Caffeine: Characterization and Properties
S Oestreich-Janzen, CAFEA GmbH, Hamburg, Germany
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
In the minds of most Europeans caffeine is strongly associated
with coffee. Indeed, this compound was first isolated in coffee
200 years ago, and this isolation led to its naming. Caffeine
occurs in plants of different families, with traditional styles of
human consumption. It has a mild stimulating effect on the
central nervous system. Caffeine-containing coffee and tea
plants are important contributors to the global economy.
Since the seventies of the last century, the biosynthesis of
caffeine, its in-plant transfer, and degradation have been thor-
oughly investigated.
Studies on caffeine’s mechanism of action, stimulation, and
health effects are ongoing. New fields of use have provoked
increasing research. The next article in this encyclopedia is
dedicated to caffeine consumption and related health effects.
Discovery and Naming
In 1819, the German poet Goethe presented some coffee beans
to the chemist F.F. Runge in Jena for investigation. Runge was
successful in this task. The aqueous extraction of the beans,
both green and roasted, the precipitation of coffee acids, and
separation eventually allowed the crystallization of the sub-
stance he called ‘Kaffeebase,’ the base of coffee, identifying its
activity on salamanders. He published his methodology and
product description in early 1820. Much later, in 1866, Runge
recounted the start-up story, with his introduction to Goethe.
Concurrent research on coffee was done using similar pro-
cedures at some other centers in Europe. Within a short interval
of time, multiple investigators had identified the same
substance. Later in 1820, F. von Giese published his findings
on coffee in present-day Estonia, calling the substance ‘Kaf-
feestoff.’ In 1821, the French senior pharmacist P. J. Robiquet
verbally presented his results to the pharmaceutical society in
Paris, and he used the word ‘caféine,’ which he had adopted
from an unconvincing attribution by R. Chenevix, some years
earlier. This name was accepted by scientific consensus, and in
1822, it was accepted for a short entry in a dictionary of
medicine by P.J. Pelletier. The following year, the term caffeine
came out at the Royal Academy of Science in Paris, in a report
on alkaloids by Pelletier, in which he mentioned Robiquet’s
earlier note. Pelletier and his companion B. Caventou had
ceased their own research on coffee in favor of Robiquet. In
1823, the latter published the details of his extraction as part of
another dictionary’s article on coffee, and his procedure
became the standard methodology of its time, being discussed
in depth in the French pharmaceutical journal (Journal de
Pharmacie et des Sciences Accessoires) in 1826.
The community researching caffeine in Paris was open to
guest scientists from all over Europe, and members communicat-
ed with corresponding societies via the exchange of letters and
printed proceedings, posted per stagecoach.
556 Encyclopedia of Food and Health
Reporting to the Swedish academy about the research in
France and the related notes of 1826, with all his authority, J.J.
Berzelius acknowledged the seniority of Runge on the isolation
of caffeine, thus disclosing his significance to the European
scientific community.
In the following years, the search for the active mechanisms
of other stimulating plants of human interest and use resulted
in several discoveries of substances that were later identified to
be identical to caffeine. In 1826, guaranine was detected in
guaraná seeds by T. Martius. In 1827, V. Oudry identified
theine in tea leaves, and its equivalence to caffeine was clarified
by Martius in 1840 and C. Jobst in 1838. In 1836, an alkaloid
was found in maté leaves by B. Trommsdorf, and this sub-
stance was identified as caffeine by J. Stenhouse in 1843. In
1865, caffeine was detected in the kola nut by W.F. Daniell,
and in cocoa beans by E. Schmidt in 1883. All these researchers
thoroughly described and characterized the compound, based
on its physical and chemical properties, as crystalline needles,
slightly bitter tasting, and soluble in hot water.
Caffeine’s molecular formula, C8H10N4O2, was determined
by C. H. Pfaff and J. Liebig in 1832. In the century’s last quarter,
the eventual structural formula of caffeine was fixed (Figure 1),
first proposed by L. Medicus of Wuerzburg, Germany, based on
cleavage reactions in 1875, and later confirmed via synthesis
from well-known educts by E. Fischer and L. Ach in Berlin in
1898. The total synthetic pathway is shown in Figure 2.
Dimethylurea reacts with malonic acid to form the
pyrimidine ring with properly placed methyl and keto groups;
nitrosylation with nitrous acid, with subsequent reduction on
platinum, introduces the amino group in position 5, where
reaction with potassium cyanate provides the missing atoms
for the imidazole part, followed by acidic ring closure to form
dimethyl uric acid. Via chlorination with phosphorus pen-
tachloride in position 8, as well as reduction with fuming hydro-
iodic acid, the dimethyl xanthine theophylline is produced. The
last step is methylation with methyl iodide in position 7 to yield
caffeine. The synthesized caffeine was checked against naturally
occurring caffeine, using mixed melting point methods.
Nomenclature
In the nomenclature of the twenty-first century, the molecule
caffeine is referred to as 1,3,7,trimethyl-2,6-dihydro-purin-2,6-
O
H 3C CH3
N N
O N N
CH3
Figure 1 Caffeine – structural formula.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00098-2
 Caffeine: Characterization and Properties 557

OH O O
O H
H3C H3C H3C N NH2
NH O H3C
N 1. NO
+
N
NH2 N
KOCN
O
O O OH O O N O
NH O N O O N
2. Red.
CH3 CH3 CH3
H3C

Dimethylurea Malonic acid

O O O O
H3C H3C CH3
NH N NH H3C H3C
+ N PCI5 HI N NH H3CI N N
H
O CI
O N N O N N O N N O N N
H
CH3 CH3 CH3 CH3
Dimethyluric acid Caffeine

Figure 2 Reaction scheme of Fischer’s synthesis of caffeine 1898 (edited from Kunz, H., (2002). Emil Fischer – unequalled classicist, master of organic
chemistry research, and inspired trailblazer of biological chemistry. Angewandte Chemie, International Edition, 41, 4447).

dione, or by the shortened form 1,3,7-trimethylpurine-2,6- 6 H

5
dione. Caffeine is accepted as synonymous, however. The par- N7
1 N
ent skeleton is purine, a bicyclic system, which consists of two 8
fused heterocycles, the five- and six-membered imidazole and 2
N
N 4
pyrimidine, respectively. The multiplicative prefix trimethyl 3 9

marks the substituents, and the suffix dione indicates the func-
tional keto groups in caffeine. Purine is a constituent of nucleic
acids.
The name purine was created by Emil Fischer in 1884. In
1897, he proposed its ring numbering, as shown in Figure 3.
This customary naming and numbering was used in the stan-
dard reference works of that time, the German Beilstein Hand-
book of Organic Chemistry and in the American Chemical
Abstracts since their first editions. In 1957, the parent name
purine was declared to be a ‘retained trivial name’ in the rules
for the nomenclature of organic chemistry established by the
International Union of Pure and Applied Chemistry (IUPAC),
which was based in Oxford, United Kingdom, at the time, and
in the same paper Fischer’s customary numbering system for
purine was accepted as an exception to the systematic number-
ing. These naming conventions were then confirmed repeat-
edly. In December 2013, with a new approach in organic
nomenclature, purine was upgraded to a ‘preferred IUPAC
name’ (PIN), a decision published in the IUPAC’s actual Blue
Book. The IUPAC’s naming principles continue to “allow alter-
natives, in order to preserve the diversity and adaptability of
the nomenclature to daily activities in chemistry and in science
in general,” as stated in the Blue Book, that is Favre and Powell.
There is no doubt that this modus operandi would matter to
purine (PIN) and caffeine. ‘Caffeine’ is allowed, without explicit
IUPAC mentioning, as an alternative for the trimethylated
purine dione. The more comprehensive name is 3,7-dihydro-
1,3,7-trimethyl-1H-purine-2,6-dione. Another trivial name
widely used for the unmethylated purine dione is xanthine; its
subsequent methylation forms part of the well-studied metabo-
lism involved in caffeine biosynthesis and degradation. Caffeine
may also be named 1,3,7-trimethyl-xanthine. Figure 4 shows
the covalent structure of caffeine – the customary skeleton num-
bering is marked at the locants. Classic mesomeric zwitterionic
structures using the valence bond model (depicted in Figure 4(a))
have stepped down in favor of the molecular orbital view;
Figure 3 Purine – structural formula with customary numbering.
computational methods with software package allow calculation
of partial charges (marked in Figure 4(b) and visualization
of molecules.
Physical and Chemical Property and Safety Data
The chemical and physical data for caffeine are well known. It
is a solid, odorless, slightly bitter-tasting, sublimating sub-
stance that crystallizes into white needles. In solution, it
absorbs in the ultraviolet region, and solubility differences
aid analytical separation and purification. Caffeine is both
hydrophilic and lipophilic, thus easily passing through biolog-
ical membranes and barriers. Its bitterness is perceived
throughout the oral cavity; it is described as long lasting, and
it is used as a standard for bitter-tasting calibrations. When
crystallized from aqueous solutions, caffeine forms a nonstoi-
chiometric stable hydrate. Caffeine salts, like citrate, are part of
pharmaceutical compounds. Anhydrous caffeine occurs in two
modifications. The low-temperature-stable form is the b mod-
ification, and at 141
C, it is transformed to the a modification,
which has a higher rate of dissolution. On cooling, the a phase
converts very slowly back to the b phase, which is important for
pharmaceutical preparations.
Several spectroscopic methods are available for caffeine
analysis and authenticity control. The basic spectra are refer-
enced in the analytical section.
Caffeine in solutions undergoes self-assembly: When a crit-
ical aggregation concentration is exceeded, caffeine molecules
in solution cluster predominantly to dimers, with coplanar
positions of the caffeine moieties, staggered at about 90
,
with a distance of 3.5 Å.
The association of caffeine with the different phenolic com-
pounds of caffeine plants follows the same principles. The
558 Caffeine: Characterization and Properties

