FACULTY OF RESOURCES SCIENCE AND TECHNOLOGY

Department of Chemistry

Determination of Caffeine Content in Local Coffee Products

Nur As Assyira Bt. Hasi

(63584)

Supervisor: AP Dr. Sim Siong Fong

Abstract

Caffeine (1,3,7-trimethylxanthine) is a natural alkaloid that widely consumed through

a wide range of dietary products like coffee, soft drinks, tea, cocoa beverages and energy

drinks which acts as a stimulant for central nervous system, respiratory and cardiovascular.

The two types of coffee which dominated the world coffee production are namely Coffea

arabica and Coffea robusta. However, there is no aunthentic data that show the caffeine

content in local coffee products. So, the objectives of this study are to determine the caffeine

content in local coffee products and to compare amount of caffeine content in different types

of coffee products. In this study, choloroform will be used as the medium for the extraction

process. Once the caffeine is obtained, Fourier-Transform Infrared (FTIR) spectroscopy will

be used to characteriterize the caffeine content.

DETERMINATION OF CAFFEINE CONTENT IN LOCAL COFFEE PRODUCTS

1. Introduction

Caffeine (1,3,7-trimethylxanthine) is a natural alkaloid that can be found in the seeds,

leaves and fruits of more than 63 species of plants worldwide (Violeta et al., 2010). Caffeine

is widely consumed through a wide range of dietary products like coffee, soft drinks, tea,

cocoa beverages and energy drinks. The appearance of caffeine is white, fleecy masses,

odorless powder with a slightly bitter taste (Komes et al., 2009).

Caffeine acts as a stimulant for central nervous system, respiratory and

cardiovascular. It is used to reduce physical fatigue and to restore alertness. Caffeine has the

ability to increase wakefulness, focus and flow of thought to give better body coordination

(Sethuraman et al., 2013). Moreover, caffeine does not accumulate in the body over the

course of time. It is normally excreted within several hours of consumption.

Various manufacturers market caffeine tablets, claiming that using caffeine of

pharmaceutical quality improves mental alertness. These effects have been borne out by

research that shows that caffeine use (whether in tablet form or not) results in decreased

fatigue and increased attentiveness. These tablets are commonly used by students studying

for their exams and by people who work or drive for long hours (Pullicino, 2008).

Young adults, particularly college students, are frequent consumers of caffeine,

especially energy drinks that are specifically marketed to them (Pettit & DeBarr, 2011).

There are many reasons why caffeine consumption may be popular among college students

including erratic schedules, peer influence, social situations, and lack of sleep. Like other

drugs, one can become dependent on caffeine and even build a tolerance to its psychomotor

stimulant effects, which would explain why some individuals rely heavily on caffeine to

complete routine daily functions (Rudolph et al., 2012).

2. Problem Statement

In Malaysia, the studies on caffeine content had been done for some beverages and

food: HPLC determination of methylxanthines and polyphenols levels in cocoa and chocolate

products, and phenolic and theobromine contents of commercial dark, milk and white

chocolates (Nazaruddin, et al., 2006; Ramli, et al., 2001), but not on local coffee products.

Since almost everyday people consume the coffee especially student which they claimed that

coffee can help them to not sleep, the caffeine content in coffee are important to known as it

can effects our daily activities if it is overtaken. There is no study that show the data of

caffeine content in local coffee products.

3. Objectives

The objectives of this study are;

i. To determine the caffeine content in local coffee products.

ii. To compare amount of caffeine content in different types of coffee products.

4. Literature Reviews

4.1 Caffeine

4.1.1- Background

Caffeine is the most widely consumed legal drugs worldwide that exhibits a

psychomotor stimulant effect (Yang, Palmer, & de Wit, 2010). Caffeine is naturally occurring

in a variety of substances such as coffee beans, tea leaves, and the cacao bean; the most

common sources. Caffeine is found in over 60 different plant sources (Heckman, Weil, & De

Mejia, 2010; Rudolph, Farbinger, & Konig, 2012). Synthetic caffeine, chemically identical to

natural caffeine, is added to products such as yogurt, chocolate, energy drinks, soft drinks, as

well as, some pharmaceuticals (Heckman et al., 2010). Caffeine is associated with an increase
in attention, concentration, mood, and arousal (Rudolph et al., 2012). Due to the stimulatory

effects of caffeine, it is quite popular and its consumption is widespread throughout all

different age groups. Caffeine is regarded as GRAS upto a level of 200 ppm (Da Silva, 2011).

