma Aldrich, HPLC grade methanol was obtained from Sigma Aldrich
.Ultra-pure water (Milli Q water) obtained from analytical laboratory LIT.
Samples: Cadbury Bournville Cocoa Powder, Barry’s Tea Gold Blend Tea bags, Maxwell
Spouse Rich Blend Coffee were obtained from local supermarket,
A SPE vacuum system, an HPLC –UV system with C, an HPLC –UV system with C18
column and A GC-Mass spectrometry.
Preparation of mobile phase
1 litre of beaker was used to measure 600mL of ultra-pure water and 400mL of methanol to
achieve 60:40% water: methanol. This was made up in 1litre of volumetric flask. The
solution was filtered through a 0.45um nylon filter. The solution was stored in a mobile phase
storage bottle.
Preparation of Caffeine
10mg of caffeine was weighed out using an electric balance and this was transferred into a
100mL volumetric flask. The mobile phase in 2.2 was used to dissolve the caffeine and made
up to the mark. Sonication was applied to completely dissolve the caffeine and was labelled
the stock solution. From the stock solution, 10ml of stock solution into 100mL making
100ppm and this was labelled as the working stock. Five standards were made from the
working standard from 10ppm to 100ppm concentration of caffeine.
Preparation of Theobromine
Experimental procedure for the determination of caffeine at 2.3 is used for theobromine sand
theophylline.
Sample Preparation
0.5g of Cocoa powder was dissolved in ultra-pure water. The solution was left to heat up for
about 15mins at 80°C in a water bath. After the sample have been heated. The sample was
placed on sequential conditioning of SPE tube. Firstly, the SPE tube in the vacuum was
conditioning with 1mL methanol for activation and after wards 1mL of ultra-pure water for
Equilibration.
The second step is loading the sample; 0.5mL of cocoa was loaded into the SPE tube, the third
step was washing all the cocoa into the tube using 1mL of ultra- pure water. The fourth step is

elute the compound needed from the cocoa. 2 times with 2.5mL of methanol was used to elute
the compounds and the second elution was placed on different vial.
The eluted compound was characterised using HPLC.
All the other samples (Coffee and Tea0 undergo the same procedure as 2.5).
Instrument
2.6.1 HPLC
Automated Liquid Chromatography
Mobile phase: H2O: Methanol 60:40%
Flow rate: 1.0ml/min
Column: 150×4.6mm, 5-um particle size
Retentions time: 10minutes
Wavelength: 275nm
Injection volume: 10ul
Temperature: 25
2.6.2 GC-Mass spectrometry
See the Appendix for the method for GC-Mass for Caffeine.

Validation of the Chromatographic Method
3.1.1 Linearity and Range
The linearity of the method was tested to demonstrate a direct proportional relationship of
response (Area) versus caffeine concentration over working range. The FDA guideless
specified a minimum of five concentration levels with the acceptance criteria linear regression
coefficient should be greater than or equal to 0.995. From the excel linearity of caffeine, the R2
value obtained is 0.998. From regression line, there is an excellent relationship between the
Peak area and concentration of caffeine shown in figure. The data obtained from linearity
experiment are presented in figure.
Table T: showing the linearity of caffeine standards
Figure 12: Showing the graph linearity of caffeine standards.
y = 28.341x + 11.333
R² = 0.998
0
500
1000
1500
2000
2500
3000
3500
0 20 40 60 80 100 120
PeakArea
Concentration in ppme
A linearity realationship of Peak Area verus the Caffeine
Concentration
Standard concentration 20ppm 40 60 80 100
1 644.1326 1113.493 1644.223 2307.699 2863.823
2 642.8065 1114.525 1643.076 2314.959 2861.409
3 642.2367 1112.266 1641.667 2308.696 2857.434
4 643.1059 1110.955 1641.633 2310.67 2862.757
Average 643.0704 1112.81 1642.65 2310.506 2861.356
Standard deviation 0.794601 1.543085 1.245783 3.215384 2.79457
%RSD 0.12356 0.138665 0.075839 0.139163 0.09766