−
O CH3
H3C + N
N −0.054
O −0.470
CH3 O CH3
O N N −0.008
H3C N H3C
N1
6
7 CH3
−0.432 C −0.402
N −0.033
5
8 N 0.610 C 0.216

2 4
O CH3 C H
0.170
9 0.505 0.357
3 H3C N C C
O N N N −0.342
N
−0.454O N −0.566
− + N
CH3 O N
O CH3 0.093 CH3
H 3C + N CH3
N

O− N N

(a) CH3 (b)

Figure 4 Caffeine – covalent structure with customary numbering, mesomeric structures (a) and atomic partial charges (b), the latter with
data from Tavagnacco, L., Schnupf, U, Mason, P. E., et al., (2011). Molecular dynamics simulation studies of caffeine aggregation in aqueous solution.
Journal of Physical Chemistry B. 115, 10957–10966.

complexes are investigated since the fifties of the last century,
inter alia by using ultraviolet (UV), infrared (IR),
crystallographic, and nuclear magnetic resonance (NMR)
data. With the latter method, the associates are observed by
changes in the NMR chemical shift of caffeine’s methyl pro-
tons, studied both in model solutions and in coffee brew.
Table 1 shows the property figures of caffeine, outlined in
the Chemical Abstracts Register, which contains references offer-
ing additional information.
In regard to occupational health effects, the vendors of chemi-
cals are obliged to provide a material safety data sheet for the
substances in their portfolio, with property and safety data as well.
For caffeine, the acute toxicity estimates on the possible route of
intake reported are low, and the risk of accidental ingestion,
contact, or inhalation at the worksite is minor. Thus, in the hazard
classes under scrutiny, caffeine is assigned to the least hard cate-
gories. The respective labeling is at the lowest warning level. For
transport, no dangerous goods marking is necessary.
Attempts to manage this information led to a compilation
of International Chemical Safety Cards, a system in which
caffeine is included with a peer review status, as of 1998, with
partial updates in 2005. To enter the system, the substance
name is requested, or its number in the registry of the Chemical
Abstracts Service, CAS, in the Registry of Toxic Effects of Chem-
ical Substances, RTECS, or the number in the Hazardous mate-
rials description of the United Nations (UN number).
Health-related property data for caffeine, with a focus on
carcinogenicity, were covered by a monograph of the Interna-
tional Agency for Research on Cancer of the World Health
Organization (IARC) in 1991. Caffeine is in Group 3, not
classifiable as to its carcinogenicity to humans.
In 1958, the American Food and Drug Administration
(FDA) declared caffeine as ‘generally recognized as safe,’
when added to cola-like beverages up to 200 mg caffeine per
liter of beverage — the famous status ‘GRAS,’ confirmed in
1978 with a report. Since then, newly designed compositions
came to the market. In November 2010, FDA sent a warning
letter to producers of caffeinated alcoholic beverages, inform-
ing them, that caffeine co-consumption with alcohol would
not be covered by the GRAS definition of cola-like drinks.
The scientific opinion of the European Food Safety
Authority on the safety of caffeine, issued in May 2015, evalu-
ates the published studies on the topic. The conclusion was,
that single doses up to 200 mg caffeine and habitual intakes up
to 400 mg per day from all sources (3 or 5.7 mg per kg body-
weight, respectively) do not give rise to safety concerns for
adults. For pregnant women, the habitual consumption should
be halved. For children and adolescents, a first approach is
based on the bodyweight frame, until specific data for this
age group will become available.
Caffeine Sources and Uptake
Caffeine is said to be the most widely consumed psychoactive
substance. It is used as a stimulant in both food and
pharmaceuticals, and its consumption and effects are considered
in the next article in this encyclopedia.
Sources of caffeine include plants and direct chemical
synthesis.
Industrial Sources
For pure caffeine, data on industrial production are rare. The
Organization for Economic Cooperation and Development
(OECD) in Paris has submitted summaries in a 2002
“Screening Information Data Set” (SIDS) of high-volume pro-
duction chemicals. The data are repeatedly cited: “1999 the
estimated world production amounted to 10.000–15.000 tons
including 3.000–4.000 tons of natural caffeine” from decaffeina-
tion. Since then, a moderate increase in production has occurred.
In 2011, China was the main supplier of synthetic caffeine, and
the country’s leading manufacturer reports a production volume
of 8000 tons.
In the early 1900s, industrial decaffeination from caffeine
plants was developed in Europe, with a focus on the produc-
tion of decaf coffee. At about the same time, caffeine produc-
tion via the decaffeination of imported tea wastes started in the
United States, in response to soft drink demand. This process is
still in use, as recently reported from Turkey and India, which
were number 6 and number 2 in 2013 world tea production.
The decaffeination of coffee pulps (i.e., the coffee waste in
coffee-producing countries) is also being developed.
The changed priorities for coffee decaffeination are best
illustrated by the title of a scientific article on decaf in 2012
“Which is the by-product: caffeine or decaf coffee?” In beverage
 Caffeine: Characterization and Properties 559

Table 1 Properties of caffeine, from the CAS Registry (if not otherwise marked); website source www.cas.org accessed February 2015, identifying
the references therein

Property Value

Molecular formula C8H10N4O2

Molecular mass 194.19 Da
Status at ambient conditions Solid, dimorphic
Appearance, taste and smell Colorless (‘white’!), bitter tasting, odorless
Crystalline formsa Hydrate 0.8 H2O, stable up to 51.5
C, dehydration to stable b phase
Anhydrous b phase
Anhydrous a phase (stable up to sublimation/melting)
Metastable a phase; on cooling down from stable a phase, back-transformation to the b
phase is kinetically restrained
Dimers in aqueous solutionb Self-associated caffeine dimers, coplanar, staggered, at concentrations above the
solubility limitc
Stability point of hydrate 0.8 H2O 51.5
(quadrupol point)d
Transformation point b ! a 141
Ca
Sublimation point 178
C
Melting point (in fused capillary) 236
C
Density [g cm3] 1.45 at 25
C/a phasee
1.23 at 18.7
Cf
Ultraviolet absorption maximum lmax (nm) 274 (aqu.)
Apparent molar absorption coefficienth ea (l mol1 cm1) 9.750  75 (aqu., c ¼ 0001%), lit. averageg
Dissociation constant pKa 10.4 at 40
C (acid–base constant)
Mass solubility aqueous (mass ratio, %) Concentr (%) Temp (
C) Remarks
2.2 20.0 pH 6.9
46.8 83.9
81.0 100.3 Extrapol. value
Mass solubility, other solvents Ethanol: slightly
Ether: insoluble
Chloroform: very good
Dipole moment, D 3.7 (benzene)i
Octanol/water partition ratio 0.091 at 23
C (log Pow)

