CHAPTER 1

INTRODUCTION

1.0 Background of the Study

Caffeine (1,3,7-trimethylxanthine) is a stimulant that is generally derived from the seeds of the coffee
tree i.e coffee bean (Abdul Mumin et al., 2006; Nour Violeta et al., 2008). It is notable for it's drug-like
properties to induce Central Nervous System activeness, and mental alertness. According to previously
sighted statistics, Coffee (which has the highest percentage of caffeine content) is the second most
consumed beverage in the world — following water. In most developing countries, like Nigeria, caffeine
is imported for it's uses in various industrialization processes due to the low coffee-farming systems, and
in view of been a world-known raw material in different industrial sectors, there's a cusp of a likely
upheaval in obtaining caffeine for food & drug productions. Over the years, research has painted a vivid
picture of local kola nut (Cola acuminate) to contain an appreciable amount of caffeine content and as
such, the choice of kola nut in this work is to establish an option for the extraction, and local production
of caffeine from kola. With the right steps and processes, characterization should also be achievable on
kola nut; as a way to bring about the extraction of caffeine through isolation (Liquid-liquid phase), and
purification (dry solid phase). Likewise, the extraction of caffeine from shampoo — the choice of hair
product, should be achievable through correlative processes to confirm the activeness of this ingredient,
and also to clarify the unhindered reaction between caffeine and various chemical mixtures in hair
shampoo. And as such, this will also affirm the positive effects of caffeine in hair beautification.

1.1 Statement of Problem

Based on the ineptitude to locally produce caffeine (which is a prominent stimulant) in Nigeria, this work
is an attempt to extract & characterize caffeine from both a food product — Kola nut (Cola acuminate)
and a hair product — shampoo, in other to apprise individuals on caffeine alternatives, and to effectuate
a wider use of the products in developing countries.

1.2 Aim and Objectives of Study

AIM

This study aims at determining caffeine contents and the significance of caffeine from locally consumed
Cola acuminate, and is as well an attempt to obtain a pure and refined method of extracting caffeine
from both a food & hair product using techniques such as liquid-liquid extraction and solid-phase
extraction.

OBJECTIVES
• To extract caffeine from kola nut and shampoo by solid-phase extraction and Liquid-liquid extraction
methods.

• To characterize the obtained caffeine from each product sample by Chemical Engineering Unit
Operations.

• To promulgate an easily adaptable method for the qualitative analysis of caffeine.

1.3 Scope of Study

The outlook for this research project is to discover another viable source of caffeine other than the
coffee bean, and to confirm if the chemical & physical properties (from both the food product (kola nut)
& hair product — shampoo) of the yielded caffeine meets it's distinctive characteristics.

Mac-Adonai Ogagaoghene Victor

CHE/2016/344
 CHAPTER 2

LITERATURE REVIEW

2.0 Caffeine

Caffeine (1,3,7-trimethylxanthine or 3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione) is a

naturally-occurring purine alkaloid of the methylxanthine class. It is majorly sourced in the
leaves, seeds or fruits of tea & coffee, with proofs of it's occurrence in cocoa, kolanut and more
than 60 other plants (Abdul Mumin et al., 2006; Nour Violeta et al., 2008). Pure caffeine is
odourless and has a bitter taste (M.J. O’Neil et al., 2001; J.A.T. Pennington., 2005). Caffeine is
one of the legally permitted additives in beverages, with a tolerated upper limit of 0.015%
(w/w) (Ministerio de Agricultural et al., 1982). Recreationally, caffeine is used to provide an
upswing of energy or sudden mental alertness which brings about vivacity. It works as a
psychoactive substance by stimulating the central nervous system (CNS). As such, caffeine is
added to soft drinks as a flavoring agent, and in dietary sources, it is the most frequently and
widely consumed central-nervous-system stimulant today3.

2.1 Chemical Structure and Properties of Caffeine

Some basic properties of caffeine include it's pure anhydrous form, caffeine tastes bitter, it is
white in colour and also an odorless powder with melting point 235—237OC (Royal society of
chemistry, 2017). Caffeine is moderately soluble in water at ambient temperature (2g/100ml),
but very soluble in boiling water (66g/100ml) with a chemical formula C8H10N4O2and chemical
structure as shown in Figure 1. It is slightly soluble in petroleum ether and Benzene, but
moderately soluble in alcohols and acetone solvents. Pure caffeine sublimes at 178OC and is
weakly basic with a pH range of 6.5 to 10.4 requiring strong acid to protonate it (Vallambroso,
2006; and Belay, 2011).