3.1.2 Precision – percentage RSD value
Standard
Concentration
(60ppm)
Injection
number
Peak Retention
time
Area Height
1 1 Caffeine 3.175 1641.66724 249.24017
2 2 Caffeine 3.165 1643.07500 251.93997
3 3 Caffeine 3.165 1644.22290 253.71841
4 4 Caffeine 3.162 1641.63281 250.34247
5 5 Caffeine 3.162 1641.79968 250.92393
6 6 Caffeine 3.163 1643.94043 251.18338
Mean 1642.72301
SD 1.18417929
%RSD 0.07%
Table 5: Showing the Precision of Caffeine standard on HPLC
Repeatability Precision: The values of relative standard deviation of six replicate injections of
the standard solutions lie well within the limit (%RSD ≤ 0.07), indicating the injection
repeatability of the method (Table). The repeatability results was taken over a short period of
time under the same conditions. The acceptance criteria for an assay method for instrument
should be %RSD≤1. The value obtained from table is %RSD ≤ 0.07.
3.1.3 Accuracy

Table
6:showing the Accuracy method validation of caffeine using HPLC instrument.
The next validation carried out was accuracy. This test use the same data as the linearity and
precision in six replicates of standard 20ppm- 100ppm were injected into the HPLC system
and data obtained were analysed.
Standard concentration 20ppm 40 60 80 100
1 644.1326 1113.493 1644.223 2307.699 2863.823
2 642.8065 1114.525 1643.076 2314.959 2861.409
3 642.2367 1112.266 1641.667 2308.696 2857.434
4 643.1059 1110.955 1641.633 2310.67 2862.757
Average 643.0704 1112.81 1642.65 2310.506 2861.356
Standard deviation 0.794601 1.543085 1.245783 3.215384 2.79457
%RSD 0.12356 0.138665 0.075839 0.139163 0.09766
% Recovery 20ppm 40ppm 60ppm 80ppm 100ppm
1 110% 98% 96% 101% 100%
2 110% 98% 96% 101% 100%
3 111% 97% 96% 101% 100%
4 110% 97% 96% 101% 100%

3.1.4 Limit of Detection
The limit of detection is the lowest amount of an analyte to be detected by the method
developed. The signal to noise must be 3:1
The baseline noise height from the blank = 0.7 cm
S= signalled obtained from the peak = 2.5
S/N = 2.5/0.7 = 3.28
The LOD is 3.28
Figure 13 Blank of the solution (mobile phase)
Figure 14-Limit of detection of Caffeine

3.1.5 Limit of Quantification
The limit of Quantification is the lowest amount of analyte to be quantified by the method.
The signal nosed is 10:1
The baseline noise height from the blank is = 0.7cm
The signaed obtained from the peak = 5.822
S/N = 5.822/ 0.7 = 8.317
LOQ is 8.317
Figure 15 Limit of Quantitation of Caffeine solution.

3.1.6 Specificity
Specify is the ability to distinguish an analyte using a chromatogram.
The chromatogram showed below is Caffeine
Figure 16the chromatogram of Caffeine
Caffeine is showed with the retention value of 3.064 minutes

Discussion
Method development and validation of Caffeine
Method development has to be validated. Method development involves a strategic approach
to an analytical problem through numerous ways. Before separation of any analysis,
information (physical and chemical properties) of the compounds interest must be gathered.
On figure 1 the chemical structures of theobromine, theophylline and caffeine are showed.
From the structure it was showed that methylxathines have similar structure; they only differ
due to the position of CH3 and NH.
After the knowledge based is established from an initial experiment protocol of a literature on
the methylxanthines. In this research a method development for caffeine was carried out
successfully. The chosen mobile phase was a mixtures of Water and Methanol in a 60: 40
composition. The column used was a Hichrom NC100-5C18-1723(150×4.6mm, 5-um particle
size) reversed phase column and detector was a UV-VIS absorbance detector. The caffeine
standards were ranged from 20 to 100ppm. The ideal HPLC conditions are
HPLC Conditions
Automated Liquid Chromatography
Mobile phase: H2O: Methanol 60:40%
Flow rate: 1.0ml/min
Column: 150×4.6mm, 5-um particle size
Retentions time: 10minutes
Wavelength: 275nm
Injection volume: 10ul
Temperature: 25
The ICH guidelines give a description on how to carry out each test and the acceptance criteria
which must be met.
Under this Conditions,
Linearity,
Precision,
Accuracy,
Specificity,
Limit of Detection
Limit of Quantitative.

