determination of caffeine concentrations of ethiopian export

determination of caffeine concentrations of ethiopian export
DETERMINATION OF CAFFEINE CONCENTRATIONS OF ETHIOPIAN
EXPORT STANDARD COFFEE SAMPLES AND THE INVESTIGATION
OF
OPTICAL
AND
QUANTUM
MECHANICAL
TRANSITIONAL
PROPERTIES OF CAFFEINE MOLECULE BY UV/VIS-ABSORPTION
SPECTROSCOPY
BY:
ALENE SEYOUM
A THESIS SUBMITTED TO THE SCHOOL OF GRADUATE STUDIES
ADDIS ABABA UNIVERSITY IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS
FOR
THE DEGREE OF MASTER OF SCIENCE IN PHYSICS
ADDIS ABABA, ETHIOPIA
JUNE 2017
© Copyright by Alene Seyoum (2017)
ADDIS ABABA UNIVERSITY
COLLAGE OF NATURAL AND COMPUTATIONAL SCIENCE
SCHOOL OF GRADUATE STUDIES
This is to certify the thesis prepared by ALENE SEYOUM MITIKU entitled of
‘‘Determination of caffeine concentrations of Ethiopian export standard coffee samples and the
investigation of Optical and Quantum mechanical transition properties of caffeine molecule by
UV/Vis- absorption spectroscopy’’ submitted in partial fulfillment of the requirements for the
degree of Master of Science in Physics. Compiles with the regulations of the university and
meets the accepted standards with respect to originality.
Signed by the Examining committee:
Supervisor: Professor A.V.P Gholap Signature: _________ Date: ___________
Examiner: ____________________ Signature: __________
Date: ___________
Examiner: ____________________ Signature: __________
Date: ___________
JUNE 2017
ADDIS ABABA UNIVERSITY
Date: June 2017
AUTHOR:
ALENE SEYOUM
TITLE: Determination of caffeine concentrations of Ethiopian export standard coffee samples
and the investigation of Optical and Quantum mechanical transition properties of caffeine
molecule by UV/Vis- absorption spectroscopy
Department:
PHYSICS
Degree: M.Sc.
Convocation: June 2017
Permission is here with granted to Addis Ababa University to circulate and to have copied for
non-commercial purposes, at its discretion, the above title upon the request of individuals or
institutions.
__________________
Signature of Author
DECLARATION
This thesis is my original work, the results reported in this work were obtained by research
carried out by me under the supervision of my Advisors in the College of Natural Sciences,
Department of Physics, Addis Ababa University.
This thesis has not been presented for a degree in any other university and that all sources of
materials used for the thesis have been duly acknowledged. No part of this work shall be
published in scientific journals or reported in the media or presented at a conference without the
knowledge and consent of my advisors, who are the principal scientists responsible for any
publication. Furthermore if the work is published the institutional address given should be that of
the Physics Department, AAU.
Name of author: Alene Seyoum
Signature__________
Date: 12 June 12, 2017
This thesis has been submitted for examination with my approval as university advisor.
Prof. A.V.P Gholap
Signature ______________ Date : 12 June 12, 2017
Date of submission June 12, 2017
DEDICATION
This Work is dedicated to:
My Father: Seyoum Mitiku
And
My mother: Belaynesh Alemayehu
STATEMENT OF THE AUTHOR
The author reserves other publication rights, and neither the thesis nor extensive extracts from it
may be printed or otherwise reproduced without the author’s written permission.
The author attests that permission has been obtained for the use of any copyrighted material
appearing in this thesis (other than brief excerpts requiring only proper acknowledgement in
scholarly writing) and that all such use is clearly acknowledged.
Alene Seyoum
June 2017
CONTENT
Page
Content…………………………………………………………………………………………………………………………………………………..i
List of figures …………………………………………………………………………………….iii
List of table………………………………………………….……………………………………………………………………………………...iv
Acronyms……………………………………………………………………...…………………..v
Acknowledgements ……………………………..……………………………………………….vii
Abstract………………………..…………………………………………………………………vii
Chapter 1…………………………………………………………………………………………..1
1.Introduction……………………………………………………………………………….....…..1
1.1. Background……………………………………………………………………………….…..1
1.2.Literature Review………………………………………………………………………….…..4
1.3. Scope of the study…………………………………………………………………………….5
1.4.Objective of the study…………………………………………………………………………5
1.5. Statement of the problem……………………………………………………………………..6
1.6. Significance of the study……………………………………………………………………...6
Chapter 2 ………………………………………………………………………………………….8
2. Theory…………………………………………………………………………………………..8
2.1. Spectroscopy………………………………………………………………………………….8
2.2. Band of Spectrum…………………………………………………………………………….8
2.3. Nature of Molecular Absorption…………………………………………………………..…9
2.4. Transition Dipole Moment ………………………………………………………………..…9
2.5. The Einstein relation and Einstein B Coefficient……………………………………...……11
2.6.Optical and Quantum mechanical transition property of molecule…………………….……12
2.7. Beer-Lambert’s Law (BLL) ………………………………………………………………...12
2.8. Integrated Absorption Technique (IAT) …………………………………………………...13
Chapter 3…………………………………………………………………………………………15
3. Materials and methods ………………………………………………………………………..15
3.1. Material……………………………………………………………………………………...15
3.1.1. Chemicals and samples……………………………………………………………………15
3.1.2. Instrumentation……………………………………………………………………………15
3.1.3. Components of UV/Vis- spectroscopy……………………………………………………16
3.1.4. UV/Vis cut-off…………………………………………………………………………….17
3.1.5. Preparation of standard solutions………………………………………………………….17
3.1.6. Coffee sample preparation…………………………………………………………….…..19
3.1.7. Data collection …………………………………………………………………………....20
3.2. Methods……………………………………………………………………………………...20
3.2.1. Beer Lambert’s Method of measuring caffeine…………………………………………...20
3.2.2. Integrated Absorption technique (IAT) of Measuring Caffeine…………………………..20
3.2.3. Least Square Method……………………………………………………………………...21
3.2.4. Non-linear curve fitting Gaussian function……………………………………………….21
i
Chapter 4………………………………………………………………………………………...23
4. Results and Discussion……………………………………………………………………….23
4.1. Calibration curve of pure caffeine in dichloromethane and in distilled water……………..23
4.2. Absorption versus concentration relation…………………………………………………..24
4.3. UV-Vis Absorption of Caffeine…………………………………………………………….26
4.3.1. Integrated absorption coefficient ………………………………………...…………….…26
4.3.2. Transition dipole moment (µ21) of caffeine……………………………….………………26
4.3.3. Einstein B Coefficient ……………………………………………………………….……27
4.4. Optical and Quantum mechanical Transition Properties of caffeine molecule in Beer
Lambert’s Law (BLL)……………………………………………………………………………28
4.4.1. Integrated absorption coefficient (𝑎𝑡 ) …………………………………………………..29
4.4.2. Integrated absorption cross-section (δt)………………………………………………..….29
4.4.3. Oscillator Strength (f) ………………………………………………………………….29
4.4.4. Number density of caffeine molecule (N)……………………………………………..….30
4.5. Optical and quantum mechanical Transition Properties of Caffeine by integrated absorption
technique (IAT)………………………………………………………………………………..…31
4.6. Number density of caffeine in medium roasted coffee beans…………………………….…36
4.7. Determination of Caffeine concentration in Roasted Coffee Beans by Beer Lambert’s Law
(BLL) and integrated absorption technique (IAT)………………………………………………36
5. CONCLUSIONS……………………………………………………………………......…….41
References………………………………………………………………………………………..42
APPENDICES…………………………………………………………………………………...45
ii
List of figures
Fig1.1:- Image of the coffee plant, green coffee beans, roasted coffee and cup of coffee ……….2
Figure 1.2:- The Chemical Structure of Caffeine…………………………………………………3
Fig 2.1: - Diagram of spectrum band…………………………………………………………….11
Fig 2.2:- Absorption, stimulated emission, and spontaneous emission where N1 and E1 are
number of population & energy density of the ground level and N 2 & E2 are number of
population & energy density of the excited level………………………………………………..13
Fig 2.3:- Absorbance, incident and transition of light for a given caffeine concentration in cuvette
quartz cell………………………………………………………………………………………...15
Fig 3.1:- Photograph of some experimental instruments used in UV/Vis absorption spectroscopy
measurement of caffeine…………………………………………………………………………16
Fig3.2:- Schematic diagram of double beam UV/Vis absorption spectroscopy………………...16
Fig 4.1;-Absorbance Vs concentration of caffeine in distilled water……………………….…...26
Fig 4.2:- Absorbance Vs Concentration of caffeine in DCM……………………………………26
Fig 4.3(a);- Normalized absorption spectrum of pure caffeine in DCM………………………...26
Fig 4.3(b):- Normalized Absorption spectrum of pure caffeine in distilled water………………27
Fig 4.3(c):- UV/Vis spectrum of Absorbance Vs wavelength for caffeine in DCM…………….29
Fig 4.4:-Molar decadic absorption coefficient over wave number Vs wave number of caffeine in
DCM..............................................................................................................................................29
Fig 4.5:- UV/Vis spectrum of Absorbance Vs Wavelength for caffeine in water……………....30
Fig 4.6:- UV/Vis spectrum of caffeine in distilled water……………………..………………….30
Fig 4.7:- UV/Vis spectra of standard caffeine solution in DCM………………………………...32
Fig 4.8;- UV/Vis spectra of caffeine solution in distilled water……………………….………...32
Fig 4.9(a);- Gaussian fit to the spectra of absorption coefficient Vs wave number of caffeine in
DCM……………………………………………………………………………………………..34
Fig 4.9(b):-Absorption coefficient versus wave number of caffeine in DCM after Gaussian fit..35
Fig 4.10(a):-the Gaussian function fitted to the spectrum band of Absorption coefficient versus
Wave number of caffeine in distilled water…………………………………….………………..35
Fig 4.10(b):- After Gaussian fit of the spectrum for caffeine in distilled water…………………36
Fig 4.11(a):-UV/Vis Spectrum for caffeine in distilled water after Gaussian fit ………………..36
Fig 4.11(b):-UV/Vis spectrum for Caffeine in DCM after Gaussian fit……………………...….37
Fig 4.12(a);- UV/Vis spectrum of caffeine extracted by DCM from roasted coffee before
Gaussian fit…………………………………………………………………………………...….39
Fig 4.12(a);- UV/Vis spectrum of caffeine extracted by DCM from roasted coffee after Gaussian
fit...................................................................................................................................................39
Fig 4.13:- UV/Vis spectra of medium roasted coffee disolved in distilled water……..…….....40
Fig 4.14:- UV/Vis Spectra of caffeine extracted from coffee Beans ……………………………40
iii
List of tables
Table 4.1:- Optical & quantum mechanical transition properties, number density, Einstein B
coefficient and transition dipole moments of caffeine dissolved in DCM by BLL for
corresponding concentration & absorbance……………………………………………..……….32
Table 4.2:- Optical & quantum mechanical transition properties, number density, Einstein B
coefficient and transition dipole moments of caffeine dissolved in distilled water by BLL for
corresponding concentration & absorbance……………………………………………………...32
Table 4.3:- Optical & quantum mechanical transition properties, number density, Einstein B
coefficient and transition dipole moments of caffeine dissolved in DCM by IAT for
corresponding concentration & absorbance. …………………………………………………….36
Table 4.4:- Optical & quantum mechanical transition properties, number density, Einstein B
coefficient and transition dipole moments of caffeine dissolved in distilled water by IAT for
corresponding concentration & absorbance…………………………………………………...…36
Table 4.5:- Number density and Weight by weight (w/w) concentration of Caffeine measured in
Ethiopian export standard Roasted Coffee Beans by UV-Vis Spectroscopy in the BLL & IAT..40
Table 4.6:- the bandwidths of pure caffeine in DCM and caffeine extracted from roasted
coffee……………………………………………………………………………………………..40
iv
ACRONYMS
Abrivations
Deffinations
UV/Vis
Ultra violate-visible
BLL
Beer Lambert’s Law
IAT
Integrated absorption technique
DCM
Dichloromethane
CNS
Central nervous system
TLC
Thin Layer chromatography
SPE-HPLC
Solid phase extraction-High performance liquid chromatography
ATR
Attenuated total refraction
FT-IR
Fourier transform infrared radiation
NIR
Near infrared radiation
RS
Radiation source
MC
Monochromator
PD
Photo detector
BS
Beam splitter
DA
Difference amplifier
RO
Readout
v
Acknowledgements
First, I would like to thank the Almighty God who made everything in this world possible, and
he who helped me to begin and finish this work successfully as it is expected.
