coffee roasting thesis.pdf
TRANSCRIPT
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Physicochemical Changes of Coffee Beans During Roasting
by
Niya Wang
A Thesis
Presented to
The University of Guelph
In partial fulfilment of requirements
for the degree of
Master of Sciencein
Food Science
Guelph, Ontario, Canada
© Niya Wang, April, 2012
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ABSTRACT
PHYSICOCHEMICAL CHANGES OF COFFEE BEANS DURING
ROASTING
Niya Wang Advisor:
University of Guelph, 2012 Professor Loong-Tak Lim
In this research, physicochemical changes that took place during roast
processing of coffee beans using fluidized air roaster were studied. The results
showed that high-temperature-short-time resulted in higher moisture content,
higher pH value, higher titratable acidity, higher porous structure in the bean cell
tissues, and also produced more aldehydes, ketones, aliphatic acids, aromatic
acids, and caffeine than those processed at low-temperature-long-time process.
Fourier transform infrared (FTIR) spectroscopy and chemometric analysis
showed that clusters for principal components score plots of ground coffee,
extracted by a mixture of equal volume of ethyl acetate and water, were well
separated. The research indicated that variations in IR-active components in the
coffee extracts due to different stages of roast, roasting profiles, and
geographical origins can be evaluated by the FTIR technique.
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ACKNOWLEDGMENTS
I am most grateful to Prof. Dr. Loong-Tak Lim for giving me the opportunity
to work in his group. I have always appreciated his far-sighted guidance,
continued support, and constructive evaluation throughout my research and in
many aspects of my life. Further, I am much indebted to my advisory committee
members Dr. Lisa Duizer, and Dr. Massimo Marcone for their unlimited
confidence on my research work and helps during the writing of the thesis.
Special thanks to Natural Sciences and Engineering Research Council of
Canada (NESRC) and Mother Parkers Tea & Coffee Inc., for their essentialfinancial support, without which this research will not be possible. Many thanks to
my Packaging and Biomaterials Group sisters and brothers: Ana Cristina Vega
Lugo, Solmaz Alborzi, Suramya Minhindukulasuriya, Roc Chan, Grace Wong,
Alex Jensen, Khalid Moomand, Qian Xiao, Xiuju Wang, and Ruyan Dai for their
assistance, friendship, patience, and bringing colourful life for these years. Many
thanks are also going to Dr. Yukio Kakuda, Dr. Sandy Smith, and Bruce Manion
for their technical assistance along the way.
I would like to take this opportunity to express my deepest gratefulness to
my parents, my husband Dr. Yucheng Fu, my son Stanley Fu, and other family
members for their infinite love, support and encouragement throughout these
years of my studies at Guelph.
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TABLE OF CONTENTS
ACKNOWLEDGMENTS.……………………………………………………… .….......iii
TABLE OF CONTENTS.………………………………………………………… ...…..iv
LIST OF FIGURES.……………………………………………………………… ...…..vi
LIST OF TABLES.…..………………………..…………………………………...…..viii
LIST OF ABBREVIATIONS.………………………………………………… .…...…..ix
1 INTRODUCTION ............................................................................................... 1
2 LITERATURE REVIEW ..................................................................................... 4
2.1 THE GREEN COFFEE BEANS ............................................................................. 4
2.2 ROASTING OF COFFEE BEANS .......................................................................... 8
2.3 AROMA COMPOUNDS IN ROASTED COFFEE ...................................................... 14
2.4 FOURIER TRANSFORM INFRARED (FTIR) SPECTROSCOPY ................................ 19
2.5 CHEMOMETRICS ........................................................................................... 21
3 JUSTIFICATION AND OBJECTIVES .............................................................. 26
4 FEASIBILITY STUDY ON CHEMOMETRIC DISCRIMINATION OF ROASTED
ARABICA COFFEES BY SOLVENT EXTRACTION AND FOURIERTRANSFORM INFRARED SPECTROSCOPY ................................................... 27
4.1 INTRODUCTION ............................................................................................. 27
4.2 M ATERIALS AND METHODS ............................................................................ 29
4.2.1 Chemicals ............................................................................................ 29
4.2.2 Coffee Beans and Roasting Conditions ............................................... 29
4.2.3 Degree of Roast as Determined by Color Measurements ................... 30
4.2.4 Solvent Extraction of Ground Coffee ................................................... 30 4.2.5 ATR-FTIR Analysis .............................................................................. 31
4.2.6 Data Analysis ...................................................................................... 32
4.3 RESULTS AND DISCUSSIONS .......................................................................... 32
4.3.1 Optimization of Solvent Extraction for FTIR-ATR ................................ 33
4.3.2 Color Analysis ..................................................................................... 38
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4.3.3 PCA Analysis of Solvent Extracts of Coffee Beans ............................. 40
4.3.4 PCA Analysis for Coffees According to Degree of Roast .................... 47
4.3.5 SIMCA Analysis ................................................................................... 52
5 EFFECTS OF DIFFERENT TIME-TEMPERATURE PROFILES ON COFFEEPHYSICAL AND CHEMICAL PROPERTIES ...................................................... 54
5.1 INTRODUCTION ............................................................................................. 54
5.2 M ATERIALS AND METHODS ............................................................................ 57
5.2.1 Chemicals and materials ..................................................................... 57
5.2.2 Green Beans and Roasting Conditions ............................................... 57
5.2.3 Degree of Roast as Determined by Color Measurements ................... 58
5.2.4 Moisture Content of Ground Coffee ..................................................... 58
5.2.5 pH Value .............................................................................................. 59
5.2.6 Titratable Acidity .................................................................................. 59
5.2.7 Solvent Extraction and ATR-FTIR Analysis of Ground Coffee ............. 59
5.2.8 Chemometric Analysis ......................................................................... 60
5.2.9 Scanning Electron Microscopy (SEM) Analysis ................................... 60
5.3 RESULTS AND DISCUSSION ........................................................................... 60
5.3.1 Evolution of physical and chemical properties during roasting ............ 60
5.3.2 Changes in coffee at various stages of roast ....................................... 66
5.3.3 Effects of roast temperature on changes in coffee .............................. 72
5.3.4 Microstructural analysis ....................................................................... 74
6 CONCLUSIONS AND FUTURE WORKS ........................................................ 78
7 REFERENCE ................................................................................................... 82
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LIST OF FIGURES
Figure 1 Chemical composition of green, roasted, and brewed coffee (Barter
2004) ..................................................................................................................... 9
Figure 2 Schematic diagram of a typical FTIR spectrometer ............................. 20
Figure 3 Vibrational absorbance due to common bands .................................... 20
Figure 4 Schematic diagram of PCA analysis .................................................... 24
Figure 5 Air temperature (in roast chamber) profiles of the fluidized bed hot air
coffee roaster ...................................................................................................... 30
Figure 6 Appearance of coffee extracts by dichloromethane, hexane, ethyl
acetate, acetone, ethanol, and acetic acid (the right vial represent the extracts by
Method #1) .......................................................................................................... 35
Figure 7 FTIR spectra of coffee extracts obtained with hexane, dichloromethane,
ethyl acetate, acetone, ethanol, or acetic acid using method #1 (with water) and
method #2 (no water) .......................................................................................... 38
Figure 8 Selected FTIR spectra of dark roast coffee extract obtained with
dichloromethane as a solvent (using method 1#)................................................ 41
Figure 9 PCA of FTIR data for hexane, dichloromethane, ethyl acetate, and
acetone extracts of medium roast coffee. Row A: Two-factor score plots. Row B:
Loading plots of PC1. Row C: Corresponding FTIR raw spectra ........................ 42
Figure 10 PCA of FTIR data for hexane, dichloromethane, ethyl acetate, andacetone extracts of dark roast coffee. Row A: Two-factor score plots. Row B:
Loading plots of PC1. Row C: Corresponding FTIR raw spectra ........................ 43
Figure 11 PCA of FTIR data for dichloromethane extracts of coffee (from the
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same origin) with two degrees of roast. Row A: Two factor score plots. Row B:
Loading plots of PC1. Row C: Corresponding FTIR raw spectra ........................ 50
Figure 12 PCA of FTIR data for ethyl acetate extracts of coffee (from the same
origin) with two degrees of roast. Row A: Two factor score plots. Row B: Loading
plots of PC1. Row C: Corresponding FTIR raw spectra ...................................... 51
Figure 13 Changes in lightness, moisture content, pH value, and titratable acidity
of coffee beans processed to different roast stages (A). The same data are
plotted as a function of actual roast time (B). Roasting occurred isothermally at
210, 220, 230 and 240
o
C .................................................................................... 62
Figure14 PCA analysis for coffees during roasting. Column A: Two-factor score