Property data taken from the CAS Registry online (February 15th, 2015) and the following:
a
Bothe, H., Cammenga, H. K. (1979). Phase transitions and thermodynamic properties of anhydrous caffeine. Journal of Thermal Analysis 16, 267—275.
b
Iza, N., Gil, M., Montero, J. L., and Morcillo, J. (1988). Self-association of caffeinein aqueous solution. Study of dilute solutions by normal and second derivative UV absorption
spectroscopy. Journal of Molecular Structure, 175, 25–30.
c
Sanjeewa, R. and Weerasinghe, S. (2011). Study of aggregate formation of caffeine in water by molecular dynamics simulation. Computational and Theoretical Chemistry 966,
140–148.
d
Epple, M., Cammenga, H. K., Sarge, S. M., Diedrich, R., and Balek, V. (1995). The phase transformation of caffeine: investigation by dynamic X-ray diffraction and emanation thermal
analysis. Thermochimica Acta 250, 29–39.
e
Bothe, H. and Cammenga, H. K. (1980). Composition, properties, stability and thermal dehydration of crystalline caffeine hydrate. Thermochimica Acta 40, 29–39.
f
Pfaff, C. H. and Liebig, J. (1832). Ueber die Zusammensetzung des Caffeins. Annalen der Pharmacie 1, 17–20.
g
Eisenbrand, J. and Pfeil, B. (1956). Beitrag zur Bestimmung des Coffeins in verschiedenen Lebensmitteln. Z. anal. Chemie 151, 241–258.
h
Mejri, M., BenSouissi, A., Aroulmoji, V., and Rogé, B. (2009). Hydration and self-association of caffeine molecules in aqueous solution: comparative effects of sucrose and
b-cyclodextrin. Spectrochimica Acta Part A 73, 6–10.
i
Weiler-Feilchenfeld, H. and Bergmann, E. D. (1968). Israel Journal of Chemistry 6, 823.

manufacture, the eventual claim ‘natural’ might be a driving
force, to use added caffeine obtained from a plant product.
Milestones in the development of industrially feasible caf-
feine synthesis include the Traube synthesis of 1900 (too many
steps with low yields, but a backbone of further improve-
ments), the Bredereck synthesis of 1950 (developed during
World War II, facing the constraints of limited accessibility of
educts by starting with xanthosine from yeast), and the novel
Zajac synthesis of 2003, starting with inexpensive uracil. For
the latter, a reaction scheme is given in Figure 5. Uracil is
double-methylated with methyliodide, the 1,3 dimethyl-uracil
is nitrated with a mixture of nitric and sulfuric acid, and it is
then reduced with iron or hydrochloric acid in tetrahydrofuran
to yield the respective amino-uracil. The latter is acylated with
formic acid and nitrated again at the remaining 4-position of
uracil; heating with iron in acetic acid allows simultaneous
reduction and intramolecular heterocyclization to produce
theophylline, which is N-methylated to yield caffeine. For
almost a century, the fully synthetic caffeine could not compete
economically with caffeine from decaffeination, however.
Agricultural Production
Volumes of agricultural products are published by FAO, the
United Nation’s food and agriculture organization in Rome,
on a regular basis. Caffeine-containing plants deliver globally
traded commodities of high overall economic value. For the
most important caffeine products, Table 2 shows the ranking
560 Caffeine: Characterization and Properties

O O O O
CH3 H3C H3C NH2
NH NaH/DMSO/CH I NO2 N
3
N HNO3 N Fe/HCI
H2SO4
N O N O N O N
H O
CH3 CH3 CH3
Uracil
5-Amino-1,3 dimethyluracil

O
O O CH3
H3C O H
N N H3C H3C
1) HCOOH/heat N N
Fe/AcOH N NaH/DMSO/CH3I N
2) HNO3/H2SO4 H Heat
O N NO2 N N
O N O N
CH3 CH3 CH3
Theophyllin Caffeine
Figure 5 Reaction scheme for Zajac’s novel method of caffeine synthesis 2003 (edited from Zajac, M.A., Zakrzewski, A.G., Kowal, M.G. and Narayan, S.
(2003). A novel method of caffeine synthesis from uracil. Synthetic Communications: An International Journal for Rapid Communication of
Synthetic Organic Chemistry, 33, 3293.)

Table 2 Caffeine sources from plants, with plant botanical naming, caffeine contents, and average production volumes of the caffeine products

Caffeine Production
Plant content (% average (tonnes
source Botanical species d.b.) per year) Market forms, remarks Use

Guaraná Paullinia cupana 2.5–5% 3,900 Seeds, dried, powdered or pounded into Beverage/masticatory (caffeine
seeds Kunth a stick and roasted supply for other products)
Tea Camellia sinensis 2–4.5% 4,800.000 Black, oolong, green, and white tea from Beverage (dust and stalks used
leaves (L.) Kuntze different postharvest treatments to gain caffeine)
Coffee Coffea canephora 1.5–2.6% 8,600,000 Roast coffee, roast and ground coffee, Beverage (caffeine from
seeds Pierre ex instant coffee, convenience decaffeination used for other
A.Froehner preparations products)
Coffea arabica L 0.9–1.9%
Coffee Coffea arabica Arab specialty ‘Qishr’ Beverage
husks
Kola Cola nitida (Vent.) 1.5% 290,000 Dried Masticatory/beverage
nuts Schott & Endl.
Maté Ilex paraguariensis 0.5–1.5% 790,000 Dried, ground, and slightly roasted Beverage
leaves A.St.-Hil.
Cacao Theobroma 0.2% 4,500,000 Cacao beans, cocoa mass, cocoa Beverage, chocolate bars
beans cacao L. powder

Plant source: customary name.

Botanical species: The Plant List (2013). Version 1.1. Publication on the Internet; http://www.theplantlist.org/ (accessed January 1st, 2015) Caffeine content: Mass percent on
dry basis.
Production average: 2009-2013 data from FAOstat (web sourceww.faostat3.fao.org accessed February 2015); for Guaraná from IBGE (weweb sourcew.ibge.gov.br/home/estatistica/
indicadores/agropecuaria/lspa/ accessed February 2015).

in caffeine content and the volumes of agricultural production
as 5-year averages.
The data suggest that caffeine plants, roughly calculated,
can provide about 300 000 tons of the biomolecule for
human uptake per year. That is an order of magnitude above
the amount of the chemical as derived from industrial syn-
thetic production.
In the FAO definition of agricultural corps, coffee, tea, and
maté are stimulant crops because they contain caffeine, but
cacao, due to its additional nutritional value, is both a
stimulant and food crop; kola nuts are separately categorized
as edible nuts. Guaraná seeds, which are the highest in caffeine
content, are not included in the FAO statistics, because their
agriculture is negligible in volume. For the purposes of this
article, guaraná data are taken from publications of the
Brazilian Institute of Geography and Statistics (IBGE). Table 2
also mentions Qishr, husks of the coffee seed, traditionally
used in Arabian countries to prepare a tea-like beverage; coffee
husks are mentioned in the FAO commodity list, but without
reported quantities; it is not a primary crop.
 Caffeine: Characterization and Properties 561

CROP
Europe
Oceania
Tea
Africa
Asia
America
Coffee

Cocoa beans

Kola nuts

Caffeine plants production 2012

- FAOstat data -

Maté
PRODUCTION IN 2012 (TONNES)
0 1.000.000 2.000.000 3.000.000 4.000.000 5.000.000
Figure 6 Agricultural production of caffeine containing plant commodities per crop and continent, 2012 volumes in tonnes, data from FAOstat
(web sourcewww.faostat3.fao.org accessed January 2015).

Natural Occurrence and Volumes microemulsions for skin penetration, and vapor sticks to imi-
tate the idea of e-cigarettes.
In all nature, more than 60 plant species are known to contain
Caffeine is used in pharmaceutical preparations in great
caffeine, and coffee and tea are almost ubiquitously present to
volumes, primarily as an adjuvant in analgesic combinations,
consumers. Surprisingly, up to the end of the fourteenth cen-
both in over-the counter and in prescription medicine, and in
tury, Europe was one of the few regions of the world where
minimal volumes in orphan drugs for rare diseases such as
caffeine-containing drinks were entirely unknown.
neonate apnea. Caffeine is also part of some cosmetic
For an outline, the 2012 crop volumes are summarized per
formulations with transdermal uptake.
continent in Figure 6. Tea is predominantly grown in Asia.
Ingested caffeine is directly absorbed in the mouth or via
Cocoa cultivation is centered on the African continent. Coffee
the stomach and intestine within 45 min, and it readily spreads
production volumes are more balanced around the globe’s
into all body tissues. Caffeine clearance happens via catabo-
tropic belt. Kola nut and maté are produced exclusively in
lism in the liver, with successive demethylations, oxidation to
Africa and America, respectively. According to the FAO, ‘Ocea-
uric acid, cleavage of the imidazole ring with remaining uracil,
nia’ comprises Australia, New Zealand, and the Pacific islands,
and finally the break down to ammonia and exhaled carbon
which are fine but very small origins of some caffeine plants.
dioxide. Only a little caffeine is excreted unchanged.
Political and economic factors influence the long-term pro-
duction pattern: the FAO archives maintained since 1961
reveal an explosive coffee increase in Asia since 1995—
Vietnam!—a starting coffee volume in China, and a decline Dispersal
of tea from Europe. The teas from the territories of the former
Caffeine’s primary use by humans, either as a food or drink
Soviet Union are gone.
preparation or via pharmaceutical or cosmetics production,
causes the chemical to enter waste waters and landfill
leachates. The biodegradation of caffeine into constituent
Uptake
compounds requires some 3–6 weeks. Due to its biodegrad-
The ways of human uptake of caffeine vary widely. ability, bio- and geoaccumulation are not expected. Neverthe-
It is swallowed in beverages produced from caffeine- less, increasing dispersal in coastal waters is observed, and
containing plants, such as coffee, tea, maté, and chocolate caffeine is found in marine fauna of coral reefs.
(these beverages mark the main uptake in volume), and in
water-based flavored drinks, so called soft and energy drinks,
to which caffeine is added. Caffeine is eaten in products such as
chocolate bars, and it has been traditionally chewed, as with Caffeine-Growing
kola nuts. New routes of uptake arise. To facilitate on-the-go
ingestion, energy drink preparations are offered as gels to Caffeine is located in different tissues and organs within the
swallow; some energy preparations for accelerated uptake plants that produce it; in these locations, it experiences synthe-
also include chewing gums, patches with gels or sis, storage, transfer, metabolism, and degradation.
562 Caffeine: Characterization and Properties

Biosynthesis In the respective complexes, different interactions take place.