Secondly, I would like to thank my advisor Prof.A.V.Gholap even if I do not have adequate
words to express my feelings of gratitude whose benevolent guidance and constant
encouragement that helped me to complete the present research work successfully. He is the
person who has always helped me. His constant encouragement made me strong enough to face
every difficulty with confidence during this research study.
Thirdly, I would like to thank Ethiopian coffee storage and liquoring board who gave me all
kinds of export standard coffee samples and to Ministry of agriculture specifically agricultural
laboratory who helps me in grinding of my coffee samples.
Lastly, I would like to give my special thanks to my country Ethiopia and my family to teach me
without having enough knowledge and who were supporting me in every aspects.
vi
Abstract
In this thesis, determination of caffeine concentration in aqueous solution of medium roasted
export quality coffee bean samples performed by using UV/Vis absorption spectroscopy. Using
UV/Vis absorption spectroscopy the molar decadic absorption coefficients and transitional dipole
moment of pure caffeine in distilled water and DCM were obtained at 273 and 276 nm. The
molar decadic absorption coefficients of caffeine in water and dichloromethane at these
wavelengths were 1125.035 and 376.06m2 mol-1, respectively. The calculated values for the
transitional dipole moment of caffeine in distilled water and in DCM were 6.70 x 10-30and 11.63
x 10-30C m for the given concentration in wave number range of 2,800,000 to 4,100,000m-1
respectively. After characterizing caffeine in water and DCM, a simple, rapid, cost-effective, and
environmentally friendly UV/Vis absorption spectroscopy method was developed that enables to
quantify the concentration of caffeine in coffee beans. Caffeine extracted with dichloromethane
(DCM) from aqueous solution of coffee in distilled water to get UV/Vis absorption spectrum.
The inference matrix was eliminated by fitting Gaussian function to the spectrum band of the
experiment. From absorption spectrum optical & quantum transition mechanical properties such
as; integrated absorption coefficient, integrated absorption cross-section, oscillator strength,
dipole moment, and Einstein B coefficient as well as number density of caffeine molecule were
calculated in DCM and distilled water. Optical & quantum transition mechanical property shows
us the intrinsic ability of molecules to absorb light and these were proportional to the intensity of
transition. The concentration of caffeine in medium roasted Ethiopian export standard coffee
samples expressed in percentage ranged from 1.34% to 2.51% by BLL and by IAT. These
finding will help Ethiopian coffee storage & liquoring board, Ethiopian standard agency, and
Farming & agricultural ministry of Ethiopia for specifying caffeine concentration in each coffee
samples.
The highest caffeine concentration in this work was found in Teppi & washed Sidama coffee
samples and the lowest caffeine concentration was found in Limu coffee sample. The caffeine
concentration for Ethiopian export standard roasted coffee in this work was in a good agreement
with other researchers reported on roasted Arabica coffee samples by the same method.
Key words: - Export standard, optical transition, caffeine, concentration, absorbtion
spectroscopy, Einstein B coefficient
vii
Chapter 1
1.Introduction
Backgrounds related to coffee growing and compounds are presented. The chemical and physical
properties of the compounds, the physiological and psychological effects in biological systems,
and their roles in determining the quality of coffee beans are discussed. Moreover, the different
physical and chemical methods to analyze these compounds in coffee beans and their interaction
with aromatic compounds and metal ions are reviewed.
1.1. Background
Coffee plant is believed to be discovered during the 6th century in Ethiopia around KAFFA, the
name coffee is derived from the southwestern massive highlands of Ethiopia, KAFFA region.
Arabs introduced coffee to Yemenis in 13 th century. Latter it developed as a habit of drink
around 15thcentury. This habit spread all over the world and popular coffee houses became
favorite meeting places for both Social and Economic purposes starting from mid 17 thCentury.
Now coffee is primary Agricultural commodity next to Oil as a source of foreign exchange for
developing countries and it is the first source of foreign exchange for Ethiopia. In Africa Coffee
tree is indigenous and can still be found growing wild in the hills of Ethiopia. Today coffee is
cultivated over four continents and more than 80 countries and becomes the base for their
economy. The biggest coffee producing countries are Brazil, Colombia, Indonesia, Vietnam and
Mexico. The coffee tree is a tropical evergreen shrub (genus coffee) and two beans are generally
contained in each fruit, which when ripe resembles a red cheery 1, 2, 3.
The two most commercial important species of Coffee are Robusta and Coffee Arabica. Coffee
Arabica is native to the southwestern highlands of Ethiopia. It is the most cultivated coffee
species throughout the world. About 90% of the world’s coffee production is coffee Arabica and
10% is Robusta. Coffee Arabica grown at higher altitude requires less rain and its beans have
lower caffeine content than that of Robusta. It grows mainly in Central and Southern America,
East Africa, India. It is the most important foreign currency earner for more than 80 developing
countries. It is the only species occurring in Ethiopia and Robusta coffee is widely grown in
Western and central Africa, Malaysia, Brazil, and India 4, 5, 6. It originated in the humid lowland
forest of tropical Africa, which stretches from Guinea to Uganda and Angola. It has stronger
flavor than Arabica with a full body and a woody after test, which is useful in creating blends
and especially useful in instant coffee. It is grown at lower altitude 7.
Ethiopia is the 3rd largest producer of coffee in Africa and the 6th from the world next to Uganda
and Ivory Coast8. The agricultural based economy of Ethiopian is highly dependent on Arabica
.c, as it contributes more than 60 % of the country’s foreign exchange earnings. There are four
types of production system of coffee in Ethiopia: forest coffee, semi forest coffee, garden coffee
and plantation coffee. Forest coffee is found in south and south- western Ethiopia Zones. It
1
accounts 10 % of Ethiopia’s total coffee production. Semi - forest coffee production system is
also found in the south and south - western parts of the country. It accounts 35 % of Ethiopia’s
total coffee productions. Garden coffee is grown in the vicinity of farmers’ residences, mainly in
the southern and eastern parts of the country. It accounts for about 35 % of Ethiopia’s total
coffee production. Plantation coffee includes that is grown in plantations owned by the former
state and some well managed smallholder coffee farms. The former state plantation accounts
about 5 % of total production and well-managed smallholder coffee farms account 15 % of the
Ethiopia’s total production1, 9.
These Four types of production system of coffee in Ethiopia are collected and exported to other
countries which seek Ethiopian coffee because of its quality production. Ethiopian coffee export
association collects coffee from all over the country depending on the coffee standard given by
Ethiopian coffee board and liquoring center in the case of category, origin and grade. These
standards are used to identify the type of coffee for collection purpose. The type of export
standard coffees of Ethiopia in category are classified as washed and unwashed; by origin
(Yirgacheffe, Sidama, Limmu, Bebeka, Teppi, Harer, Lekempti and Djimmah) and by grade for
each unwashed category; grade 1,2,3,4,5,& under grade(UG) and for each washed category;
grade 1, grade 2 & under grade (UG)). Ethiopia has five types of coffee in Washed category viz.
Yirgacheffe, Sidama, Limmu, Bebeka, & Teppi and four types in Unwashed coffee category
which are (Harer, Sidama, Lekempti, & Djimmah). In this work, we did the determination of
caffeine concentration in Ethiopian export standard coffee samples. Coffee is a brewed drink
prepared from roasted coffee beans, which are the seeds of berries from the Coffee plant. Coffee
is a widely and extensively consumed as recreational beverage. It is a worldwide favorite.
Everybody everywhere at all times enjoys a fresh cup of coffee. Coffee is a big deal in almost all
regions of Ethiopia. In most places of the country, it is culturally prepared and served one up to
three times a day. Coffee is served in special occasions such as birth, marriage, ceremonies,
burials, and holidays.
Fig1.1:- Image of the coffee plant, green coffee beans, roasted coffee and cup of coffee
Coffee is chemically composed of over 1000 compounds such as; caffeine, minerals, trigonelline
lipids, total chlorogenic, aliphatic acids, oligosaccharin, total polysaccharides proteins and
hommic acid. The chemical composition of green coffee depends on the species and variety.
2
Other factors such as agricultural practices, degree of maturation and storage conditions
determines its chemical composition to a lesser extent. Caffeine is the most commonly found
alkaloid in coffee beans that belongs to a class of organic compounds called methylxanthines.
Caffeine or 1,3,7-trimethylxanthine is a xanthine molecule with methyl groups replacing all of
the three hydrogen’s bound to nitrogen in xanthine. In pure form, caffeine is a crystalline white
powder moderately soluble in water and a wide range of organic solvent such as ethanol, ethyl
acetate methanol, benzene, dichloromethane. This powdered form of caffeine is actually the
scientific definition of bitter, that is why many beverages containing caffeine also contains
copious amounts of sugar or other sweeteners. Its molecular weight is 194.19 g/mol. with
chemical formula C8H10N4O2, melting point is 2360C, which sublimes at 1780C at atmospheric
pressure. The advantage of drinking coffee on human depends on concentration of caffeine
consumed. Consumption of high concentration of this compound causes various physiological
and psychological problems. Caffeine acts as a mild psychoactive stimulant drug, works by
stimulating the central nervous system (CNS), heart, muscles, and the centers that control blood
pressure, may raise blood pressure However, moderate consumption is advantageous due to its
anti-oxidant property. It is also used as a painkiller for preventing and treating headaches, after
epidural anesthesia. It acts as a “water pill” that increases urine flow, for treating migraine
headaches, for asthma, gallbladder disease, shortness of breath in newborns, and low blood
pressure, for weight loss and type 2 diabetes, gastric acid secretion and dieresis Bolton, Caffeine
creams are used to the skin to reduce redness and itching in dermatitis.
As it has an advantage, caffeine may actually harm voice quality, aggravates heart disease,
carcinogenesis, kidney malfunction and asthma.
Figure 1.2:- The Chemical Structure of Caffeine
Ethiopian Coffee graded in export standard with the objective of producing the best quality and
there by securing the best price possible. However, there is no universal grading system. Each
producing country has their own national standard which fulfills the minimum export quality
requirement suggested by the market. Most probably the flavor and quality of coffee depends on
the coffee beans selected, the roasting process, the brewing method and the fineness of the
3
ground coffee used. In Ethiopia, coffee grading is conducted through the combination of two
methods. They are green coffee analysis and cup tests (liquoring). Green coffee analysis involves
visual inspection of physical characteristics of coffee bean. This includes screen analysis which
makes size assessment, defect count, appearance or color test and shape that usually refers to the
structure of beans. Cup test is based on roasted ground coffee analysis (chemical process) by
which aroma, acidity, and other flavor components are tested. From the overall grading methods
of coffee, green analysis accounts 40% and cup test accounts 60% in the quality inspection
processes. In light of these, the most economical and faster system which saves time and more
accurate chemical analysis of caffeine concentration in coffee beans is also used as an additional
tool for evaluating coffee quality by reducing observer effects of biases pertaining to the quality
standard that enhances the commercial needs4,9.
1.2.Literature Review
Caffeine is determined in coffee samples mostly with the use of HPLC 10, 11. Caffeine was also
determined using Thin Layer Chromatography (TLC), Solid Phase Extraction and High
Performance Liquid Chromatography (SPE-HPLC) 12, 13, Capillary Zone Electrophoresis (CZE),
Fourier Transform Infrared (FT-IR) and electrochemical methods have been reported14, 15, 16.
Although these methods are used widely for caffeine determination, the methods require very
costly instrumentation, higher skilled technician, complicated, and time consuming procedures.