plots. Column B: Loading plots of PC2. Column C: Representative FTIR spectra
at the start-of-second-crack ................................................................................ 69
Figure15 The expanded 2910-2850 cm-1, and 1800-1500 cm-1 regions of the
spectra of coffee roasted at 230oC ...................................................................... 71
Figure16 PCA analysis for coffees collected at the same sampling point. Row A:
Two-factor score plots. Row B: Loading plots of PC1. Row C: Representative
FTIR spectra at 230oC ........................................................................................ 73
Figure 17 SEM micrographs of internal texture for coffee beans collected at
different stages of roast. The temperatures indicated on each row of micrographs
were the roast temperature ................................................................................. 76
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LIST OF TABLES
Table 1 Chemical composition of green Arabica and Robusta coffee beans
(g/100g) ................................................................................................................ 7
Table 2 Potent odorants in Arabica coffee from Colombia ................................. 16
Table 3 Physical properties of the investigated solvents (Pagni 2005) ............... 34
Table 4 Evaporation time of the coffee extracts.................................................. 36
Table 5 L* Value of Roasted Ground Arabica Coffee Beans .............................. 39
Table 6 Turkey method for L* value comparisons .............................................. 40
Table 7 SIMCA Classification Results for Coffees from Different Geographic
Origins ................................................................................................................ 52
Table 8 SIMCA classification results for coffees according to degree of roast ... 53
Table 9 Time Taken to Achieve Different Stages of Roasting at Four Different
Final Roast Temperatures .................................................................................. 58
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LIST OF ABBREVIATIONS
FTIR Fourier transform infrared
ATR Attenuated total reflectance
PTR-MS Proton transfer reaction-Mass spectrometry
PAS Photoacoustic spectroscopy
PCA Principal component analysis
HCA Hierarchical cluster analysis
PLS Partial least squares
PCR Principal component regression
PLS-DA Partial least squares-discriminant analysis
KNN K-nearest neighbour
SIMCA Soft independent modeling of class analogy
PCs Principal components
HS-SPME Headspace solid phase microextraction
NMR Nuclear magnetic resonanceGC-MS Gas chromatography-mass spectrometry
GC Gas chromatography
L* Lightness
SEM Scanning electron microscope
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1 INTRODUCTION
Coffee is one of the most popular beverages in the world. Nearly 25 million
farmers in 50 countries around the world depend on coffee for a significant part of
their livelihoods (Cague et al. 2009). Coffee is the most traded commodity
second after oil (Ponte 2002). Among coffee drinkers, the average consumption
in the United States is 3.2 cups of coffee per day versus 2.6 cups in Canada
(Canada 2003).
A good quality cup of coffee is depended on many factors, such as the
quality of green beans, the roasting conditions, the time since the beans are
roasted, and the type of water used for brewing. More than 800 volatile
compounds have been identified in roasted coffee, whereof around 30
compounds are responsible for the main impression of coffee aroma
(Baggenstoss et al. 2008).
The overall quality and chemical composition of green coffee beans are
affected by many factors, such as the composition of the soil and its fertilization,
the altitude and weather of the plantation, the cultivation, and the drying methods
used for the beans. Coffee plants are mainly grown in tropical and subtropical
regions of central and South America, Africa and South East Asia, in temperate
and humid climates at altitudes between 600 and 2500 m (Schenker 2000). The
genus coffee belongs to the botanical family of Rubiaceae and comprises more
than 90 different species (Davis 2001). However, only C. arabica, C. canephora,
and C. liberica are of commercial importance (Schenker 2000). As a result of
modem breeding techniques some hybrids of C. arabica and C. canephora have
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recently been introduced with success. Usually roasted coffee beans from
different origins are blended at specific ratios to provide coffee of unique flavour
profiles. Often time, coffee beans are blended for the purpose of cost saving.
Coffee cherries are harvested each year when they are bright-red, glossy,
and firm. After removing the outer hull, the seeds inside of the cherry are
commonly called "green coffee beans". The quality of the green coffee beans is
dictated by a number of parameters, including bean size, color, shape, method of
drying, crop year, and presence of defects (crack, withered bean, bean in
parchment, mouldy bean, etc.).
The unique aroma profiles of coffee are closely related to the time-
temperature profile used during roasting. The roasting profiles are chosen to
produce high quality coffee which are unique to specific brands and must be
strictly controlled to meet consumers’ expectations. Coffee producers rely on
sensory and physicochemical characteristic evaluations to assure that roasting
takes place at the target process parameters. Industrial scale roasting of coffee
beans is mainly achieved by conventional drum roasting, in which beans are
heated with hot gas in a horizontal drum, or vertical drums equipped with paddles.
Roasting time can range from 3 to 12 min, depending on the temperature used,
which is typically between 230 to 250oC. By contrast, fluidized bed roasting is
achieved by directing high velocity hot air towards the beans, usually from the
bottom of the roaster, to suspend the beans in turbulent air. The hot air
temperature ranges from 230 to 360oC (Eggers & Pietsch 2001). The roast
temperature determines both flavour formation and structural product properties.
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Different temperature profiles affect dehydration and the chemical reaction
conditions in the bean which control gas formation, browning and flavour
development. In general, the use of roasting temperature of greater than 200°C
is required in order to result in desirable chemical, physical, structural, and
sensorial changes in the coffee beans (Clarke & Macrae 1988; Schenker 2000;
Schenker et al. 2002; Baggenstoss et al. 2008). Color change and weight loss
are frequently used as a measure of the degree of roast, and both are directly
related to the final roasting temperature (Sivetz 1991; Illy & Viani 1995). Other
methods, such as the ratios of free amino acids (Nehring & Maier 1992), andchlorogenic acids content (Illy & Viani 1995) have also been used.
Researchers have reported the effects of time-temperature profile on coffee
aroma properties. In general, low-temperature-long time roast processes result
in sour, grassy, woody, and underdeveloped flavour properties. In comparison,
high-temperature-short-time produced the higher quality coffee in terms of
producing more aroma volatiles and higher brew yield (Schenker et al. 2002;
Lyman et al. 2003). Reviewing these and other literature, one can conclude that
the complex changes in coffee during roasting do not solely depend on physical
parameters at the start and end point of the thermal process, but rather a path-
dependent phenomenon. Therefore, to gain insight into the changes of
physicochemical properties of coffee during roasting, the green beans must be
roasted under controlled conditions.
The overall objective of this study is to apply a chemometric technique, in
conjunction with Fourier transform infrared spectroscopy, to elucidate the effects
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of time-temperature effects on physical and chemical properties of coffee from
different grown regions during fluidized-bed roasting.
2 LITERATURE REVIEW
2.1 The green coffee beans
The overall quality and chemical composition of green coffee beans are
affected by many factors, such as the composition of the soil and its fertilization,
the altitude and weather of the plantation, and the final cultivation and dryingmethods used. Coffee plants are grown in tropical and subtropical regions of
central and South America, Africa, and South East Asia, mainly in regions with
temperate and humid climates (Schenker 2000). Brazil is by far the largest
grower and exporter of green coffee beans in the world followed by Vietnam,
Colombia, Indonesia, Ethiopia and India – producing nearly 2.5 million tons of
green coffee beans per year (Franca & Oliveira 2009).
The genus coffee belongs to the botanical family of Rubiaceae and
comprises more than 90 different species (Davis 2001). However, only Coffea
Arabica (Arabica), Coffea canephora (Robusta), and Coffea liberica are of
commercial importance (Schenker 2000). Arabica accounts for approximately 64%
while Robusta accounts for about 35% of the world’s production; other species
with not much commercial value like Coffea liberica and Coffea excelsa represent
only 1% (Rubayiza & Meurens 2005). Due to its more pronounced and finer
flavour qualities, Arabica is considered to be of better quality and accordingly
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The aroma profile of roasted ground coffee is also related to the origin and
variety of the beans. In general, blends with greater Arabica content tend to carry
more fruity notes due to the aldehydes, acetaldehyde, and propanal, while the
pyrazines give the earthy odor. In comparison, Robusta beans carry stronger
“roasty” and “sulphury” note due to the presence of greater amount of sulphur-
containing compounds (Sanz et al. 2002). Thus, Arabica is often added for the
aroma effect while Robusta is used for enhancing the body, earthy and phenolic
notes of the coffee blend (Parliment & Stahl 1995). Besides contributing to
balanced flavour profiles, Robusta coffee is often blended with Arabica for costreduction purpose. Robusta beans are lower in cost since the crops are more
hardy to grow (more resistant to infestation) and easier to harvest (grown in
regions of low elevation) than the Arabica counterpart.