Caffeine does not accumulate in the body over the course of time and is normally excreted

within several hours of consumption (Barone and Roberts, 1996).

4.1.2- Structure of caffeine

Caffeine is an alkaloid from the family of methylxanthine. The structure of caffeine

(1, 3, 7 – trimethylxanthine) is shown in Figure 1. Other naturally occurring methylxanthines

are theobromine and theophylline. Caffeine and its related compounds contain an imidazole

ring fused to a pyrimidine ring. Their structures are shown in Figure 2.

Figure 1: Structural formula of caffeine

Figure 2: Derivatives of caffeine

4.1.3- Uses of caffeine

Caffeine is used as a drug on the basis of its effect on respiratory, cardiovascular and

the central nervous system. It is included with aspirin in some preparations for treatment of

headaches as it decreases cerebral eye blood flow. It is included with ergotamine in some

antimigrane preparations, the object being to produce a mildly agreeable sense of alertness

(Lawrence, 1986).

Caffeine is administered in the treatment of mild respiratory depression caused by

central nervous system depressants such as narcotic (Jeanne, 1987). Caffeine may also be

used in the treatment of acute circulatory failure. In either beverage or in nonprescription

tablet form, it may be used to relieve fatigue since it increases the amount of urine flow. In

fact there are about 2000 non-prescription and about 1000 prescription drugs containing

caffeine (Jeanne, 1987).

4.1.4- Effects of caffeine

Caffeine acts as a natural stimulant in central nervous system that give the effects of

reducing drowsiness and increasing alertness. Many people seek to reap the psychomotor

effects of caffeine, which is the primary reason caffeine intake is so common. There are

specific genetic, metabolic, and thermogenic responses in the body that have an effect on

individual intake (Yang, Palmer, & de Wit, 2010; Astrup et al., 1990). Yang et al (2010)

determined that genetics have a direct effect on an individual’s response to caffeine and

personal preference. Genetics also affect the metabolism and thermogenic responses induced

by caffeine. Genetic influence on caffeine consumption changes as people age, being most

pronounced through adolescent years and then more stable in middle adulthood (Yang et al.,

2010). Genetic influence is more pronounced in heavier caffeine consumers. Twin studies
have show a large heritability component to taste preference, internal effects of caffeine, and

dependence to caffeine (Yang et al., 2010).

4.2 Coffee

4.2.1- Background

Coffee’s popularity and worldwide appeal stems from its unique flavour. It makes

coffee one of the most desirable and frequently consumed beverages as well as psychoactive

substance. According to Hecimovic et al. (2011), processing conditions of coffee affect the

caffeine content. The coffee quality used to prepare beverages is related to the chemical

composition of the roasted beans, which is in turn affected by the chemical composition of

the green beans. Furthermore, the post-harvest processing conditions such as drying, storage,

roasting and grinding also affect the quality of coffee. In placing more emphasis, Ayelign and

Sabally, (2013), said that high coffee roasting temperatures would result in loss of caffeine

which is in smaller amount by sublimation.

4.2.2- Types of coffee beans

The most important commercial species of coffee are from the genus of Coffea

arabica, Coffea robusta or Coffea canephora and Coffea liberica. The two types of coffee

which dominated the world coffee production are namely Coffea arabica and Coffea robusta.

Reports also showed that Coffea arabica and Coffea robusta constitutes 66% and 34% of the

world coffee production, respectively (Mekuria, et al., 2004).