Figure 1: Structure of Caffeine

Environmental effects such as local climate (humidity/sunlight) soil and agricultural factors may
be less important than generic variation in the control of caffeine contents in both tea leaves
and coffee beans (Belay, 2011).

2.2 Importance of Caffeine

Since caffeine works as a psychoactive substance — by stimulating the central nervous system,
it is widely used as a strong stimulant, especially among athletes. Besides, it is used for boosting
energy levels, improving mental alertness, increasing urine flow, treating migraine headaches,
curing breathing problems in newborns, etc. Caffeine has also been found to be effective in
ADHD, OCD, asthma, hepatitis C, Parkinson’s disease, type 2 (ii)diabetes, weight loss, and so on.

2.3 Sources of Caffeine

Caffeine is an organic chemical compound that is found in many plants and can also be
produced synthetically. The most popular sources of caffeine are:

•Coffee (Rubiaceae . Coffea) [coffee seeds/bean; caffeine content depends on the type of
coffee]

•Tea leaves (Camellia sinensis - especially green tea)

•Cocoa (Theobroma cacao; theobromine (3,7-dimethylxanthine)

•Leaves of the holly (yerba maté)

•Guarana seeds (Paullinia cupana)

Other natural sources of caffeine include guarana berries, kola nuts (C. nitida), and guayusa
(Ilex guayusa). Caffeine is added to a variety of well-known popular soft drinks, and is also a
component of many pharmacological preparations and over-the-counter medicines including
analgesics, diet-aids, and cold/flu remedies.

2.4 Kola nuts

Kola nut is the edible seed or nut of evergreen tree of the genus kola with prima species, Cola
acuminate and cola nitida. Cola acuminate is an ever green tree of about 20 meters in height
and has long ovoid leaves pointed at both the end with a leathery texture. Such trees have
yellow flower with purple spot, and star shaped fruit with about a dozen round or square seeds
which can be found in a typical white seed shell. The tree is commercially grown around the
world, particularly in tropical zones like Nigeria, Sri lanka, and Brazil. The tree is propagated
through seed and, C. nitida and C. acuminate can easily be interchanged with similar species.
Kola nut is extremely popular amongst the inhabitant because of it's caffeine-like properties
when consumed. It is also known for its sweet & rose-like aroma, and for it's likeness to a
bittersweet delicacy upon chewing. Kola nuts can be eaten whole or they can undergo size
reduction into powder in other to be mixed with drinks & beverages.

2.4.1 Importance of Kola nuts.

The uses of kola nut, like some other edible plants appears to have ancient origin. It is chewed
in many West African cultures, individually or in social settings to restore vitality, and to also
ease hunger pangs. Kola nut is an important part of the traditional practice of culture and
religion in West Africa, particularly Nigeria (Somadhi, 2004). Kola nut is used as a religious
object and scared offering during prayers, ancestors’ venerations and significant life events.
This archaic practice is currently causing a surge to the increased growth in geopolitical regions
like the United States and the Caribbean. Various medicinal and pharmacological values have
been further observed in this species of cola. Fresh kola nuts have stimulant action that are
similar to that in caffeine-containing plants, and as such their action is indistinguishable from
that of other caffeine drugs (Jayeola, 2001). Due to this stimulant action, the consumption of
kola nuts have been evidently employed to treat whooping cough and asthma. The caffeine
contents present in kola nut acts as a bronchodilator, expanding the bronchial air passages
(Jayeola, 2001), and kola nut is likewise known to stimulate the central nervous sy5stem, heart,
and muscles; just like caffeinated foods.

2.4.2 Proximate and nutritional composition of kola nut

Kola is a natural source of caffeine. The two common species being C. nitida and Bitter kola
have relatively comparable nutrient compositions5 with high moisture, crude fat and crude
fibre contents respectively. Due to the caffeine content in Kola nuts, it's effect when consumed
should subsume inducing wakefulness when arising from sleep or to prevent falling asleep at
specific times, and to relieve the boredom of quotidian routines.
Table 2: Mineral Composition of Kola nut.

2.5 Caffeine (Hair) Shampoo

Caffeine shampoo is a treatment that is designed to stimulate hair growth. Such shampoo
contains caffeine extract, which is thought to help stimulate hair growth and help with hair loss.
Caffeine shampoo stimulates hair growth with the use of caffeine extract, and fundamental
proofs will tally with caffeine stimulating hair growth in a laboratory dish. Caffeine can help to
promote hair growth by targeting DHT — which is the hormone responsible for hair loss.