From the test carried out in Method validation section, it was concluded that the method for
the analysis of Caffeine and Methylxanthines was validated successfully as all the results
obtained within the requirements set out by the ICH.
Before the method was validated, series of tests were carried out using different composition
of mobile phase and different flow rate. Hichrom NC100-5C18-1723(150×4.6mm, 5-um
particle size), the conditions used for this initial attempt consisted of 90%:10% composition of
water/menthol, column temperature of 25°C flow rate of 0.8ml/min, and UV detection
wavelength of 274nm. The figure (17) below shows the peak obtained from the HPLC
conditions outlined. From the peak, it was observed that the peak was broad due to the mobile
phase composition and the flow rate. Using sigma Aldrich HPLC troubleshooting guide, it was
understood from there that I need to change the composition of mobile phase and mobile phase
flow rate is too low.
Figure 17showing the HPLC analysis of caffeine at a concentration of 20ppm with a Hichrom
NC100-5C18-1723(150×4.6mm, 5-um particle size) column and HPLC conditions of 95%
water: 10% methanol, flow rate of 0.8ml/min, column temperature of 25°C and a UV
wavelength of 273.compound labelled.

The variations were mobile phase and flow rate. The flow rate was adjusted from 0.8ml/min to
1.0ml/min and the mobile phase was changed from water/methanol 90:10% to 69:40%
composition. As the conditions were changed, the peak became more shaper with shorter
retention time. The adjusted conditions for the analysis of caffeine is shown below in figure ()
Figure 18 showing the HPLC analysis of caffeine at a concentration of 20ppm with a Hichrom
Once the method for the analysis of Caffeine was developed and validated, it was decided that
the method should be use to separate methylxanthines. Before separation, the standards of
theophylline and theobromine were ran on HPLC with the same conditions as caffeine.
The chromatograms below showed the standards of theophylline and theobromine.

Figure 19 showing the HPLC analysis of theobromine at a concentration of 20ppm with a
Hichrom NC100-5C18-1723(150×4.6mm, 5-um particle size) column and HPLC conditions
of 60% water: 40% methanol, flow rate of 1.0ml/min, column temperature of 25°C and a UV
Figure 20 showing the HPLC analysis of theophylline at a concentration of 20ppm with a
Hichrom NC100-5C18-1723(150×4.6mm, 5-um particle size) column and HPLC conditions
of 60% water: 40% methanol, flow rate of 1.0ml/min, column temperature of 25°C and a UV

After the methylxanthines have been analysed separately on RP-HPLC, the retention time of
the three compounds were obtained. From the introduction, we were ask how we can separate
the three compounds. The diagram below showed the separation of the three methylxanthine
standards using the same HPLC parameters as above.
Figure 21showing the separation of methylxanthines at a concentration of 100ppm with a Hichrom
Based on this chromatogram, it shows that theobromine came out first followed by theophylline
and the caffeine. The elution of all this compounds is based on their structure properties. As
explained in the introduction about log P. log P explains how to measure an analyte partitions
between the immiscible. The log P of theobromine was found to be -0.78, the Log P of
theophylline is found to be -0.77 and caffeine was found to be -0.55. From the log P
theobromine is more polar than theophylline and caffeine. More polar molecule elute faster
than less polar. Also it explained that the higher the log P, the more hydrophobic the molecule.
Hydrophobic is non-polar.
After gathering all the information on different parameters for the analysis of methylxanthines,
a full standards on different concentrations of theobromine, theophylline and caffeine is done

to check for the system suitability. On the each standards, the theoretical plates and tailing
factor were put into consideration before analysing it on samples.
Theobromine
standards
concentrations
Retention time in
seconds
Width of the
peak in second
Theoretical plate
number
Tailing Factor
20ppm 126.9 4.884 10,801.70 1
30ppm 126.84 4.800 11,172.49 1
50ppm 126.9 4.77 11,324.17 1
100ppm 126.9 4.728 11,526.26 1
Table 7showing the standard concentrations of theobromine with the theoretical plate number
and tailing factor.
Theophylline
standards
concentrations
Retention time in
seconds
Width of the
peak in second
Theoretical plate
number
Tailing Factor
20ppm 160.56 5.424 14,020.24 1
30ppm 160.50 5.382 14,229.27 1
50ppm 160.26 5.34 14,410.79 1
100ppm 160.02 5.262 14,796.77 1
Table 8showing the standard concentrations of theobromine with the theoretical plate number
and tailing factor
Caffeine standards
concentrations
Retention time in
seconds
Width of the
peak in second
Theoretical plate
number
Tailing Factor
20ppm 183.84 5.898 15,544.99 1
30ppm 183.84 5.904 15,513.41 1
50ppm 183.96 5.82 15,985.30 1
100ppm 183.90 5.82 15,974.88 1
Table 9: showing the standard concentrations of theobromine with the theoretical plate number
and tailing factor
From the FDA, the acceptance criteria should be > 2000. From the tables above it showed
that the theoretical plate number is greater than 2000. This shows that the column is
efficiency, the factors that can affect the N is flow rate of the mobile phase, particle size in
column, and these have been corrected. Also the tailing factor for each standards is 1, the
FDA acceptance criteria should be < 2. This system is acceptable for the analysis of samples.