4.2.3- Local coffee products

Almost all coffees produced in Malaysia are coffee mixtures comprising of roasted

coffee, sugar, margerine and wheat. Food Regulations 1985 states that coffee mixtures must

contain not less than 50 percent coffee beans. The caffeine content of a coffee mixture is

usually used to indicate its coffee level. Stringent labelling regulations exist but cases of

coffee manufacturers not complying with the requirements still arise. Therefore, it is
necessary from a technico-legal point of view to detect and quantify the coffee bean content

as well as to determine if labelling laws are adhered to. Malaysian Standard MS 1360

assumes that all coffee species contain 0.9% caffeine, although this is certainly not the case.

This difference in caffeine content may result in inaccuracy of coffee level determinations as

well as poses complications in enforcement practices.

4.3- Determination of caffeine using several methods

Numerous studies of biological, environmental, plants and food had been published in

determining the caffeine content with different matrices. Fernandez-Maestre and Hill (2009),

employed the optical techniques of ultraviolet (UV) for quantification of caffeine with the

limit of detection (LOD) of 5×10-4 M, whilst Ito et al. (2008) and Kovela et al. (2008),

applied FT-infrared and FT-Raman with LOD of 2×10-4 M and 9×10-5 M respectively. Del

Campo et al. (2010), on the other hand used Nuclear Magnetic Resonance (NMR)

spectroscopy with LOD of 7×10-3 M. The separation method such as gas chromatography

(GC) is used for the analysis of mixtures containing caffeine and other drugs or metabolites

(Jafari et al., 2011).

The most sensitive techniques for determination of caffeine are chromatographic

methods hyphenated with mass spectrometry. In an observation-based study with the method

of gas chromatography-ion trap tandem mass spectrometry done by Verenitch et al. (2006),

caffeine was observed with very low detection limits of 1.1×10-9 M. Belay (2010),

determined caffeine from spectral methods using Ultraviolet-Visible (UV-VIS) spectroscopy

with detection limit of 8.5×10-6 M. However, these techniques are very expensive and the

analysis can be lengthy including other additional steps such as derivatization, extraction and

purification. Fourier Transform Infrared (FTIR) spectroscopy is considered a low-cost, rapid,

accurate and reliable method for caffeine determination.

5. Materials and Methods

5.1- Sample Collection

Five different types of coffee will be obtained from local market in Kuching,

Sarawak. The brand of coffee is Mixed Coffee, Cap Tangan, Bunga Mas, Alicafe, Ah Huat

Coffee, Sin Sing Coffee O, and Teck Lee Seng. The sample will be kept in room temperature

along the experiment conduct. The standard caffeine (Sigma-Aldrich) will be traceable to

National Institute of Science and Technology, US (Shrestha et al., 2016) while the chemicals

and reagents that will be used in this study will be of analytical grade.

5.2- Caffeine Extraction

Firstly, 2 g of coffee samples will be diluted in 100 mL of boiling water (boiled for 5

min). Then, 10 mL of coffee samples that have diluted will be taken and put it into separating

funnels followed by the addition 10 mL of chloroform to each sample. Next, the separating

funnel will be shaken vigorously for 5 min. The solutions will be allowed to separate for 10

min at room temperature. Each experiment will be repeated by three times (Paradkar &

Irudayaraj, 2002).

5.3- FTIR Analysis

For the FTIR analysis, a Thermo Nicolet iS10 spectrometer equipped with an

overhead ATR accessory (Horizontal Attenuated Total Reflectance Accessory with multiple

reflections), will be used. The infrared radiation is directed to a detachable ATR diamond

crystal mounted in a shallow trough for sample containment. For the background spectra,

pure chloroform will be used and 256 codded scans will be taken at a resolution of 16 cm-1.

The samples single beam spectra, 4000 to 400 cm-1 will be obtained and corrected against the

background spectrum of chloroform, to present the spectra in absorbance units. Then, pure
chloroform will be used to clean the ATR crystal between measurements. After each

experiment, the ATR crystal will be dried with nitrogen gas to assure the best possible sample

spectra and reduce noisy spectra. The spectra will be collected in triplicates (Paradkar &

Irudayaraj, 2002).