The use of attenuated total reflection (ATR) accessories in conjunction with Fourier transform
infrared (FT-IR) spectrometers provides for the non-destructive measurement of samples and
mid-infrared approaches have a huge potential for gaining rapid information about the chemical
composition and related properties of coffee17. In addition to its ability for effectively
quantifying and characterizing caffeine content of coffee in aqueous solution, it is also able to
measure multiple chemical constituents simultaneously avoiding extensive sample preparation.
On the other hand, the derivative spectrophotometer is relatively easy18 ; however, it is not
reliable for the determination of small concentration of caffeine in samples. With HPLC
methods, the use of expensive equipments and demand of more operator awareness for
application in small industrial laboratories results to perform few analyses in each day. Other
methods such as FT infrared, Raman spectroscopy and NIR reflectance spectrometry are equally
versatile for the measurement of caffeine and do not require expensive chemicals however, such
instruments are expensive and are not available in most laboratories 19, 20, 21. The method of
UV/Vis-absorbance spectroscopy is available in most laboratories. Moreover the method, is easy
fast and cheap for the determination of the caffeine contents in coffee beans 22, 23, 24, 25, 26, 27, 28.
In this thesis one of such best method UV/Vis-absorption spectroscopy used for roasted,
grounded and dissolved coffee solutions. UV/Vis-absorption spectroscopy is an applied
spectroscopic technique that used to study the physical system when it interacts with
electromagnetic radiation (EMR). More over it is a spectroscopic technique that studies about the
4
Absorption, transmission and detection of light with relation to the optical properties of a
material medium. The methods are characterizing pure caffeine in distilled water and
dichloromethane, and based on these several techniques were developed to determine caffeine in
coffee beans. These are extracting caffeine from coffee using dichloromethane solution. After
caffeine is extracted from coffee using dichloromethane, still there exist some interfering
substances from chlorogenic-acid related compounds. Finally, these interfering matrices
eliminated by fitting the Gaussian function.
1.3. Scope of the study
The scope of this study includes the following
 Extraction of caffeine from medium roasted coffee samples
 Analyzing of spectrum for measuring absorbance of pure caffeine by varying the
concentration
 Calculating the molar decadic absorption coefficient
 Finding the concentration of caffeine in roasted coffee samples
 Finding optical and quantum mechanical transition property of caffeine
1.4.Objective of the study
In the agricultural production of export standard coffee quality of Ethiopian coffee;
coffee users in all over the country, association of coffee exporters and the countries of
Ethiopian coffee importer needs to know how much caffeine is found in the coffee to
ensure the quality of products which was not done yet for all export standard level. One
of the requirements to be a good coffee is whether it contains a reasonable and moderate
quantity of caffeine or not. Therefore, quantitative determination of the amount of
caffeine in roasted coffee of Ethiopian export standard coffee is necessary.
The general objective of this study is:
Determination of caffeine concentration in Ethiopian export quality coffee samples.
Investigation of optical and quantum mechanical transition properties of caffeine
molecule by UV/Vis-absorption spectroscopy.
The specific objective of this study is to :
Develop simple, rapid, cost-effective, and environmentally friendly UV/Vis-absorption
spectroscopy method for the quantitative determination of caffeine concentration in
medium roasted coffee samples, which are prepared in export quality
Investigate the optical & quantum mechanical transition property of caffeine molecules.
Compare the results of this work with results reported by literatures .
5
1.5. Statement of the problem
Like many other foods, the composition of coffee that we eat and drink are complex. However,
coffee contains a very wide range of macro and micronutrients and as one of the most popular
beverages consumed worldwide it has nutritional contribution to our diet. Many compounds in
coffee are often thought to have implications upon human health. These include caffeine,
micronutrients, chlorogenic acid and other bioactive components. In this study, caffeine has been
studied as a compound of interest due to the reason that consumed daily in our real life through
different beverages.
1.6. Significance of the study
The aim of this study is to produce a quick and cost effective method for the routine analysis of
caffeine in coffee beans. Developing a system based on UV/Vis absorption spectroscopy to
assess the coffee quality and quantity parameters would bring economical benefits to the coffee
industry by increasing consumer’s confidence in the quality products. Since there is no report in
literature the amount of caffeine directly determined in aqueous solution of coffee beans by using
spectroscopic techniques for example in the UV-Vis absorption spectroscopy is difficult to
quantify directly in aqueous solution of coffee beans owing to matrix effect 23.
In this research work, water and DCM have been used as a solvent for caffeine determination in
roasted coffee beans using optical methods. DCM and water used for coffee extraction are
environmentally friendly, decrease cost, toxicity and time. Due to this reason, it is desirable to
develop methods of caffeine determination in aqueous solution, which is similar with the actual
caffeine intake through coffee beverages.
The thesis is organized as follows. In Chapter 1, Backgrounds related to coffee growing and
compounds are presented. The chemical and physical properties of the compounds, the
physiological and psychological effects in biological systems, and their roles in determining the
quality of coffee beans are discussed. Moreover, the different physical and chemical methods to
analyze these compounds in coffee beans and their interaction with aromatic compounds and
metal ions are reviewed. In Chapter2, The derivation of electromagnetic radiation from
Maxwell’s equations and the quantum mechanical derivation that is often used for qualitative
and quantitative understanding how transitions are induced in molecular system when it interacts
with electromagnetic radiation are presented. The Schrodinger’s equation is introduced and
simple problems to illustrate its relation to quantities that are important in UV-Vis absorption
spectroscopy are solved.
In Chapter 3, The materials and methods used in this work are presented. This chapter has two
sections. the first section of this chapter deals with the description of various chemicals, samples
and instruments used to carry out this research and the second section of this chapter deals with
6
the methods of measuring the optical & quantum mechanical transition properties of as well as
the mathematical and experimental procedures used to analyze these compounds in coffee beans
are presented. In Chapter 4, The results and discussion of the thesis are presented. The
calculated values of molar decadic absorption coefficients, the optical and quantum mechanical
transition properties (transitional dipole moment, oscillator strength, integrated absorption crosssection and Einstein B coefficient) as well as number density of caffeine are reported. In
addition, the caffeine concentration measured in coffee beans both in Beer-Lambert’s law and
integrated absorption coefficients techniques are presented using UV/Vis absorption
spectroscopic method. Finally, conclusion is given in Chapter 5.
7
Chapter 2
2. Theory
The derivation of electromagnetic radiation from Maxwell’s equations and the quantum
mechanical derivation that is often used for qualitative and quantitative understanding how
transitions are induced in molecular system when it interacts with electromagnetic radiation are
presented. The Schrodinger’s equation is introduced and simple problems to illustrate its relation
to quantities that are important in UV-Vis absorption spectroscopy are solved.
2.1. Spectroscopy
Spectroscopy is the study of how light interacts with matter. Spectroscopy is an experimental
charting of the energy level structure of physical systems for the purpose of transition processes,
spontaneous or induced between different energy states are studied, and it is normally a means of
analysis for various types of electromagnetic radiation or particle emission17, 28, 38. Spectroscopy
is a tool for studying the structure, dynamics of molecules & for exploring the micro world of
atoms and molecules. The structure of atoms and molecules are studied based on spectroscopic
investigations38.
2.2. Band of Spectrum
Molecules produce band spectrum and are the groups of lines, which are closely spaced to one
another. These spectra are characteristics of molecular gases or chemical compounds. For
molecules, the UV/Vis absorption usually occurs over a wide range of wavelengths because
molecules have many excited modes of vibration and rotation at room temperature. In fact the
vibration of molecules cannot be completely frozen out even at absolute zero. Consequently,
molecules have their members in many states of vibration and rotational excitation. The energy
levels for these states are quite closely spaced, corresponding to energy differences considerably
smaller than those electronic levels. The rotational and vibration levels are thus super-imposed
on the electronic levels. A molecule may therefore undergo electronic and vibration or rotational
excitation simultaneously. Because there are so many possible transitions that one differing from
others by only a slight amount & each band spectrum consists of a vast number of lines very
spaced to each other closely at the sharp edge but spaced at the end. What observed from these
types of combined transitions is the UV/Vis-absorption spectrum of a molecule usually consists
of a broad band of absorption23, 28.
8
Fig 2.1: - Diagram of spectrum band
2.3. Nature of Molecular Absorption
When continuous radiation passes through a transparent material, a portion of the radiation may
be absorbed. If that occurs the residual radiation when it passed through a prism, yields a
spectrum with gaps in it, called an absorption spectrum. Because of absorption of energy, atoms
or molecules pass from a state of lower energy to a state of higher energy. The absorbed
electromagnetic radiation has excited energy state and ground energy states. In the case of
ultraviolet and visible Spectroscopy, the transition results absorption of electromagnetic radiation
in region of the spectrum are transitions between electronic energy levels. As a molecule absorbs
energy, an electron was promoted from an occupied orbital to an unoccupied orbital of higher
potential energy. Molecular absorption is more complex than atomic absorption because many
more potential transitions exist. When a molecule interacts with photons in UV/Vis region, the
absorption of energy results in displacing an outer electron of the molecule and the quantum
energy hѵ equals the energy difference between the two energy levels at resonant condition as a
result the atom gains a quantum of energy.
2.4. Transition Dipole Moment
The Transition dipole moment is the electric dipole moment associated with the transition
between the two states. It is complex vector quantity include the phase factors associated with
the two states. Its direction gives the polarization of the transition, which determines how the
system will interact with an electromagnetic wave of a given polarization and while the square of
the magnitude gives the strength of the interaction due to the distribution of charge within the
system.
The transition dipole moment for the 2
1 is given by the relevant off-diagonal element of the
dipole matrix, which can be calculated from an integral taken over the product of the wave
functions of the initial and final states of the transition,
9
μ12 = −𝑒 ∫ ѱ1∗ (𝑟)(𝑟⃗)𝛹2 (𝑟)𝑑3 𝑟 = ⟨𝛹1 |(μ)⃒𝛹𝑛 ⟩
(2.1)
where Ψ1and Ψ2 are the wave functions of states 1 and 2, respectively, μ is dipole moment
operator, μ21 is molecular transition of dipole moment, e is elementary electron charge and r⃗ is
the position vector.
The rate of transition probability that any molecule will go to excited sate 2 because of
absorption under the perturbing effect of the electric field related to transition dipole moment by
the following equation
d
dτ
(C2 (t)C2∗ (t)) = π
ρ(ѵ)
3ε0
h2
|μ12 |
2
(2.2)
Where C(t) are the coefficient of time dependent probability amplitudes of the two molecular
states of 1 and 2, C*(t) is the conjugate part, ρ(ѵ) is the incident energy density of the sample at
frequency ѵ of electromagnetic radiation per volume, h is Plank’s constant and ε0 is
permeability constant of vacuum.
In order to compare the theoretical expressions with the experimentally measurable quantities
d
consider the following. If dτ (C2 (t)C2∗ (t)), is the rate of probability for a single molecule to change
as a result of absorption of radiation under perturbing effect of electric field radiation then,
d
dτ
(C2 (t)C2∗ (t))Ndl is the number of molecules excited in a layer dl with energy absorption.
Therefore, the loss in intensity becomes30, 31
d
−dI = dt (C2 (t)C2∗(t))NhѴ12dl
(2.3)
Further, the loss in intensity of light when light passes through material whose concentration is
N
the ratio of number density with number of Avogadro, C =N , and molar decadic absorption
a
coefficient, ε given by
N
- dI = cρln(10)ε(ν) N dl
a
N
(2.4)
= 2.303cρε(ν) N dl
a
From comparison of equations (2.3), and (2.4) we can express rate of probability as follows,
d
dt
(C2 (t)C2∗ (t)) =
2.303ε(ν)cρ
(2.5)
Na hѴ
If the energy density assumed to be constant throughout the bands, the total rate of probability
for the entire absorption band obtained by integrating over the entire frequency range. The total
intensity of the band are obtained by measuring ε in the region of absorption usually determined
by integrating the area under the graph31, 32, 33.
So the integrated area due to transition for purely theoretical expression of transition dipole
moment related to experimentally measurable quantities of molar decadic absorption coefficient
is given by the following equation 32, 33.
