OBJECTIVE:

To determine the concentration of caffeine in tea bag by using UV-VIS

spectrophotometric method.
To study the right techniques in handling UV-VIS Instrument.

INTRODUCTION:

Caffeine is a central nervous system stimulant

Figure 1 : Structure of caffeine
themselves to no more than 100mg of caffeine per day.
of the methyl xanthine class. It is the world most
widely consumed psychoactive drug, but unlike
many other psychoactive substances it is legal
and unregulated in nearly all parts of the world.
Caffeine is most fond in the bean of coffee plant
and the leaves of bushes. Up to 400 milligrams
(mg) of caffeine a day appears to be safe for
most healthy adults. Thats roughly the amount
of caffeine in four cups of brewed coffee, 10 cans
of cola or two energy shot drinks. Although
caffeine use may be safe for adults but not a
good idea for children. Adolescents should limit

Even among adults, heavy caffeine use can cause unpleasant side effects. Caffeine are not
good for those who take certain medications. Heavy daily caffeine use more than 500 to
600mg a day may cause side effects such as insomnia, nervousness, restlessness, irritability,
stomach upset, fast heartbeat and muscle tremors. Some people may be more sensitive to
caffeine than others. If you are susceptible to the side effects of caffeine, just small amounts
of it may prompt unwanted effects, for example sleep problems. How you react with caffeine
may be determined in part of how much caffeine you consumed. People who did not take
caffeine regularly tend to be more sensitive to its negative effects. Others factors include
body mass, age, medication use and health conditions such as anxiety disorder. Research also
suggest that men may be more susceptible to the effects of caffeine than women.

Caffeine works by stimulating the central nervous system (CNS), heart, muscles and the
centre that control blood pressure. Caffeine can also rise the blood pressure, but might not
have this effect in people who are used to it. Caffeine can also act like a water pill thats
increase the urine flow. But again, it may not have this effect in people who consume caffeine
regularly. Also, drinking coffee during moderate exercise is not likely to cause dehydration.
 UV-VIS Molecular Absorption Spectrometry is the most common analytical technique in
the analytical laboratory in which involves the absorption of ultraviolet and visible radiation
for quantitative purposes. Absorptions commonly occurs with many organic molecules,
metals and metal-organics complexes. In UV-VIS, absorption involves bonding (outer
valence) electrons. UV-VIS spectrophotometer are made up of stable source of radiant
energy, a transparent sample container, a device for isolating specific wavelength, a radiation
detector which converts transmitted radiation into a useable signal and a signal processor or
readout. UV-VIS spectra can be used to detect the presence of absorbing functional groups or
chromophores.

UV-VIS spectroscopy is valid, simple and cost effective method in determining the
concentrations of absorbing species if applied to pure compounds and used with the
appropriate standard curve. A standard curve relating absorbance to concentration can be
developed for any compounds and used to determine the concentration of sample containing
the same compound. The analysis should be done at a wavelength of maximum absorbance
and located in relatively flat region of the spectra. This is to ensure that the absorbance will
be high and constant in narrow range around the chosen wavelength. The optimal wavelength
should be selected on the good absorbance of the analyte and low absorbance by other
species presence in the solution. This wavelength will allow valid absorption measurements
to be made on analyte samples that contain mixture of materials.

A radiation sources of spectroscopy must generate a beam with sufficient power,

wavelength range and stability for detectable and reproducible results. Many UV-VIS
spectrophotometer use a deuterium lamp for the UV region and switch to tungsten filament
lamp at 350nm for the visible region. The electrical excitation of deuterium at low pressure
results in continuous spectrum of emitted radiation from 160nm to the beginning of visible
region which is in 375nm. At around 350nm, the instrument switches its radiation source that
a black body and thus is temperature dependant.