Defective beans (black or brown, sour, immature, insect-damaged, split),
which represent about 11-20% of coffee production, can impact the flavour of the
roasted products. Mazzafera compared the chemical composition of defective
beans and non-defective beans. The researcher found that non-defective beans
were heavier, had higher water activity, and lower titratable acidity than the
defective beans. The content of sucrose, protein, 5-caffeoylquinic acid, and
soluble phenols were also higher in non-defective coffee beans (Mazzafera 1999).
Nevertheless, the antioxidant level in the defective beans, especially chlorogenic
acids, remains high which may be a good source of antioxidant or radical
scavenger for other food applications (Nagaraju et al. 1997).
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Table 1 Chemical composition of green Arabica and Robusta coffee beans
(g/100g)
Component Arabica coffee Robusta coffee
Polysaccharides 49.8 54.4
Sucrose 8.0 4.0
Reducing sugars 0.1 0.4
other sugars 1.0 2.0
Lipids 16.2 10.0
Proteins 9.8 9.5
Amino acids 0.5 0.8
Aliphatic acids 1.1 1.2
Quinic acids 0.4 0.4
Chlorgenic acids 6.5 10.0
Caffeine 1.2 2.2
Trigonelline 1.0 0.7
Minerals (as oxide ash) 4.2 4.4
Volatile aroma traces traces
Water 8 to 12 8 to 12
After harvesting, green coffee beans should be dried to 10-14.5% moisture
content and stored below 26oC under dry environment (50-75% RH) to maintain
the bean quality and to prevent the growth of mould (Gopalakrishna Rao et al.
1971; Kulaba 1981; Betancourt & Frank 1983). Under optimal storage conditions,
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green coffee beans may be stored for more than 3 years (Bucheli et al. 1998).
Usually, green coffee beans are packaged in natural jute, sisal or burlap bags,
although high quality beans may be packaged in high barrier synthetic vacuum
packages fabricated from synthetic thermoplastic polymers. Cupping is a method
to detect the early stages of coffee deterioration. Bucheli and others (Bucheli et al.
1996) reported that glucose was a sensitive marker for green coffee bean quality
during storage. Glucose is present only in trace amount of good quality green
coffee, and the content will increase when deterioration occurs (Wolfrom & Patin
1965; Bucheli et al. 1996).
2.2 Roasting of coffee beans
Green coffee beans provide neither the characteristic aroma nor flavour of
brewed coffee until they are roasted. Moreover, the roasting process increases
the value of coffee beans, by 100-300% of the raw material (Yeretzian et al.
2002). Roasting of coffee beans typically takes place at 200-240°C for different
times depending on the desired characteristics of the final product. Events that
take place during roasting are complex, resulting in the destruction of some
compounds initially present in green beans and the formation of volatile
compounds that are important contributors to the characteristic of coffee’s aroma.
The chemical compositions of green, roasted, and brewed coffee are shown in
Figure 1 (Barter 2004).
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Figure 1 Chemical composition of green, roasted, and brewed coffee (Barter
2004)
Briefly, as temperature increases to about 100oC, green coffee beans
undergo moisture loss from 8-12% in green coffee beans to about 5% in the
roasted coffee beans (Illy & Viani 1998). The smell of the beans changes from
herb-like green bean aroma to bread-like, the color turns from green to yellowish,
and the structure changes from strength and toughness to more crumbly and
brittle. When the internal temperature of beans reaches 100oC, the color
darkened slightly for about 20-60 s due to the vaporization of water. At 160-
170oC, the beans become lighter in color for about 60-100 s. As roasting
cellulose
(non Hyd)
18%
cellulose
(Hyd)
13%
starches and
pectins
13%
soluble
carbohydrates
9%
water
12%
non volatile acids
7%
caffeine
1%
protein
12%
ash
3% oil
11%
trigonelline
1%
Green coffee beans
caffeine
1%
water
2%
starches and
pectins
14%
CO22%
cellulose(Hyd)
14%
cellulose
(non Hyd)
17%trigonelline
1%
oil
13%
ash
4%
protein
13%
non volatile acids
7%
soluble
carbohydrates
10%
Roasted coffee beans
oil 1%
soluble
carbohydrates
37%
non volatile
acids
31%
caffeine
6%
protein
5%
ash
16%
trigonelline
4%
Brewed solubles
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continues at this temperature, Maillard and pyrolytic reactions start to take place,
resulting in gradually darkening of the beans (Hernandez et al. 2007). The
buildup of water pressure, along with the large amount of gases generated
causes the cellulose cell wall to crack, giving rise to the so called “first crack”. As
heating continues at the roasting temperature (160-170oC), the coffee becomes
darker and more rapid popping of coffee bean occurs (“second crack”) as the
carbon dioxide (CO2) buildup exceeds the strength of the cellulosic walls of the
bean. Finally, after roasting, the fresh roasted coffee beans are quickly cooled to
stop roasting (Yeretzian et al. 2002).
The final quality of roasted coffee is influenced by the design of the roasters
and time-temperature profiles used. Although heat transfers during roasting can
involve conduction, convection, and radiation, convection by far is the most
important mode of heat transfer that determines the rate and uniformity of
roasting (Baggenstoss et al. 2008). Coffees roasted in fluidized-bed roaster that
is almost exclusive based on convective heating can result in low density and
high yield coffee (Eggers & Pietsch 2001). On the other hand, coffees roasted in
drum roaster that involves mainly conductive heat transfer have less soluble
solids, more degradation of chlorogenic acids, more burnt flavour, and higher
loss of volatiles than the fluidized bed roasters (Nagaraju et al. 1997).
The effects of time-temperature profile on coffee aroma properties have
been reported by Lyman et al. (Lyman et al. 2003). They observed that the
medium roasted process (6.5 min to the onset of the first crack and 1.0 min to the
onset of the second crack) resulted in good balance of taste and aroma with
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citrus flavour . However, the “sweated process” (4.5 min to the first crack and 6.5
min to the second crack) resulted in non-uniform bean color and the coffee was
“sour, grassy, and underdeveloped”. Reducing the heating rate further by using
the “baked process” (11 min to the first crack and 18 min to the second crack)
produced coffee of “flat, woody with low brightness and acidity” (Lyman et al.
2003). In another study, Schenker et al. reported that LHC process (150 to 240oC
in 270 s; 240oC for 55 s) resulted in the formation of the highest quantities of
aroma volatiles, while the long time low temperature (LTLT) approach (isothermal
heating at 220
o
C for 600 s) generated the lowest aroma volatiles. Moreover, thedistribution of the 13 volatile compounds monitored was considerably different
depending on the roasting profiles used (Schenker et al. 2002).
Depending on the extent of heat treatment, coffee can be largely
categorized as light, medium or dark roasts. Light roast process tends to give
non-uniform bean color with sour, grassy, and underdeveloped flavour, while
medium roast process produces a balanced taste and aroma with citrus flavour.
By contrast, dark roast process produces coffee of low acidity sensory profiles
(Lyman et al. 2003). Physical characteristics such as temperature, color, and
weight-loss are often used as indicators of roast degree. However, these
parameters only allow assessment of the flavour profile for coffee roasted under
narrow process conditions (Sivetz 1991; Illy & Viani 1995). Other analytical
methods for quantifying the degree of roast include ratio of free amino acids
(Nehring & Maier 1992), alkylpyrazines (Hashim & Chaveron 1995), and
chlorogenic acids content (Illy & Viani 1995). Fobe and others (Fobe et al. 1968)
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studied changes in chemical composition of Arabica coffee roasted at 230°C at
different process times. They reported that as the roasting time increased, the
following changes occurred: (1) sugar contents first increased, and then
decreased; (2) minimal change in caffeine content; (3) proteins decreased
continuously; (4) free fatty acids increased; and (5) unsaponifiable compounds
decreased (Fobe et al. 1968).
2.3 Changes in Chemical Compositions during Roasting
Roasting causes a net loss of matters in the forms of CO2, water vapor, and
volatile compounds. Moreover, degradation of polysaccharides, sugars, amino
acids and chlorogenic acids also occurred, resulting in the formation of
caramelization and condensation products. Overall, there is an increase in
organic acids and lipids, while caffeine and trigonelline (N-methyl nicotinic acid)
contents remained almost unchanged (Buffo & Cardelli-Freire 2004). The main
acids present in green beans are citric, malic, chlorogenic, and quinic acids.