At current, on-going research works will show that using a shampoo that contains caffeine or
coffee bean extracts stimulates hair growth. So, hair washing processes with this type of
cosmetic for about two minutes should significantly improve the condition of the hair as
caffeine is absorbed even after rinsing the caffeine shampoo out. This is due to the factor that
active ingredient will quickly move through the hair shaft and directly into hair follicles. As such,
if the shampoo is left on the hair for a longer period, caffeine will also penetrate the scalp.

2.5.1 The role of caffeine in the cosmetics industry.

Caffeine is not only beneficial for our health but also has a wide range of applications in the
cosmetics industry. Manufacturers use the unique properties of caffeine to remove cellulite and
flabby skin. This compound improves blood circulation, lymphatic outflow, and helps to get rid
of toxins. In addition, it gives the skin energy and allows it to maintain its firmness. Caffeine is
also valuable for the hair, as it stimulates micro-circulation of the scalp, which in turn promotes
hair growth and prevents hair loss. In addition, cosmetics rich in caffeine, such as hair shampoo,
also have a very favorable impact on physical appearance thus significantly improving hair
quality, and enhancing hair color.

2.5.2 Benefits of Caffeine Shampoo (DIY Coffee Shampoo)

Caffeine shampoo, uses an organic active ingredient of coffee — which is an example of
caffeine shampoo. It has been proven to stimulate hair follicles into producing healthy hair
growth and can also penetrate the scalp, enabling the hair to shine and provide added
condition. Coffee powder is really great for the hair, because of the significant (high) caffeine
content. When ground coffee is incorporated in hair treatments, it works directly on the scalp
and does wonders for the locks. Following are some of the benefits of caffeine/coffee shampoo:

• Caffeine increases blood circulation throughout the scalp, thereby stimulating hair follicles
and boosting hair growth significantly. Thus, caffeinated shampoo will bring about speedy hair
growth.

• Coffee is enriched with antioxidants that are invigorating for the hair. If damaged hair is
treated with coffee regularly, the antioxidant properties will repair the hair structure, and
prevent further damage.

• Coffee promotes the vice for a healthy scalp, which is of utmost importance for a healthy and
beautiful mane. It makes the scalp free from product buildups and sanitizes hair in a safe and
natural way.

• Hair (pH 3.67) and scalp (pH 5.5) are naturally acidic. Coffee is also acidic in nature, and thus
helps in rebalancing the pH levels of the hair and scalp.

• Coffee can make the mane look naturally healthy and radiant by adding a beautiful
shine/sheen to it. Hence caffeine shampoo makes up for expensive hair styling products to give
glossy lustrous hair.

• Coffee works as a natural hair dye that is absolutely safe and beneficial to use. It can even
bring out wonderful highlights in brown locks.

• Caffeine shampoo turns dry frizzy hair softer, smoother, and organizes dehydrated locks.

In caffeine shampoo products, the caffeine present depends on the origin or source and the
caffeine contained in the various products may also cause different effects. This is due to other
substances contained in the plant from which the caffeine is extracted, as each of them posses
different properties. The raw material, in this case,

2.6 Caffeine Extraction

Extraction is the process employed in the separation of a substance from a cell matrix. Such a
process can also be referred to as a decoction, or solution that is obtained by drawing out from
any substance which gives both an essential and characteristic virtue. In simpler terms, it means
to withdraw by expression, distillation, or other mechanical or chemical processes.
2.6.1 Extraction of caffeine as a Bio-active Compound

The choice of the extraction method is dependent on the bio-active compound to be isolated
and the nature of the subject from where the extract will be obtained. The objective of the
extraction should be clear since the bioactive compounds of interest to be isolated could be
unknown, known, or structurally similar to a group of known compounds.

Table 2.6.1: Examples of Bio-active Compounds and

the Solvents Commonly Used for Their Extraction.

The extraction by solvents could follow the principle

of either “liquid-liquid” or “solid-liquid” extractions.
The liquid-liquid extraction is the classical technique in chemistry to isolate a target component
from a mixture. Thus, the selective partitioning of such components of interest into one of the
two immiscible phases results from the choice of the most adequate extraction solvent, as
shown in Table 3.3. When the optimal conditions are not applied, low recoveries are achieved
and further extraction should be made in order to find the optimal combination of extraction
solvents to obtain high recovery and higher purity of the liquid–liquid extraction.

2.6.2 Extraction Review

The practice of extracting organic compounds from solid matrices using solid–liquid extraction
can be described with a Soxhlet extractor — which brings about the extraction of organic
compounds from biological samples that typically require large volumes of organic solvents. A
significant advance in organic extraction techniques from solid matrices, specifically involves
pressurized liquid extraction (PLE).

