CHM 580

SPECTROCHEMICAL METHODS OF ANALYSIS

LABORATORY REPORT
EXPERIMENT 2 : ULTRAVIOLET-VISIBLE SPECTROSCOPY

NAME INAS SYUHAIDAH BINTI CHAIRIL ANWAR

STUDENT ID 2017420012
CLASS AS245 4M3C
NAME OF PARTNERS  NURUL AFIQAH BINTI MAT AZMAN
(2017413102)
 MUHAMMAD SHUKERI BIN RAZMAN
(2017420096)
 NURFAZA BINTI ZAIMON (2017419966)
 NURFARRAH ATIQAH BINTI ROSLAN

NAME OF LECTURER MADM HALIZA BINTI KASSIM

DATE OF EXPERIMENT 13 MARCH 2019
DATE OF SUBMISSION 12 APRIL 2019
1. TITLE

DETERMINATION OF CAFFEINE IN TEA BAG USING SECOND

DERIVATIVE UV SPECTROMETRY

2. ABSTRACT

The sample used in this experiment is a bag of tea where the caffeine was
extracted from it. The sample was determined by using the Ultraviolet-visible
spectrosocopy to know the concentration and the amount of caffeine in the sample. The
data was analysed by using background-correction method and second derivative. The
sample has been triplicate to ensure the data more accurate and precise. The samples
were diluted with dilution factor of 10 to ensure the amount of caffeine in samples is in
the standard range. The standard solutions of 10ppm, 20ppm, 30ppm, 40ppm and 50ppm
was prepared from 100ppm using dilution technique. The concentration of the three
samples were 10.80 ppm, 10.0 ppm and 10.85 ppm with the relative standard deviation of
2.2361. The amount of the caffeine in the sample 1, sample 2 and sample 3 was
0.027000g , 0.027125g and 0.0271125 respectively with the confidence limit ± 0.9232 g.
The weight percent was 1.09% for sample 1 and 3 while 1.10% for sample 2.
3. INTRODUCTION

Caffeine, a xanthine alkaloid, occurs in teaplants, coffew, mate leaves, guarana

paste and cola nuts. It is also commonly found in beverages such as cola drinks and tea.
Beverages containing caffeine are ingested to relieve or prevent drowsiness and to
improve performance. To make these drinks, caffeine is extracted by steeping the plant
product in water, a process called infusion. Decaf black tea usually contains about 2-10
mg of caffeine. Green tea. usually contains around 25 milligrams of caffeine per 8-ounce
serving. This was the structure of caffeine :

Caffeine can have both positive and negative health effects. It can treat and
prevent the premature infant breathing disorders. Some people experience sleep
disruption or anxiety if consume caffeine. affeine can produce a mild form of drug
dependence which associated with withdrawal symptoms such as sleepiness, headache,
and irritability.

The UV-Visible molecular absorption spectroscopy was used to analyse the

concentration and the amount of caffeine in the tea bag samples. This instrument was
measured the absorbance of light energy or electromagnetic radiation at 298.6 nm, which
excites electrons from the ground state to the first singlet excited state. The principle of
absorbance spectroscopy was Beer’s Law. The formula was A= εbc. From the law, the
absorbance was directly proportional to the concentration. Therefore, increasing of
absorbance will increase the concentration too.
4. LITERATURE REVIEW

