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DETERMINATION OF CAFFEINE, CHLOROGENIC ACID ANDNICOTINIC ACID IN COFFEE BEANS BY USING HPLC

NOR HANISAH BINTI MAT YASSIN

BACHELOR OF SCIENCE (Hons.) CHEMISTRYFACULTY APPLIED SCIENCESUNIVERSITI TEKNOLOGI MARA

NOVEMBER 2008


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DETERMINATION OF CAFFEINE, CHLOROGENIC ACID AND

NICOTINIC ACID IN COFFEE BEANS BY USING HPLC

NOR HANISAH BINTI MAT YASSIN

Final Year Project Report Submitted in

Partial Fulfilment of the Requirements for theDegree of Bachelor of Science (Hons.) Chemistry

in the Faculty of Applied Sciences

Universiti Teknologi MARA

NOVEMBER 2008


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ACKNOWLEDGEMENTS

Alhamdulillah and thanks to Allah S.W.T for his blessing which enable me to completemy final project. Firstly, I would like to thank my supervisor, Pn. Haliza Kassim for her

support, guidance and inspiring suggestions during the preparation for final year project

until the end my project. My appreciation also goes Dr. Famiza Abdul Latif as a

coordinator of final year project, for her information and guidance to this course. To my

parents, En. Mat Yassin Abdullah and Wan Mainumanah Wan Abd Hamid, thank as

much for supporting during my final project. Finally, I would like to express my gratitude

to those who helped me directly and indirectly involved in this final year project.

Nor Hanisah Mat Yassin


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TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS iiiTABLE OF CONTENTS iv

LIST OF TABLES vi

LIST OF FIGURES viiLIST OF ABBREVIATIONS viii

ABSTRACT ix

ABSTRAK x

CHAPTER 1 INTRODUCTION

1.1 Background 11.2 Significance of study 11.3 Objectives of study 3

CHAPTER 2 LITERATURE REVIEW

2.1 Coffee 42.2 Roasting 42.3 Chemical effects 5

2.3.1 Caffeine 62.3.2 Chlorogenic acid 72.3.3 Nicotinic acid 8

2.4 Method development 92.4.1 Sample preparation 102.4.2 HPLC analysis 10

CHAPTER 3 METHODOLOGY

3.1 Chemicals 113.2 Coffee samples 113.3 Sample preparation 11

3.3.1 Standard 11

3.3.2 Roasted coffee beans 11

3.3.3 Brewed coffee beans 123.4 HPLC analysis 12

CHAPTER 4 RESULTS AND DISCUSSION

4.1 Effect of roast time in coffee samples 14

4.2 Different amount of chlorogenic acid in roasted andbrewed coffee samples 16


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CHAPTER 5 CONCLUSION AND RECOMMENDATIONS 19

REFERENCES 20

APPENDICES 23CURRICULUM VITAE 37


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LIST OF TABLES

Table Caption Page

4.1 Roasting characteristic of the coffee used 144.2 Amount of chlorogenic acid 16


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LIST OF FIGURES

Figure Caption Page

2.3.1 Structure of caffeine 72.3.2 Structure of chlorogenic acid 72.3.3 Structure of nicotinic acid 83.0 Flow chart of experiment 134.1 Chromatogram of roasted robusta coffee 15

(A) for eight minute and (B)five minute at

106

0

C

4.2 Chromatogram of roasted robusta coffee 17

(A) and (B) for brewed coffee sample


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LIST OF ABBREVIATIONS

CGA : Chlorogenic acid

HPLC : High Performance Liquid Chromatography

m : Micrometer

l : Microliter

mg : Miligram

ml : Mililiter

mm : Millimeter

mM : Milimolar

Er : Relative error

RP : Reversed Phase

v/v : Volume per volume

: Wavelength


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ABSTRACT

DETERMINATION OF CAFFEINE, CHLOROGENIC ACID AND

NICOTINIC ACID IN COFFEE BEANS BY USING HPLC

Coffee usually comes in two types of coffee beans. They are coffee arabica andcoffee robusta. These coffees contain many chemicals that are affected during the

roasting process. The coffee contains caffeine, chlorogenic acid and nicotinic acid