During roasting the first three acids decrease while quinic acid increases as a
result of the degradation of chlorogenic acids (Ginz et al. 2000). Formic and
acetic acids yields increase up to the medium roasting degree and then begin to
fall as roasting is continued. According to Balzer (Balzer 2001), a rapid increase
in titratable acidity during roasting was observed from green to medium roast,
followed by a smaller decrease as roasting proceeded.
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The reaction products formed are highly dependent on the roasting time-
temperature profile used. Excessive roasting produces more bitter coffee lacking
satisfactory aroma, whereas very short roasting time may be insufficient to
develop full organoleptic characteristics (Yeretzian et al. 2002; Lyman et al. 2003;
Buffo & Cardelli-Freire 2004). Although the majority of phenolic antioxidants
naturally occurring in coffee bean are lost during roasting, the formation of other
antioxidants from Maillard reactions during roasting can enhance the antioxidant
activity of coffee. Compared to medium roast coffee, dark roast coffee exhibited
lower radical scavenging activity than medium roasted coffee due to thedegradation of polyphenol, and thus the antioxidant activity will also depend on
roasting severity and type of coffee (Giampiero Sacchetti 2009).
The profile of organic compounds generated during roasting is very
dynamic and complex. Using Proton transfer reaction-Mass spectrometry (PTR-
MS) technique, Yeretzian et al. (Yeretzian et al. 2002) simultaneously monitored
the evolution of 8 volatile compounds at isothermal conditions as a function of
time. They observed a distinctive increase in acetic acid, methyl acetate, and
pyrazine concentrations in the headspace, all occurred at the same time.
Concomitantly, there was a rapid decrease in water vapor and methanol
concentrations. Moreover, these peaks shifted in synchronous manner with the
roasting condition. For instance, at 190o
C, the above observed changes took
place at 19 min but shifted to 30 min when the beans were roasted at 180 oC
(Yeretzian et al. 2002). Similar observations were observed by Hashim and
Chaveron, who concluded that methylpyrazine may be used as an indicator to
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monitor the roasting progress of coffee beans (Hashim & Chaveron 1995). It has
been suggested that the pressure buildup within intact bean cells is comparable
to inside an autoclave, which can further complicated the chemical reactions
occurred in coffee bean during roasting (Buffo & Cardelli-Freire 2004).
Chemical reactions happened during coffee roasting are very complex,
which have not been fully understood. Based on the literature reviewed, we can
conclude that the quality of roasted coffee cannot be solely described in terms of
physical parameters at the start and end point of roasting, but rather it is
dependent on the path taken during the roasting process. To reach a specific
flavour profile, not only that precise control of roasting time and temperature is
needed, the variety/quality of green beans, cooling, and degassing conditions are
expected to be important as well.
2.4 Flavour compounds in roasted coffee
Chemical compounds present in roasted coffee can be roughly grouped into
volatile and non-volatile, some of the former being responsible for aroma and the
latter for the basic taste sensations of sourness, bitterness and astringency
(Buffo & Cardelli-Freire 2004). Russwurm reported that carbohydrates, proteins,
peptides and free amino acids, polyamines and tryptamines, lipids, phenolic
acids, trigonelline, and various non-volatile acids in the green coffee beans were
involved in the flavour formation during roasting (Russwurm 1970). For example,
chlorogenic acid contributes to body and astringency; sucrose contributes to
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color, aroma, bitterness, and sourness; minor protein components like free amino
acids are highly reactive by interacting with reducing sugars, which make the
Maillard reaction happen; triogenlline generates pyridine and may be
consequently be responsible for some objectionable flavours; and caffeine can
be contributed to the bitterness (Flament 2002).
Maillard reactions have been identified to be the major pathway in the
formation of volatile compounds in coffee roasting (Shibamoto 1991). In the
Maillard reaction, reducing sugars such as glucose or fructose react with free
amino acids to form N -substituted glycosylamine adducts, which are then
rearranged to aminoketones and aminoaldoses by Amadori and Heynes
rearrangements. A complex reaction cascade of Amadori and Heynes
rearrangement products leads to numerous volatile compounds and complex
melanoidins.
More than 800 volatile compounds have already been identified in roasted
coffee, among which, about 40 compounds are responsible for the characteristic
aroma of coffee (Belitz et al. 2009). Some of these compounds are summarized
in Table 2, showing the odorant groups that they are being categorized to
(Semmelroch et al. 1995; Czerny et al. 1999; Mayer et al. 2000).
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Table 2 Potent odorants in Arabica coffee from Colombia
Sweet/caramel-like group
Methylpropanal
2-Methylbutanal
3-Methylbutanal
2,3-Butandione
2,3-Pentandione
4-Hydroxy-2,5-dimethyl-3(2H)-furanone
(HD3F)
5-Ethyl-4-hydroxy-2-methyl-3(2H)-
furanone (EHM3F)
Vanillin
Sulfurous/roasty group
2-Furfurylthiol
2-Methyl-3-furanthiol
Methional
3-Mercapto-3-methylbutyl-formiate
3-Methyl-2-butene-1-thiol
Methanethiol
Dimethyltrisulfide
Earthy group
2-Ethyl-3,5-dimethylpyrazine
2-Ethenyl-3,5-dimethylpyrazine
2,3-Diethyl-5-methylpyrazine
2-Ethenyl-3-ethyl-5-methylpyrazine3-Isobutyl-2-methoxy-pyrazine
Smoky/phenolic group
Guaiacol
4-Ethylguaiacol
4-Vinylguaiacol
Fruity group
Acetaldehyde
Propanal
(E)-β-Damascenone
Spicy group
3-Hydroxy-4,5-dimethyl-3(5H)-furanone
(HD2F)
5-Ethyl-3-hydroxy-4-methyl-2(5H)-
furanone(EHM2F)
The non-volatile components in roasted coffee are made up of mainly the
following:
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(1) Proteins, peptides and amino acids: Crude protein content is relatively
stable during roasting, while the free amino acids decrease by 30%, with dark
roast espresso reaching up to 50% (Belitz et al. 2009). Protein content plays an
important role in espresso coffee as it affects the foamability of the beverage that
the foamability increased generally with increase total protein concentration until
a maximum value is reached (Nunes et al. 1997). The composition of the amino
acids vary dependent on their thermal stability and reactions involved. For
instance, changes in glutamic acid content are less dramatic as compared to
cysteine and arginine. The latter amino acids tend to deplete rapidly duringroasting due to their involvement in Maillard browning reactions (Illy & Viani
2005).
(2) Carbohydrates: Only traces of free mono and disaccharides in green
coffee remain after roasting. Cellulose, hemicellulose, arabinogalactan and
pectins play important roles in the retention of volatiles and contribute to coffee
brew viscosity. It is reported that in espresso coffee, the foam stability is related
to the amount of galactomannan and arabinogalactan (Nunes et al. 1997).
(3) Non-volatile lipids and lipid-solubles: Triglycerides, terpenes,
tocopherols and sterols contribute to brew viscosity. The lipid fraction tends to be
stable and survive the roasting process with only minor changes. Linoleic and
palmitic acids are the predominant fatty acids in coffee. Cafestol and kahweol are
diterpenes that degrade by the roasting process. Another diterpene, 16-O-
methylcafestol, is present in Robusta but not Arabica coffee, making it a suitable
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indicator for detecting Robusta content in coffee blend (Speer et al. 1991; Belitz
et al. 2009).
(4) Caffeine: Caffeine is of major importance with respect to the
physiological properties of coffee, and also in determining the strength, body and
bitterness of brewed coffee. The caffeine content of green coffee beans varies
according to the species that Robusta coffee contains about 2.2%, and Arabica
about 1.2%. Environmental and agricultural factors appear to have a minimal
effect on caffeine content. During roasting there is no significant loss in terms of
caffeine (Ramalakshmi & Raghavan 1999). However, caffeine content per 177
mL (6 oz) of coffee range from 50 to 143 mg, depending on the mode of
preparation(Rogers & Richardson 1993; Bell et al. 1996). Bell and others (Bell et
al. 1996) reported that more coffee solids, larger extents of grinding, and larger
volumes of coffee prepared at a constant coffee solids to water ratio led to
significantly higher caffeine content. Home-grinding yielded caffeine content
similar to that of store-ground coffee, and boiled coffee had caffeine contents
equal to or greater than filtered coffee (Bell et al. 1996).