Caffeine is recognized as an addictive substance by the World Health

Organization (WHO). Caffeine is classified by the US Food and Drug
Administration as generally recognized as safe (GRAS). It is the most widely consumed
beverage after water (Alan and Iris, 2004). It is probably the most frequently ingested
pharmacologically active substance in the world, found in common beverages such as
coffee, tea, soft drinks, products containing cocoa or chocolate and medications,
including headache or pain remedies and over-the-counter stimulants. Coffee is also the
primary source of caffeine in the diet of adults in some European countries, such as
Finland, Sweden, Denmark and Switzerland. Brewed coffee contains the most caffeine
(56–100 mg/100 ml), followed by instant coffee and tea (20–73 mg/100 ml). (Murphy
and Benjamin 1981, IARC 1991b, Dlugosz and Bracken 1992, Carrillo and Benitez
1996). Excessive consumption of caffeine with lead to negative health effects. But, death
due to excessive caffeine ingestion is not common. Death has been reported after
ingestion of 6.5 g caffeine, but survival of a patient who allegedly ingested 24 g caffeine
was also reported (Stavric 1988, James 1991). It is now widely believed that habitual
daily use of caffeine about four to seven cups of coffee or seven to nine cups of tea
represents a significant health risk and may therefore be regarded as ‘abuse. According to
the U.S. Food and Drug Administration (FDA), about 90 percent of the world’s
population ingests some form of caffeine. In the United States, about 80 percent take in
some caffeine every day. The determination of caffeine in various natural products is also
very important aspect from an economic point. The most widely used methods for the
determination of caffeine in beverages include various analytical techniques such as
derivative spectrophotometer (Alpdo-gan, Karbina, & Sungur, 2002). The derivative
spectrophotometer is relatively easy however, it is not reliable for the determination of
small concentration of caffeine in samples. According to Abebe Belay, Kassahun Ture,
Mesfin Redi , Araya Asfaw a method for measuring caffeine content is reported using
UV/vis spectrophotometer, which is available in most laboratories. Moreover the
methods are easy, fast and cheap for the determination of the caffeine content.
5. EXPERIMENTAL PROCEDURE

A. Preparation of caffeine standard solution

From 100ppm caffeine stock solution, standard solutions 10ppm, 20ppm, 30ppm,
40ppm and 50ppm were prepared. Then, each of it was diluted with distileed water
until the calibration mark in a 50ml volumetric flask. The formula of C1V1=C2V2
was applied for the dilution.

B. Preparation of copper (II) acetate solution and sodium hydroxide solution

Copper (II) Acetate solution was prepared by adding 2.495g of copper (II) acetate
anhydrous into 50ml volumetric flask and diluted with distilled water up to the
calibration mark.

Sodim hydroxide solution was prepared by adding 0.4g of solid hydroxide into a
10ml of volumetric flask and diluted with distilled water up to the calibration mark.

C. Preparation of samples solutions

About 2g of tea sample weighed was boiled with 200ml of distilled water on hot plate
for 15 minutes. Then, cooled the sample at room temperature filtered using Buchner
funnel funnel and diluted it with distilled water in 250ml volumetric flask until
calibration mark. Lastly, the sample solution was diluted again by adding 5ml of tea
sample prepared into a 50ml volumetric flask and diluted up to the calibrated mark.

For the analysis, eight centrifudges were used. The tubes consists five standards
solution and three sample solution. Each tube was filled with 1ml of sodium
hydroxide, 4 ml of copper(II) acetate solution and 5 ml of sample tea solution. The
solutions were analysed by UV Spectrophotometry Lambda 35.
 6. RESULTS

SAMPLE CONCENTRATION WEIGHT (g) ± CL RANGE OF WEIGHT

(ppm) CONFIDENCE PERCENT
LIMIT (%)
1 10.80 0.027000 ± 0.92329 -0.89669, 0.95069 1.09
2 10.90 0.027250 ± 0.92329 -0.89664, 0.95094 1.10
3 10.85 0.027125 ± 0.92329 -0.89656, 0.95082 1.09

Table 1 : The amount of caffeine in teabag using second derivatives ultraviolet spectroscopy

UPPER LIMIT LOWER LIMIT STANDARD RELATIVE STANDARD

DEVIATION (s) DEVIATION (%RSD)
1.31684 -0.52974 0.88 2.2361

Table 2 : Statistical value of the sample

7. DISCUSSION

The absorption spectra of caffeine in tea bag was measured second derivative
using UV-visible spectrometer. The second derivative of caffeine was measured at
298.6nm. There are three preparations required for this experiment which is sample
solution preparations, tea sample preparations and copper (II) acetate solutions. The
concentration of caffein in the test samples was determined by the calibration curve and
comparison with the standard solution of caffeine. Calibration graph was obtained
between the absorbance intensity on y–axis and concentration of caffeine (mg/L) on x–
axis followed Beer-Lambert’s law over the concentration range of standard solution
measured 0.0-50.0 ppm.