which give a variety of the effect that is both beneficial and harmful to human.This research will determine the amount of compound such as caffeine,

chlorogenic acid and nicotinic acid present in coffee after roasting. The coffee

beans were roasted in an oven and grounded coffee was brewed in freezer. Thesample preparation was quite simple involving only heating water extraction and

filtration. Analytical HPLC unit consists of Water 717 plus Autosampler andWater 600 Controller, Water 2487 Dual Absorbance detector, a 150 x 4.6 mm

i.d. Merck Superspher 100 Reversed Phase (RP), C-18 column with 5 m particlesizes. The analysis is uses reversed phase (RP) column with gradient elution of 10

mM citric acid (A) and methanol (B). Robusta coffee beans contained the highest

amount of caffeine compared with arabica were, 36.0290 mg/ml and 35.2950

mg/ml respectively. The amount nicotinic acid in roasted coffee arabica for five

minute was 0.5859 mg/ml and roasted coffee robusta was 0.7000 mg/ml. The

amount of chlorogenic acid in brewed coffee samples is smaller than roastedcoffee sample. In roasted coffee sample, the amount of CGA in arabica and

robusta were 10.8936 mg/ml and 15.1398 mg/ml respectively.


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ABSTRAK

PENENTUAN JUMLAH CAFFEINE, CHLOROGENIC ACID AND

NICOTINIC ACID DALAM BIJI KOPI MENGGUNAKAN HPLC

Kebiasaannya kopi hadir dalam dua jenis biji kopi. Ianya adalah kopi arabica dankopi robusta. Kopi tersebut mengandungi banyak bahan kimia yang akan

memberi kesan semasa proses memanggang. Kopi mengandungi caffeine,

chlorogenic acid and nicotinic acid dimana akan memberi pelbagai faedah dan bahaya kepada manusia. Kajian ini akan menentukan jumlah sebatian seperti

caffeine, chlorogenic acid dan nicotinic acid yang hadir selepas dipanggang. Biji

kopi dipanggang di dalam ketuhar dan diperam dalam peti sejuk. Persediaan bahan adalah ringkas yang hanya melibatkan perahan melalui pemanasan air.

HPLC analisis mengandungi Water 717 plus Autosampler dan Water 600Controller, Water 2487 Dual Absorbance pengesan, a 150 x 4.6 mm i.d. Merck

Superspher 100 Reversed Phase (RP), C-18 kolum dengan 5 m saiz partikel.Analisis ini menggunakan kolum fasa berbalik dengan kecerunan oleh 10mM

citric acid (A) dan methanol (B). Kopi robusta mengandungi jumlah caffeine

yang tinggi berbanding dengan arabica adalah 36.0290 mg/ml dan 35.2950mg/ml. Jumlah nicotinic acid dalam kopi arabica yang dipanggang selama lima

minit adalah 0.5859 mg/ml dan untuk kopi robusta adalah 0.700 mg/ml. Jumlah

chlorogenic acid (CGA) dalam kopi yang diperam adalah sedikit berbanding kopiyang dipanggang. Kopi yang dipanggang, jumlah CGA dalam arabica dan

robusta masing-masing adalah 10.8936 mg/ml dan 15.1398 mg/ml.


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CHAPTER 1

INTRODUCTION

1.1 Background and problem statement.

Coffee is one of the worlds most popular beverages. It also produces pleasant

taste and aroma. Coffee usually comes in two types of coffee beans. They are

Coffee Arabica and Coffee Robusta. These coffees contain many chemicals that

are affecting during the roasting process. The roasting process is a process that is

time and temperature dependent whereby chemical changes are induced in the

coffee beans. There are several analytical methods available for determination of

these components in coffee. High Performance Liquid Chromatography (HPLC)

is usually used by researchers to study the presence of biochemical changes in

coffee beans. Determination of caffeine, chlorogenic acid and trigonelline in

coffee was developed by using different reverse phase (RP) High Performance

Liquid Chromatography (HPLC) methods (Trugo et al., 1983) and chlorogenic

acid analysis (Duijn and van der Stegen, 1980; Macrae and Trugo, 1984) but not

for the simultaneous analysis of all three compounds. These chemicals in coffee

give the side effect to human drinking it in large amounts.

