Introductory Chemistry

SCHOOL OF SCIENCE AND HEALTH

Introductory Chemistry
Laboratory 5

Analytical Chemistry:
Extraction of Caffeine
from Common Beverages

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 Extraction of Caffeine
AIMS

 To extract isolate determine the yield and identify an extracted

component from a range of drinks.
 To quantitatively determine which drinks contain most caffeine
 To identify the caffeine extract using a standard laboratory procedure
(TLC)
 To determine the chemical yield of the extraction
 To compare the relative amounts of caffeine in various beverages

BACKGROUND

Aromatic tea, freshly brewed coffee and refreshing cola soft drink on ice are popular
beverages that share one common ingredient: caffeine. The history of caffeine is as
colourful as its many current uses. A naturally occurring plant extract, it is found in the
leaves, seeds and/or fruit of more than 63 plant species growing around the world. Since
ancient times, people have consumed caffeine in foods and beverages.

In Australia, regulatory authorities permit a maximum of 145mg of caffeine per kilogram

(ppm) of cola-type soft drink. Amounts used by manufacturers are usually considerably
lower, averaging about 100mg per litre, or 20mg per 200ml serving.

Small amounts of caffeine have been used in various soft drinks for over a century. It was
part of the original formula for cola soft drinks, found in one of the flavouring extracts.
Like other foods and beverages, the flavour and taste of soft drinks depend on subtle
amounts and combinations of ingredients. Caffeine contributes to the overall flavour
profile of cola type soft drinks by providing a slightly bitter flavour to the product.

Energy drinks are designed to deliver a heightened state of alertness and a

combination of ingredients are used to perform this function. The caffeine is known as
a stimulant that affects cognitive and mental performance.

The caffeine content of an average serving of an energy drink is 80mg (this varies
between brands). Is this the same as a cup of coffee and twice that of tea or cola soft
drink as the literature suggests? In today’s practical, we will test this claim,

CAFFEINE

Caffeine and theobromine (Found in chocolate) are both xanthine stimulants, and
theobromine is the metabolite of caffeine, which is in fact methyltheobromine. Caffeine’s
IUPAC name is however 7-methyltheophylline.
 Fig 1 some of the better-known xanthine compounds

Caffeine has the molecular formula C8H10N4O2.

Theobromine’s molecular formula is C7H8N4O2

Caffeine is an alkaloid which can be obtained from the wastes of tea, or coffee or from
the dried leaves of Camellia sinesis, or it may be prepared synthetically. It is also present
in guarana, maté and kola. Both caffeine and theobromine are naturally occurring
members of the methylxanthine family and whilst caffeine has an effect on predominately
the central nervous system, theobromine is associated with diuretic responses.
Methylxanthines are not particularly potent, needing to be administered in gram doses.

Caffeine can kill. Although this is not often realised, it does have a toxicity level, which
varies from person to person but is typically in the region 5 to 50 g. For comparison, a
typical cup of coffee contains approximately 100 mg of caffeine. This would mean
needing to drink at least fifty cups of coffee in one go for it to be toxic. It should also be
noted that these are the toxicity levels for humans. The levels for horses could be vastly
different. Although it is not easy to say whether they could ingest more or less, as their
body weight is much greater, this might imply the ability to ingest more. Caffeine is not a
regular part of a horse’s diet whereas for humans it is. Therefore, this suggests humans
may be more tolerant of caffeine.

THIN LAYER CHROMATOGRAPHY - TLC

TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick answer
as to how many components are in a mixture. TLC is also used to support the identity of
a compound in a mixture when the Rf of a compound is compared with the Rf of a known
compound (preferably both run on the same TLC plate).
A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a
solid adsorbent (usually silica or alumina). A small amount of the mixture to be analysed
is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of
a solvent in a developing chamber so that only the very bottom of the plate is in the
liquid. This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate
by capillary action.

As the solvent moves past the spot that was applied, an equilibrium is established for
each component of the mixture between the molecules of that component which are
adsorbed on the solid and the molecules which are in solution. In principle, the
components will differ in solubility and in the strength of their adsorption to the adsorbent
and some components will be carried farther up the plate than others. When the solvent
has reached the top of the plate, the plate is removed from the developing chamber,
dried, and the separated components of the mixture are visualized. If the compounds are
coloured, visualization is straightforward. Usually the compounds are not coloured, so a
UV lamp is used to visualize the plates. (The plate itself contains a fluorophore which
fluoresces everywhere except where an organic compound is on the plate.)

