AS245: Bachelor in Science Applied Chemistry

Title:

Analysis of Caffeine in Tea Bag using Second

Derivative UV Spectrometry

Course: Spectrochemical Methods of Analysis

(CHM580)
Name: Mohamad Nor Amirul Azhar b. Kamis
Matrix no:

2014647344 (AS245 3S)

Date of experiment: 8th April 2015

Date of submission: 29th May 2015
Group members: 1. Nurnailah bt. Noorazlan
2. Nor Amirah bt. Ahmad Azuan
3. Noramira bt. Saad
It is my responsibility as a student of UiTM to adhere
to truthfulness and avoid dishonesty, fraud or deceit
of any type in connection with write up and conduct
of this experiment.
Signature:__________
Date:

TITLE:

Determination of caffeine in tea bag using second derivative UV

Spectrometry

ABSTRACT:
The sample used in this experiment is a bag of tea where the caffeine was extracted
from. The sample was analysed by using the UV-Visible Molecular Absorption
Spectrometry to know the concentration and the amount of caffeine in the sample.
The sample has been triplicate to ensure the accuracy and getting more accurate
data. In order to ensure the amount of caffeine in samples is in the range of standard
solutions, the samples were diluted with dilution factor of 10. The average
concentration of caffeine in the triplicate samples is 123.4ppm (actual concentration)
with relative standard deviation of 8.97. The amount of caffeine in the samples is
30.85mg and the with 1.31 w/w%.
INTRODUCTION:
Caffeine with molecular formula of C 8H10N4O2 is a legal psychoactive drug that most
widely consumed. It is found in the seeds, nuts or leaves, for this experiment is the
tea leaf. Beverages containing caffeine are ingested to relieve or prevent drowsiness
and to increase the energy level. Commonly, there is about 80-175mg of caffeine in a
cup of coffee. Overdose amount of caffeine consumed will lead to unpleasant
symptoms such as nervousness, irritability, restlessness, insomnia, headaches and
palpitations. For most of tea brands, the amount of caffeine in a tea bag is about 30100mg. The UV-Visible molecular absorption spectroscopy was used to analyse the
amount of caffeine in the tea bag sample. This instrument measure the absorbance
that is directly proportional to the path length, b and concentration, c. This can be
expressed in the Beers Law: A = bc, where is the constant molar absorptivity.
The absorption of UV or visible radiation corresponds to the excitation of outer
electrons which can be divided into 3 types of electronic transition that caused the
absorption of the electromagnetic radiation. The electronic transitions are involving p,
s and n-electrons, involving charge-transfer electrons and involving d and felectrons. From the absorbance data, the concentration of sample can be obtained
by plotting the calibration curve.

OBJECTIVES:
1. To develop a simple background correction method which enable the
determination of caffeine in tea bags by using second derivative UVSpectrometry.
2. To determine the concentration and amount of caffeine in tea bag sample.

EXPERIMENTAL:
A. Preparation of reagents:
1. Preparation of 0.025M copper (II) acetate solution:
a. About 0.25g of Cu(CH3COO)2 solid was weighed and then diluted in
distilled water.
b. The solution was then transferred into a 50mL volumetric flask and
distilled water was added until the mark.
2. Preparation of 1.0M NaOH solution:
a. About 2g of NaOH solid was weighed and then diluted in distilled water.
b. The solution was then transferred into a 50mL volumetric flask and
distilled water was added until the mark.
B. Preparation of standard solutions:
Caffeine stock solution = 100ppm
Standard

Concentration (ppm)

Volume of 100ppm
caffeine stock solution

pipetted (mL)
Blank
0
0
1
10
5
2
20
10
3
30
15
4
40
20
5
50
25
a. The volume of 100ppm caffeine stock solution was pipetted into each
standard of 50mL volumetric flasks based on the table above.
b. The distilled water was used to mark up the solution until the calibration
mark of 50mL.
Sample calculation of standard solution 1:
C1V1 = C2V2
100V1 = 10(50)
V1 = 5mL

c. Each standard solution was prepared for analysis by mixing 1mL of 1.0M
NaOH solution, 4mL of 0.025M Cu(CH 3COO)2 and 5mL of standard
solution in 5 different vials.
d. All vials were shaken and centrifuge for 10 minutes at 20rpm.
C. Preparation of sample:
a. 2.3635g of tea was added into 200mL distilled water and was heated for
15 minutes.
b. The mixture was let to cool at room temperature.
c. The cooled sample then was transferred into 250mL volumetric flask and
distilled water was added until the calibration mark.
d. 5mL of the sample solution prepared was pipetted into a 50mL volumetric
flask and distilled water was added until the calibration mark (working
sample solution).
e. The working sample solutions was prepared for analysis by mixing 1mL of
1.0M NaOH solution, 4mL of 0.025M Cu(CH 3COO)2 and 5mL of sample
solution in 3 different vials (triplicate sample).
f. All vials were shaken and centrifuge for 10 minutes at 20rpm.