Sample
Cocoa powder (Bourville),
Coffee powder (Richman
blend) and Tea bags
(Barry’s gold) were
analysed on RP-HPLC
using the same conditions
as the standards.
Cocoa sample analysed on
RP-HPLC. It was
observed that there is a lot
of theobromine in cocoa
and just traces of caffeine and theophylline in cocoa.
Figure 22 showing the cocoa extract using HPLC with the same above HPLC conditions
Coffee sample
anaysised on RP-
HPLC to obsereve if
there is any
methylxanthines. It
was observed that
there is no
theophylline in
coffee sample
Figure 23: showing the Coffee sample extract using the same above HPLC condtions

Tea bags was analysed
on RP-HPLC using the
same condition above
It was observed that
there just traces of
theophylline in tea bags
Figure 24: showing the Tea extract using HPLC with the same HPLC conditions above
Quantitative Analysis
Three different samples Cocoa, Coffee and Tea bags of theobromine, theophylline and
caffeine has been quantified in this project. Results of the quantitative of three extracts per
sample are shown in Table ().
Ingredient Cocoa in 100g of
sample
Coffee in 100g of
sample
Tea in 100g of
sample
Theobromine 1.5g 0.5622g 0.338g
Theophylline Trace - Trace
Caffeine 0.05788g 1.2g 0.7554
Table 10: showing the amount of methylxanthine in each samples analysed
.

Gas Chromatography
Caffeine was analysed on Gas chromatography. The method development has already been
validated by the technical in Limerick Institute of Technology.
Gas Chromatography separate compounds based on the boiling point. The lower the boiling
point, the more polar the compound it is on the GC, the faster the retention time.
The chromatogram is the mixtures of methylxathaines and it was analysed on GC –Mass
spectrometry.
Figure 25: showing the sepration of Methylxanthine on GC-mass spectrometry
From the chromatogram, it was observed that Caffeine elute out first, based on the boiling
point, the boiling point of caffeine is 178°C, the boiling point for theobromine is 357°C and
the theophylline boing point is 454°C. Based from the boiling point, Caffeine has the lowest
boiling point a caffeine elute out first followed by theobromine and the last is theophylline.

The chromatogram outlined can be identified through the mass spectrometry, the figure
below is the mass spectrometry of the mixtures. To identify the compound, the use of the
molecular weight is used to obtain the compounds from the chromatogram.
The first fragment is caffeine, Caffeine has a molecular weight of 194g/mol.
Figure 27: showing the caffeine from the coffee sample.
Figure 26: showing the spectrum of the Caffeine from Mass- spectrometry

Figure 28: showing the fragment of Theobromine in the mass spectrum of the mixtures

5 Conclusion
The main goal for this project was to develop a method using HPLC system and also to
determine Methylxanthine. The method was developed for the analysis of methlyxanthine
and validated according to FDA.
This project focused on the determination of methylxanthine using HPLC system. The SPE
extraction for the sample were successfully extracted .
The validated HPLC method for the quantification caffeine, theobromine and theophylline in
beverages was found to be sensitive and precise.
Cocoa powder was found to contain Theobromine mostly in the container, about 1.5g.
6 Future work
If this project was to be done again, some more tests and analysis is suggested, for example
more work to be done for the GAS chromatography mass spectroscopy. I found it interesting
but there was not enough time to go further on the analsying of methylxanthine using GC-mass
Spectrometry.
Also the use of automated SPE will be a great advantage

7 Calculations
Theobromine
Concentration of Theobromine
standards
Peak Area of Theobromine
standards
0 0
10 332.85
20 440.57
30 904.51
50 1617.96
100 3087.03
Cocoa sample 2344.80
Coffee sample 847.65
Tea sample 579.56
Figure –Standard curve for caffeine as analysed by HPLC using C-18 columns and 60%/40%(
v/v) water and methanol as mobile phase detected at 275nm.
From the graph, the equation line is
Y = 31.37x-34.131
y = 31.37x - 34.131
R² = 0.9947
-500
0
500
1000
1500
2000
2500
3000
3500
0 20 40 60 80 100 120
Theobromine