10
IA = ∫
ε(ν)
Ѵ
⃒µ12 ⃒2
dѵ = S
(2.6)
3
Where, (S = 2.9352 × 1060C−2mol−1 the cross-section of the incident beam) and Na is number of
Avogadro 6.022 x 1023/mol, h is Plank’s constant 6.27 x10-34Js & ћ is 1.055 x 10 -34Js, IA is
integrated area & μ12 transition dipole moment and ε molar decadic absorption coefficient. eq
(2.7) relates experimentally measured molar decadic absorption coefficient with the quantum
mechanical expression of the transition dipole moment. The transition dipole moment is a vector
that depends on both ground state and excited states of wave function and couples the transition
to the electric field of light. This transition dipole moment was expressed from eq (2.6) as
3
(2.7)
µ12 =√ 𝑆 IA
2.5. The Einstein relation and Einstein B Coefficient
The stimulated emission of radiation in a two-level atomic system, characterized by the wave
function Ψ1 of the lower level and Ψ2 of the upper level for an electric dipole transition is
determine by the dipole matrix element
μ21 = -∫ Ψ2∗ erΨ1dѴ, where dѴ is volume element.
(2.8)
We are looking for relations between the Einstein coefficients in the interaction of a two-level
atomic system when radiation occurs via absorption, stimulated and spontaneous emission. By
describing the three processes of rate equations that correspond to differential equations of first
order, we can describe Einstein B coefficient as
𝛍𝟐
B =𝟔𝜺 𝟐𝟏ћ𝟐 , Where μ21 is molecular transition of dipole moment
𝟎
(2.9)
where, B is Einstein coefficient of stimulated emission and equal to Einstein coefficient of
absorption
E2, N2
Absorption
Photon
Stimulated emission
E1, N1
Spontaneous emission
Fig 2.2:- Absorption, stimulated emission, and spontaneous emission where N 1 and E1 are
number of population & energy density of the ground level and N 2 & E2 are number of
population & energy density of the excited level.
11
2.6. Optical and Quantum mechanical transition property of molecule
Optical & quantum mechanical transition property is an optical absorption and emission that
occurs through the interaction of optical radiation with electrons in a material system that defines
the energy levels of the electrons. Depending on the properties of a given material, electrons that
interact with optical radiation can be either those bound to individual atoms or those residing in
the energy-band structures of a material such as a semiconductor. The absorption or emission of
a photon by an electron is associated with a resonant transition of the electron between a lower
energy level |1> of energy E1and an upper energy level |2> of energy E 2.
In this thesis since the optical & quantum mechanical transition properties of the molecules are
important to characterize an electron transition and to interpret the absorption spectra, we see the
properties of electrons interacting with optical radiation which residing in the energy-band of
caffeine molecules. The optical & quantum mechanical transition properties (oscillator strength,
dipole moment, integrated absorption coefficient, integrated absorption cross-section and peak
absorption cross-section) calculated in different solvents are the intrinsic ability of molecules to
absorb light and they are proportional to the intensity of transition. Experimental determinations
of the optical &quantum mechanical transition probabilities are important for direct applications
to absorption, emission and dispersion radiation.
2.7. Beer-Lambert’s Law (BLL)
The BLL allows us to measure the absorbance of a particular sample and to deduce the
concentration of the solution from that measurement. We can measure the concentration of a
particular chemical species in a solution as long as we know the species absorbs light of a
particular wavelength. In optics, BLL relates the absorption of light to the properties of
materials through which the light travels. Firstly, BLL considered the changes in the intensity of
radiation passing through the absorbing material of the system. Further, Beer modifies the
change in the intensity of radiation also depend on the concentration of the absorbing material.
Here law states that there is a logarithmic dependence between the transmission T of light
through substance, the absorption coefficient of molecule (αλ ), and the distance of light travel
through the material (the path length l). The absorption coefficient can in turn be written as a
product of either a molar decadic absorption coefficient (𝝴) of the absorber, path length (l) and
the concentration (C) of the absorbing species in material or the absorption cross-section (σ λ),
path length and number density N of the absorbers as eq (2.9) and (2.10).
For liquids and solutions, these relations usually written as
I
T =I t = e−αt l = eεCl = 10−αt l = 10εCl
(2.10)
0
Where, I0 , It , αt , l, ε and C are the intensities of the incident light, intensity of the transmitted
light, absorption coefficient, path length of the substance, molar decadic absorption coefficient,
and concentration of the substance respectively. The transmission for liquid substances expressed
12
in terms of absorbance as follows and Absorbance is a direct measure of how much light
absorbed by our sample.
I
1
A = log ( I0 ) = log (T) = log (eεCl ), since it is easier to use logarithm to the base ten rather than
t
natural logarithm as:
I
A = log ( I0 ) = log (10εCl )
(2.11)
t
A = εCl
C, A
I0
It
Fig 2.3:- Absorbance, incident and transition of light for a given caffeine concentration in cuvette
quartz cell.
An important extension of Beer’s law is the “law of additivity” which states that the absorption
of the radiation by one species will be unaffected by the presence of other materials, whether
they absorb or not.
A =∑ni εi Ci l , where i = 1, 2, 3 , ... n
(2.12)
From equation (2.10), absorption coefficient can be expressed as
1
I
l
It
(2.13)
α(λ) = log ( 0 )
The absorption cross-section σ(λ) is related to the absorption coefficient α(λ) at a single frequency
for N number of molecules per unit volume expressed by the following relation34,35,36.
(2.14)
α(λ) = σ(λ)N
However, in a UV/Vis-absorption spectroscopy, the absorption of molecules in a liquid occurs
over a certain range of frequencies rather than at a single frequency. Therefore, absorption
coefficient measured at any single frequency may not express the true intensity of the molecular
transition in BLL. Due to this limitation of BLL, we can use another technique.
2.8. Integrated Absorption Technique (IAT)
IAT is the preferable technique of finding integrated absorption coefficient, which is the sum of
absorption coefficients for all frequencies in the band of spectrum. In such cases, the technique is
useful for different applications since it is independent of the line function, which may vary by
parameters like pressure, temperature, concentration of the solute and solute-solvent interaction.
13
In addition, the technique is very important in the absence of a high-resolution spectroscopy.
Therefore, in liquids and solutions where the above effects observed, the true integrated
absorption intensity of a band should be defined by the following equations:
αt =∫ αdν
(2.14)
By using equation (2.10), we can rewrite the integrated absorption coefficient (α t) as
1
I
αt = l ∫ log ( I0 )dν
and
t
1
I
(2.15)
1
σ(t) = Nl ∫ log ( I0 )dν = N ∫ αdν
(2.16)
t
Where σ(t) is integrated absorption cross-section, N is number density of the molecule and l is
path length
On the other hand, oscillator strength was considered as the other useful parameter providing the
intensity of transition. It expresses the relative strength of electron transition. Oscillator strength
is one of the most fundamental quantities in analytical absorption spectroscopy. In practice, it
determines the sensitivity of a given atomic resonance line and needs to be accurately known if
one needs to relate the magnitude of the absorption signal to its concentration. Oscillator strength
can be determined directly through absolute emission, absorption or dispersion measurement.
Oscillator strength is related to the molar decadic absorption coefficient by the following
equation
f = 4.32 × 10−9mol cm L-1 ∫ ε(v)dν = 4.32 × 10−10mol m-1 ∫ ε(v)dν
(2.17)
Measurements of emission, absorption and dispersion intensities of the molecules give their
number density and oscillator strength. Absorption and dispersion measurement involves the
number density of the lower level of the transition, and emission measurement involves that of
the upper level. An equation relating integrated absorption coefficient with number density and
the oscillator strength given by
∫ α(ν)dν =2.65×10−6Nf
(2.18)
Where N is number density in molecules m−3, α(ν) in m−2and ν in m-1, and f is dimensionless
oscillator strength of the transition molecule. From equation (2.18), we can express the number
density of caffeine molecule as follows;
1
N = f 1062.65 ∫ α(ν)dν =3.774 x 105 ∫ α(ν)dν
(2.19)
14
Chapter 3
3. Materials and methods
The materials and methods used in this work are presented. This chapter has two sections. the
first section of this chapter deals with the description of various chemicals, samples and
instruments used to carry out this research and the second section of this chapter deals with the
methods of measuring the optical & quantum mechanical transition properties of as well as the
mathematical and experimental procedures used to analyze these compounds in coffee beans are
presented.
3.1. Material
3.1.1. Chemicals and samples
Commercially bought Chemical Dichloromethane (Aldrich, Germany) and distilled water (Dallul
pharmaceuticals plc, Ethiopia) were used. For standard solution preparation, caffeine sample that
was (Evan, England) bought from the local market.
Arabica Coffee samples were obtained from Ethiopia coffee & Tea development and marketing
authority for coffee quality inspection and storage board, Ethiopia Four of these were unwashed
(Harer, Sidama, Nekempti, & Djimmah) and other five were washed (Yirgacheffe, Sidama,
Limmu, Bebeka, & Teppi). All samples were export quality. These coffee samples collected
from the association of coffee exporters and farmers in Ethiopia and stored at the center. The
coffee samples used for this research medium roasted level at 215 oc for 15 minute. Then the
roasted coffee beans were grounded and sieved with micro sieve.
3.1.2. Instrumentation
The laboratory instruments used for the experiment are the following. Carbolite Oven
(Bamford,sheffield®, ENGLAND,S302AU) to roast coffee, Beakers, measuring cylinders,
pipettes, volumetric flasks, spatula, magnetic stirrer with hot plate, funnel, separatory funnel,
glass filter, filter paper, 1cm quartz cuvette and 300µm sieve are some of the apparatus used. All
glassware thoroughly cleaned, rinsed with distilled water and dried before use. For measuring the
mass of caffeine and coffee samples digital electro balance with maximum measuring of 0.5g
were used. For electronic absorption measurement of standard solutions and coffee samples a
double monochromator UV-Vis-NIR spectrophotometer, Perkin Elmer Lambda 19 (PerkinElmer,
D-7770 Ueberlingen, Germany) with wavelength ranges of 170-3200 nm was used. The
instrument operated by a powerful software package termed UVCSS. It provides a wide range of
operating mode for the instrument and it also includes comprehensive data handling and file
management capabilities. Scanning speed of UV-Vis-NIR spectrophotometer was 266.75 nm per
min and slit width 2nm were used during spectral data acquisition. The instrument is a PC-driven
15
spectrometer. The spectrum was interfaced with computer, which was operated by origin®2015
software.
Fig 3.1:- Photograph of some experimental instruments used in UV/Vis absorption spectroscopy
measurement of caffeine.
3.1.3. Components of UV/Vis- spectroscopy
The basic components of double beam spectroscopy are light sources, monochromator, focusing
devices, sample cell compartment. The sources are a deuterium lamp, which covers ultraviolet
(UV) range and tungsten-halogen lamp for visible (VIS) and near infrared (NIR) ranges39. The
monochromator isolate radiant energy of desired wavelength by dispersing the beam in to its
components and placing the light path in a slit that only passes through a narrow wavelength
band. The focusing devices are a combination of lenses, slits, and mirrors inserted in to light path
to render the light rays parallel or to isolate narrow portion of the light beam or its spectrum. The
detectors are photosensitive materials, mostly a photomultiplier tube for UV and VIS ranges.
When light strikes the PMT, it generates electron that passes through a series of dynode that
amplify the signal several times.
SH1
RS
S
PD1
BS
RO
DA
SS
SH2
PD2
Ss
Fig 3.2:- Schematic diagram ofSdouble beam UV/Vis absorption spectroscopy.
sS
SS
In the UV/Vis spectrophotometer,
there are two types of radiation sources from the entire
MC
ultraviolet to near IR region31. For the UV region, the radiation source is Deuterium discharge
lamp that emits polychromatic UV- radiation, which can filtered into monochromatic UV radiation. For the visible region, the radiation source should change to a tungsten filament similar
to the ones found in a common incandescent light bulb. The monochromator or wavelength
selector disperses the light from radiation source into separate wavelength. The wavelength
selector consists of an entrance slit, collimating lenses, dispersing device a focusing lens and an
16
exit slit. A radiation of only a particular wavelength leaves the monochromator through an exit
slit. The monochromatic light that emerges from exit slit is pulsed by a chopper and split into
sample and reference beams by the beam splitter. A reference beam passes through a sample
holder or a quartz cuvette that contains only a solvent. The sample beam passes through a sample
holder or a quartz cuvette that contains a sample solution. The radiation beams that pass through
the detectors is amplified by different amplifiers, and finally reaches the recorder (RO), where
the results are recorded digitally, in a personal computer attached to a spectrophotometer.