(5) Acids: Acids are responsible for acidity, which together with aroma and
bitterness is a key contributor to the total sensory impact of a coffee beverage.
Carboxylic acids, mainly citric, malic and acetic acids are responsible for acidity
in brewed coffees. Arabica coffee brews are more acidic (pH 4.85-5.15) than
Robusta brews (pH 5.25-5.40) (Vitzthum 1975).
(6) Melanoidins: The final products of the Maillard reaction between amino
acids and monosaccharides, are the brown-coloured substances that impart to
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roasted coffee its characteristic color, possess antioxidant activity, and affect on
the flavor volatiles (Hofmann & Schieberle 2001; Del Castillo et al. 2002; Vignoli
et al. 2011).
2.5 Fourier transform infrared (FTIR) spectroscopy
FTIR spectroscopy is a powerful tool for identifying types of chemical bonds
in a molecule by producing an infrared (IR) absorption spectrum. Interferometer
is one of the key components in a FTIR spectrometer. It consists of IR light
source, fixed mirror, moving mirror, beam splitter, and detector (Figure 2). The
principle of the FTIR spectroscopy is that the beam splitter splits the light beam
from the IR source and sends half of the IR radiation to the fixed mirror and the
other half to the moving mirror. The split beams recombine to form overlapping
radiation waves that interact with the sample, resulting in an infrared spectrum.
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Figure 2 Schematic diagram of a typical FTIR spectrometer
The radiation emerging from an IR source passes through the
interferometer and to a sample before reaching a detector. Upon amplification of
signal, the data are transformed to the digital type by an analog-to-digital
converter and transferred to a computer for Fourier-transformation. FTIR
measures the absorbance of IR active species over a range of wavenumbers in
the IR region that are absorbed by a material. IR spectral regions can be divided
into three parts, which are near-IR (13000-4000 cm-1), mid-IR (4000-400 cm-1),
and far-IR (400-10 cm
-1
). The bonds involved in the near-IR are usually due to C-H, N-H or O-H stretching. Typical vibrational absorbance for common bonds in
the mid-IR is shown in Figure 3.
Figure 3 Vibrational absorbance due to common bands
Sampling methods in FTIR include transmission, reflectance, and micro-
sampling (Stuart 2003). The transmission method is based on the absorption of
IR radiation as it passes through a sample. It can be used to analyze solid, liquid,
and gaseous sample. The reflectance method can be used for samples that are
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difficult to analyze by transmission method. Attenuated total reflectance (ATR)
spectroscopy uses total internal reflection phenomenon to analyze a sample. In
many applications, it successfully replaces constant path transmission cells and
salt plates used for the analysis of liquid and semi-liquid materials. Because of
the reproducible effective path length, ATR is well suited for both qualitative and
quantitative applications. Some other spectroscopy such as specular reflectance
spectroscopy, diffuse reflectance spectroscopy, and photoacoustic spectroscopy
(PAS) are also very useful in analyzing samples. Micro-sampling method is used
for very small samples (microgram or microlitre) by the help of an IR microscope.If a microscope facility is not available, some other special sampling accessories
such as a beam condenser or a diamond anvil cell can be used (Stuart 2003).
Various FTIR techniques have been used for coffee research. For instance,
FTIR has been used for caffeine determination in roasted coffee in the mid-IR
range (Garrigues et al. 2000; Ohnsmann et al. 2002), for discrimination of coffee
varieties (Kemsley et al. 1995; Briandet et al. 1996b; Garrigues et al. 2000), and
for detection of adulteration in instant coffees by sugars, starch, or chicory
(Briandet et al. 1996a). Moreover, FTIR-ATR has been successfully used in the
analysis of brewed coffee to study the effects of roasting conditions on coffee
aroma. Lyman et al. investigated the 1800-1680 cm-1 region of IR spectrum,
which contains carbonyl vibration bands that can be used to correlate vinyl
esters/lactones, esters, aldehydes, ketones, and acids (Lyman et al. 2003).
2.6 Chemometrics
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Chemometrics can be generally described as the application of
mathematical and statistical methods to improve chemical measurement
processes, and extract more useful information from chemical and physical
measurement data (Workman et al. 1996; Paul 2006; Fu 2011).
In general, there are three categories of chemometric analysis (InfoMetrix
2010):
(1) Exploratory data analysis is often used to reveal hidden patterns in
complex data by reducing the information to a more comprehensible form, to
expose possible outliers, and to indicate whether there are patterns or trends in
the dataset. Principal component analysis (PCA) and hierarchical cluster analysis
(HCA) are some of the exploratory algorithms.
(2) Continuous property regression is used to develop calibration models
that correlate the information in a set of known measurements to the property of
interest. Partial least squares (PLS) and principal component regression (PCR)
are two algorithms commonly used for regression and are designed to avoid
problems associated with noise in the data.
(3) Classification modeling is applied in scenarios where samples are
required to be classified into predefined categories or "classes". A classification
model is used to assign a sample's class by comparing the sample to a
previously analyzed data set, for which its categories are already known. PLS
discriminant analysis (PLS-DA), k-nearest neighbor (KNN) and soft independent
modeling of class analogy (SIMCA) are some of the primary chemometric
workhorses in classification modeling.
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Among the chemometric analyses used, PCA by far is the most commonly
used. It is a linear and non-parametric pattern recognition technique which
reduces multidimensionality by correlating data to two or three dimensions (Anil
et al. 2004). The goal of PCA is to visualize the inherent data structure and reveal
how different variables change in relation to each other. This is achieved by
transforming correlated original variables into a new set of uncorrelated
underlying variables, known as principal components (PCs), using the covariance
matrix. The new variables are linear combinations of the original ones. The
principle of PCA can be illustrated using a simple dataset, where the 3 variablesneeded to describe the dataset are represented by three axes in the data-space
(Figure 4). PC1 has a direction that takes into account as much variance in the
data as possible. PC2, orthogonal to PC1, has a direction where the second
largest variance occurs. The objects are then projected down to the plane of the
two PCs. A large data-set may therefore be represented by only a few PCs,
which describe a large part of the variance in the data as a linear combination of
the original variables. PCA is very useful for solving pattern recognition problems
arising from chromatographic and spectroscopic data (Hagman & Jacobsson
1990).
On other hand, PLS is a useful multivariate regression technique for
correlating two or more blocks of data with each other, or predicting a value of
one block by using the data from the other block that is easier to measure
(Gerlach et al. 1979). PLS can handle more than one dependent variable and is
not significantly influenced by the correlation between the independent variables.
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In addition, it can tolerate missing values in the data-matrix (Geladi & Kowalski
1986). In the PLS method, X (independent) variables are related to a block of Y
(dependent) variables through a process where the variance in Y-block
influences the calculation of PCs of X-block (Hagman & Jacobsson 1990).
Figure 4 Schematic diagram of PCA analysis
Many researchers have used chemometrics to study various phenomena in
coffee. Briandet and others adopted PCA to analyze FTIR spectra of coffee
extracts. They showed that 100% correct classifications for both training and test
samples for Arabica and Robusta in Instant Coffee. They also applied PLS to
predict the relative Arabica and Robusta contents in their coffee samples by
analyzing the FTIR spectra (Briandet et al. 1996a). Bicchi and others
characterized different roasted coffees and coffee beverages by applying PCA to
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chromatographic data obtained by headspace solid phase microextraction (HS-
SPME), and the results showed that coffees from different origins can be
successfully separated (Bicchi et al. 1997). In another study, Charlton and others
applied PCA to analyze Nuclear magnetic resonance (NMR) spectra from 98
coffee samples obtained from three different producers (Charlton et al. 2002). In
their study, 99% of the samples were correctly classified accordingly to their
manufacturers. Also, blind testing of the PCA model with a further 36 samples of
instant coffee resulted in a 100% success rate in identifying the samples from the
three manufacturers.
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3 OBJECTIVES
Currently, integrated studies are lacking on elucidating the effects of bean
variety and roast degree, under different time-temperature conditions, on the
physical and chemical properties of coffee. The objectives of this study are:
To analyze coffee from different geographical origins (Colombia, Costa
Rica, Ethiopia, and Kenya) processed to medium and dark roasts, using
FTIR spectroscopy and chemometric analysis.
Developing the understanding of the effects of time-temperature
conditions on the physicochemical properties (color, moisture contents, pH,
titratable acidity, and microstructure) of coffee from Brazil.
To study the physicochemical changes (color, moisture contents, pH,
titratable acidity, and microstructure) in coffee beans at different stages of
roast using FTIR spectroscopy and chemometric analysis.