5.4- Recovery

Different amount of coffee samples will be spiked with a known amount (0.5 g) of

analytical standards of caffeine. The standard caffeine solution in water (20 mL) will be

thoroughly mixed with coffee solution after leaching. To ensure homogenization, the spiked

samples will be shaken by using mechanical shaker for 30 min. After that, the solvent

extraction will be carried out as mentioned before. The whole process will be done only for

standard caffeine with the amount of 0.5 g for the method validation. For the unspiked coffee

sample, similar treatment will be done (Abdul Mumin et al., 2006).

The reproducibility of this method will be checked by determining the percentage

recovery of known amount of standard caffeine in the sample. The percentage recovery will

be calculated by using this equation;

% Recovery = [X’ / (X + 5) ] x 100%

Where,

X= actual caffeine content in sample

X+5= actual caffeine content in sample with addition 5ppm standard caffeine

X’= percentage recovery

5.5- Data Analysis

All measurements and analysis will be carried out in triplicates. The statistical

analysis will be done by using IBM SPSS Statistics software version 24.0. The statistical

analysis will be the analysis of variance (ANOVA) with multiple comparisons by using

Tukey’s test in order to determine any significant difference between the means at the

significant level of 95%. Next, Pearson’s correlation coefficient analysis will be conducted to

evaluate the relationship between the two variables.

6. Expected Outcome

From this study, the caffeine content in local coffee products will be extracted and

determined. All the types of coffee products will be compared based on the amount of

caffeine content. The data about caffeine content in local coffee products will be produced.

7. Workplan

2019

Year(month)/ Item Feb Mar Apr May June July Aug Sept Oct

Literature Review

Sample collection in local market

Sample Preparation

Sample Extraction

Determination of Caffeine

Data Analysis

Report Writing

Preparation for Presentation

Presentation
8. References

1) Abdul Mumin, M., Akhte, K. F., Abedin, M. Z., & Hossain, M. Z. (2006). Determination

and characterization of caffeine in tea, coffee and soft drinks by solid phase extraction

and high performance liquid chromatography (spe-hplc). Malaysian Journal of

Chemistry, 8(1), 45-51.

2) Ayelign A., & Sabally K. (2013). Determination of cholorogenic acids (cga) in coffee

beans using hplc. American Journal of Research Communication, 1(2), 78-91.

3) Bacsik, Z., Mink, J., & Keresztury, G.(2004). FTIR spectroscopy of the atmosphere. i

.principles and methods. Applied Spectroscopy Reviews, 39(3), 295-363.

4) Belay, A. (2010). Measurement of integrated absorption cross-section, oscillator strength

and number density of caffeine in coffee beans by integrated absorption coefficient

technique. Food Chemistry, 12, 585–590.

5) Cheng, C.M., Mhd Jalil, A. M., & Ismail, A. (2009). Phenolic and theobromine contents

of commercial dark, milk and white chocolates on the malaysia market. Molecules, 14,

201-209.

6) Craig, A. P., Franca, A. S., & Oliveira, L. S. (2012). Evaluation of the potential of FTIR

and chemometrics for separation between defective and non-defective coffees. Food

Chemistry, 132, 1368-1374.

7) Farah, A. (2012). Coffee constituents in: coffee emerging health effects and disease

prevention. Yi-Fang C, Ed Wiley-Blackwell Oxford. 1, 21-25

8) Fernandez-Maestre, R., & Hill, H. H. (2009). Ion mobility spectrometry for the rapid

analysis of over-the-counter drugs and beverages. International Journal for Ion Mobility

Spectrometry, 12, 91–102.

9) G., Berregi, I., Caracena, R., & Zuriarrain, J. (2010). Quantitative determination of

caffeine, formic acid, trigonelline and 5-(hydroxymethyl) furfural in soluble coffees by

1h nmr spectrometry. Talanta, 81, 367–371

10) Hecimovic, I., Belscak-Cvitnovic, A., Horzic D., & Komes D. (2011). Comparative

study of polyphenols and caffeine indifferent coffee varieties affected by the degree of

roasting. Food Chemistry, 129, 991-1000

11) Ito, M., Suzuki, T., Yada, S., Kusai, A., Nakagami, H., & Yonemochi, E. (2008).