3.1.4. UV/Vis cut-off
Every solvent has a UV/Vis absorbance cut-off wavelength. The solvents cut-off is the
wavelength below which the solvent itself absorbs the entire incident light. So when choosing a
solvent we should be aware of its absorbance cut-off that tells us where the compound under
investigation is to be absorbing. With this procedure, we can get the free spectral range of our
compound caffeine in the two solvents. In this experiment, the solvents that we used are distilled
water and dichloromethane (DCM). The UV cut-off for DCM is 233nm and for water is 190nm
as (Burdick & Jackson https://www.researchgate.net/post/What_is_the_UV_cut-off). However, in
our finding, the UV/Vis cut-off for DCM is 228nm and for distilled water is 196nm.
3.1.5. Preparation of standard solutions
For standard solution preparation, commercially bought caffeine mass of 0.0045g dissolved in
distilled water and in DCM. The volume was raised to 50ml. The solutions were stirred for
30min using magnetic stirrer. We prepare the working solution from the above prepared stock
solution with a series of one millimeter (1ml, 2ml, 3ml, 4ml, 5ml and 6ml) and dissolved again
each working solution until the volume of each working solution became to 25ml with DCM.
The concentration for working solution of caffeine in DCM calculated as follows and these were
listed in appendix A.
Concentration(C) =
Given mass of caffeine (g)
g
Molar mass of caffeine (mole ) x Volume of solution (L)
Given mass of caffeine = 0.0045g
Molar mass of caffeine = 194.19g/mol
Volume of solution = 50ml = 0.05liter
C =?
C=
0.0045g
194.19
g
mol
x0.05L
C = 4.635 x 10-4
mol
L
, since 1L = 0.001m3 then the concentration is given by
17
(3.1)
C = 463.5 x 10-3 mol m-3
(3.2)
This was the concentration of the stock solution for caffeine standard in DCM and we calculated
the working solutions with the solvent DCM which was used to lowering the concentration by
applying the following equation;
CV = C1V1
(3.3)
Where C was the concentration of the stoke solution, V is volume of our concentration that we
took as working solution from stoke solution, V1 is the amount of solution we want to reach
which was the sum of V and additional volume of solvent DCM that can used to lowering our
concentrations.
Then we can calculate the concentration (C1, C2, C3, C4, C5, C6) of working solutions as follows;
C1 = CV /V1
(3.4)
Here C & V1 had constant value for each calculations 463.5 x 10 -3 mol m-3 and 25 ml
respectively. The only change is in the value of V for each working solutions.
When we have V = 1ml
1ml
C1 = 463.5 x 10-3(mol m-3) x25ml
C1 =18.4 x 10-3 mol m-3
C1 = 1.84 x 10-2 mol m-3
(3.5)
By the same procedure we can calculate the concentrations of the working solution at V 2ml,
3ml, 4ml, 5ml & 6ml and these values are presented in appendix A.
Similarly, stock solution for 0.1g mass of caffeine dissolved in one liter of distilled water was
stirred for 30 minutes using hotplate magnetic stirrer. Then working solution were prepared with
in a series of 4 milliliter which are 1ml, 4ml, 8ml, 12ml, 16ml and 20ml dissolved in distilled
water till its volume will be 30ml. The concentration for standards caffeine in distilled water was
calculated as follow by using equation (3.1)
C=
0.1g
194.19
g
mol
x 1L
C = 5.15 x 10-4mol/L, since 1L = 0.001m-3 then the concentration is given by
C = 515 x 10-3 mol m-3
(3.6)
This was the concentration of the stoke solution for caffeine standard in distilled water and we
calculated the concentrations of each working solutions with the solvent which was used to
lowering the concentration by applying equation (3.4).
18
Here also C & V1 have constant value 515 x 10-3 mol m-3 and 30 ml respectively. The only
change is in the value of V.
When we have V = 1ml
C1 = 515 x 10-3 mol m-3 x1ml/30ml
C1 =1.762 x 10-2 mol m-3
C1 = 176.2 x 10-4 mol m-3
(3.7)
By the same procedure, we calculated the concentration at 4ml, 8ml, 12ml, 16ml & 20ml and
these values were represented in appendix B.
After preparing the desired concentration of that standard caffeine, solutions for both solvents
were stirred gently with magnetic stirrer for 30 minutes and the absorbance versus wave length
of each solution was measured by UV/Vis- absorption spectroscopy at room temperature in the
wavelength range of 200nm-500nm. The maximum wavelength (λ max) for maximum absorbance
of caffeine standards in DCM and distilled water were 276nm and 273nm respectively.
From the maximum absorbance and the corresponding concentrations of caffeine standards the
molar decadic absorption coefficient was obtained in DCM and water as in appendix C.
3.1.6. Coffee sample preparation
Coffee beans from the samples are medium roasting level at 215 oc for 15 minutes by Carbolite
Oven (Bamford, sheffield ®, ENGLAND, S302AU). The roasted coffee was ground and the
powder was sieved through 300μm sieve to get a uniform texture. An accurately weighed amount
of sieved coffee 0.050g for each sample dissolved in 25 ml of distilled water. The solution stirred
gently for 30 minutes using a stirrer of 80oC magnetic hot plate to remove caffeine easily from
the solution. In addition, the solutions filtered by using glass filter and filter paper to get rid of
particle from solution. In this case, for extraction of caffeine distilled water and DCM were used
as solvents. Because, it had been observed that many interfering matrices were extracted with
water than dichloromethane. The efficiency of DCM to extract caffeine from coffee beans is 98 99 % and caffeine is 140 mg·ml-1 times more soluble in DCM than it is 22 mg·m-1 times in
water3, 23, 26, 29, 37. To extract caffeine from coffee, the coffee solution prepared above under
coffee sample preparation should mixes with 25ml DCM. Then first, this mixture of the solution
was stirred for 10 minutes for each sample. After this process by using separatory funnel,
caffeine extracted with DCM from the solution and coffee with water remains as a residue. This
is due to the density difference between the two solvents and caffeine is more soluble in DCM,
the extracted caffeine with DCM easily separated from coffee with water solution. The extraction
of caffeine repeated for three times with 25 ml dichloromethane at each round. The caffeine
extracted by dichloromethane at each round was stored in one volumetric flask. Finally, the
absorbance of caffeine was measured by UV/Vis-absorption spectroscopy in the wavelength
19
range of 200nm – 500nm against the corresponding blank of DCM. The total content of caffeine
extracted from coffee samples was stored in the same container. After extraction, there is still
interfering substances that eliminated by Gaussian function based on non-linear curve fitting.
Coffee samples with their maximum absorbance at peak wavelength of 276nm represented in
appendix C.
3.1.7. Data collection
The data for the six standard caffeine concentrations in DCM and distilled water with their
measured maximum absorbance at peak wavelength of each solvent and the data for nine roasted
coffee samples concentration & their measured maximum absorbance at peak wavelength of
276nm collected and listed at the Appendices.
3.2. Methods
3.2.1. Beer Lambert’s Method of measuring caffeine
Beer Lambert’s Law (BLL) based on peak absorbance was the most popular method to
investigate the concentration of the targeted molecule in a sample. Recently, the caffeine
concentration of coffee beans had been reported using UV-Vis absorption spectroscopy with
BLL by extracting caffeine from coffee solution with solvent DCM. In this method, there is
change in the intensity of radiation passing through the caffeine molecule & the change in
intensity of radiation depends on the concentration of the absorbing material. The results
obtained using BLL are satisfactory, cost-effective, timely and reproducible at room temperature
for particular wavelength.
However, in a UV/Vis- absorption spectroscopy, the absorption of molecules in a liquid occurs
over a certain range of frequencies rather than at a single frequency. Therefore, integrated
absorption coefficient measured at any single frequency may not express the true intensity of the
molecular transition in BLL. In addition, when there is no high-resolution spectroscopy since a
finite slit width is used, the radiation is not monochromatic under these conditions; thus, the band
determined experimentally comprises no true physical constants of the absorbing molecule but
depends on the instrumental conditions employed. Due to these limitations of BLL, we can use
another technique.
3.2.2. Integrated Absorption technique (IAT) of Measuring Caffeine
Measuring the intensity of absorption by the IAT provides additional information about the
nature of the absorbing molecules and establishes accurate evaluation of UV-Vis absorption band
intensity. Recently, an IAT becomes alternative methods instead of BBL to determine the
concentration of caffeine in coffee beans optical & quantum mechanical transition property and
number density of caffeine in coffee beans. It is independent of the line function, which may
vary by parameters like pressure, temperature, concentration of the solute and solute-solvent
interaction. In liquids and solutions, IAT used to improve the interference matrix and we can
20
deconvolute the overlapped spectra of caffeine to determine the area under peak of Gaussian
function that fitted to each bands of spectrum for caffeine.
Therefore, in this research, IAT proposed to determine optical & quantum mechanical transition
property and number density of caffeine molecules in different standards and roasted coffee
beans. In IAT we calculate the area under the spectrum band for optical & quantum mechanical
transition property and number density of caffeine depending on their variables. after we
obtained the area deconvolution of Gaussian fit of Gaussian function for each spectrum bands to
improve interference matrices related to chloregenic acid in roasted coffee of Ethiopian export
standard coffee beans34, 35, 40.
In this study, we used Origin 2015 software for data analysis of the spectra; for integrated the
absorption coefficient, integrated absorption cross-section and molar decadic absorption
coefficient across the entire absorption band.
3.2.3. Least Square Method
One does not accept the molar decadic absorption coefficient value determined from one known
concentration; rather, several concentrations are prepared and the corresponding absorbance
value plotted against concentrations. In any real experiment there will be a random errors arising
from the limitation of the experiment.
Based on Beer-Lambert’s equation absorbance A = εCl, it is customarily assumed that
concentrations are known (i.e. more accurately than the absorbance value). The deviation of
experimental values from the equation written as
Ai – εCil = ei
(3.8)
the deviations ei, are squared and the sum of them for all the experimental points. It is required
that this sum of the squares of the deviation be minimum. This achieved by setting the derivative
with respect to the adjustable parameter, ε or εl equal to zero.
∂
∂εl
∑ni=1 ei2 =
∂
∂εl
∑i(A2i - 2εlCiAi + εlCi2 ) = 2 ∑i(εlCi2 − Ci2 A2i ) = 0
(3.9)
The least square condition for molar decadic absorption is therefore,
ε=
2 2
∑n
i Ai Ci
(3.10)
2
l ∑n
i Ci
Where A is absorbance, ε is the molar decadic absorption coefficient, l is a distance in the
absorbing medium (the path length) and C is the concentration of the absorbing caffeine
molecule. By this criterion the molar decadic absorption coefficient of caffeine (the ability of
caffeine molecule to absorb light) in DCM and water was calculated.
3.2.4. Non-linear curve fitting Gaussian function
In this research, there were many interfering bands in caffeine spectrum from the other coffee
components extracted by DCM and the peak of these interfering bands observed at the
21
wavelength below 246nm & above 312nm for roasted coffee beans. The compound responsible
for these peaks is related to chlorogenic acid compounds. This interfering band had an effect on
the maximum peak of caffeine. Therefore, the interference matrix was eliminated by fitting the
following equation of Gaussian fit to the experimental data.
−2(
y = y0+ Ae
x−xc 2
)
w
(3.11)
Where y0 represents the minimum point, A is amplitude, xc is the central wavelength and w is full
width at half maxima of the given spectrum. It was fitted by Non-linear curve fitting based on the
Origen 2015 software. The four quantities of Gaussian function (y0, A, xc, & w) served as
searching parameters in order to achieve minimum discrepancy between the experimental data
and Gaussian function. Thus, the peak absorbance for calculating the concentration of caffeine
obtained after subtracting the fitted Gaussian function from the total caffeine spectrum.