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4 FEASIBILITY STUDY ON CHEMOMETRIC DISCRIMINATION OF
ROASTED ARABICA COFFEES BY SOLVENT EXTRACTION AND
FOURIER TRANSFORM INFRARED SPECTROSCOPY
4.1 Introduction
Coffee is one of the most popular beverages in the world due to its unique
aroma, taste, and stimulating effects of caffeine. The quality of brewed coffee is
affected by many parameters. Depending on the species (Arabica, Robusta, or
Liberica) and method used to process the coffee cherries (dry vs wet), the overall
quality and chemical composition of coffee bean can vary considerably. By and
large, the Arabica coffees have more pronounced and finer flavor profiles that are
considered better quality and, accordingly, command a higher price than the
Robusta and Liberica coffees (Davis 2001). The composition of the soil and its
fertilization, the altitude and weather of the plantation, and the final cultivation
and drying methods used will all affect the green bean quality (Costa Freitas &
Mosca 1999). Roasting, the final processing step before grinding and brewing,
ultimately determines the organoleptic properties of the coffee beverage. During
the roasting process, the reactions that occur in the coffee bean are complex and
strongly dependent on the time-temperature profile used (Lyman et al. 2003;
Baggenstoss et al. 2008).
Grading of whole roasted coffee beans is relatively easy as compared to
ground coffee due to the presence of visual clues in the former (size, shape,
defect, etc.). By contrast, these indicators are absent for ground coffees;
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infrared spectral data of these extracts, in conjunction with PCA and SIMCA, to
discriminate four Arabica ground coffees from different origins (Colombia, Costa
Rica, Ethiopia, and Kenya) that had been roasted to two roast degrees (medium
or dark).
4.2 Materials and Methods
4.2.1 Chemicals
Hexane was purchased from Sigma-Aldrich Ltd. (St. Louis, MO).Dichloromethane, ethyl acetate, acetone, and acetic acid were purchased from
Fisher Scientific (Ottawa, Canada). Ethanol was purchased from Greenfield
Ethanol Inc. (Brampton, Canada).
4.2.2 Coffee Beans and Roasting Conditions
Wet-processed green coffee beans (Arabica variety) from Colombia, Costa
Rica, Kenya, and Ethiopia were purchased from Green Beanery (Toronto,
Canada). Green coffee beans (45 g) were roasted in a fluidized bed hot air
roaster (Fresh Roast SR 500, Fresh Beans Inc., Park City, UT). Two isothermal
roasting programs were used for preparing dark and medium roast coffees
(Figure 5). The roasted beans were stored in hermetic glass bottles in the dark at
15°C before grinding.
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Figure 5 Air temperature (in roast chamber) profiles of the fluidized bed hot air
coffee roaster
4.2.3 Degree of Roast as Determined by Color Measurements
Roasted coffee beans were ground using a coffee grinder (Bodum Antigua
Electric Burr Grinder, Bodum, Inc., Copenhagen, Denmark) at the medium grind
setting. The color of the ground coffee was measured in the L*, a*, b* system
using a Konica Minolta CM-3500d spectrophotometer (Konica Minolta Sensing,
Inc., Osaka, Japan) in the reflectance mode. Before analysis, the instrument was
calibrated on a white standard tile. Measurements were taken in triplicate.
4.2.4 Solvent Extraction of Ground Coffee
After grinding, coffee grounds were extracted with dichloromethane, ethyl
acetate, hexane, acetone, ethanol, or acetic acid, following two extraction
25
50
75
100
125
150
175
200
225
250
0 50 100 150 200 250 300 350 400 450 500 550
R o a s t i n g T e m p e r a t u
r e ,
o C
Roasting Time, S
Medium roast profile
Dark roast profile
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procedures. In the first procedure (method #1), 0.2500 g of ground coffee was
accurately weighed into a glass vial, and 1 mL deionized water was added to wet
the sample. The glass vial was shaken for 1 min with an IKA-VIBRAX-VXR
vibrator (Janke & Kunkel, Inc., Staufen, Germany) at the 200 dial setting; 1 mL of
organic solvent was added and the mixture was shaken for an additional 5 min.
The organic phase was then transferred to another vial and allowed to rest for 10
min before ATR-FTIR analysis. In the second procedure (method #2), a similar
procedure was used except that water was not added prior to solvent extraction.
All extractions were performed in triplicate.
4.2.5 ATR-FTIR Analysis
The coffee extract was scanned using an FTIR spectrometer (IR Prestige-
21; Shimadzu Corp., Tokyo, Japan) equipped with a deuterated triglycine sulfate
detector and a KBr beam-splitter. A MIRacle ATR accessory equipped with a
diamond crystal (Pike Technologies, Madison, WI) was used for sampling. The
background spectrum was collected using an empty ATR cell. To collect each IR
spectrum, coffee extract (6 μL) was placed onto the ATR crystal, and the solvent
was allowed to evaporate until no further changes through consistently controlling
evaporation time during the experiment in IR spectrum were observed. This
technique removed interference from the solvent signals and increased the
sensitivity of chemometric analysis. The times required for complete evaporation
of solvent were different due to the different solubilities of each solvent in water.
Samples were scanned from 600 to 4000 cm−1 at 4 cm−1 resolution. Each
spectrum was an average of 20 scans. For each extract, 3 FTIR spectra
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replicates were scanned. Between samples, the ATR crystal was carefully
cleaned with 95% (v/v) aqueous ethanol solution, and dried with lint-free tissue
paper. The spectral baseline was examined visually to ensure that no residue
from the previous sample was retained on the crystal. All spectra were recorded
at room temperature (23 ± 0.5 °C).
4.2.6 Data Analysis
Statistical comparison of color values of ground coffee samples was
conducted based on Tukey pairwise comparisons using R software (www.r-
project.org). For chemometric analysis, FTIR spectra were exported as ASCII
format, organized in Excel spreadsheets, and then analyzed using Pirouette v.4.0
software (Woodinville, WA). During PCA, second derivative and mean-center
were applied to FTIR spectra to reduce baseline variation and enhance spectral
features. Nine spectra (3 extracts for each coffee and 3 replicate spectra for each
extract) for each coffee were divided into two groups: 6 spectra from the first two
extracts were used to calibrate the SIMCA model, while the remaining 3 spectra
from the third extract were used for validation to evaluate the prediction accuracy
of the calibrated SIMCA model. The optimum number of PCs in each class was
selected on the basis of the lowest number of PCs giving minimum value of
variance.
4.3 Results and Discussions
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while hexane, ethyl acetate, acetone, ethanol, and acetic acid phases were on
top (Figure 6). Three layers (solvent, water, ground coffee phases) were
observed when dichloromethane, hexane, and ethyl acetate were used as a
solvent because they were immiscible or slightly soluble in water. The three
layers observed were likely caused by the different densities of ground coffee,
water, and solvent. However, for acetone, ethanol, and acetic acid extractions,
only two phases were observed since these solvents were miscible with water.
For coffee extracted by method #1, coffee grinds were all in one layer. On the
other hand, in the presence of organic solvent alone (Method #2), the extractlayers were hazy, and tended to contaminate with grind particulates. This may be
due to the fact that when the samples were wetted with water, the entrapped air
in the ground coffee matrices was readily displaced by the solvents, thereby
reducing the buoyancy of the grind particulates.
Table 3 Physical properties of the investigated solvents (Pagni 2005)
SolventSolubility in water, at
20°C
Polarity
index (P)
Density,
g/mL
Dichloromethane Immiscible (1.3 v/v) 3.1 1.326
Hexane Immiscible(0.0013 v/v) 0.1 0.659
Ethyl acetate Slightly soluble (8 v/v) 4.4 0.895
Acetone Miscible (infinitely) 5.1 0.786
Ethanol Miscible (infinitely) 5.2 0.789
Acetic acid Miscible (infinitely) 6.2 1.049
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Table 4 Evaporation time of the coffee extracts
Evaporation time (s)
Solvent With H2O No H2O
Dichloromethane extract 60 60
Hexane extract 60 60
Ethyl acetate extract 180 180
Acetone extract 600 60
Ethanol extract 760 280
Acetic acid extract 840 780
Selected FTIR spectra of solvent extracts obtained by methods #1 (with
water) and #2 (no water) are shown in Figure 7. The 3100 to 2750 cm -1 region in
the majority of spectra (except acetic acid, acetone, and ethanol extracts
obtained with extraction method #1) were typical for the fatty acid moiety of lipids
due to asymmetrical C-H stretching (2920 cm-1), symmetrical C-H stretching
(2850 cm-1), and methylene asymmetrical stretching band (weak shoulder at
2954 cm-1) (Innawong et al. 2004). In the presence of water, the absorbance
around 3676-3028 cm-1 for acetic acid, acetone, and ethanol extracts can be
attributed to the O-H stretching band. The 1800 –800 cm-1 region contained
absorbance bands due to C=O (ester, aldehydes, and ketones) stretching, C-H
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(methylene) bending (scissoring), and C-O (esters and alcohol), CH2
stretching/bending (Innawong et al. 2004). These regions contained fingerprint
information that may be important for discriminating coffee samples from different
origins.