Caffeine biosynthesis in plants is well established during the
last 40 years, with comprehensive overviews since 2000, nota-
bly by Ashihara. It follows a general pathway, starting with the
DNA’s purine base xanthosine. This is methylated at N-7 using
xanthosine-methyltransferase, which depends on S-adenosyl-L-
methionine (SAM) as a methyl donor, itself turning to S-aden-
soyl-homocystein (SAH). Next, the ribosyl unit is removed
with the help of N-methylnucleosidase. The resulting 7-
methylxanthine is methylated at N-3, catalyzed by another
enzyme, 7-methylxanthine transferase (theobromine
synthase), again with SAM as methyl donor, to produce theo-
bromine. Further methylation with 3,7-dimethylxanthine
transferase (caffeine synthase) at N-1 of the xanthine eventu-
ally leads to caffeine. The first methylation at the xanthosine
level is the rate-determining stage.
The structures of the involved enzymes are known, their
encoding genes have been consolidated in a multicentered
work on the coffee genome in 2014 (’provides insight into
the convergent evolution of caffeine biosynthesis’, says the
title). The main biosynthetic pathway is shown in Figure 7,
which marks the involved enzymes together with their enzyme
code numbers. Additionally, minor routes via theophylline or
paraxanthine exist. Methylxanthines from the side pathways
of biosynthesis may accompany caffeine in the composition of
caffeine-plant products. They form plant-specific methylxan-
thine patterns, providing a useful tool for plant-source
identification.
In-plant Localization
In the plant cells, caffeine is synthesized in the cytoplasm,
translocated, and stored in intracellular membrane-bound
compartments, the central vacuoles. It is in tight complexation
with phenolic molecules of the respective plant.
These phenolics are chlorogenic acids for coffee and mate;
catechins for cocoa, kola nut, and guaraná; and tannins for tea.
Equilibrium constants are determined. The caffeine chlorogen-
ate complex is described as an 1:1 type, p-molecular complex
stabilized by hydrophobic interactions.
The release of caffeine from its complex in plant products
was an analytical challenge of the early encounters with caf-
feine in coffee: Runge achieved the separate preparation of
Kaffeesäure (coffee acid) and Kaffeebase (caffeine) by breaking
the complex via the precipitation of the acid with lead acetate
and the isolation of caffeine from the filtrate. Robiquet
achieved the same result with magnesium acetate.
Basic research was done, comprising the localization of
xanthines in the tissues and organs of the plant, and their
ways and distribution during plant development. The key
aspects are similar in all caffeine plants.
Caffeine as a Secondary Metabolite
For plant biology, caffeine is classified as a secondary metabo-
lite, that is, it is not primarily essential and active for growth
and reproduction, but it is useful for adaptation to the envi-
ronment. Caffeine may serve as a chemical defense against
predators, or it might contribute to the allelopathic prevention
of competing up-growth, or it might help attract supporters – a
fashionable keyword is ‘bioecology.’
As the plant accumulates caffeine in direct proportion to the
risk of predation, it is synthesized in the outer parts of young
leaves and fruits, and on their epicuticular waxes. On the
ground surrounding the individual plant, the phytochemical
is released by the seed coat of dropped fruits or from fallen
leaves, which may prevent the germination of competing seeds
on the same ground. On the other hand, the caffeine content of
the blossom’s pollen and nectar acts as an incentive to polli-
nators attracted by the scent; and crosspollination by bees may
help to maintain the genetic diversity of caffeine-containing
plants. The experimental confirmation of the presumed
intention-and-effect chains requires a sophisticated study
design, however. With the turn of the century, the general

O O CH3
H N H N+
N N

N 7-methylxanthosine N N-methyl nucleosidase

O N O N
synthase (EC 2.1.1.158) (EC 3.2.2.25)
H H
HO HO
O O
SAM SAH H2O D-ribose
HO OH HO OH
xanthosine 7-methylxanthosine

O CH3 theobromine synthase O CH3 caffeine synthase O CH3

H N H N H 3 C N
N (EC 2.1.1.159) N (EC 2.1.1.160) N

O N N O N N O N N
H SAM CH3 SAM CH3
SAH SAH
7-methylxanthine 3,7-dimethylxanthine 1,3,7-trimethylxanthine
(theobromine) (caffeine)
Figure 7 Main pathway of caffeine biosynthesis in plants compiled from different sources: (1) Ashihara, H., Kato, M. and Crozier, A. (2011)
Distribution, biosynthesis and catabolism of methylxanthines in plants. In Fredholm, B.B. (ed.) Methylxanthines. p.17. Berlin: Springer. 2) Kanehisa, M.,
(2013). KEGG database, Pathway map Caffeine metabolism. Kyoto, Japan (web source http://www.genome.jp/kegg/ accessed June 2015).
 Caffeine: Characterization and Properties 563

emphasis of research changed to field studies in order to con-
vince the producers to adopt the ideas of bioecology and
moderate agroforestry. Follow-up crop management is covered
in the articles on coffee, tea, and cocoa.
Caffeine-Containing Plants
The most important caffeine-containing plants are named in
Table 2. Of these, coffee, tea, and cocoa are itemized in indi-
vidual articles of this encyclopedia: two articles are dedicated to
cocoa, four deal with coffee, and three articles describe tea.
Maté, kola nut, and guaraná are discussed in more detail in
this article. They are the next most commonly produced
caffeine-containing plants. In the years 2011–13, their use as
herbal medicine in the European Union was assessed, with a
focus on the use of maté, kola nut, and guaraná as powdered
leaf or seed, all of which appear to be nontoxic preparations for
oral use.
Other caffeine synthesis happens in some citrus plants,
where nectar and pollen may contain caffeine.
Caffeine plants grow in the tropic zone north and south the
equator. Further climatic requirements limit their cultivation
to distinct environments, altitudes, and geographic regions.
Weather conditions in the crop year are connected to strong
up- and downturns in the outcomes of sensitive countries, with
big economic impacts.
Figure 8(a)–8(d) shows the 2013 worldwide distribution
of the six main caffeine crops, using the basic data from the
FAO, aggregated by country, as well as Brazilian statistics.

Figure 8 (a–d) Agriculture on caffeine products 2013, crop- and country wise marked on world maps, subtitles within the figures data from FAO and
(for Guaraná) Brazil statistics. Web-sourcesFAOstat, http://faostat3.fao.org/download/Q/QC/E (accessed 11 Jan 2015 and 6 March 2015), and IBGE,
http://www.ibge.gov.br ‘Levantamento Sistemático da produção Agrı́cola’, LSPA 2014 (accessed 6 March 2015).
564 Caffeine: Characterization and Properties
Figure 8—cont’d
The figures visualize the respective cultivation belts around the
globe in four crop dedicated maps. The basic grids are world
maps with political borders, with mark-ups on the surface of the
countries that cultivate the respective caffeine-containing plants;
the differences in production volumes are discretely implied by
coloration. The place of the plants’ genetic origin is indicated, as
established by history and sophisticated phylogeographic analy-
sis, lastly by Anthony for coffee in 2010.
Figure 8(a) shows the 80 countries that maintain coffee
crops; Brazil is by far the biggest producer; Ethiopia, the cradle
of Arabica coffee, is no. 7; on the Northern rim of the tropics,
China is emerging with her Southern regions (only those are
marked).
Figure 8(b): Fifty countries cultivate tea, outreaching the
tropic belt to both sides; China is the top producer.
Figure 8(c): Sixty countries have cacao agriculture, with an
eye-catching concentration very near the equator, in West
Africa (Ivory coast!) and Indonesia.
Figure 8(d) the production sites of kola nut, mate and gua-
raná do not overlap, thus they are visualized together in Figure
8(d). The productions are retained at the genetic origin of the
plants. Kola nut is grown in some coastal countries of West
Africa. Mate is harvested in an even smaller region in the triangle
of Argentina, Brazil, and Paraguay, where only the really produc-
ing districts are marked. Guarana has spread from its Amazonas
origin to neighboring Brazilian states; only those are marked.