TLC ADSORBENT

Here we use silica gel plates (SiO2) almost exclusively. (Alumina (Al2O3) can also be
used as a TLC adsorbent.) The plates are aluminium-backed and you can cut them to
size with scissors. Our plates are purchased ready-made from EM Sciences or from
Scientific Adsorbents. The adsorbent is impregnated with a fluorophore, zinc sulfide. The
fluorophore enables most organic compounds to be visualized when the plate is held
under a UV lamp. In some circumstances, other visualization methods are used, such as
charring or staining.

TLC SOLVENTS OR SOLVENT SYSTEMS

Choosing a solvent is a question of solubility: choosing a phase that will carry the
component on interest away from other parts of the mixture. This is therefore a question
of the degree of interaction between solvent and solute and therefore a question of the
relative polarities of the solvents under consideration.

INTERACTIONS OF THE COMPOUND AND THE ADSORBENT

The strength with which an organic compound binds to an adsorbent depends on the
strength of the following types of interactions: ion-dipole, dipole- dipole, hydrogen
bonding, dipole induced dipole, and dispersion forces. With silica gel, the dominant
interactive forces between the adsorbent and the materials to be separated are of the
dipole-dipole type. Highly polar molecules interact fairly strongly with the polar Si—O
bonds of these adsorbents and will tend to stick or adsorb onto the fine particles of the
adsorbent while weakly polar molecules are held less tightly. Weakly polar molecules
thus generally tend to move through the adsorbent more rapidly than the polar species.
TLC - RETENTION FACTOR (RF)

The retention factor, or Rf, is defined as the distance travelled by the compound divided
by the distance travelled by the solvent.

Rf = distance travelled by the compound/distance travelled by solvent For example, if a

compound travels 2.1 cm and the solvent front travels 2.8 cm, the Rf is 0.75:

Provided that the chromatography conditions below are also constant the Rf for a
compound is a constant from one experiment to the next:

 solvent system
 adsorbent
 thickness of the adsorbent
 amount of material spotted
 temperature

Since these factors are difficult to keep constant from experiment to experiment, relative
Rf values are generally used in the literature. “Relative Rf” means that the values are
reported relative to a standard, or in other words it means that you compare the Rf values
of analytically pure standard compounds run on the same plate at the same time.

The larger the Rf of a compound, the larger the distance it travels on the TLC plate.
When comparing two different compounds run under identical chromatography
conditions, the compound with the larger Rf is less polar because it interacts less strongly
with the polar adsorbent on the TLC plate. Conversely, if you know the structures of the
compounds in a mixture, you can predict that a compound of low polarity will have a
larger Rf value than a polar compound run on the same plate.

The Rf can provide corroborative evidence as to the identity of a compound. If the identity
of a compound is suspected but not yet proven, an authentic sample of the compound, or
standard, is spotted and run on a TLC plate side by side (or on top of each other) with the
compound in question. If two substances have the same Rf value, they are likely (but not
necessarily) the same compound. If they have different Rf values, they are definitely
different compounds. Note that this identity check must be performed on a single plate,
because it is difficult to duplicate all the factors which influence Rf exactly from
experiment to experiment.
VISUALIZING THE SPOTS

If the compounds are coloured, they are easy to see with the naked eye. If not, a UV
lamp is used.

EXPERIMENTAL PROCEDURE

In this experiment we will evaluate the caffeine content of a range of energy drinks and
teas. You will be required to calculate and discuss the yield of the extract.

CAFFEINE EXTRACTION:

1. Place 4.8 g of pre-weighted calcium carbonate into a 400mL beaker with a magnetic
stirring bar.

1. For Tea: Add 4 tea bags into the beaker and pour 100mL of distilled water
over the tea.

Do not break open the tea bags as they become very difficult to filter.

2. Place the mixture onto a hotplate/stirrer. Stir the mixture for 1 minute.
Continue to stir, turn the heat element on, and boil the mixture. Once boiling,
switch off the heater and leave to react for 15 minutes.

3. Remove from the hotplate. Place a thermometer into mixture. Cool the reaction to
55°C. While you are waiting prepare the TLC plate by ruling a 1cm line on the
bottom of edge of a thin layer plate (5 cm × 10 cm) using a faint pencil. Mark two
pencil spots on the 1cm line. Label them A and B. Do not break the silica surface of
the TLC plate.

4. Filter the mixture under vacuum, using a Buchner filtration flask and Buchner funnel

5. Rinse the Buchner funnel thoroughly by adding cold deionised water and then
turning on the vacuum once more.

6. Pour the cold mixture into a separating funnel.

7. To extract any residual caffeine left behind pour 15mL of dichloromethane (DCM)
into the Buchner flask, and then add this rinse solution to the separating funnel.