22
Chapter 4
4. Results and Discussion
The results and discussion of the thesis are presented. The calculated values of molar decadic
absorption coefficients, the optical and quantum mechanical transition properties (transitional
dipole moment, oscillator strength, integrated absorption cross-section and Einstein B
coefficient) as well as number density of caffeine are reported. In addition, the caffeine
concentration measured in coffee beans both in Beer-Lambert’s law and integrated absorption
coefficients techniques are presented using UV/Vis absorption spectroscopic method.
4.1. Calibration curve of pure caffeine in dichloromethane and in distilled water
The molar decadic absorption coefficient of caffeine in water and dichloromethane obtained by
measuring the intensity of the absorption for a series of caffeine concentration in DCM and
distilled water. Caffeine concentration of (1.84 x 10-2 – 11.04 x 10-2) mol·m-3 and (1.762 x 10-2 –
34.3 x 10-2) mol·m-3 was prepared in DCM and distilled water respectively for calibration curves.
From the analysis of calibrations (Figure 4.1 & 4.2) are good in linear relationships (R =
0.99993, R2 = 0.99783 & S.D = 0.01611 and R = 0.99913, R2 = 0.9998 & S.D = 0.00463) were
observed for variance concentrations of caffeine in distilled water and in DCM respectively for
absorbance versus concentration of caffeine molecule, which is convenient for the determination
of caffeine in coffee beans. From the analysis of calibrations, a linear dataset was obtained.
Where, R = linear regreation coefficient and S.D = standard deviation of the measurement.
Similarly, with peak height measurements, a linear fit with (R = 0.999) was obtained. Therefore,
the methods are valid in terms of sensitivity. Over all method of repeatability was also
determined by calculating the coefficient of variance (C.V) and a value ranged from (0.99 - 5.56)
% were obtained. These results suggested that the proposed method is valid in terms of precision.
The molar decadic absorption coefficient for caffeine molecule in distilled water at a maximum
peak wavelength (λ max = 273nm) was calculated from appendix B by using equation (3.10)
ε=
A1 C1 +A2 C2 +A3 C3 +A4 C4 +A5 C5 +A6 C6
(4.1)
l(C21 +C22 +C23 +C24 +C25 +C26 )
ε = 376.06 m2 mol-1
(4.2)
It is molar decadic absorption coefficient of caffeine in distilled water.
Similarly the molar decadic absorption coefficient measuring the intensity of optical absorption
for caffeine in DCM at a maximum wavelength of (λ max = 276nm) can be calculated from
appendix A as
ε=
A1 C1 +A2 C2 +A3 C3 +A4 C4 +A5 C5 +A6 C6
l(C21 +C22 +C23 +C24 +C25 +C26 )
ε = 1228.035m2 mol-1
(4.3)
23
1.4
1.4
Absorbance
Line fit
2
4
6
8
10
10
1.2
8
1.0
1.0
Absorption(a.u)
Absorbance(a.u)
1.2
0
0.8
0.6
6
0.8
0.6
0.4
0.4
0.2
0.2
4
2
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.0
0.00
0.35
0.02
0.04
0.06
0.08
0.10
0
0.12
Concentration(mol/m3)
Fig 4.2:- Absorbance Vs Concentration of caffeine in DCM
Concentration (mol/m3)
Fig 4.1;-Absorbance Vs concentration of caffeine in distilled water
4.2. Absorption versus concentration relation
To determine the concentration of caffeine in solutions of roasted coffee beans different
concentrations of standard caffeine in DCM & distilled water were prepared and the maximum
absorbance of the standard solutions was recorded to find molar decadic absorption coefficient at
λmax= 276 & 273nm respectively. In this work absorbance versus wavelength graphs of different
concentrations of standard caffeine are normalized for both solvents shown in Figure 4.3(a) and
4.3(b).
1.3
0.0184
0.0328
0.0552
0.0736
0.092
0.1104
1.2
1.1
Absorbance (a.u)
1.0
0.9
0.8
mol
mol
mol
mol
mol
mol
m-3
m-3
m-3
m-3
m-3
m-3
0.7
0.6
0.5
0.4
0.3
0.2
WAvelength (in nm)
0.1
0.0
200
300
400
Fig 4.3(a);- Normalized absorption spectrum of
pure caffeine in DCM
24
500
0.343 mol m-3
0.275 mol m-3
0.206 mol m-3
0.137 mol m-3
0.0687 mol m-3
0.0176 mol m-3
1.2
Absorbance ( a.u)
1.0
0.8
0.6
0.4
0.2
Wavelength (in nm)
0.0
200
300
400
Fig 4.3(b):- Normalized Absorption spectrum of pure
caffeine in distild water
500
4.3. UV-Vis Absorption of Caffeine
Fig 4.3(c) shows absorbance versus wavelength of caffeine in DCM has a maximum absorbance
at 276 nm. The intensity of caffeine in DCM drops to zero for wavelengths greater than 312 nm
and rise below this wavelength, on the other hand it has a shoulder at 246nm and a new peak
absorption is noticed at a wavelength below 246nm. This new spectrum is due to the solvent
interference. The maximum peak absorbance of caffeine observed in DCM at wavelength 276nm
is 1.30409 for the concentration of C = 1.1 × 10-1 mol m-3. The molar decadic absorption
coefficient for the intensity of optical absorption at λ max= 276 nm was calculated using Equation
(4.3). This molar decadic absorption coefficient of caffeine in DCM is ε max =1228.035 m2 mol-1.
Similarly, Fig 4.5 shows caffeine has peak at 273nm in distilled water. The intensity of caffeine
in distilled water drop to zero for wavelength greater than 302nm and start to rise below this
wavelength, more over a new peak absorbance was noticed at a wavelength below 245 nm. This
new spectrum was due to the solvent interference. The maximum peak absorbance of caffeine
observed in distilled water at wavelength 273nm is 1.2907 for concentration of C = 3.43 x 10 1
mol m-3. The molar decadic absorption coefficient for the intensity of optical absorption at λ
max=273 nm was calculated by Equation (4.2). This molar decadic absorption coefficient value of
caffeine in distilled water is ε max = 376.06 m2 mol-1. From these discussions, molar decadic
absorption coefficient of caffeine in DCM is greater than that of in distilled water.
The wavelength of caffeine in DCM and distilled water found in this work matches with
Atomssa and Gholap et al 2011.
25
4.3.1. Integrated absorption coefficient
The total intensity of the band is obtained by measuring ε(ν) in the region of absorption for
caffeine molecules due to transition of electromagnetic radiation in the spectrum band and
usually determined by integrating the area under the graph of molar decadic absorption
coefficient over wave number versus wave number. It is also an important to express transition
dipole moment of caffeine molecule. So the integrated absorption coefficient due to transition
can be express as eq (2.6)30, 31, 33
The integrated absorption coefficient I A is calculated from the spectra of caffeine standard
dissolved in DCM [Fig 4.4] by using the Origin 2015 software and the result is I A = 132.37227
m2 mole-1 . It was obtained by integrating the molar dicadic absorption coefficient over wave
number versus wave number in the wave number range of (2,800,000 – 4,100,000) m-1 for the
concentration
C = 1.1 × 10-1mol m-3.
Similarly, the integrated absorption coefficient of caffeine in distilled water [fig 4.6] is I A =
43.93985m2 mole-1 in wave number range of (2,800,000 – 4,100,000) m-1 for the concentration of
C = 3.43 x 10-1mol m-3.
4.3.2. Transition dipole moment (µ21) of caffeine
The transition dipole moment is a vector that depends on both ground state (1) and excited state
(2) of wave function and couples the transition to the electric field of light. The transition dipole
moment of caffeine, which related to the molar decadic absorption coefficient was calculated by
using the result of the integrated absorption coefficient of caffeine spectra with eq (2.7).
3
µ21 = √ 𝑆 IA
3
µ21 = √2.9352 x 132.37227 X 10−60 m2 C2
µ21 =11.6316222 x 10-30C m
(4.4)
The transitional dipole moment of caffeine in DCM =11.6316222 x 10 -30C m
Similarly, transition dipole moment of caffeine dissolved in water was
µ21= 6.70 x 10-30Cm
(4.5)
26
4.3.3. Einstein B Coefficient
The Einstein B coefficient for standard caffeine in DCM & in distilled water can be expressed
μ2
using the transition dipole moment of caffeine standards using eq (2.9), B = 6𝜀 21ћ2 and these are
0
given in tables (4.1- 4.4).
0
2
4
6
8
10
10
1.2
8
Absorbance(a.u)
1.0
0.8
6
0.6
4
0.4
2
0.2
200
250
300
350
400
450
0
500
Molar decadic abs coefficient per wave number(m3/mol)
Wavelength( in nm)
Fig 4.3(c):- UV/Vis spectrum of Absorbance Vs wavelength for caffeine
in DCM
3.5x10-4
3.0x10-4
2.5x10-4
2.0x10-4
1.5x10-4
1.0x10-4
5.0x10-5
Wavenumber (per meter)
0.0
2.0x106
2.5x106
3.0x106
3.5x106
4.0x106
4.5x106
Fig 4.4:-Molar decadic absorption coefficient over wave number
Vs wavenumber of caffeine in DCM
27
1.2
Absorbance(a.u)
1.0
0.8
0.6
0.4
0.2
Wavelength(in nm)
0.0
200
250
300
350
400
450
500
Molar decadic absorption coefficient over wavenumber
(in m mol-1)
Fig 4.5:- UV/Vis spectrum of Absorbance Vs Wavelength for caffeine in water
1.0x10-4
6.0x10-5
4.0x10-5
2.0x10-5
Wave number (in m-1)
0.0
2.0x106
2.5x106
3.0x106
3.5x106
4.0x106
4.5x106
Fig 4.6:- UV/Vis spectrum of caffeine in distilled water
4.4. Optical and Quantum mechanical Transition Properties of caffeine
molecule in Beer Lambert’s Law (BLL)
When an electromagnetic radiation is incident on caffeine molecules, there are transitions of
molecules from lower energy state to the higher energy state due to interaction of molecules with
incident radiation. From UV-visible absorption spectra, by using BLL we can study the optical &
quantum mechanical transition properties (integrated absorption coefficient, integrated
28
(3.64964E6,1.02765E-4)
8.0x10-5
absorption cross-section, oscillator strength) & number density of caffeine calculated in DCM
and distilled water to compare the strength of transition.
4.4.1. Integrated absorption coefficient ( 𝑎𝑡 )
In this work the integrated absorption coefficient of molecules in a liquid using UV/Visabsorbance spectroscopy was expressed as absorption coefficient over path length 33, 34.
𝑎𝑡 =
1
𝑙
𝐼
(4.6)
∫ log ( 𝐼0 )𝑑ṽ = ∫ 𝑎ṽ 𝑑ṽ
𝑡
These results were calculated from the spectrum band of absorption coefficient versus wave
number of caffeine standard in DCM and distilled water [Fig 4.3 & 4.5] & presented in Table 4.1
& 4.2.
4.4.2. Integrated absorption cross-section (δt)
The integrated absorption cross-section δt related to the integrated absorption coefficient at at a
single frequency of number density N of caffeine molecule is given by 33, 34.
δt =
𝑎𝑡
(4.7)
𝑁
4.4.3. Oscillator Strength (f)
Oscillator strength related to molar decadic absorption coefficient can described as 34, 35:
f = 4.32 × 10−10mol m-1 ∫ 𝜀(ṽ)𝑑ṽ
(4.8)
The oscillator strength of caffeine in DCM using equation (4.13) was obtained by integrating the
spectra of molar decadic absorption coefficient versus wave number at concentration C = 1.1 x
10-1 mol m-3 of Fig 4.7 from 3,200,000 m-1 – 4,100,000 m-1 is
∫ 𝜀(ṽ)𝑑ṽ = 4.97371x108m mol-1
(4.9)
f = 4.32 x 10-10 mol m-1 x (4.97371 x 108 m mol-1)
f = 0.214864272 is the oscillator strength of caffeine in DCM.
(4.10)
Similarly, by using the spectra of Fig 4.8 the oscillator strength for caffeine in distilled water at
concentration C = 3.43 x10 -1 mol m-3 from wavelength range of 3,200,000m-1– 4,100,000m-1was
0.06996672.