The following descriptions are in order of the product’s
caffeine content, as given in Table 2.
Guaraná
The seeds of the guaraná plant, Paullinia cupana, of South
America are traditionally consumed as a stimulant beverage.
Guaraná belongs to the family Sapindaceae, genus Paullinia L.;
the species is Paullinia cupana Kunth; and the chemotaxonomy
was accepted for The Plant List in 2012. Another Paullinia
species has caffeine in its bark, Paullinia yoco R. E. Schult. &
Killip, found in the frontier region of Colombia, Peru, and
Ecuador. Guarana’s caffeine is most concentrated in the
seeds, in complexes with the phenolics catechine and
epicatechine.
Guaraná is native to the tropical rainforest of the broad
Amazonian basin, where it has been gathered and used since
pre-Columbian times. In 1669, a Portuguese missionary
described the plant and its history, and named the plant.
Linné installed the genus Paullinia in 1753. Humboldt col-
lected his specimen of Paullinia cupana further northward in
present-day Venezuela in 1821, stating, “crescit in ripa obum-
brata fluminis Orinoci, prope S. Fernando de Atabapo; floret
Majo” or “is growing on the umbrageous banks of the river
Orinoco, near San Fernando de Atabapo; it is flowering in
May,” cited from Bonpland and Humboldt.
In its original form, guaraná was a woody liana, as shown in
Figure 9, reaching the forest canopy, at heights up to 12 m.
Figure 9 Guarana vine with fruits, scandent on a rainforest tree in East
Ecuador, September 2010 (courtesy of the photographer Geoffree R.
Gallice, University of Florida, Gainsvillle, USA, under collective common
license by Geoff Gallice from Gainesville, FL, USA (Guaraná) [CC BY 2.0
(http://creativecommons.org/licenses/by/2.0)], via Wikimedia
Commons; accessed March 2015).
Caffeine: Characterization and Properties 565
Over generations of crop growing, it was domesticated to a
scandent shrub, up to 2 m tall and 4 m in diameter. In their
Amazonas indigenous territory, the Sateré Mawés grow these
shrubs in orchards around their homes; they recruit new plants
by gathering seedlings from the rainforest. Colonists in the
same region cultivate the plants in plantation-style arrange-
ments, however, propagating them by cuttings.
The guaraná plant blossoms with white corollas in clusters
directly on the branches. It is monoecious with separate male
and female flowering periods even on the same branch, and
pollination is done by insects. The plant develops bunches of
eye-catching fruits, each with a red mantle, white mesocarp
flesh, and a dark seed, so that the fruit resembles an open
eyeball.
The Satare Mawes have strong traditions that guide the
processing of the fruit: they pick the bunches when the outer
skin of the fruit bursts, and the eyes open. Immediate post-
harvest treatments include roasting, and the seeds are unhar-
nessed, separated from residual testae, and pounded with
mortar and pestle. The grist is thoroughly kneaded and rolled
into a cylinder. After some weeks in the smokehouse, the
guaraná achieves its reliable storage form, the ‘baston’ or gua-
raná stick. For beverage preparation, the powder is obtained by
grating the hard stick by means of the bony tongue of a special
fish or a slab of basalt.
At the beginning of the twentieth century, guaraná was
introduced as flavoring in bottled soft drinks named ‘Guaraná,’
with several fabrication sites and nationwide acceptance. As the
use and production of Guaraná increased, an extension of the
agriculture was required, as was a simplification of the many
laborious stages of traditional processing, and an adaption to
industrial procedures. For soft drink manufacture, guaraná was
utilized as a syrup prepared via the alcoholic extraction of seeds
after simple roasting and grinding.
Over the last 70 years, international demand for guaraná
has grown, given its use as a natural source of caffeine. Culti-
vation was expanded to regions outside the Amazon, south-
ward to Bahı́a and Mato Grosso, with plants provided by
research institutes. Climatologic and edaphic conditions in
these states are similar to the Amazon Basin, and plantations
in Bahia account for some 70% of national guaraná produc-
tion (Brazilian official data acquisition for 2013), with nearly
90% national use. The discussion on the ethnobotanic heritage
of the crop is ongoing (see the 2013 article titled ‘Who owns
Guaraná?’).
Tea
Tea-leaf beverages have been consumed in Asia for thousands
of years, and tea is the world’s second largest agricultural
commodity with caffeine content by volume. The tea plant
belongs to the family Theaceae, genus Camellia, and species
Camellia sinensis (L.) O. Kuntze, with two main varieties, C.
sinensis var. sinensis and C. sinensis var. assamica, as well as some
minors. In 1753, Linné had installed this genus, calling it thea
with one species, Thea sinensis; a separate genus was the deco-
ratively flowering Camellia japonica. Later, Thea sinensis and
camellia japonica were reorganized with the common genus
name Camellia; this section deals with the species C. sinensis,
the tea plant.
566 Caffeine: Characterization and Properties
Tea was first cultivated in China, followed by Japan, some
thousand years ago, with India and Indonesia starting to culti-
vate it in the nineteenth century. On plantations, tea is an
evergreen shrub. The primary crop consists of the tender leaves.
Their postharvest treatment up to the final product is done in
the countries of origin.
The tradable commodities are the tea leaves; they are
brewed for beverages, esteemed as stimulants for their caffeine
content. The series of phenolics accompanying the caffeine in
tea provokes a specific profile of caffeine release from the
drink. The caffeine metabolism follows the general pathways.
Tea is explicitly treated in articles 685–687 of this
encyclopedia.
Coffee
Coffee beans are the most important primary product of the
caffeine-containing plants, and the second internationally
traded commodity in value, following mineral oil. The coffee
plant’s evolutionary source is Africa and Madagascar, with west
central Africa for the robusta coffee, and Ethiopia for arabica
coffee, the latter being used in beverages since about one
thousand years ago.
The coffee plant belongs to the family Rubiaceae, genus
Coffea L., with the economically important species being C.
arabica L., C. canephora Pierre ex Fröhner (the arabicas and the
robustas), and some minors. In the context of the project
’World Checklist of Rubiaceae’, its chemotaxonomy was eluci-
dated and consensually accepted, cornerstones are publica-
tions by Davis 2006 and Bremer 2009.
Since the seventeenth century, coffee cultivation has
expanded from its Afro-Arabian homelands into all climati-
cally suitable regions in the tropics. Occasionally, it succeeded
other tropical crops, which experienced an economic decay,
including sugar in Indonesia or Hawaii. Coffee plantations are
often run by smallholders. The intensity of crop management
ranges from monoculture (i.e., no shade, high inputs) to poly-
culture (shade overstory retained, fewer inputs, agroforestry).
Coffee is a tropical shrub with cherry-like fruits. They are
processed after harvest to yield the ‘naked’ dried beans, the
trade commodity green coffee, as defined in the International
Coffee Agreement between producer and consumer states.
Final treatment prior to consumption is the green beans’ roast-
ing and grinding; it takes place in the consumer country. The
last step is the preparation of the beverage.
The different aspects of coffee are covered in the coffee
articles of this encyclopedia.
Kola Nut
The kola nut has long been known as a source of caffeine
stimulation. It is native to the West African coast, very near
the equator. Cultivation centers include Sierra Leone/Liberia,
Nigeria/Cameroon, and Gabon. It has been domesticated in
West Africa, and it has likely been cultivated in the forests of
Sierra Leone since the fourteenth century. The tree is constitu-
ent of the lowland forest, requiring a hot, humid climate and
capable of withstanding 3 months of dry season. Kola may
be cultivated even in drier areas wherever ground water is
available.
The kola plant belongs to the cacao family. The classifica-
tion is in a state of flux. In 2003, it changed its place from
Sterculiaceae to Malvaceae, following the angiosperm
phylogeny group. The genus remains Cola (Vent.) Schott &
Endl., with the species are C. nitida (Vent.) Schott & Endl.,
Gbanja kola, cultivated around Ghana and Nigeria, C. acumi-
nata (P. Beauy,) Schott & Endl., Abata kola, which is distrib-
uted from Togo to Angola and cultivated in the tropical forests
of West Africa, and some minor ones.
The phenolics, accompanying the kola nut’s caffeine, are
tannic acid, catechins, and, to a lesser extent, chlorogenic and
quinic acids. The nuts have a bitter, astringent taste, getting
milder on drying. Chewing is common in the local population,
used against fatigue and hunger, and a beverage is prepared by
boiling the powdered seeds in water. Exported to Europe and
America, the kola nut is used as an ingredient in the production
of beverages in those areas.
When grown from seed, kola needs about 3 months to
germinate, but propagation from cuttings is also possible.
After 4 years, when the tree has grown to a height of 2–3 m,
the first fruits appear. Full maturity is reached in 10 years, with
harvests up to the age of 100 years. Yields of 300 nuts per tree
are considered good. Old kola trees may serve as shade trees for
cocoa plantations.
The plant is an evergreen, up to 20 m high, with a rather
short stem and a dense crown. It has large leaves, variable in
shape and size, and its flowers are male or hermaphrodite, with
wind or insect pollination. Fruits are ripe after 5–6 weeks.
Botanically, they are no nuts but comprise of a set of volumi-
nous warty follicles gathered into a star, with each follicle
containing 5–10 ellipsoid seeds, about 2.5 cm in diameter
and colored red or white depending on the variety. Ripe fruits
are dispersed by bats, birds, and squirrels.
The kola fruits are harvested before full maturity, using
knives mounted on long poles. The fruits are immediately
opened, and the seeds recovered and left in heaps for fermen-
tative decay and sun drying. After some days, the seeds are
washed and put in baskets on fresh leaves to be stored under
surveillance.
Kola nuts are habitually used in West Africa for social and
cermenonial purposes. Without kola seeds, in Nigeria and many
West African countries, traditional hospitality, cultural, and
social ceremonies are considered to be incomplete.
In a 1995 document on edible nuts as non-wood forest
products, the potential of kola nuts is discussed: “Considering
how much cola nuts are appreciated in West Africa while being
virtually unknown elsewhere, expectations for an expanding
market are reasonable.” (Wickens, 1995).
Maté
Maté is native in South America, in the woodlands of the
Paraná River in eastern Paraguay. The leaves are used to make
a tea-like beverage, and they have been collected since pre-
Hispanic times. Throughout the twentieth century, maté was
once again cultivated in the mountainous southern parts of
Brazil, northern Argentina, and Paraguay, reestablishing
the abandoned plantations that Jesuit monks had run in
eighteenth and nineteenth centuries. The traditional ‘extractive’