8. Cool the mixture by add a few pieces of ice into the separating funnel.

9. Extract the caffeine from the aqueous layer into the organic DCM solvent by
inverting the funnel and swirling gently for 2 minutes.
 Whilst swirling- ENSURE THAT YOU INVERT THE FUNNEL and DO NOT
SHAKE VIGOROUSLY. You may need to release the pressure in the funnel
periodically. Your demonstrator will show you how to do this without losing the
extract

10. Remove the lower DCM layer by draining it from the funnel into a dry beaker.
Add another 10mL of DCM to the separating funnel where the aqueous layer
remains.

11. Invert the funnel and swirl the solutions gently for 2 minutes.

12. Add the second DCM (lower) layer to the first extract.

13. Rinse the separating funnel with water, and empty the funnel completely.

14. Add the two combined DCM layers to the separating funnel and add 20mL of
deionised water.

15. Invert the funnel and swirl the solutions gently for 2 minutes.

16. Drain the lower DCM layer into a clean 100mL conical flask.

17. Add a small amount of anhydrous sodium sulfate to the DCM layer; this will be
used to remove any residual water and thus dry the solution.

18. Allow the solution to stand for 5 minutes in the fume hood then filter the solution
using gravity filtration into another 100mL pre weighed 50mL beaker.

19. For spot A, using a clean capillary tube, draw the solution up the capillary tube. Apply this
solution to the first pencil spot.

Do not exceed 3mm in diameter of the circle formed.

Allow the solution to dry on plate and repeat the addition of the solution in the same
spot.

20. Place onto the steam bath to evaporate all of the DCM. A crude solid caffeine will
form at the bottom of the beaker.

Mass of flask + extract =

Mass of flask =

Mass of extract =

21. For spot B, a standard solution of pure caffeine will already be prepared. Using a
new clean capillary tube draw up the solution as above. Apply this solution to the
second spot
 Do not exceed 3mm in diameter of the circle formed.

Allow the solution to dry on plate and repeat the addition of the solution in the
same spot.

22. To develop the plate carefully place the plate, base line down, into a developing
chamber (jar) containing a mixture of ethyl acetate and ethanol (6:4). Screw the
cap on the jar and allow the solvent to rise up the plate until the solvent front is
about 1cm from the top of the plate. Quickly remove the plate from the developing
jar and before the solvent evaporates, mark the position of the solvent front with a
pencil.

23. Place the developed plate under the ultraviolet lamp (254nm). The plate
fluoresces and reveals the position of the extract as separated dark spots. Mark
these spots with a pencil.

This is the end of the Experimental Section

Identify the caffeine peak by comparison with the standard. Are there any other peaks?

What might they be caused by?

Calculate the Rf values for caffeine.

Height of caffeine
above baseline

Height of solvent

Retention Factor (Rf)

Write the mass of caffeine extracted on the board

Calculate the average mass of caffeine extracted for your particular beverage

My beverage

Class average mass mg

Calculate the yield of caffeine for your drink as follows:

4 bags represents four cups of tea which has a volume of 1200 mL yield mg per Litre
(ppm) = extract per 4 teabags (1200 mL) / 1.2

Beverage Mass extract Concentration

mg ppm

Which beverage provides the greatest caffeine hit/ drink?

TOTAL MARKS FOR LABORATORY PRACTICAL (22 MARKS)

PRE-WORK (0-1 marks)

No preparation of lab book Correct preparation of Lab book

0 1

TECHNIQUE (0-5 marks)

Errors in technique (includes No errors in technique,

Did not complete experiment
late arrival in practical) separations good
0 1-4 5

LABORATORY BOOK (0-3 marks)

Correct legible record

Little data or
of all data including
experimental Not all data recorded or
No Laboratory Book procedure and
observations and illegible incorrectly recorded
particularly
records
observations
0 1 2 3

The marks for the results of the laboratory must be submitted online through vUWS.
These will be collated and assessed using the following criteria.

Quality of TLC (assessed by demonstrators)

Plot contains insufficient data to Average quality Good results close to Rf to

define equivalence (too big spots etc.) Standard clear spots
0 1-4 5

Calculation Rf

Calculation Rf Arithmetic errors Calculation correct

0 1-2 3

Calculation and yield

Calculation error Arithmetic errors poor yield Calculation correct, high yield
0 1-4 5