From the table,
System Suitability Results
Theoretical number is calculated for theobromine standards using the USP method at 10ppm
concentration
N= 16(Ve/ Wb)2
N = number of the theoretical plates
Ve= Retention time
Wb= Width of the peak at half peak height
N = 16 (
126.96
4.98
)
2
= 10,399.08
From others concentration the theoretical plates and Tailing factor are
Theobromine
standards
concentrations
Retention time
in seconds
Width of the
peak in second
Theoretical
plate number
Tailing
Factor
20ppm 126.9 4.884 10,801.70 1
30ppm 126.84 4.800 11,172.49 1
50ppm 126.9 4.77 11,324.17 1
100ppm 126.9 4.728 11,526.26 1
Tailing factor
Tailing Factor is calculated for theobromine standards using the USP method at 10ppm
concentration
T = (A+B)/2A.
T = tailing factor (measured at 5% of peak height)
b = distance from the point at peak midpoint to the trailing edge
a = distance from the leading edge of the peak to the midpoint
A = 0.4

B = 0.4
T = (
0.4+04
2(0.4)
) = 1
Theoretical number
Theoretical number for cocoa sample is calculated to be
N = 16 (
126.72
5.214
)
2
= 9450.79
The tailing factor
Tailing factor for the sample is
A = 0.25
B = 0.25
T = (
0.3+0.3
2(0.3)
) = 1
Resolution
Resolution between the peak 1 and peak 2
Peak 1 = theobromine
Peak 2 = caffeine
Rs = (tR2 – tR1) / ((0.5 * (w1 + w2)) (1)
Tr1 = 2.112, Tr2= 3.040, W1= 0.6, W2 = 0.5
Rs=
3.040−2.112
0.5(0.5+0.6)(1)
= 1.68 = 1.7
Retention Factor
K =
𝑡𝑅−𝑡𝑂
𝑡𝑂
tR= Retention time

tM = time taken for the mobile phase to pass through the column.
K =
2.112−0.75
0.75
= 1.816
Cocoa peak area = 2344.80
The sample peak area is obtained, the absolute amount of the sample is calculated as follows:
Y = 31.37x-34.131
Interpolated amount(x) = 𝑥 =
2344.80+34.131
31.37
= 75.83ppm concentration of theobromine
From the SPE extraction,
The original dilution factor from 20ml of sample solution took 0.5ml of solution into SPE is 40
dilution factor.
1 HPLC vial contains 2.5ml of elution.
75.83ppm solution of theobromine in 2.5ml vial elution
75.83mg 1000ml
75.83
1000
× 2.5 × 40 0.5g of sample
7.5mg of theobromine in 0.5g of cocoa.
From the Bonriville 100g cocoa powder,
1 g of cocoa powder = 15mg of theobromine
In 100g of cocoa powder there is 1.5g of theobromine.

Coffee peak area = 2344.80
Y = 31.37x-34.131
847.65+34.131
31.37
= 28.11ppm concentration of theobromine
The original dilution factor from 20ml of sample solution took 0.5ml of solution into SPE is 40 dilution
factor.
28.11mg 1000ml
28.11
1000
× 2.5 × 40 0.5g of sample
2.811mg of theobromine in 0.5g of coffee sample.
From the Richman blend 100g coffee powder,
1 g of coffee powder = 5.622mg of theobromine
In 100g of coffee powder there is 0.5622g of theobromine.

Theophylline
Concentration of Theophylline
standards
Peak Area of Theophylline
standards
0 0
10 445.2551
20 911.1568
30 1313.886
50 2293.057
100 3928.322
Cocoa sample 18.79980
Coffee sample -
Tea sample -
Figure –Standard curve for Theophylline as analysed by HPLC using C-18 columns and
60%/40 %( v/v) water and methanol as mobile phase detected at 275nm.
Y = 39.446x+101.35
From the table,
y = 39.446x + 101.35
R² = 0.9926
0
500
1000
1500
2000
2500
3000
3500
4000
4500
0 20 40 60 80 100 120
AreaPeak
Concentration (ppm)
Theophylline