The sample container, that we called cuvette or cell, must be constructed of a material that
is transparent to radiation in the wavelength range of interest. Containers of quartz or fused
silica are necessary in the UV range and can be used in the 700 -3000nm infrared region.
Glass and plastic can be used in the visible region as well. The wavelength selector are filters
and monochromators. The detector are phototube, photomultiplier tube (PMT), photodiode,
photodiode array.
RESULT AND CALCULATIONS:

Table 1: Preparation of Standard Caffeine Solution

Standard Concentration (ppm) Volume From 1000ppm

Caffeine Stock Solution (ml)
Blank 0 0
1 10 0.25
2 20 0.50
3 30 0.75
4 40 1.00
5 50 1.25

Calculation of preparation Standard Caffeine:

100mg = 100 x 10-3g

100 x 10-3g = 1000ppm

100mg

Dilution Equation: M1V1=M2V2 thus;

V1 (1000ppm) = 10 ppm (25ml)

V1 = 0.25 ml

V1 (1000ppm) = 20 ppm (25ml)

V1 = 0.50 ml

V1 (1000ppm) = 30 ppm (25ml)

V1 = 0.75 ml

V1 (1000ppm) = 40 ppm (25ml)

V1 = 1.00 ml

V1 (1000ppm) = 50 ppm (25ml)

V1 = 1.25 ml

Table 2: Absorbance of Caffeine Standard Solution at max

Concentration of Standard Solution (ppm) Absorbance
10 0.019
20 0.055
30 0.085
40 0.105
50 0.130

Tea Sample Absorbance

1 0.130
2 0.055
3 0.105

Calibration Curve of Caffeine Standard Solution

1.4

1.2 f(x) = 0x + 0.96

R = 0.91
1

0.8
Absorbance 0.6

0.4

0.2

0
0 20 40 60 80 100 120 140 160

Concentration

Figure 1: Calibration curve of caffeine standard solution

Table 3: Absorbance of Tea Sample at max

 Table 4: Concentration of tea sample based on the calibration curve equation

Tea Sample Absorbance Concentration (ppm)

1 0.130 48.630
2 0.055 20.851
3 0.105 39.370

Calculation of tea sample concentration based on the Beers Law:

y= mx + c where;

y = absorbance, m = gradient, x = concentration, c = y-axis

From graph: y = 0.0027x - 0.0013 thus;

Tea sample 1:

Y = mx + c

0.130 = 0.0027x 0.0013

X = 48.630 ppm

Tea sample 2:

Y = mx + c

0.055 = 0.0027x 0.0013

X = 20.851 ppm

Tea sample 3:

Y = mx + c

0.105 = 0.0027x 0.0013

X = 39.370 ppm

Average concentration of tea sample:

(48.630 + 20.851 + 39.370) ppm = 36.284 ppm

 3
Actual concentration of tea sample:

Average concentration x dilution factor = 36.284 ppm x 10

= 362.84 ppm

Amount of caffeine in triplicate sample:

362.84 mg x 0.5L = 181.42 mg

L
Percentage weight (w/w) of caffeine in triplicate sample:

(Mass of solute mass of solution) x 100 %

(181.42 mg 2000 mg) x 100% = 9.07 %

Table 5: Molar absorptivity of tea sample

Tea sample Absorbance Concentration (ppm) Molar Absorptivity ()

1 0.130 48.630 2.67 x 10-3
2 0.055 20.851 2.64 x 10-3
3 0.105 39.370 2.66 x 10-3

Calculation for the tea sample molar absorptivity using the equation:
A = bc where;
A = absorbance, = molar absorptivity, b = length of cuvette, c = concentration of analyte

Tea sample 1:
0.130 = (1.00) (48.630)
= 2.67 x 10-3

Tea sample 2:
0.055 = (1.00) (20.851)
= 2.64 x 10-3

Tea sample 3:
0.105 = (1.00) (39.370)
 = 2.66 x 10-3

DISCUSSION:

In this experiment, the spectrum for standard caffeine and caffeine from the tea sample were
obtained. The maximum peak of absorption was measured at peak 285nm, this is because the max
is the wavelength that gives the maximum value applied in the experiment. The peak
maximum correspond to the maximum value of molar absorptivity and hence the sensitivity
of the analysis.
Beers Law typically applied in this experiment. Beers law is strictly valid only where the
molar absorptivity changes little wavelength. The linearity of the Beers Law is limited to
chemical and instrumental factors. Beers law describe absorption behaviour of relatively low
analyte concentration in a solution as a limiting law. At high concentration will involve
solute-solvent interaction, solute-solute interaction and hydrogen bonding which will affect
the analyte concentration and its absorptivity. Beers law applies monochromatic radiation
with use of continuous source and a monochromator which will provide a very narrow band
with an effective bandwidth. Therefore, the selection of the wavelength should be made at the
max.
In this experiment, we can observed that there are few chromophores presence in the
caffeine molecule by the presence of carbonyl group (C=O) in it. A chromophore is the
functional group of an organic molecule that responsible for the molecule to be coloured.
This particular chromophore will undergo an n * transition around 280-290 nm.
Therefore, by this fact it is clearly stated why the peak of 285nm wavelength was chosen as
the taken into account. Secondly, there were possibilities that the structure undergo - *
transition. This energy transition was due to the presence of double bond and will occur in the
near UV region. According to Pavia, Lampman, Kriz and Vyvyan (2015), the energy
happened in this type of transition is rather a high energy level and their positions are
sensitive to the presence of substitution.
The absorbance values that we obtained from the spectrum of standard caffeine was 0.019,
0.055, 0.085, 0.105 and 0.130 according to their respective concentration. Meanwhile, for the
sample we analysed, the absorbance value that take was taken are 0.130, 0.055 and 0.105
respectively. After calculations had been made, we obtained the percentage weight (w/w)
with the value of 9.07%. This came from the result in plotting the Beers law graph to figure
out the quantitative value of the caffeine from the tea samples we had prepared.
The experiment been made, but we cannot said it is totally succeed even though from the
graph we obtain, the R2 = 0.9926 is nearly to 1. This is because, the sample container or the
cuvette is not matched and it is not clear and transparent to let the light to penetrate through it
completely. Other than that, to get a good result of the Beers law plot, the concentration of
the sample analysed must be diluted to become a sample solution that is very low
concentration. If the solution contains many solute, it could lead to a greater scatter effect of
the source of radiation. In liquids, the extinction coefficient will usually changes slowly with
the wavelength. The rate of change in absorbance with wavelength is the smallest at the
maximum peak of absorbance curve. Therefore, the measurement will be made at this
particular peak to minimize the errors produced by errors in wavelength in the instruments
that is errors due to having a different extinction coefficient than assumed.
CONCLUSION:
Caffeine in the tea sample was successful analyse by using UV-VIS
spectrophotometer. Next, the concentration of caffeine in the tea sample was
successful determined by graph. The average concentration of caffeine in
triplicate sample is 362.84ppm and the amount of caffeine presence in it is
181.42mg. The Beers law equation for this analysis is y = 0.0027x + 0.0013.

REFERENCES:
1. Skoog, D.A., Holler, F.J. & Crouch, S.R. (2007). Principle of Instrumental
Analysis
(6th Edition). Belmont, CA: Brooks/Cole, Cengage learning.

2. Dobrinas, S., Soceanu, A., Popescu, V. (2013). Optimization of a UV-VIS

Spectrometric Method for Caffeine Analysis In Tea Bag, Coffee And Other
Beverages. Scientific study &Research. Chemistry & chemical engineering,
Biotechnology, food industry, 14(2), 71-78.

3. Atomssa, T. & Ghollap, A.V. (2010). Characterization of Caffeine and

Determination of Caffeine in Tea leaves Using UV-VIS Spectrometer.
African Journal of Pure Applied Chemistry, 5(1), 1-8.

4. Bhawani, S.A., Fong, S.S., & Mohamad Ibrahim, M.N. (2015). Spectrometric
Analysis of Caffeine. International Journal of Analytical Chemistry.
Retrived from http://dx.doi.org/10.1155/2015/170239.

5. Pavia, D.L., Lampman, G.M., Kriz, G.S., & Vyvyan, J.R. (2015). Introduction to
Spectroscopy. (5th Edition). Singapore: Cengage learning Asia Pte Ltd.
QUESTIONS:

1. What is the advantage of applications of derivative spectra?

Derivative spectrometry is very useful to separate the signal of analytical compound

from background or other compound signal. Besides, it is also make peak appears
sharper and easier to take information.

2. Background-correction methods have been developed for the determination of

caffeine in beverage, coffee and tea by second derivative ultraviolet spectrometry. The
second derivatives of caffeine is measured at 298.6nm. What is the purpose of this
method?

Background-correction method is to minimize the derivations and enhancing the

signal from the analyte. This is because there are other signal detected by the detector
from things other than sample resulting a loss in spectral detail such as broadening of
peak and peaks in place other than where the sample absorb.

3. Why does caffeine of tea increase as we boil it for more time?

Leaving the tea bag longer will make a stronger cup of tea. The concentration of
caffeine (along with the flavour molecules and everything else) will slowly trend
towards an equal concentration in the leaf and in the water. The longer the tea bag in
hot water, the closer to equilibrium you will get.