Pressurized Liquid Extraction

(PLE) is a solid–liquid extraction technique capable of extracting a wide range of organic
compounds from a wide variety of biological matrices. PLE improves extraction efficiency,
sample through-put, and automation. Thus, PLE can be viewed as an extension of supercritical
fluid extraction, and utilizing organic solvents instead of CO2 as PLE holds solvents near their
supercritical region, which is where solvents have their elevated extraction properties and still
remain in a liquid state. Hence, PLE utilizes solvents at elevated temperature and pressure,
which builds on Soxhlet, sealed and unsealed microwave-assisted extraction, and subcritical
water extraction techniques.

Throughout literature, PLE is also described as pressurized fluid extraction, pressurized hot
solvent extraction, high-pressure solvent extraction, and subcritical solvent extraction. A brief
description of the attributes and benefits of an automated PLE system are described below:

• Typically, cellulose or glass fiber filters are placed at the bottom of the extraction cell to
prevent bulk matrix from damaging the PLE system. Sample homogenates are packed into the
extraction cells and the empty space (above the packed homogenate) can be filled with
dispersant matrix or dehydrating agents, such as anhydrous sodium sulfate. PLE parameters
include extraction temperatures (25–200 °C), pressure (500–3000 psi), solvents (organic to
weak acids), number of extraction cycles, duration of static cycles, and rinse volume.

• PLE of target analytes from solid matrices is kinetically and thermodynamically driven.
Improved performance at elevated temperature and pressure is mainly due to the disruption of
surface equilibrium as well as solubility and mass transfer effect. The disruption of surface
equilibrium occurs when thermal energy overcomes cohesive (solute–solute) and adhesive
(solute–matrix) energy by lowering the activation energy required for the desorption processes.

• The increased solubility of water in organic solvents at elevated temperatures can facilitate
the extraction of analytes found inside water-sealed pores of the sample matrix.

• Faster diffusion rates at elevated temperatures and the addition of fresh solvent during static
extraction increase the mass transfer in PLE similar to the mass transfer in Soxhlet.

• In addition, increased temperatures result in decreased surface tension and solvent viscosity,
which enhance the solvent’s ability to wet and penetrate the matrix. Elevated pressure allows
the use of liquid solvent at temperatures above their boiling points. The high pressure also
allows solvent to solubilize air bubbles, which improves the solvents capability to come in
contact with the entire sample (especially analytes trapped within matrix pores).

• PLE techniques reduce solvent consumption and extraction time, with increased efficiency
and analyte recovery precision compared to previous methods.
On average, Soxhlet extractors consume 100–500 mL of organic solvents over a 12 to 24 hours
extraction period. Typically, PLE extractions require between 50 to 150 mL of solvent per
sample over a 15 to 40-minutes total extraction time. As a result of these improvements, PLE
can be approved. The yield of a chemical extraction using the solid-liquid process can depend
on the type of solvents with varying polarities, pH, extraction time, and temperature as well as
on the chemical composition of the sample. Under the same conditions of time and
temperature, the solvent and the chemical properties of the sample become the two most
important factors.

The main advantages of the use of solvent extraction are its low processing cost and the ease of
operation. On the other hand, the drawbacks are low selectivity, low extraction efficiency,
production of solvent residue, and environmental pollution.

2.6.3 Caffeine extraction from Kola nut

In other to extract caffeine from kola nuts, the fresh Kola nuts are to be subjected to processes
such as drying, grinding, flavour-charging &: extraction, filtration, and purification; as caffeine
isolation from kola nut will expressly involve the solid-liquid extraction techniques. On another
view-point for the extraction of caffeine from caffeinated shampoo, the liquid-liquid extraction
technique will be employed so as to separate crude caffeine from other solutions in the form of
either a binary or tertiary mixture (i.e component separation in different layers/phases), before
evaporation, drying, and purification processes will commence. The process to be used for
caffeine extraction from kola nut is “Johnson's Method” — which is a solid-liquid extraction
technique, and that to be employed for the extraction of caffeine from shampoo is the “Liquid-
liquid extraction method.”

2.7 Johnson's Extraction Method

Extraction methods using solid-phase extraction (SPE) cartridges is generally preferred over
traditional liquid-liquid extraction methods for caffeine isolation from kolanut. Johnson's solid-
phase extraction method will require a less preparative work, thus increasing sample through-
put. This SPE method will develope the process to extract the accurate concentration of crude
caffeine (1,3,7-trimethylxanthine). Generally, Johnson's extraction method is divided into the
following processes or stages: Drying, Grinding, Boiling/Flavour-charging, Filteration,
Distillation, Evaporation.