1.2 Significance of study

Like many other foods we eat and drink, the composition of coffee is very

complex. It depends on the species and variety of plant, the way they are grow


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and the way beans are picked and processed. However coffee contains a very

wide range of macro and micro nutrients and, as one of the most popular

beverages consumed worldwide, it is worth considering what nutritional

contribution the coffee can make to our diet. There are many compounds in coffee

that are often thought to have implications upon human health. These include

caffeine, micronutrients and chlorogenic acid. There are several types of side

effects produced such as physiological effect, energy metabolism, psychoactive

and neurological effect. Drinking a cup of coffee is therefore helpful in

counteracting sleepiness during the day and mental sluggishness, brought about

long concentration and mental effort such as in a job. More importantly, caffeine

has been shown to induce a positive effect and it is able to increase an

individuals mood that makes coffee an important source of pleasurable activity

and individual happiness.The nutritional, flavor and toxicological properties of

coffee are affected by roasting or thermal degradation of several compounds. The

coffee contains caffeine, chlorogenic acid and nicotinic acid which give a variety

of the effect either beneficial or risky to human. The excessive amount of coffee

can give bad side effects to those who drink it.


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1.3 Objectives of study

1. To determine the amount of chlorogenic acid, caffeine and nicotinic acid

in two different type of coffee beans.

1. Coffee Robusta

2. Coffee Arabica

2. To determine if roasting time changes the amount of caffeine, chlorogenic

acid and nicotinic acid present.

3. To determine the amount of chlorogenic acid in brewed coffee samples.


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CHAPTER 2

LITERATURE REVIEW

2.1 Coffee

Coffee is most popular around the world. There are Coffee Arabica and Coffee

Canephore usually known as Coffee Robusta. Arabica or Robusta beans or blends

of them are commercial coffee beverage, the Arabica being considered of better

quality (Macrae, 1985). There are many compounds in coffee that are often

thought to have implications upon human health. These include caffeine,

micronutrients and chlorogenic acid. The coffee beverage is rich in biologically

active substances such as nicotinic acid, trigonelline, quinolinic acid, tanninc acid,

pyrogolic acid and caffeine (Minamisawa et al., 2004).

2.2 Roasting

Roasting is a complex process from the chemistry point of view since hundreds of

chemical reactions occur simultaneously. The roasting process is integral to

producing a savory cup a coffee. The green coffee bean expands to nearly double

its original size, changes in color, taste, smell and density. Some examples such as

Maillard and Strecker reactions, degradation of proteins, polysaccharides,

trigonelline and chlrogenic acid (Alviano, Aquino Neto, De Maria, Moreira and

Trugo, 1996). The pharmacological effect of coffee has been related with caffeine


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(Wurzner, 1988) and both chlorogenic acid (CGA) and trigonelline have been

associated with flavor formation and production of aroma during the roasting time

(Clifford, 1985). Niacin in particular is formed in great amounts from trigonelline

during roasting process (Casal et al., 2000; Czok, 1977). The nutritional, flavor

and toxicological properties of coffee are affected by roasting or thermal

degradation of several compounds. Natural nitrogen compounds such as

trigonelline (N-methylbetaine of pyride-3-carboxylic acid), nicotinic acid

(pyridine-3-carboxylic acid) and caffeine (1, 3, 7-trimethylxanthine) are present in

coffee beans (Clifford, 1985; Macrae, 1987) that change chemically during

roasting procedure. Thermal degradation of chlorogenic acids will result in the

production of phenolic substances that will contribute to bitterness of the coffee

(Clifford, 1985). The highest amount of transformation from chlorogenic acid to

the corresponding lactones during the medium roasting condition, suggests that

this process reduced the amount of chlorogenic acid in coffee (Farah et al., 2005).

2.3 Chemical effects

According to Vatten et al. (1990), the coffee consumption reduces the incidence

of breast cancer in lean women, whereas it might have opposite effect in relatively

obese women. Coffee-drinking had no significant effect on the lung cancer risk of

cigarette-smoking lung cancer patients compared with matched controls

(Mendilaharsu et al., 2008). There are also several types of physiological effects,


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energy metabolism, psychoactive and neurological effects of coffee drinking. The

various components such as caffeine and chlorogenic acid or caffeic acid in coffee

can affect the gastrointestinal tract and liver also the biliary system. The

stimulating effect on these organs can be caused by direct or indirect liberation of

gastin (Czok, 1977). Coffee drinking was also associated to decrease risk of

alcohol associated pancreatitis (Morton et al., 2004). According to Naismith et al.