From the graph of second derivative of absorbance versus the wavelength, the
result for standard 1 until standard 5 and sample 1, sample and sample 3 was obtained.
The method of linear regression was used for data evaluation and to determine the
concentration of sample 1, sample 2 and sample 3. The graph representing the second
derivative of absorbance versus the concentration of standard was plotted. The
concentration of sample 1, sample 2 and sample 3 are 10.80ppm, 10.90ppm and
10.85ppm respectively. Hence, by using the suitable formula, the original amount of
caffeine in tea bag in 250ml was obtained. The amount of caffeine for sample 1, sample
2 and sample 3 are 27.00 mg, 27.25mg and 27.125mg respectively. The reading was far
away from expected. The weight percent (w/w%) of were calculated. From calculation,
weight percent of sample 1 and sample 3 was 1.09% while sample 2 was 1.10%. The
value of weight percent is lower because of the amount of caffeine in the samples was out
of the range from the original amount of caffeine.

Precision of the method was determined by three replications of each sample. The
precision (%RSD) of the replications should less than two which is indicative of a precise
method. The smaller the value of relative standard deviation to xero indicates the more
accurate sample preparation. But, the relative standard deviation of this experiment is
higher than two which is 2.2361. Therefore, there is errors when doing this experiment or
the preparations of sample.

The samples need of improvement in the sample preparation. Therefore, there is

precaution steps need to be taken when doing this experiment. Firstly, used the pipette in
correct ways to ensure the reading is correct. It also affect the volume of samples taken
that lead to different concentration. Beside, the samples is centrifuged completely to
make sure all the precipitate. Next, make sure the same person doing the dilution to avoid
various error at the result.
8. CONCLUSION

The concentaration of the sample 1, sample 2 and sample 3 were 10.80 ppm, 10.0 ppm
and 10.85 ppm respectively with the relative standard deviation of 2.2361. The amount of
the caffeine in the sample 1, sample 2 and sample 3 was 0.027000g , 0.027125g and
0.0271125 respectively with the confidence limit ± 0.9232 g. The weight percent was
1.09% for sample 1 and 3 while 1.10% for sample 2.

9. REFERENCES

 Goodwin, L. (2019, March 26). How Much Caffeine Is in Your Drink of Choice?
Retrieved April 7, 2019, from https://www.thespruceeats.com/caffeine-in-coffee-
tea-cola-765276
 Marina, S. (n.d.). UV-VIS SPECTROPHOTOMETRIC TRACE LEVEL
DETERMINATION OF CAFFEINE IN 'RED BULL'. Retrieved April 7, 2019,
from https://www.academia.edu/9264278/UV-
VIS_SPECTROPHOTOMETRIC_TRACE_LEVEL_DETERMINATION_OF_C
AFFEINE_IN_RED_BULL?auto=download
 Caffeine. (2019, April 03). Retrieved April 7, 2019, from
https://en.wikipedia.org/wiki/Caffeine
 A., Azhar. (n.d.). UV-Visible Spectroscopy. Retrieved April 7, 2019, from
https://www.scribd.com/document/267919454/UV-Visible-Spectroscopy
10. QUESTION

1. Write the wavelength ranges for the ultraviolet and visible region of the
spectrum?
Ultraviolet : 180nm-380nm
Visible : 380nm-780nm

2. List the molecular or structural features that give rise to absorption of the
ultraviolet/visible (UV/VIS) radiation in organic species. Give specific sample
example of an organic compound that would not absorb UV/VIS radiation.

 Double and triple bonds and those bond involve the pi bond was absorb
UV/VIS radiation. For example, alkene, alkyne and aromatic compound.

 Single bond such as carbon and hydrogen in alkanes and those are sigma
bonds does not absorb UV/VIS radiation. For example is hexane C6H6
3. Why is it important to determine caffeine in tea using background correction
and second derivative ultraviolet-visible spectrophotometry?

To remove the interference in the absorption wavelength that may cause by other
substances such as tannis that maybe found in tea leaves. Therefore, the second
derivative is required in this experiment. To minimize the tannis the sodium
hydroxide is used.