29
4.4.4. Number density of caffeine molecule (N)
The number density of caffeine molecule dissolved in DCM & in distilled water calculated by
using the integrated absorption coefficient and the oscillator strength of each bands of spectrum.
∫ 𝑎 𝑑ṽ
It is given by N = 𝑓 2.65 𝑥ṽ 10−6
(4.11)
The number density & oscillator strength of caffeine in DCM and in distilled water given in
Table 4.1 & 4.2 respectively.
1x103
1x103
Molar decadic absorptioncoefficient
(in mol m-2)
9x102
8x102
7x102
6x102
5x102
4x102
3x102
2x102
1x102
Wave number
0
2x106
3x106
4x106
(in m-1)
5x106
-2
Molar decadic absorption coefficient (in mol m )
Fig 4.7:- UV/Vis spectra of standaed caffeine solution in DCM
350
300
250
200
150
100
50
Wavenumber (in m-1)
0
2x106
3x106
4x106
5x106
Fig 4.8;- UV/Vis spectra of caffeine solution in distilled water
30
The value of the above optical & quantum mechanical transition properties, number density,
Einstein B coefficient and transition dipole moments of caffeine dissolved in DCM & distilled
water by BLL expressed below in table 4.1 & 4.2 respectively.
Table 4.1:- Optical & quantum mechanical transition properties, number density, Einstein B
coefficient and transition dipole moments of caffeine dissolved in DCM by BLL for
corresponding concentration & absorbance.
Concentra Absorban
tion (C in ce
(A
mol/m-3)
in
arbitrary
unit)
1.84 x10-2
0.28
-2
3.28 x10
0.48
5.52 x10-2
0.71
7.36 x10-2
0.89
9.20 x10-2
1.09
1.10 x10-1
1.30
Integrat
ed area
(IA
in
m2/mol)
183.37
173.94
158.16
110.64
117.12
132.37
Transiti
ondipole
moment
(μ21in1030
cm )
13.69
13.33
10.71
10.63
10.94
11.63
Einstein
Bcoefficie
nt
(in S-1 )
Integrated
absorption
coefficient
(at in m-1 )
3.17 x1080
3.01 x1080
1.94 x1080
1.91 x1080
2.03 x1080
2.29 x1080
1.23x107
2.11x107
3.22x107
3.01x107
4.06x107
5.49x107
Number
density of
caffeine
(N in m-3 )
Absorptio
n crosssection (δt
in m2)
1.61 x1013
2.87 x1013
4.82 x1013
6.43 x1013
8.04 x1013
9.65 x1013
0.766x10-6
0.737x10-6
0.668x10-6
0.470x10-6
0.506x10-6
0.570x10-6
Integrated
value of
∫ 𝜀(ṽ)𝑑ṽ
(in m/mol
)
6.71 x108
6.44 x108
5.83 x108
4.09 x108
4.41 x108
4.97 x108
Oscillat
or
strength
(f)
0.290
0.278
0.252
0.177
0.191
0.215
Table 4.2:- Optical & quantum mechanical transition properties, number density, Einstein B
coefficient and transition dipole moments of caffeine dissolved in distilled water by BLL for
corresponding concentration & absorbance.
Concentra
tion (C in
mol/m-3)
1.76 x10-2
6.87 x10-2
1.37 x10-1
2.06 x10-1
2.75 x10-1
3.43 x10-1
Absorba
nce (A
in
arbitrary
unit)
0.08
0.27
0.51
0.77
1.02
1.29
Integrat
ed area
(IA in
m2/mol)
49.61
45.65
44.75
43.87
43.43
43.94
Transitio
n dipole
moment
(μ21 in
10-30cm )
7.12
6.83
6.76
6.70
6.66
6.70
Einstein
B
coefficient
(in S-1 )
Integrated
absorption
coefficient
(at in m-1 )
8.58 x1079
7.89 x1079
7.73 x1079
7.59 x1079
7.51 x1079
7.59 x1079
3.21x106
1.16x107
2.27x107
3.34x107
4.41x107
5.56x107
Number
density of
caffeine
(N in m-3 )
Absorption
crosssection (δt
in m2)
Integrated
value
of
𝜀(ṽ)𝑑ṽ
∫
(in m/mol )
1.54 x1013 0.208 x10-7
6.00 x1013 0.193x10-6
11.96x1013 0.190 x10-6
17.98x1013 0.186 x10-6
24.07x1013 0.183 x10-6
29.95x1013 0.186 x10-6
1.82x108
1.69x108
1.66x108
1.62x108
1.60x108
1.62x108
4.5. Optical and quantum mechanical Transition Properties of Caffeine
by integrated absorption technique (IAT)
From UV-visible absorption spectroscopy by using IAT, we can calculate the optical & quantum
mechanical transition properties (integrated absorption coefficient, integrated absorption crosssection, oscillator strength) & number density of caffeine in DCM and distilled water after
deconvolution of each spectrum bands of Gaussian fit of Gaussian function to improve matrix
related to chloregenic acid. Since in IAT all calculations performed on the calculated area of the
31
Oscilla
tor
strengt
h (f)
0.079
0.073
0.072
0.070
0.069
0.070
spectrum band after Gaussian fit of each bands & IAT is independent of the line function, which
may vary by parameters like pressure, temperature, concentration of the solute & solute-solvent
interaction. These make integrated absorption technique different from Beer Lambert’s
technique. Then we can calculate the optical & quantum mechanical transition properties with
the same procedure as we calculated in BLL, but after deconvolution of the spectrum by
Gaussian fit of Gaussian function.
For deconvoluted bands of spectrum after Gaussian fit for caffeine molecules dissolved in DCM
and distilled water, integrated absorption coefficient [Fig 4.9(b) & 4.10(b)] in the wave number
regions 2,500,00m-1 to 4,100,00m-1, integrated absorption cross-section, oscillator strength [Fig
4.11 (a & b)] in the region of 3,200,000 m-1 – 4,100,000 m-1 and the number densities are given
in table 4.3 & 4.4.
Absorption coefficient (m-1)
120
Spectra of caffeine
Gaussian fit
100
80
60
40
Wavenumber (m-1)
20
0
2x106
3x106
4x106
5x106
Fig 4.9(a);- Gaussian fit to the spectra of absorption coefficient Vs
wavenumber of caffeine in DCM
As the standard concentration of caffeine in DCM & distilled water increases the absorbance,
integrated absorption coefficient and number density are increased. On the other hand as
concentration of caffeine in DCM & distilled water increases the transition dipole moment,
Einstein B coefficient, integrated absorption cross-sections and oscillator strengths are decreased
in both solvents of the two methods.
32
Absorption coefficient (m-1)
125
(3.6814E6,128.07114)
100
75
50
25
Wave number (m-1)
0
6
6
2x10
4x106
3x10
5x106
Fig 4.9(b):- Absorption coefficient versus wave number of caffeine in
DCM after Gaussian fit
Caffeine spectra
Gaussian fit
Absorption coefficient (m-1)
55
50
45
40
35
30
25
20
15
10
5
0
2x106
Wave numbe (m-1)
3x106
4x106
5x106
Fig 4.10(a):-the Gaussian function fited to the spectrum band of Absorption
coefficient versus Wave number of caffeine in distilled water
33
absorption coefficient (m-1)
55
50
45
40
35
30
25
20
15
10
5
0
2x106
(
Wave number m-1
3x106
Molar decadic absorption coefficint (mol m -3
Fig 4.10(b):- After Gaussian fit of the spectrum
for caffein in distilled water
4x106
)
5x106
400
350
300
250
200
150
100
50
0
2000000
Wave number (m-1)
3000000
4000000
5000000
Fig 4.11(a):-UV/Vis Spectrum for caffeine in distilled water after Gaussian fitt
34
(3.67787E6,1032.45058)
Molar decadic absorption coefficient (mol m-3)
1000
500
Wave number (m-1)
0
2.5x106
3.0x106
3.5x106
4.0x106
Fig 4.11(b):-UV/Vis spectrum for Caffeine in DCM after Gaussian fitt
Table 4.3:- Optical & quantum mechanical transition properties, number density, Einstein B
coefficient and transition dipole moments of caffeine dissolved in DCM by IAT for
corresponding concentration & absorbance.
Concentra Absorba
tion (C in nce (A
mol/m2)
in
arbitrary
unit)
-2
1.84 x10
0.28
3.28 x10-2
0.48
5.52 x10-2
0.71
-2
7.36 x10
0.89
9.20 x10-2
1.09
1.10 x10-1
1.30
Integrat
ed area
(IA in
m2/mol)
183.37
173.94
158.16
110.64
117.12
132.37
Transition
dipole
moment
(μ21 in 1030
C m)
13.69
13.33
10.71
10.63
10.94
11.63
Einstein
B
coefficient
(in S-1)
Integrated
absorption
coefficient
(at in m-2 )
3.17 x1080
3.01 x1080
1.94 x1080
1.91 x1080
2.03 x1080
2.29 x1080
1.24 x107
2.12 x107
3.29 x107
3.03 x107
4.03 x107
5.47 x107
Number
density
(N in m-3 )
1.59 x 1013
2.84 x 1013
4.94 x 1013
6.55 x 1013
7.98 x 1013
9.61 x 1013
Integrated
absorption
crosssection (δt
in m1)
0.77 x 10-6
0.74 x 10-6
0.66 x 10-6
0.45 x 10-6
0.50 x 10-6
0.56 x 10-6
Integrate
d value
of
∫ 𝜀(ṽ)𝑑ṽ
(in /mol )
6.81x108
6.51 x108
5.82 x108
4.04 x108
4.41 x108
4.97 x108
Oscilla
torstrength
(f)(unit
less )
0.294
0.281
0.251
0.175
0.191
0.215
Table 4.4:- Optical & quantum mechanical transition properties, number density, Einstein B
coefficient and transition dipole moments of caffeine dissolved in distilled water by IAT for
corresponding concentration & absorbance.
Concentra Absorb
tion (C in ance
mol/m2)
(A in
arbitrar
y unit)
1.76 x10-2
0.08
6.87 x10-2
0.27
1.37 x10-1
0.51
-1
2.06 x10
0.77
2.75 x10-1
1.02
3.43 x10-1
1.29
Integrat
ed area
(IA in
m2/mol)
49.61
45.65
44.75
43.87
43.43
43.94
Transitio
n dipole
moment
(μ21 in
10-30C m)
7.12
6.83
6.76
6.70
6.66
6.70
Einstein
B
coefficient
(in S-1 )
Integrated
absorption
coefficient
(at in m-2 )
Number
density (N
in m-3)
8.58 x1079
7.89 x1079
7.73 x1079
7.59 x1079
7.51 x1079
7.59 x1079
2.68x106
1.09x107
2.18x107
3.25x107
4.32x107
5.45x107
1.46 x 1013
5.88 x 1013
11.80 x 1013
17.78 x 1013
23.72 x 1013
29.63 X 1013
35
Integrated
absorption
crosssection (δt
in m1)
0.184x10-6
0.185 x10-6
0.185 x10-6
0.183 x10-6
0.182 x10-6
0.184 x10-6
Integrated
value
of
𝜀(ṽ)𝑑ṽ
∫
( in m/mol )
Oscillato
r strength
(f)
1.60x108
1.62x108
1.61x108
1.59x108
1.59x108
1.61x108
0.069
0.069
0.069
0.069
0.069
0.069
4.6. Number density of caffeine in medium roasted coffee beans
The number densities of caffeine in coffee beans were calculated by fitting the Gaussian function
to the spectra of absorption coefficient versus wave number of caffeine Fig 4.12(a & b) and to
the spectra of molar decadic absorption coefficient versus wave number of caffeine extracted
from coffee solutions Fig 4.14. From the area of Gaussian function fitted to the spectra in Fig
4.12(a & b) and Fig 4.14, the number density of caffeine was calculated in the frequency region
of 2,500,000-4,100,000m−1 using eq (2.19). The Number density of caffeine molecule extracted
from roasted coffee beans based on BLL and IAT given in table 4.5 below.
4.7. Determination of Caffeine concentration in Roasted Coffee Beans by Beer
Lambert’s Law (BLL) and integrated absorption technique (IAT)
In this work by UV/Vis absorption spectroscopy a direct measurement of roasted Ethiopian
export standard coffee samples were impossible owing to the matrix effect of UV/Vis absorbing.