Spectra from method #1 extracts were relatively more complex than those
from method #2 extracts, especially when dichloromethane and ethyl acetate
were used for extraction. For instance, dichloromethane extract from method #1
resulted in many additional peaks that were absent for those from method #2,
including 1487 cm
-1
(C=C, C-H deformation), 1398 cm
-1
(CH3 symmetricdeformation), 1323 cm-1 (symmetric vibrations of COO- groups), and 1284 cm -1
(Amide III band components of proteins) (Movasaghi et al. 2008). In terms of
band shape and intensity, different spectral features were observed in the 1720-
1203 and 1064-940 cm-1 regions. With method #1, water-induced swelling of the
coffee particles might have facilitated the extraction of additional compounds. A
similar enhancement in spectral features was observed for the dichloromethane
and ethyl acetate coffee extracts. For the hexane and acetic acid extracts,
minimal spectral differences were observed between methods #1 and #2. The IR
spectra of the hexane extracts were similar to lipid (Hennessy et al. 2009)
indicating that lipids may be the main components extracted when hexane was
used as a solvent. Overall absorbance values were considerably stronger for the
acetone and ethanol extracts probably due to the contribution from water present
in the extracts. The spectra of acetic acid extracts and pure acetic acid were
similar (data not shown), indicating that acetic acid is not an effective solvent for
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Tukey pairwise comparison analysis (Table 6) confirmed that differences in L*
values were not significant between ground samples for dark or medium roasted
beans, implying that samples from the same degree of roast exhibited the same
lightness.
Table 5 L* Value of Roasted Ground Arabica Coffee Beans
Roast degree Coffee bean sample Lightness [L*]
Dark Colombian 19.83 ± 0.05
Costa Rican 19.61 ± 0.18
Ethiopian 19.46 ± 0.21
Kenyan 19.72 ± 0.06
Medium Colombian 25.21 ± 0.16
Costa Rican 25.35 ± 0.29
Ethiopian 25.64 ± 0.06
Kenyan 25.28 ± 0.09
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Table 6 Turkey method for L* value comparisons
Comparison95% SCI
(Dark roast)
95% SCI
(Medium roast)
Different
from 0?
Colombian VS. Costa Rican (-0.176, 0.616) (-0.326, 0.866) No
Colombian VS. Ethiopian (-0.026, 0.766) (-0.036, 0.896) No
Colombian VS. Kenyan (-0.286, 0.506) (-0.396, 0.536) No
Costa Rican VS. Ethiopian (-0.246, 0.546) (-0.176, 0.756) No
Costa Rican VS. Kenyan (-0.286, 0.506) (-0.396, 0.536) No
Ethiopian VS. Kenyan (-0.136, 0.656) (-0.106, 0.826) No
Dark roast: MSE = 0.022908, HSD (t, αF) = 0.396; Medium roast: MSE = 0.03175,
HSD (t, αF) = 0.466
4.3.3 PCA Analysis of Solvent Extracts of Coffee Beans
Typical FTIR spectra of dichloromethane extracts (method #1) of dark roast
coffee beans from various regions are presented in Figure 8. As shown, although
variances between spectra exist, the differences are subtle and data
interpretation is difficult. To extract relevant information from the data, PCA was
employed to reduce the dimensionality of the IR spectra and facilitate the
visualization of the inherent structure of the dataset (Figures 9 and 10).
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Figure 8 Selected FTIR spectra of dark roast coffee extract obtained with
dichloromethane as a solvent (using method 1#)
75010001250150017502000225025002750300032503500375040001/cm
-0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
Abs
HIGH T-COLOM2-2HIGH T-KENYA 1-1
HIGH T-ETHIOPIAN 1-1HIGH T-COSTA 1-1
FTIR Measurement
Costa Rican
Colombian
Kenyan
Ethiopian
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Figure 9 PCA of FTIR data for hexane, dichloromethane, ethyl acetate, and acetone extracts of medium roast coffee.
Row A: Two-factor score plots. Row B: Loading plots of PC1. Row C: Corresponding FTIR raw spectra
A
B
C
-0.003
-0.002
-0.001
0.000
0.001
0.002
-0.010 -0.005 0.000 0.005 0.010
2 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Colombian Costa Rican Ethiopian Kenyan
Hexane Extract
-0.003
-0.002
-0.001
0.000
0.001
0.002
-0.005 -0.003 0.000 0.003 0.005
3 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Colombian Costa Rican Ethiopian Kenyan
Dichloromethane Extract
-0.003
-0.002
-0.001
0.000
0.001
0.002
-0.005 -0.003 0.000 0.003 0.005
2 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Colombian Costa Rican Ethiopian Kenyan
Ethyl acetate Extract
-0.002
-0.001
0.000
0.001
0.002
-0. 003 -0 .00 2 -0. 001 0. 000 0. 001 0. 002
3 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Colombian Costa Rican Ethiopian Kenyan
Acetone Extract
-0.15
-0.05
0.05
0.15
0.25
8001600240032004000
PC 1 Loading (89.4%)
-0.15
-0.05
0.05
0.15
0.25
8001600240032004000
PC 1 Loading (24%)
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
2850
1741 1678
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
16681548
1028
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
Wavenumber, cm-1
2850
2920
1741 1726
-0.15
-0.05
0.05
0.15
0.25
8001600240032004000
PC 1 Loading (59.4%)
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
2920
2850
1236
1550
16431697
1743
-0.15
-0.05
0.05
0.15
0.25
8001600240032004000
PC 1 Loading (26.4%)
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Figure 10 PCA of FTIR data for hexane, dichloromethane, ethyl acetate, and acetone extracts of dark roast coffee. Row
A: Two-factor score plots. Row B: Loading plots of PC1. Row C: Corresponding FTIR raw spectra
Hexane Extract
Ethyl acetate Extract
-0.003
-0.002
-0.001
0.000
0.001
0.002
-0.010 -0.005 0.000 0.005 0.010
2 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Colombian Costa Rican Ethiopian Kenyan
-0.15
-0.05
0.05
0.15
0.25
8001600240032004000
PC 1 Loading (85.8%)
-0.002
-0.001
0.000
0.001
-0.002 -0.001 0.000 0.001 0.002
3 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Colombian Costa Rican Ethiopian Kenyan
-0.15
-0.05
0.05
0.15
0.25
8001600240032004000
PC 1 Loading (60%)
-0.002
-0.001
0.000
0.001
0.002
-0.004 -0.002 0.001 0.004
2 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Colombian Costa Rican Ethiopian Kenyan
-0.002
-0.001
0.000
0.001
0.002
-0.010 -0.005 0.000 0.005 0.010
3 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Colombian Costa Rican Ethiopian Kenyan
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
2920
2850
1741 1726
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
1550
1683
-0.15
-0.05
0.05
0.15
0.25
8001600240032004000
PC 1 Loading (51.3%)
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
2920
2850
16471697
1236
1550
1741
-0.15
-0.05
0.05
0.15
0.25
8001600240032004000
PC 1 Loading (83.7%)
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
1514
1662
1649
1481
A
B
C
Dichloromethane Extract Acetone Extract
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extracted. The polarity effect can be observed in the original spectra (Figures 9
and 10, row C). The spectral region from 3676-3028 cm-1 is mainly due to the O-
H stretching band from water. As shown, the absorbance intensity in this region
progressively became stronger for hexane, dichloromethane, ethyl acetate, and
acetone in ascending order. This result is consistent with the polarity for these
solvents.