production is practiced in parallel, with leaves gathered from
‘wild’ forest trees, before regular postharvest treatment.
In plant taxonomy, maté belongs to the family Aquifolia-
ceae, genus Ilex L., species I. paraguariensis A. St.-H. (called
yerba maté). Linné named the genus in 1753, and Auguste
Saint-Hilaire gave the maté a description in 1822. Other spe-
cies are I. vomitoria Ait. (the yaupon holly of the southern
United States, archaeobotanically identified as ‘black drink’
from 1000 years ago) and I. guayusa Loes., cultivated in Ecua-
dor, with evidence of its use for 1500 years. The decorative
Christmas holly known in Europe, I. aquifolium, is a relative
without caffeine. Maté leaves contain 0.5–1.5% caffeine, and
the accompanying phenolics are the chlorogenic acids.
The maté tree is 8–15 m high, evergreen, and dioecious,
flowering from October to November, fruiting from March to
June, and pollinated by insects. The perennial leaves are about
8 cm long, olive-green, and leathery, with slightly crenate den-
tate margins. The plant needs constant, moderate rainfall and
average temperatures of about 22
C, although it can tolerate
extremes down to 6
C. The conditions are met in its region.
For harvest, older leaves and tender branches are taken. Soon
after, the crop is flash-dried through direct heat for 1 min at
300
C (blanching, zapecado). In subsequent processing stages,
the leaves are dried with hot air (secado) and coarsely ground. A
seasoning storage for several months can be added (aging,
Maté (llex paraguariensis)
A1 inflorescence; A2 flower; A3 fruit; A4 gourd and tube for consuming
the infusion
A3
A1
Figure 10 Maté, ensemble of twig with leaves, inflorescence, flower, fruit and consumption utilities. (Source: Hernández Bermejo, J.E. and León, J.
(1994). Neglected crops: 1492 from a different perspective, p.247. Rome: FAO Publications Division. Reproduced with permission of the Food and
Agriculture Organization of the United Nations.)
Caffeine: Characterization and Properties 567
maturation), followed by final grinding and sieving. Modifica-
tions are developing, both with economic and ecologic aims. They
have influence on the products’ appearance and composition.
In 2010, an Argentine study on the bioactive compounds
variation during maté processing steps revealed that these steps
lead to an increased caffeine content in the product, as com-
pared to the green leaves after harvest. The marked augmenta-
tion was observed after the zapecado step, from 0.9 to 1.5% in
caffeine, with parallel increase of chlorogenic acid (1.8 to 2.1%
for the mono-CQA and similar for di-CQA’s). As possible
reason, a degradation of nucleic acids is discussed, with release
of purins for biosynthesis of caffeine.
In Latin America, the beverage is prepared in a special gourd
with hot or cold water. Maté is often drunk as part of the
consumption ritual in a social setting, using a metal straw
called a ‘bombilla’; the straw ends in a closed bulb of teaspoon
size with perforations, to avoid the aspiration of fines from
powdered maté leaves when the infusion is sucked up through
it. The drawing in Figure 10 incorporates maté leaves, blos-
soms, fruit, and the classical drinking equipment. Maté is
predominantly consumed in South America, and export is
preferentially done in final consumer packaging, such as indi-
vidual tea bags (1–2 g). For use as an ingredient in the food or
dietary supplement industries, it is also supplied as maté tea
concentrate.
A2
A4
568 Caffeine: Characterization and Properties

Cocoa an item governed by the International Standard Organization

(ISO), with emphasis on caffeine as a main constituent. For
Cacao beans were the first caffeine-containing plant products
ISO coffee, the determination of caffeine in coffee was one of
that Columbus encountered in 1502. At that time, they were
the first standards, published 1983, following an American
cultivated by the Maya in Central America. Cacao is a neotropic
AOAC procedure of 1979, which relied on liquid chromato-
plant, possibly originating from the lowland rainforests of the
graphic separation and ultraviolet spectrometric quantification
Amazon Basin. Today, it is spread within a narrow zone 10
of caffeine via UV absorption at 276 nm. The method was
degrees of latitude south and north of the equator around the
explicitly described in a European directive of that time, even
globe. Cacao belongs to the family Malvaceae, genus
with an illustration of the chromatographic columns.
Theobroma, species Theobroma cacao L. As with the kola nut,
When liquid chromatographic procedures developed
the classification of cacao was merged, in 2003, with
toward high-performance liquid chromatography (HPLC), caf-
Sterculiaceae. The reorganization was carried out by the Angio-
feine determination was soon affected. The standard technique
sperm Phylogenetic Group, and it is on the way to be accepted.
is now HPLC on reversed phase columns, with quantification
The caffeine content of cacao in the chocolate mass (ground
again via UV absorption, as suggested with Figure 11, where
beans) averages 0.2%, the lowest in the series we look at
the UV spectrum is an insert to the HPLC chromatogram.
here, and it is clearly overshadowed by cacao’s theobromine
Actually, ISO provides HPLC/UV methods both for tea and
content of 1.2%.
coffee, with each one applicable to the specific products on
Cacao is an understory tree, propagated by seed or cuttings
the market. Chromatograms allow separation of the other
and reaching maturity with 4–5 years of age; it grows up to
methylxanthines accompanying caffeine in the same run. The
10–12 m in height, with leathery leaves and small, pinkish
specific distribution patterns of the different caffeine plants
flowers directly on the trunk and branches. Here, the fruits or
may serve as an analytical tool for authenticity proof, and
‘pods’ develop, each yielding 20–50 cacao beans, which equate
“although several detection modes have been recently pro-
to 20–30 pods per harvest. The beans are ground and processed
posed, UV detection is still the first choice in the liquid chro-
to make chocolate. Pulp and pods are also used for food and
matography analysis of these analytes due to its simplicity and
forage. Cocoa has its own articles in this encyclopedia.
reliability,” as emphasized in a Venezuelan research article on
methylxanthines in cocoa beans in 2007. The HPLC chromato-
gram for this section originates there.
Analytics Other classic spectroscopic figures transport further consti-
tutive information:
Since the early seventies of the last century, the analytical Caffeine’s infrared spectrum, shown in Figure 12, provides
characterization of caffeine is preferentially done via chro- the two C]O valence bands of C-2 and C-6, well separated at
matographic separation and spectroscopic identification. This 1700 and 1650 cm1, in 2013 with molecular calculations
preference represents a return to the roots of caffeine research: attributed to in-phase and out-of-phase stretching vibrations
the first to observe chromatographic separations was Runge, of the C¼O bonds respectively, and in the fingerprint region,
the caffeine man, in 1850. the methyl C–H separated from the ring C–H vibrations.
As coffee and tea are globally traded commodities, their The proton nuclear magnetic resonance spectrum, in
characteristics are important to international trade. They are Figure 13, shows the signals of the methyl proton groups, with