Theoretical number is calculated for theobromine standards using the USP method at 10ppm
concentration
N= 16(Ve/ Wb)2
Ve= Retention time
N = 16 (
160.44
5.466
)
2
= 13784.91
From others concentration the theoretical plates and Tailing factor are
Theophylline
standards
concentrations
Retention time
in seconds
Width of the
peak in second
Theoretical
plate number
Tailing
Factor
20ppm 160.56 5.424 14020.24 1
30ppm 160.50 5.382 14229.27 1
50ppm 160.26 5.34 14410.79 1
100ppm 160.02 5.262 14796.77 1
Tailing factor
Tailing Factor is calculated for theophylline standards using the USP method at 10ppm
concentration
T = (A+B)/2A.
A = 0.4
B = 0.4
T = (
0.4+04
2(0.4)
) = 1

Caffeine
0 0
10 350.5
20 741.66
30 1063.4
50 1984.98
100 3443.49
Cocoa sample 144.49
Coffee sample 105.61
Tea sample 1360.73
Figure –Standard curve for caffeine as analysed by HPLC using C-18 columns and 60%/40%(
v/v) water and methanol as mobile phase detected at 275nm.
Y = 34.87x + 43.562
From the table,
y = 34.87x + 43.562
R² = 0.9936
0
500
1000
1500
2000
2500
3000
3500
4000
0 20 40 60 80 100 120
AreaPeak
Concnetration(ppm0
Caffeine standards

Theoretical number is calculated for Caffeine standards using the USP method at 10ppm
concentration
N= 16(Ve/ Wb)2
Ve= Retention time
N = 16 (
184.32
6.03
)
2
= 14949.62
From others concentration the theoretical plates are
Caffeine standards
concentrations
Retention time in
seconds
Width of the peak
in second
Theoretical plate
number
20ppm 183.84 5.898 15,544.99
30ppm 183.84 5.904 15,513.41
50ppm 183.96 5.82 15,985.30
100ppm 183.90 5.82 15,974.88
Tailing factor
Tailing Factor is calculated for Caffeine standards using the USP method at 10ppm
concentration
T = (A+B)/2A.
A = 0.4
B = 0.4
T = (
0.4+04
2(0.4)
) = 1

Cocoa peak area = 144.49.
Y = 34.87x + 43.562
144.4875−43.562
34.87
= 2.894ppm concentration of caffeine
dilution factor.
2.894mg 1000ml
2.894
1000
× 2.5 × 40 0.2894g of sample
0.2894mg of caffeine in 0.5g of cocoa.
From the Bonville 100g cocoa powder,
1 g of cocoa powder = 0.5788mg of caffeine
In 100g of cocoa powder there is 0.05788g of caffeine.

Theoretical number for cocoa sample
Theoretical number for cocoa sample is calculated to be
N = 16 (
182.4
6.36
)
2
= 13,159.99
The tailing factor Tailing factor for the sample is
A = 0.25
B = 0.25
T = (
0.25+0.25
2(0.5)
) = 0.5
Resolution
Resolution between the peak 1 and peak 2
Peak 1 = theobromine
Peak 2 = caffeine
Rs = (tR2 – tR1) / ((0.5 * (w1 + w2)) (1)
Tr1 = 2.112, Tr2= 3.040, W1= 0.6, W2 = 0.5
Rs=
3.040−2.112
0.5(0.5+0.6)(1)
= 1.68 = 1.7
Retention Factor
K =
𝑡𝑅−𝑡𝑂
𝑡𝑂
tR= Retention time
tM = time taken for the mobile phase to pass through the column.
K =
3.040−0.75
0.75
= 3.05

Coffee samples
From Maxwell house rich blend products
2221.26 = 34.87x + 43.562
2221.26−43.562
34.87
= 62.452ppm concentration of caffeine
dilution factor.
62.452mg 1000ml
62.452
1000
× 2.5 × 40 6.2452g of sample
6.2452mg of caffeine in 0.5g of coffee.
From the Maxwell house rich 100g coffee,
1 g of coffee = 12mg of caffeine
In 100g of cocoa powder there is 1.2g of caffeine

Tea samples
From Barry’s tea gold blend.
1360.73= 34.87x + 43.562
1360.73−43.562
34.87
= 37.77ppm concentration of coffee in 0.5ml.
dilution factor.
37.77mg 1000ml
62.452
1000
× 2.5 × 40 3.777mg of sample
3.777mg of caffeine in 0.5g of tea.
From the Barry tea bag 100g,
1 g of Barry tea bag = 7.554mg of caffeine
In 100g of Barry tea bag, there is 0.7554g of caffeine

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