(1970), increased coffee consumption reduced the plasma glucose level. The

effect of coffee drinking in obese and control subjects was studied by Acheson et

al., (1980). The metabolic rate was increased and fat oxidation were observed

only in the control group. According Zahorska-Markiewiez (1980), the increased

metabolic rate flows were from the breakfast ingestion of caffeinated coffee. The

effect of coffee consumption on the nervous system goes beyond alertness and

mood changes. The consumption of a few cups of coffee strengthens the central

information processing, specifically the monitoring of ongoing cognitive process,

foe signs of erroneous outcomes. Smith et al. (2003) suggested caffeine added to

coffee showed positive effects on the speed of encoding of new information.

2.3.1 Caffeine

Caffeine is a chemical compound that is made by plants. Caffeine is classified as

a methylxanthine alkaloid. It is a central nervous system stimulant. It is a white

powder in its pure form that is odorless with a slightly bitter taste. There are 63

different species of plants present known to contain caffeine. Historically, the

most common food sources of caffeine in the human diet have been found in

coffee.


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N

N

O

ON

N

H3C

CH3

CH3

Figure 2.3.1 Structure of caffeine.

There has been no association established between moderate caffeine drinking

and increased risk to health. Caffeine may produce a mild withdrawal syndrome

on discontinuance of regular use such as drowsiness and headache. Caffeine

exerts most of its effects through the antagonism of caffeine are elevated blood

pressure, an increase in ones metabolic rate and stimulation of the central

nervous system.

2.3.2 Chlorogenic acid (CGA)

Chlorogenic acids are family of esters formed between trans-cinnamic and quinic

acid. Usually the individual chlorogenic acid is formed between caffeic acid and

qunnic acid .

OH

OH

OO

OH

OHHO

O

HO

Figure 2.3.2 Structure of chlorogenic acid (CGA).


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Chlorogenic acid is also a major phenolic compound in coffee and have been

found in several plants and can be isolated from the leaves and fruit. This

compound long known as an antioxidant also slows the release the glucose into

the bloodstream after a meal (Clifford, 2003). Both chlorogenic acid and caffeic

acid are strong antioxidants in vitro (Rice-Evans, 1996). Chlorogenic acids (CGA)

are known for their contribution to the final acidity, astringency and bitterness of

the beverage. CGA and their lactones also not only contribute to coffee flavor but

also may be of potential biopharmacological importance to human. The

pharmacological activities of phenolic compound such as CGA have been related

with antioxidant properties because they are thought to have positive effects on

depression, alcoholism, chronic degenerative diseases, cardiovascular disease and

cancer.

2.3.3 Nicotinic Acid

Nicotinic acid (niacin) is a form of vitamin B3 which can be found naturally in

meat, eggs and dairy products also found in coffee. It is also available as an over

the counter supplement as vitamin B3 or niacin.

N

O

OH

Figure 2.3.3 Structure of nicotinic acid.


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Niacin is precursor to NADH, NAD, NAD+

and NADP which play essential

metabolic roles in living cells (Nelson and Cox, 2003). Niacin is sometimes used

in the treatment of high cholesterol. Nicotinic acid is also formed by the

pyrolysis of trigonelline (Viani and Horman, 1974). According to Taguchi and

Shimabayashi (1983), the trigonelline found in dimetylating enzyme activity

widely in nature but the activity is generally low for nicotinic acid formation.

Accordingly, the trogonelline content in coffee beans and the process of heating

coffee bean at higher thermal conversion of trigonelline into nicotinic acid were

investigated. Based on biological synthesis, humans do have the ability to

synthesize sufficient nicotinic acid; this means that it is an essential component of

balanced diet. If deficiency occur a disease caused by a lack of nicotinic acid is

known as Pellagra. This disease affects epithelia and nervous system.