Development of a method for the determination of caffeine anhydrate in various

designed intact tables by near infrared spectroscopy: a comparison between reflectance

and transmittance technique. Journal of Pharmaceutical and Biomedical Analysis, 47,

819–827.

12) Islam, M. S., Rahman, M. M., & Abedin, M. Z. (2002). Isolation of caffeine from

commercially available tea and tea waste. Journal of Science, 25, 9.

13) Jafari, M. T., Rezaei, B., & Javaheri, M. (2011). A new method based on electrospray

ionization ion mobility spectrometry (esi-ims) for simultaneous determination of caffeine

and theophylline. Food Chemistry, 126, 1964–1970.

14) Koleva, B. B., Kolev, T. M., Tsalev, D. L., & Spiteller, M. (2008). Determination of

phenacetin and salophen analgetics in solid binary mixtures with caffeine by infrared

linear dichroic and raman spectroscopy. Journal of Pharmaceutical and Biomedical

Analysis, 46, 267–273.

15) Komes, D., Horzic, D., Belscak, A., Kova K.C., Ganic, C., & A. Baljak, K. (2009).

Determination of caffeine content in tea and maté tea by using different methods. Czech.

Journal Food Science, 27, 213-216

16) Leloup, V. (2006). Evaluation of the nutritive value of soluble coffee. In Proceedings of

21st colloquium ASIC, Montpellier, France, p. 80-87

17) Lyman, D. J., Benck, R., Dell, S., Merle, S., & Murray-Wijelath, J. (2003). FTIR-ATR

analysis of brewed coffee: effect of roasting conditions. Journal of Agricultural and

Food Chemistry, 51, 3268–3272.

18) Mazzafera, P., & Silvarolla, M. B. (2010). Caffeine content variation in single green

Arabica coffee seeds. Seeds Science Research, 20, 163-167

19) Murkovic, M., & Pichler, N. (2006). Molecular Nutrition and Food Research, 50, 842–

846.

20) Nazaruddin, R., Seng, L. K., Hassan, O., & Said, M. (2006). Effect of pulp

preconditioning on the content of polyphenols in cocoa beans (theobroma cacao) during

fermentation. Industrial Crops and Products, 24, 87-94.

21) Paradkar, M. M., & Irudayaraj, J. (2002). A rapid ftir spectroscopic method for estimation of

caffeine in soft drinks and total methylxanthines in tea and coffee. Journal of Food Science,

67, 2507-2511

22) Ramli, N., Mohd Yatim, A., Said, M., & Heng, C. H. (2001). HPLC determination of

methylxanthines and polyphenols levels in cocoa and chocolate products. Malaysian

Journal of Analytical Sciences, 7(2), 377-386.

23) Ribeiro, J. S., Salva, T. J., & Ferreira, M. M. (2010). Chemometric studies for quality

control of processed brazilian coffees using drifts. Journal of Food Quality, 33, 212–227.

24) Sereke-Brhan, H. (2017). Coffee, culture, and intellectual property: lessons for africa

from the ethiopian fine coffee initiative. Retrieved Sept 5, 2018, from

https://open.bu.edu/bitstream/handle/2144/22672/Pardee-Paper-11-Ethiopian-Fine

Coffees.pdf

25) Shrestha, S., Rijal, S. M., Pokhrel, P., & Rai, K. P. (2016). A simple hplc method for

determination of caffeine content in tea and coffee. Journal of Food Science, 9, 74-78.
26) Verenitch, S. S., Lowe, C. J., & Mazumder, A. (2006). Determination of acidic drugs and

caffeine in municipal wastewaters and receiving waters by gas chromatography-ion trap

tandem mass spectrometry. Journal of Chromatography, 1116, 193–203.

27) Violeta, N., Trandafir, I., & Ionica, M. E. (2010). Chromatographic determination of

caffeine contents in soft and energy drinks available on the romanian market, 11, 351-

358.