Drying of kola nut

Fresh Kola nut are to be weighed first before beginning the extraction method so as to avert
result alteration. Basically, this will be done with an evaporating dish and a weighing balance
before drying will begin in a thermostatic oven (80°C) for 48 hours, which at the end of 48
hours, the dry weight of the Kola nut will then be obtained by re-weighing.

Grinding of dry Kola-nut.

This is the second stage of caffeine extraction and dry Kola nut will undergo size reduction by
using a manual grinder to reduce them to fine particles.11

Boiling/Flavour-charging, filtration, and distillation processes will all be carried out in

continuous steps with the mixing of reagents simultaneously, before evaporating the portioned
mixture to dryness to extract crude caffeine. Reagents like 2M (conc.) Sulphuric acid [H2SO4],
Ammonia (NH3), Lead Acetate Monohydrate, and Chloroform [CHCl3] will also aid the
implementation of this caffeine extraction method.

2.7.1 Caffeine extraction from caffeinated-shampoo using Liquid-liquid extraction.

Liquid–liquid extraction (LLE) is a solvent extraction technique that can be employed to

separate compounds based on their relative solubilities in two different immiscible liquids,
which can be typically be water (polar), and another organic solvent (non-polar). Generally,
there is a net transfer of one or more species from one liquid into another liquid phase (from
aqueous to organic). Such transfers are driven by chemical potential which implies that once
the transfer is complete, the overall system of the chemical components that make up the
solutes and the solvents will be in a more stable configuration/lower free energy. The solvent
that is enriched in the solute(s) is then called ‘extract,’ with the feed solution that will be
depleted in solute(s) termed as 'raffinate.' LLE is performed using a variety of apparatus,
ranging from separatory funnels to counter-current distribution equipment known as mixer
settlers, and this type of process is commonly performed after a chemical reaction as part of a
work-up.

Solvent extraction

The term partitioning is commonly used to refer to the underlying chemical and physical
processes involved in liquid–liquid extraction, but on another note this may be fully
synonymous with it. The term solvent extraction also refers to the separation of a substance
from a mixture by preferentially dissolving that substance in a suitable solvent. With this view
at hand, a soluble compound can be separated from an insoluble compound or a complex
matrix.
The PEG–NaCl system will therefore be effective at partitioning small molecules, such as
peptides and nucleic acids. These compounds are often flavorants or odorants. The system will
then be used to isolate or eliminate particular flavors and caffeine extraction from caffeinated
shampoo will be done using liquid–liquid extraction, specifically direct and indirect liquid–liquid
extraction (Swiss Water Method).

2.8 Caffeine Characterization

2.8.1 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 14th century. The tree is a constituent of the
low land forest, requiring a hot, humid climate and is capable of withstanding up to three
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 classification 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 with the species are C. nitida cultivated around Ghana, Nigeria, e.t.c
and C. acuminata which is distributed 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 thus calling for the bitter & astringent taste in the nuts.
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.

2.8.2 Shampoo

Shampoo is a hair care product, typically in the form of a viscous liquid, that is used as a hair
cleaning product. Shampoo is used by the application to wet hair, massaging into the scalp, and
then rinsing it out. Less commonly, shampoo is available in bar form, like a bar of soap.
Shampoo is generally made by combining a surfactant, most often sodium lauryl sulfate or
sodium laureth sulfate, with a co-surfactant, most often cocamidopropyl betaine in water to
form a thick, viscous liquid. Other essential ingredients include salt (sodium chloride), which is
used to adjust the viscosity, preservatives and fragrance. Other ingredients like caffeine are
generally included in shampoo formulations to maximize qualities such as: minimal skin and eye
irritation, pleasing foam, pleasant fragrance (from coffee shampoo), low toxicity, good
biodegradability, slight acidity (pH less than 7), no damage to hair, and repair of damage
already done to hair. Many shampoos are pearlescent and such an effect is achieved by the
addition of tiny flakes of suitable materials, e.g. glycol distearate, chemically derived from
stearic acid, which may have either animal or vegetable origins. Many substantial caffeinated-
shampoo products are purported to protect hair from damage due to ultraviolet radiation
though caffeine sources can limit the concentration of such ingredients in other to be effective.

Caffeine shampoo is a treatment that is designed to stimulate hair growth. Such shampoo
contains caffeine extract, which is thought to help stimulate hair growth and help with hair loss.
Caffeine shampoo stimulates hair growth with the use of caffeine extract, and fundamental
proofs will tally with caffeine stimulating hair growth in a laboratory dish. Caffeine can help to
promote hair growth by targeting DHT — which is the hormone responsible for hair loss.