2.4 Method Development

Caffeine, trigonelline and 5-Caffeoyilquinine acid concentrations were also

measured of these substances could vary with coffee quality (Franca et al., 2004).

The compounds analyzed simultaneously were trigonelline, nicotinic acid and

caffeine by a HPLC/diode array detector method (Casal et al., 1998).


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2.4.1 Sample preparation

All chemicals used were of analytical grade (Merck, Darmstadt). The extracted

samples were filtered and vacuumed (Bell, Grand and Wetzel, 1996). An aliquot

of the solution was filtered through a 0.2 m pore size membrane. Moisture was

determined by drying the samples at 1030C until constant weight. A 2.0 g portion

of grounded coffee was extracted with 100 mL of boiling water (Casal, Ferreira

and Oliviera, 1998).

2.4.2 HPLC analysis

The simultaneous determination of caffeine and trigonelline in coffee (Trugo et

al., 1983) and chlorogenic acid analysis (Dujin and van der Stegen, 1980; Macrae

and Trugo, 1984) have been developed by different reversed phase (RP) HPLC

method. A solvent gradient was formed with phosphate buffer 0.1 M (pH 4.0) and

methanol (Casal et al., 1998). Caffeine and chlorogenic acid contents were

evaluated by means of reversed phase (RP) HPLC with UV detection at 254 nm.

The procedure is a modification at previously reported works (Macrae and Trugo,

1984) performed at isocratic mode using a mobile phase 20% v/v methanol/water

at pH 4.5. The retention time (tR), 4 minute 36 second for nicotinic acid and tR, 20

minute 10 sesond for caffeine (Casal etal., 1998) was obtained using diode array

detector


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CHAPTER 3

METHODOLOGY

3.1 Chemicals

Caffeine, chlorogenic acid and nicotinic acid standards were purchased from local

vendors. All other chemicals are used of analytical grade.

3.2 Coffee sample

The samples of coffee beans were purchased from local market.

3.3 Sample preparation3.3.1 Standard

10.0 mg of nicotinic acid and caffeine for a single standard and 15.0 mg of

caffeine and 0.60 mg of nicotinic acid for mixture which contained the

proportions usually present in coffee was placed in beaker and was roasted in

oven at 1060C for 5 and 8 minutes. 10.8 mg of chlorogenic acid was roasted to

compare with chlorogenic acid in brewed coffee sample. 0.3838 g of chlorogenic

acid in brewed coffee was prepared in methanol.

3.3.2 Roasted coffee beans

The coffee beans were roasted in an oven at 1060C for 5 minute and 8 minute.

The coffee beans are then ground into fine powder to pass through a 25-mesh


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sieve/0.75 mm sieve. The sample was dried at 1030C until constant weight was

obtained. 2.0 g of ground coffee was mixed with 20 mL of water in an

Erlenmeyer flask and heat with stirring for five minutes. The solution is then

transferred to a volumetric flask and diluted to the mark. The solution was filtered

by 0.45m syringe filter paper.

3.3.3 Brewed coffee beans

2.5 g grounded-roasted coffee was brewed with 90 mL deionized water. The

grounded coffee was mixed with 20 mL of water in an Erlenmeyer flask and boil

with stirring for five minutes. The solution was filtered by 0. 45m syringe filter

paper. The brewed coffee was immediately cooled to room temperature in an ice

bath, after which sample were stored at 50C until required for HPLC analysis.

3.4 HPLC analysis

Analytical HPLC unit consists of Waters 717 plus Autosampler and Waters 600

Controller, a variable wavelength Waters 2487 Dual Absorbance detector, a 150

x 4.6 mm i.d. Merck Superspher 100 RP (Reversed Phase) 18 column (5 m

particle size). The mobile phase consists of two composition, mobile phase A

which is 10 mM citric acid and mobile phase B is methanol. The gradient mode is

initially set at A: B ratio of 85:15 from 0 to 5 minutes and will increase to 60:40 at

40 to 85 minutes. The detector is set up at 325 nm for chlorogenic acid, 264 nm

for nicotinic acid and 276 nm for caffeine.


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Figure 3.0 Flow chart of experiment.

Roasting

Grind

Drying

Extract

Filter

HPLC analysis

Coffee

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