To further investigate regions of spectra that contribute to the variance of
samples, the loading plots for a corresponding PC were inspected. Here we
focused on PC1 since it explained the maximum variance existing in the dataset
(Figures 9 and 10, row B). The percent variance accounted by PC1 was also
indicated on each loading plot. Regions of each spectrum with a relatively large
loading score (>0.1) were highlighted as red dotted lines. As shown, the loading
plots for hexane extracts were markedly different than those of the other three
solvent extracts, due to the non-polar nature of hexane. The loading plots of
hexane extracts for medium and dark roasts were similar, except that
absorbance at region 1741-1726 cm-1, which is due to C=O stretching band
mode of fatty acid esters, was higher and wider in the medium roast compared
with the dark roast coffee (Yoshida et al. 1997). For dichloromethane extracts,
the most prominent difference in loading plots for dark and medium roast coffees
was in the region of 2920-2850 cm-1
, which can be attributed to CH2
asymmetrical stretching vibrations of hydrocarbon methyl groups (Eliane
Nabedryk 1982). The medium roast coffees exhibited significant loading score
around this region, but negligible for dark roast coffees. A similar trend was
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observed for the region around 1741-1678 cm-1 The minimal changes observed
for these spectral regions for the dark roast samples could be caused by a
decrease in protein and lipids due to the Maillard reaction and pyrolytic cleavage,
respectively (De Maria et al. 1994; Yeretzian et al. 2002).
For ethyl acetate extracts, loading plots for medium and dark roast coffees
were comparable, indicating that the compounds extracted by ethyl acetate from
medium and dark roast coffees were similar, although subtle differences did exist.
The main regions that contribute to the differences between samples are 1743-
1741, 1647-1643, and 1697 cm-1. The band at 1697 cm-1 is due to isolated
carbonyl stretching of C=O bonds, and the band at 1647 cm -1 is due to
conjugated carbonyl stretching of C=O bonds of caffeine compounds (Falk et al.
1990). Garrigues et al. (Garrigues et al. 2000) and Ohnsmann et al. (Ohnsmann
et al. 2002) also utilized absorbance at 1659 and 1704 cm-1 to determine the
caffeine content in coffee and tea, respectively. In these cited studies, the C=O
bands investigated shifted to higher frequencies due to the different solvent used
(i.e., chloroform). Based on this information, it is conceivable that the separated
clusters observed were partly caused by the different caffeine contents of among
the various coffee samples.
Other important vibration bands that contributed to the separated clusters
for dichromethane extracts were at 1705 cm -1 (C=O stretching vibrations of
ketones), 1655 cm-1 (C=O stretching of caffeine compounds), 1599 cm-1 (-NH
group), and 1548 cm-1 (N-H bending of peptide groups). These bands were also
detected in ethyl acetate and acetone extracts with some shifts (1701, 1651,
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1604, and 1552 cm-1 for ethyl acetate; 1699, 1647, 1599, and 1558 cm -1 for
acetone) (Magidman 1984; Mishra & Kumar 2002). For hexane extracts, the most
prominent spectral difference between the medium and dark roast coffees is that
the latter showed a stronger overall absorbance, implying that more lipids (1600-
1700 cm-1) and fatty acid esters (1700-1800 cm-1) were being extracted from the
dark roast coffee.
4.3.4 PCA Analysis for Coffees According to Degree of Roast
Roasting results in many physical changes and chemical reactions in the
coffee beans. Depending on the extent of the roast, which is time-temperature
dependent, the quality and sensory properties of the resulting coffees can vary
considerably. Medium roast coffee has a more full-bodied flavour, a balance of
taste and aroma, and carries citrus taste. In comparison, dark roast coffee has a
heavier sweet taste, with a lingering aftertaste of chocolate (Schenker et al. 2002;
Lyman et al. 2003).
Dichloromethane and ethyl acetate extracts were tested for the
discrimination of dark and medium roast coffees. As shown in Figures 11 and 12
(Row A), two-component score plots resulted in well-separated clusters
corresponding to dark coffees (right clusters) and medium coffees (left clusters)
from the four origins. The loading plots for dichloromethane extracts showed that
all coffee samples, except the Columbian coffee, exhibited strong loading scores
at 2920, 2850, and 1743 cm-1 due to CH2 asymmetrical stretching of methyl
groups, C-H symmetrical stretching of methyl groups, and C=O stretching of
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aliphatic esters (Hennessy et al. 2009; Wang et al. 2009). For the Colombian
coffee, the bands that correspond to significant loading scores at 1550, 1510,
and 1481 cm-1 can be attributed to N-H bending of peptide groups, C=N
stretching of amino groups, and benzene absorption bands, respectively (Mishra
& Kumar 2002; Zhang et al. 2005; Barua et al. 2008).
For ethyl acetate extracts, the loading plots revealed that spectral regions
that contributed to cluster separation were mainly at 2850-2920 cm-1 due to CH2
asymmetrical stretching and C-H symmetrical stretching of methyl groups
(Hennessy et al. 2009) as well as 1650-1750 cm -1 due to C=O stretching
vibrations and C=N stretching (Paradkar & Irudayaraj 2002). For coffee, this
region has been assigned to a number of important compounds, including
aromatic acids (1700-1680 cm-1), aliphatic acids (1714-1705 cm-1), ketones
(1725-1705 cm-1), aldehydes (1739-1724 cm-1), and aliphatic esters (1755-1740
cm-1) (Bellamy 1975; Keller 1986; Socrates 1994). Absorbance in the 2850-2920
cm-1 region was mainly due to lipids (Hennessy et al. 2009).
Overall, roasting coffee from a medium to a dark degree causes increases
in esters/lactones (1788 cm-1), aldehydes/ketones (1739-1722 cm-1), ketones
(1725-1705 cm-1), aromatic acids (1700-1680 cm-1), and aliphatic acids (1714-
1705 cm-1), but a decrease in caffeine content (1700-1692 cm-1, and 1647-1641
cm-1) (Lyman et al. 2003; Movasaghi et al. 2008; Wang et al. 2009). Others have
also observed decreases in the amount of lipids (around 1736, 1740, 1745, and
1750 cm-1), polysaccharides and hemicelluloses (1739 cm -1), esters (1751-1740
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cm-1), and lipids/proteins (2935-2847 cm-1) (Lyman et al. 2003; Movasaghi et al.
2008; Wang et al. 2009).
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Figure 11 PCA of FTIR data for dichloromethane extracts of coffee (from the same origin) with two degrees of roast. Row
A: Two factor score plots. Row B: Loading plots of PC1. Row C: Corresponding FTIR raw spectra
-0.002
-0.001
0.000
0.001
0.002
-0.003 -0.001 0.001 0.003
2 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Dark roast Medium roast
-0.002
-0.001
0.000
0.001
0.002
-0.003 -0.001 0.001 0.003
2 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Dark roast Medium roast
-0.002
-0.001
0.000
0.001
0.002
-0.003 -0.001 0.001 0.003
2 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Dark roast Medium roast
-0.002
-0.001
0.000
0.001
0.002
-0.003 -0.001 0.001 0.003
2 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Dark roast Medium roast
Colombian Costa Rican Ethiopian Kenyan
A
-0.1
0.0
0.1
0.2
8001600240032004000
PC 1 loading (75.9%)
-0.1
0.0
0.1
0.2
8001600240032004000
PC 1 loading (67.8%)
-0.1
0.0
0.1
0.2
8001600240032004000
PC 1 loading (46.6%)
-0.1
0.0
0.1
0.2
8001600240032004000
PC 1 loading (29.2%)
B
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
1481
1510
1550
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
2850
17432920
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
2850
1743
1465
2920
1678
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
2850
1743
1695
2920
860
829C
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Figure 12 PCA of FTIR data for ethyl acetate extracts of coffee (from the same origin) with two degrees of roast. Row A:
Two factor score plots. Row B: Loading plots of PC1. Row C: Corresponding FTIR raw spectra
C
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
2920
2850
1741
2355-2347
16741647
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
2928-2916
2850
1741
16741701
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
2920
2850
1741
1674
1030
0.00
0.04
0.08
0.12
0.16
0.20
8001600240032004000
A b s o r b a n c e
Wavenumber, cm-1
2920
2850
1741
1701
1674
1647
1550
1236
-0.002
-0.001
0.000
0.001
0.002
-0.003 -0.001 0.001 0.003
2 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Dark roast medium roast
Kenyan
-0.002
-0.001
0.000
0.001
0.002
-0.003 -0.001 0.001 0.003
2 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Dark roast Medium roast
Colombian
-0.002
-0.001
0.000
0.001
0.002
-0.003 -0.001 0.001 0.003
2 n d P r i n c i p a l C o m p o n e n t
1st Principal Component
Dark roast Medium roast
Costa Rican
-0.002
-0.001
0.000
0.001
0.002
-0.003 -0.001 0.001 0.003
2 n d P r i n c i p a l C o m p o n e n t
1st Pri