RESULT:
Absorbance at 270nm wavelength:

Blank
10ppm standard
20ppm standard
30ppm standard
40ppm standard
50ppm standard
Sample 1
Sample 2
Sample 3

Absorbance
-0.0001
0.3258
0.5059
0.7411
0.9921
1.1984
1.5281
1.5023
1.4376

Absorbance at maximum wavelength of 298.61nm second derivative order:

Absorbance

Blank
10ppm standard
20ppm standard
30ppm standard
40ppm standard
50ppm standard
Sample 1
Sample 2
Sample 3

0.0000
0.0864
0.1609
0.2328
0.2613
0.3831
0.1002
0.1103
0.0953

Graph of Absorbance versus standard concentration at 298.61nm wavelength

0.45
0.4
0.35

f(x) = 0.01x + 0.02

R = 0.97

0.3
0.25
Absorbance

0.2
0.15
0.1
0.05
0
5

10

15

20

25

30

35

40

45

50

55

Standard concentration, ppm

FIGURE 1:

Graph of absorbance versus standard concentration at 298.61nm

wavelength

Sample calculation of determination of sample concentration:

Sample 1: absorbance = 0.1002
y = 0.0069x + 0.0168
0.1002 = 0.0069x + 0.0168
x = 12.09ppm
Sample 1
Sample 2

Absorbance
0.1002
0.1103

Concentration (ppm)
12.09
13.55

Sample 3

0.0953

11.38

Average concentration of sample =

12.09+13.55+11.38
3

= 12.34ppm
*See appendix for graph that show how the concentration of sample is obtained.

Standard deviation =

1
( x imean)2

N 1 i
1
2
2
2
[( 12.0912.34 ) + ( 13.5512.34 ) + (11.3812.34 ) ]
31

= 1.1064

Relative standard devation, %RSD

standard deviation
mean

1.1064
12.34

100%

100%

= 8.97

Actual concentration of sample (average)

= 12.34ppm 10 (dilution factor)

= 123.4ppm

Amount of caffeine in triplicate sample

= 123.4

mg
L

= 30.85mg
w/w% of triplicate sample =

30.85 mg
2363.5 mg

100%

0.25L

= 1.31 w/w%

DISCUSSION:
The analysis is done at maximum wavelength where the highest absorbance can be
read which is at 298.61nm. The maximum wavelength is in the range of UV region
which we desired for this experiment. Figure 1 shows the graph of absorbance
versus standard concentration at maximum wavelength from the result by the
instrument analysis. The graph shows value of R 2 of 0.9687 that indicate there is
error in the preparation of the standard solutions. The good standard solutions will
give the value of R2 nearer the value of 1. The error may occur while pipetting the
stock solutions or from any other contaminants from the apparatus.
The actual concentration of the sample in average after taking the dilution factor into
account is equal to 123.4ppm. With that value, the amount of caffeine in the triplicate
sample calculated to be 30.85mg. This amount of caffeine is in the range of the
amount of caffeine in a tea bag by most brands of tea producers. The amount of
caffeine analysed also in the range of safe consume-able amount with less
probability of overdose. In order to ensure the accuracy between the triplicate
samples, the relative standard deviation was calculated. The calculated value of
relative standard deviation of the triplicate sample is 8.97. The smaller the value of
relative standard deviation to zero indicates the more accurate sample preparation.
For this triplicate samples, there is need of improvement in the sample preparation.

CONCLUSION:
The actual average concentration of caffeine in tea bag sample is 123.4ppm that
corresponds to 30.85mg with 1.31 w/w%. The amount of caffeine is in the range of
suggested amount.

REFERENCES:

1. F. J. Holler, D. A. Skoog, S. R. Crouch (2007), Principles of Instrumental

Analysis 6th ed.), United States, Brooks/Cole Cengage Learning.
2. Caffeine, Retrieved April 20, from http://en.m.wikipedia.org/wiki/Caffeine
3. UV-Vis Absorption Spectroscopy Theoretical principles, Retrieved April 20,
from http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab1.htm