HPLC Chromatogram of methylxanthines TB, TP and Caf: 8, 2, and 2 mg/ml water, inj.vol. 20 ml
C-18, methanol/water 1/5 (v/v) at 1, 4 ml/min, ambient temperature (22 ⬚C), UV detection 274 nm

0.5 Theobromine
0.06 0.4
Absorbance (arbitray units)
UV absorbance (AU)

0.3 Caffeine UV spectrum

in distilled water
0.04 0.2

0.1
Theophylline Caffeine
0.02 0
200 250 300 350
Wave length (nm)

0.00

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
HPLC Retention time (minutes)
Figure 11 HPLC chromatogram of methylxanthines caffeine, theophylline, and theobromine in standard solution,A with UV spectrum of caffeineB as
insert edited from several sources: ABrunetto, M.R., Gutiérrez, L., Delgado, Y. et al. (2007). Determination of theobromine, theophylline and caffeine
in cocoa samples by a high-performance liquid chromatographic method. Food Chemistry 100, 463, BBelay, 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 121, 587.
 Caffeine: Characterization and Properties 569

IR spectrum of caffeine IR-NIDA-82055 : KBR 018c KBr disk

100

Transmittance (%)

A
O CH3
50 H3C C
N C N
B C H
C C
O N N

CH3

A B

0
4000 3000 2000 1500 1000 500
Wavenumber (cm-1)
from SDBS, Spectral Database for Organic Compounds, by courtesy of National Institute of Advanced Industrial Science
and Technology (AIST), Japan, published online at SDBSWeb: http://sdbs.db.aist.go.jp (accessed Jan. 25th, 2015)

Figure 12 Infrared spectrum of caffeine in KBr disc, adapted from AIST spectra base, Japan; caffeine formula inserted, relation to the C-O-bands
marked.

1H NMR spectrum of caffeine at 399.65 MHz 0.016 g caffeine : 0.5 ml CDCI3

C D

Caffeine

O CH3 (B)
(C) H C
3 C C N
N
C H (A)
C C
O N N
CH3
(D)

9 8 7 6 5 4 3 2 1 0
HR200600973NS ppm
SDBS, Spectrum Database for Organic Compounds, by courtesy of National Institute of Advanced Industrial Science
and Technology (AIST), Japan, published at SDBSWeb: http://sdbs.db.aist.go.jp (accessed Jan. 25th, 2015)

Figure 13 Nuclear magnetic resonance spectrum of caffeine in CDCl3 solution, adapted from AIST spectra base, Japan; caffeine formula inserted,
relation to the proton chemical shifts marked.
570 Caffeine: Characterization and Properties
100 Mass spectrum of caffeine
MS2006-03097WA
80
Relative intensity
60
40
20 55
42
0
20 40
from SDBS, Spectral Database for Organic Compounds, by courtesy of National Institute of Advanced Industrial
Science and Technology, (AIST), Japan, published online SDBSWeb: http://sdbs.db.aist.go.jp (access ed Jan.
25th, 2015)
Figure 14 Mass spectrum of caffeine, adapted from AIST spectra base, Japan; main fragments marked.
individual chemical shifts, magnetically shielded from each
other. The single-ring proton, with its characteristic aromatic
chemical shift, has a weak spin–spin-coupling to the protons of
the adjacent 7-methyl group, reducing their signal height by
splitting into a doublet, and splitting itself into a quartet.
In addition to 1H-NMR, caffeine can be analyzed by 13C and
15
N nuclear magnetic resonance, and, further on, with two-
dimensional resonance techniques such as heteronuclear
multiple bond coherence (HMBC) and heteronuclear single
quantum coherence (HSQC), to investigate interactions between
protons and distant carbons or nitrogens, including 13C and 15N.
In the mass spectrum, Figure 14, the last of the basic
spectra, the molecular ion peak, dominates as the base signal,
with only a few fragmentations. Remarkably, no methyl frag-
ments are present: the MS-fragmentation starts with a ring
cleavage at the pyrimidine part, with a cut off of methyl isocy-
anate (m/z 55), leading to the fragment m/z 137 and, after CO
elimination, to the dominant fragment ion m/z 109. The rest of
the MS are the broken bits.
Mass spectrometry is commonly used following the chro-
matographic separation of the analyte mixture, with GC/MS or
HPLC/MS/MS as well established combinations. The mass spec-
trum may serve as a proof of authenticity for the caffeine origin
in caffeinated soft drinks and energy drinks. A 2012 research
article was entitled “Caffeine in your drink: natural or
synthetic?” The isotopic composition of carbon reveals whether
the caffeine in the drink is truly natural, as might be claimed, or
synthetic with a carbon isotope pattern of fossil times.
Caffeine in Registries, Inventories, and Databases:
Identifiers and Registry Codes
Caffeine is defined in several scientific and administrative
registries. These registries have their roots in individual or
direct inlet, 20 eV, source 250 ⬚, sample 20 ⬚C
m+
194
109
67
82
137 165
60 80 100 120 140 160 180 200
m/z
corporate private initiatives or in regulatory intentions, and
they are administered by either private or governmental ser-
vices. The actually most accessed registers are listed in Table 3,
together with the individual numerical identifiers for caffeine
they provide.
By far, the most widely used is the Chemical Abstracts
Service (CAS) registry, with caffeine having CAS registry num-
ber 58-08-2. The low numbers of this registry ID indicates the
very early uptake of caffeine into the registry. Two later codes
had to be deleted. The CAS number may often serve as an
entrance to online work with elaborate regulatory listings.
The CAS has been run by the American Chemical Society
since 1907. Caffeine is placed in the abstracts section
“Biomolecules and their synthetic analogs.” The first CAS
entries of caffeine referred to German abstracts on the early
caffeine synthesis by Fischer and Traube. In 1965, the service
with registry numbers started. With modern tools, it may easily
be accessed for pooling substance-related data and literature.
The caffeine property data in Table 1 are generated from this
source on-line.
Another important source, the Beilstein Handbook of Organic
Chemistry, was started in Germany in 1881, and it incorporated
papers from 1771 onward. It organized the scientific literature
according to distinct chemical substances. In systematic order,
caffeine had its place in Volume 26, within the nitrogen-
containing heterocycles. The print version continued until the
fifth multivolume supplement, covering the literature until
1979. The tradition was continued with the powerful Beilstein
online database, which was taken over by Elsevier and reorga-
nized in Elsevier’s Reaxys, which integrated the older data
almost completely. The Beilstein registry number is now called
the Reaxys registry number, for caffeine numerically the same.
In 2011, the International Union of Pure and Applied Chem-
istry (IUPAC) introduced the International Chemical Identifier
code (InChI). It provides structural information on chemical
 Caffeine: Characterization and Properties 571

Table 3 Inventories containing caffeine with registry codes

Inventory/register and reference Administered by Short name Code for caffeine

Chemical Abstracts Register, online services Chemical Abstracts Service CAS CAS RN 58-08-2
since 1980 Columbus, OH, USA
Reaxys, continuing the Beilstein Online Reed Elsevier London Reaxys 17705
Database Registry
Number
IUPAC International Chemical Identifier InChI Trust, Cambridge, UKa InChI code InChI = 1S/C8H10N4O2/c1-10-4-9-6-5
(10)7(13)12(3)8(14)11(6)2/h4H,1-
3H3
InChI key. IUPAC,Research Triangle Park, NC, USA InChI key RYYVLZVUVIJVGH-UHFFFAOYSA-N
Inventory on classification, labeling, and European Commission, Brussels EINECS 200-362-1
packaging of dangerous substances
(CLP)b.
Adapted by follow-up regulationsc. European Chemicals Agency (ECHA), EC No. 200-362-1
Helsinki
Index of harmonized classification and ECHA, Helsinki Index No. 613-086-00-5
labelingd
Registry of Toxic Effects of Chemical BIOVIA Foundation,San Diego, CA, USAe RTECS EV6475000
Substances, originally based in the USA
Hazardous materials description of the United Nations Committee of Experts on UN No. 1544g
United Nationsf the Transport of Dangerous Goods
Harmonized Commodity Description and World Customs Organization WCO, HS Code 2939.30
Coding System (HS)h Brusselsi
a
With members IUPAC, NIST, CAS, Elsevier, FIZ, and others
b
Tracing back to the respective European directive of 1967
c
The last (EC) 1272/2008, Annex VI
d
Annex VI of Reg. (EC) 1272/2008
e
With licenses to CAS, Can. NIOSH, Elsevier and others
f
With hazard classes for safe transportation of hazardous chemicals, based on the United Nations Recommendations on the Transport of Dangerous Goods – Model Regulations since
1957
g
The UN number 1544 is entitled ‘solid alkaloids, not otherwise specified’, and ‘alkaloid salts, not otherwise specified’; caffeine is dealt with in these items, but not explicitly mentioned.
h
With common nomenclature and classification of goods (customs tariffs) since 1968 (tracing back to the principles of the former General Agreement on Tariffs and Trade GATT);
actually in 2015 laid down in in Council Regulation (EEC) No 2658/87.
i
With regular amendments; the next HS 2017 Edition was finalized in January 2015.