This effect is clearly seen in (Fig 4.13) for spectrum band of roasted coffee beans dissolved in
distilled water. To resolve the interfering substances of this work the extraction of caffeine from
roasted coffee solution performed for three rounds with equal amount 25ml of DCM solutions.
However, the extraction technique still could not completely remove the possible interference of
caffeine spectra and it is impossible to obtain the qualitative and quantitative information from
unresolved band of spectrum. The peak of this interference bands observed below wavelength of
246 nm and above 312 nm as shown in Fig 4.12(a) for caffeine extracted from roasted coffee
beans. These peaks are due to the interference of solvent effect and chlorogenic acid.
To eliminate this interference Gaussian function given in Equation (3.11) was fitted to the
spectrum of caffeine using non-linear curve fitting by origin 2015 software. Fig 4.12(a) shows
the spectrum of caffeine extracted from roasted coffee beans by DCM and Gaussian function
fitted to this spectrum for Absorbance versus wavelength overlap together and to eliminate
another peaks due to interference we deconvoluted the spectrum as it shown in fig 4.12(b). After
the inferences eliminated, the shape and peak of the two spectra are the same. Therefore, it can
concluded that the applications of experimental and computational methods enable to determine
the concentration of caffeine in roasted coffee beans using UV-Vis absorption spectroscopy of
this study. By this method, the concentrations of caffeine in roasted Ethiopian export standard
coffee bean samples were determined. The mean percentage of caffeine concentration in roasted
coffee beans calculated for three independent measurements by Beer Lambert’s Law &
Integrated absorption techniques are given in Table (4.5).
36
Absorption coefficient (a.u)
1.00E-007
8.00E-008
6.00E-008
4.00E-008
wave number (m-1)
2.00E-008
2x106
3x106
4x106
5x106
Fig 4.12(a);- UV/Vis spectrum of caffeine extracted by DCM from roasted
coffee before Gaussian fitt
Absorption coefficient (a.u)
1.0x10-7
Gaussian fit
8.0x10-8
6.0x10-8
4.0x10-8
wave number (m-1)
2x106
3x106
4x106
5x106
Fig 4.12(a);- UV/Vis spectrum of caffeine extracted by DCM from roasted
coffee after Gaussian fitt
37
0.40
Absorbance (a.u)
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
200
Wavelength
250
300
350
400
( in m-1)
450
500
Molar decadic absorption cofficient (mol m-3)
Fig 4.13:- UV/Vis spectra of medium roasted coffee disolved in
distilled water
Gaussian fit
1500
1000
500
Wave number (m-1)
0
2x106
3x106
4x106
5x106
Fig 4.14:- UV/Vis Spectra of caffeine extracted from coffee Beans
38
Table 4.5:- Number density and Weight by weight (w/w) concentration of Caffeine measured in
Ethiopian export standard Roasted Coffee Beans by UV-Vis Spectroscopy in the BLL & IAT.
Samples
Bebeka
Harer
DJimmah
Limmu
Nekempti
Sidama unwashed
Sidamw washed
Teppi
Yirgacheffe
Maximum
Absorbance
(a.u)
1.04
0.69
0.90
0.60
0.78
0.77
1.12
1.12
0.90
Number density of caffeine in Percentage of Caffeine
roasted coffee (in 1013m-1)
roasted coffee (w/w %)
By BLL
By IAT
By BLL
2.40
8.001
8.346
1.58
5.160
6.470
2.01
7.290
7.213
1.35
4.236
4.564
1.76
6.010
6.808
1.76
12.417
15.375
2.51
3.666
3.735
2.51
7.580
7.241
2.03
6.538
7.032
concentration in
By IAT
2.38
1.51
1.96
1.34
1.71
1.72
2.48
2.50
1.98
The other basic parameters describing any individual absorption band is a bandwidth at halfmaximal intensity, defined as follows (Antonov and Nedltcheva, 2000)
ΔѴ1/2 = Ѵ1 – Ѵ2, where Ѵ1, Ѵ2 are initial and final frequencies at half of intensity maximum
In electronic spectroscopy especially in organic molecules, the transition observed in UV-visible
region is π* ← π. Thus, for pure caffeine the electronic type transition is π*← π and this
transition is the cause for absorption. It measures the anti-bonding character of the excited state.
The anti-bonding character is a function of inter nuclear distance and in some cases the change of
the bandwidth can indicate the change of inter nuclear distance. In this research, the bandwidth at
half maximum (FWHM) of caffeine calculated in DCM and roasted coffee beans are presented in
Table 4.6 from the graph of absorption coefficient versus wave number. Here we observed that
the bandwidth at FWHM of pure caffeine in DCM decreases as the concentration increases.
Table 4.6:- the bandwidths of pure caffeine in DCM and caffeine extracted from roasted coffee.
Pure
caffeine Bandwidth at (Fwhm) Samples of coffee
concentration in of pure caffeine in
DCM (mol m-3)
DCM (m-2)
1.84 
3.28 
5.52 
7.36 
9.20 
11.0 
10-2
10-2
10-2
10-2
10-2
10-2
1.09 
0.81 
0.90 
0.42 
0.41 
0.40 
106
106
106
106
106
106
Bebeka
Harer
DJimmah
Limmu
Nekempti
Sidama unwashed
Sidamw washed
Teppi
Yirgacheffe
39
Bandwidth at (Fwhm) of
caffeine extracted from
coffee
1.20
2.28
1.59
0.95
2.52
2.49
1.03
0.41
0.44









106
106
106
106
106
106
106
106
106
The percentage for caffeine concentration of Ethiopian export standard roasted coffee bean
sample varies from 1.35% -2.51 % in Beer Lambert’s Law and from 1.34% - 2.50% in integrated
absorption techniques. Our results obtained by using integrated absorption technique and BeerLambert’s laws had a good agreement. Of all sample tested Tepi and Sidama (washed) coffee
samples had the highest percentage of caffeine concentration, but Limu coffee sample had the
least percentage of caffeine concentration. From these samples based on Ethiopian coffee export
standard and liquoring board standardization Tepi and Limu are in the same category, which
were in washed & Harer was in another category, which was in unwashed. Sidama coffee that
was of the same origin with different category had different concentration of caffeine. Sidama
unwashed had caffeine concentration of 1.76% in BLL & 1.72% in IAT and sidama washed had
caffeine concentration of 2.51% in BLL & 2.48% in IAT.
Furthermore, the caffeine concentration varies from 1.34% to 2.51% of medium roasted at 2150C
in fifteen minute of Ethiopian export standard Arabica coffee samples determined by this work
were in a good agreement with the caffeine concentration reported by various analytical
techniques in literatures. The amount of caffeine concentration by UV-Vis spectrophotometer in
medium roasted Vietnam Robusta coffee bean reported to be 3.591% 43. By using UV/Visspectrophotometer & HPLC reported that the amount of caffeine in Arabica coffee was about
3.312%42. Other studies also have reported that caffeine content increased during the roasting
process from 0.96% to 1.26% 40 & from 2.04% to 2.515% and it was higher in the medium
roasting level40. By using UV-Vis spectrophotometer that coffee Arabica in light roasted coffee
concentration is 2.24%, in the medium roasted coffee concentration is 2.47% and in the medium
roasted Cherry coffee concentration is 2.52% reported 42. The caffeine content reported for
Ethiopian Arabica coffee concentration ranges from 0.46% to 2.82% by HPLC43. For caffeine,
content in Ethiopian coffee samples by using UV/Vis spectrophotometer analysis was from
0.90% to 1.27% 22 and by HPLC it was from 1.10% to 2.90% for 68 samples44 which our work is
found in this range.
.
40
5. CONCLUSIONS
In this work we determined optical and quantum mechanical parameters of caffeine for nine
samples of export quality Ethiopian coffee by UV/Vis-absorption spectroscopy. The integrated
absorption technique has advantage that it is independent of line function. The effects of line
broadening and shift in peak intensity due to, temperature and pressure variation, solute-solvent
interaction and a finite slit width of UV-Vis spectroscopy do not affect the intensity of the
absorbing molecules. Therefore, the technique is best for evaluating the UV-Vis absorption
intensity; moreover, it is also sensitive, precise, and accurate for determining caffeine in coffee
beans. In addition, the optical transition & quantum mechanical properties (oscillator strength,
dipole moment, integrated absorption cross-section and peak absorption cross-section) of pure
caffeine analyzed and the results agree with other workers and other analytical methods. The
optical transition & quantum mechanical properties calculated in dichloromethane & distilled
water are the intrinsic ability of caffeine molecules to absorb light and they are proportional to
the intensity of transition. UV-Vis spectroscopy method applied successfully for the
determination of caffeine concentration in medium roasted coffee beans extracted by
dichloromethane from nine washed and unwashed samples of Ethiopian export standard coffee.
In thesis, a significant variation in the concentration of caffeine was observed depending on the
geographical origin and category of the coffee beans of Ethiopia. In case of category among the
fife washed coffee samples Tepi & sidama coffee beans had high caffeine concentration and
Limu coffee bean had the list caffeine concentration, but among four unwashed coffee samples,
DJimmah coffee had the highest caffeine concentration & Harer coffee had the list caffeine
concentration. On the other hand, in the case of geographical origin of coffee varieties Tepi’s
coffee had the highest concentration of caffeine & Limu’s coffee had the list concentration of
caffeine. Both Tepi & Limu coffee samples that have highest and lowest concentration of
caffeine respectively found in washed coffee brands.
Generally the average values were taken for highest and lowest values of caffeine concentration
for medium roasted Ethiopian export standard coffee samples percentage (% w/w) are 2.51% in
BLL & 2.50% in IAT for Tepi and 1.35% in BLL & 1.34% in IAT for Limu. These of caffeine
concentration results of Ethiopian export standard coffee are in a good agreement with
literatures.
In this thesis we had different concentration of caffeine from the same origin but from different
category of coffee sample that is 2.50% for washed Sidama and 1.72% for unwashed Sidama.
This difference is because of the category difference of coffee that originated from the same
place. Thus, we recommended that there should be further research investigation to be done
based on category difference using UV/Vis absorption spectroscopy.
41
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44
APPENDICES
Appendix A: Raw data for absorbance measurement of standard caffeine in DCM
Working solution(V in ml)
1
2
3
4
5
6
Volume of solution in
dichloromethane(V1 in ml)
25
25
25
25
25
25
Concentration(C1 mol m-3)
1.84 x 10-2 3.28 x 10-2 5.52 x 10-2 7.36 x 10-2 9.2 x 10-2
1.104 x 10-1
Absorbance (A in a.u) at
276nm
0.28264
1.30409
0.48467
0.70854
0.89721
1.09016
Table (3.1):- The Concentration and peak absorbance at 276nm wavelength for working
solutions of standard caffeine in DCM
Appendix B: Raw data for absorbance measurement of standard caffeine in water
Working solution(V in ml)
1
4
8
12
16
20
30
30
30
30
Volume of solution
distilled water (V1 ml)
in 30
30
Concentration
solution(C1 in mol m-3)
of 1.762 x 10-2
6.87 x 10-2 13.73 x 10-2
2.06 x 10-1 2.746 x 10-1
3.43 x 10-1
0.26543
0.77233
1.29181
Absorbance (A in a.u) 0.07761
at 273nm
0.51115
1.02628
Table (3.2):-The Concentration and peak absorbance at 273nm wavelength for working solutions
of standard caffeine in distilled water
Appendix C: Raw data for absorbance measurement of caffeine extracted from coffee by
DCM
Samples
Bebeka
Concentration
In mol m-3
Absorbance
Harer
Sidamw(
washed)
1.12464
Teppi
1.04348 0.69139 0.89881
Nekempti Sidama(u
nwashed)
0.60313 0.77841
0.7749
1.11749
Yirgacheffe
0.89996
0.8497
0.04911 0.06339
0.9158
0.091
0.07328
0.0563
DJimma Limmu
0.07319
0.0631
Table 3.3:- Coffee samples with its concentration and maximum absorbance at 276nm
wavelength.
45
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