substances, using a string of ciphers and letters: a textual code fit
for search engines. Developed in cooperation with the US
National Institute of Standards (NIST), it is now held by the
InChI Trust, which incorporates other relevant media organiza-
tions. The InChI code is increasingly used, replacing the earlier,
less powerful SMILES code. Additionally, the shorter InChI key
of IUPAC works like a registry number, providing identification
and basic data for the substance requested.
The European Inventory of Existing Commercial Chemical
Substances (EINECS) number follows a regulatory approach,
with an inventory focused on any handling of the substances
concerned. The cutoff date for ‘existing’ was 1981, and caffeine
is included in it. The inventory was expanded step-by-step to
other groups of chemicals, with an increasing volume of code
numbers, and thus, a new name for the same identifier came
up: EC number.
A similar approach in the United States led to the Registry of
Toxic Effects of Chemical Substances (RTECS) number, estab-
lished in 1971 by the National Institute for Occupational
Safety and Health (NIOSH), and caffeine is included in the
RTECS as well. After several transfers and mergers since 2001,
RTECS was shifted from Accelrys, in 2014, to BIOVIA, which is
owned by the French commercial group Dassault Systèmes.
Dassault gives license other registry organizations, such as the
CAS. Even though a series of other nation’s analoga to EINECS
or RTECS exist, these registry codes have gained remarkably
broad acceptance.
Meanwhile, the EC number serves as a software key to the
Europe-wide registration of chemical substances required by
law. For caffeine, industry has submitted a dossier to the
European Chemicals Agency (ECHA), in Helsinki, to notify it
about caffeine’s property and safety data, with a focus on the
manufacturing workplace. In the EU, the comprehensive
‘chemical index number’ is included in the same regulation
as the European Communities (EC) Number, again including
caffeine.
The United Nations have a register with recommended
coding for substances and articles dangerous to transport. Caf-
feine is considered part of ‘solid alkaloids n.o.s. or alkaloids
salts n.o.s. poisonous,’ positioned in Class 6.1 Toxic, with a
common four-digit code. The abbreviation ‘n.o.s.’ stands for
‘not otherwise specified.’ For caffeine, an outside marking on
transport containers is not required.
In the Harmonized System (HS) of commodity description
and coding, maintained by the World Trade and World
Customs Organizations, ‘Caffeine and its salts’ have an HS
572 Caffeine: Characterization and Properties
Code number identifying it as an organic chemical in the
subgroup ‘vegetable alkaloids’ (From 2017 onwards, the sub-
group will be identified as ‘alkaloids.’). This classification is
independent of caffeine’s origin, whether from chemical syn-
thesis or from decaffeination. The caffeine plant crops, as
important internationally traded commodities under customs,
are placed in the HS chapters for these vegetable products.
Table 3 is a rendering of registry codes for caffeine in several
databases.
The EC Number of Table 3 need not be confused with the
Enzyme Commission’s EC number, which identifies the
enzymes catalyzing specific reactions. The number has been
systematically designed and attributed by the International
Union of Biochemistry and Molecular Biology since 1955.
Enzyme numbers for the enzymes involved in the biosynthesis
of are indicated in Figure 5.
Other mighty registers, with a history from medieval times,
are the national pharmacopeias. They identify substances for
pharmaceutical use and provide specifications and safety infor-
mation. Caffeine is included in them. Its generic name ‘caf-
feine’ serves as pharmaceutical International Nonproprietary
Name (INN) for the World Health Organization (WHO). Since
1965, the European pharmacopoeia has incorporated more and
more the national texts, as laid down in the European Pharma-
copoeia Convention. Under the WHO umbrella, harmonized
pharmacopoeial texts are exchanged with the United States and
Japanese pharmacopoeias (USP and JP, respectively).
Literature on the medical and physiological aspects of caf-
feine can easily be found in the Medline databases of the US
National Institutes of Health and the National Library of Med-
icine via the search engine Pubmed. The search on ‘caffeine’
yields some 30 000 results.
A broad list of identification numbers is presented by the
web-based information service of the Wikipedia foundation,
which is authored by voluntary contributors, and has gained
an increasing reputation during the last decade. Within the
different language versions, the English article on caffeine
dates back to 2001. Articles compile actual data, which are
cross-linked intensively.
Legislative aspects and requirements belong to the part on
consumption, and will be covered by the next article.
See also: Caffeine: Consumption and Health Effects; Cocoa:
Composition and Health Effects; Cocoa: Production, Chemistry, and
Use; Coffee: Analysis and Composition; Coffee: Decaffeination; Coffee:
Health Effects; Coffee: Types and Production; Food Colloids and
Emulsions; Tea: Analysis and Tasting; Tea: Chemistry and Processing;
Tea: Health Effects.
Further Reading
Anaya AL, Cruz-Ortega R, and Waller GR (2006) Metabolism and ecology of purine
alkaloids. Frontiers in Bioscience 11: 2354–2370.
Baumann TW, Dornelas MC, Frungillo ML, and Mazzafera P (2010) A ciência e Goethe:
cafeı́na e flores (Sciences and goethe: caffeine and flowers). Ciência e, Cultura
62: 56–59 (in Portuguese).
Belay A (2013) Self-association of caffeine (CA) and its hetero-association with
polyphenols and drugs. Journal of Biological and Chemical Research 30: 143–151.
Berger S and Sicker D (2009) Caffeine. In: Berger S and Sicker D (eds.) Classics in
spectroscopy, pp. 25–38. Weinheim, Germany: Wiley-VCH, 541–544.
Bonpland A and von Humboldt A (1821) Voyage de Humboldt et Bonpland, Nova
genera et species plantarum, vol. 5, p. 118. Paris: N. Maze.
Favre HA and Powell WH (eds.) (2014) Nomenclature of organic chemistry: IUPAC
recommendations and preferred names 2013, p 1. Cambridge: Royal Society of
Chemistry.
Hernández Bermejo JE and León J (1994) Neglected crops: 1492 from a different
perspective. FAO plant production and protection series 26: pp. 223–228. Rome:
FAO Publications Division, Ch. Paullinia, ch. Maté, pp. 245–253.
Jamieson RW (2001) The essence of commodification: caffeine dependencies in the
early modern world. Journal of Social History 35: 269–294.
McClatchey WC, Mahady GB, Bennett BC, Shiels L, and Savo V (2009) Ethnobotany as
a pharmacological research tool and recent developments in CNS-active natural
products from ethnobotanical sources. Pharmacology & Therapeutics
123: 239–254.
Mosimann G (2014) Estudios de casos. Caso 2. El guaraná de Maués, Brasil Case
studies. Case 2. The Guaraná of Maués, Brazil. In: Oyarzún MT, Riveros H, and
Vandecandelaere E (eds.) Cómo promover la calidad vinculada al origen para
contribuir al desarrollo en América Latina: enseñanzas de cuatro casos piloto, How
to promote quality linked to geographical origin, a contribution to the development
in Latin America: lessons learned from four pilot studies pp. 31–43. Rome: FAO
Publications Division, 76–82 (in Spanish).
Rosenfeld LS, Mihalov JJ, Carlson SJ, and Mattia A (2014) Regulatory status of caffeine
in the United States. Nutrition Reviews 72(s1): 23–33.
Tavagnacco L, Schnupf U, Mason PE, Saboungi M-L, Cesàro A, and Brady JW (2011)
Molecular dynamics simulation studies of caffeine aggregation in aqueous solution.
Journal of Physical Chemistry B 115: 10957–10966.
Wickens GE (1995) Edible nuts, p. 85. Rome: FAO.