technical report - determination of benzoic acid in soft drink
TRANSCRIPT
PROFESSIONAL COMMUNICATION 2 (WEB 20102)
TECHNICAL REPORT
DETERMINATION OF BENZIOC ACID/CAFFEINE IN SOFT DRINK
SUBMITTED BY:
MOHAMAD ASHAZIQ BIN ABU BAKAR
(55201210121)
AHMAD ZUHDI BIN MUNIR
(55201210327)
MOHAMAD HAZMAN ZHAFRI BIN SHAMSUDIN
(55201210210)
AHMAD FAIZ BIN MOKHTAR
(55201210212)
SUBMITTED TO:
MADAM SA’ADIAH HUSSIN
COURSE:
BACHELOR OF CHEMICAL ENGINEERING TECH. (PROCESS)
5 BCP A
DATE: 30 OCTOBER 2012
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ABSTRACT
High performance liquid chromatography (HPLC) technique is used to separate a mixture
of compounds in analytical chemistry and biochemistry with the purpose of identifying,
quantifying and purifying the individual components of the mixture. HPLC is also considered an
instrumentation technique of analytical chemistry, instead of a gravitimetric technique. Benzoic
acid and caffeine standard is prepared with different concentration. Then, both sample and
standard were tested in the HPLC. The mixture of compound that we need to separate is caffeine
and benzoic acid with the soft drinks sample. In this experiment, presence of caffeine and
benzoic acid in soft drink sample is identified and the amount of caffeine in soft drink sample
was determined. The sample was degassed by placing it in a vacuum flask before filtered through
the filter paper. Compound differentially retained in the stationary phase reach the detector at
different times to produces a set of peaks along the time line. Each component of the mixture
reaches the detector at the different time and produces a signal at the characteristic time called
the retention time. The area under a peak is related to the amount of the component present the
mixture. In this experiment, serial dilution also will be prepared to be as standard caffeine and to
determined if caffeine is present in the soda sample by use retention time. Other than that, by
using the concentration to peak area relationship, the concentration of caffeine in the soda
sample can be determined. The peak of caffeine will appeared after 2 second and by measure
caffeine peaks of the standards, the amount of caffeine in a sample can be determined. The area
is getting bigger when the concentration is increasing. This shows that the higher concentration
of caffeine will make a bigger effect.
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TABLE OF CONTENTS
CONTENTS PAGE NO
Title Page I
Abstract II
Table of Contents III
List of Tables IV
List of Figures V
List of Symbols VI
1.0 Introduction 1
2.0 Methodology 3
2.1 Procedures 5
2.2 Analysis of data 5
3.0 Results & Discussion 6
4.0 Conclusions 9
References VII
Appendices VIII
3
LIST OF TABLES
Table No. Title Page
3.1 Retention time of caffeine in standard 6
4
LIST OF FIGURES
Figure No. Title Page
2.1 How the HPLC actually works 3
3.1 Standard curve for peak area vs. concentration 7
5
LIST OF SYMBOLS
μm micrometer
μL micro liter
mL milliliter
ppm part per million
µv.s peak area (microvolume x second)
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1.0 INTRODUCTION
A soft drink is a beverage that typically contains water (often, but not always carbonated
water), usually a sweetener, and usually a flavoring agent. The sweetener may be sugar, high-
fructose corn syrup, fruit juice, sugar substitutes or a combination of these. Soft drinks are called
"soft" in contrast to "hard drinks" (alcoholic beverages). Widely sold soft drink flavors are cola,
cherry, lemon-lime, root beer, orange, grape, vanilla, ginger ale, fruit punch, and sparkling
lemonade.
Soft drinks may be served chilled or at room temperature. They are rarely heated. Soft
drinks are made by mixing dry ingredients and fresh ingredients like lemons, oranges, etc. with
water. Production of soft drinks can be done at factories or at home. Soft drinks can be made at
home by mixing either syrup or dry ingredients with carbonated water.
The main ingredients in this soft drink are usually caffeine and benzoic acid. Caffeine is a
natural component of chocolate, coffee and tea, and is added to colas and energy drinks. It is
found in varying quantities in the seeds, leaves, and fruit of some plants, where it acts as a
natural pesticide that paralyzes and kills certain insects feeding on the plants. It is most
commonly consumed by humans in infusions extracted from the seed of the coffee plant and the
leaves of the tea bush, as well as from various foods and drinks. Beverages containing caffeine
enjoy great popularity among the teenagers specially. (Wikipedia, 2011)
On the other hand, benzoic acid occurs naturally in various berries notably cranberries,
cinnamon, plums, currants, cloves, etc. It has long been used to inhibit microbial growth in
many products including non-alcoholic beverages, jams and emulsified sauces. The salt of the
benzoate is more stable than the acid form and more soluble in water making the benzoates a
favourable choice for the soft drinks industry. Benzoic acid is very effective against moulds,
yeasts and bacteria. It is particularly well suited for use in soft drinks, such as carbonated, still
and juice beverages because it works best between pH levels of 2–4. (UNESDA, 2010)
The composition of the drink therefore has an effect on its efficiency and suitability for
use. The major cause of benzene in soft drinks is the decarboxylation of benzoic acid in the
presence of ascorbic acid (vitamin C) or erythorbic acid (a diastereomer of ascorbic acid).
Benzoic acid is often added to drinks as a preservative in the form of its salts sodium benzoate,
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potassium benzoate, or calcium benzoate. Other factors that affect the formation of benzene are
heat and light. Storing soft drinks in warm condition speed up the formation of benzene.
In order to check or identify if there is a presence of caffeine or benzoic acid in the soft
drink, we used the high performance liquid chromatography (HPLC) system. Compounds are
separated by injecting a plug of the sample mixture onto the column. The different components
in the mixture pass through the column at different rates due to differences in their partitioning
behavior between the mobile liquid phase and the stationary phase. Thus a mixture component
will separate with one another. (Lindsay, 1997)
HPLC typically utilizes different types of stationary phases, a pump that moves the
mobile phase and analyte through the column, and a detector that provides a characteristic
retention time for the analyte. The detector may also provide other characteristic information (i.e.
UV/Vis spectroscopic data for analyte if so equipped). Analyte retention time varies depending
on the strength of its interactions with the stationary phase, the ratio/composition of solvent used
and the flow rate of the mobile phase.
With HPLC, a pump (rather than gravity) provides the higher pressure required to propel the
mobile phase and analyte through the densely packed column. The increased density arises from
smaller particle sizes. This allows for a better separation on columns of shorter length when
compared to ordinary column chromatography. (Lindsay, 1997)
1.1 Objective:
1.1.1 - To Identify the present of Benzoic acid/ Caffeine in soft drink sample
1.1.2 - To determine amount of caffeine in soft drink sample
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2.0 METHODOLOGY
The equipment required for this experiment is an isocratic HPLC system with UV
detector, C18 column, vacuum and 0.45μm filter paper. In addition, the following equipment
along with the specified technical data is also required:
Additional Equipment:
A 0.45 μm filter syringe
A 100 μL syringe
A 60 mL syringe
A Volumetric flask
Other than that, the experiment also used the chemical substances which is caffeine 1000ppm
standard (stock solution), methanol (HPLC grade), double distilled water that filtered with
0.45μm filter paper and the soft drink sample.
The HPLC works as shown in Figure 2.1;
Figure 2.1: How the HPLC actually works
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A reservoir holds the solvent that’s called the mobile phase, because it moves. A high-
pressure pump is used to generate and meter a specified flow rate of mobile phase. An injector is
able to introduce by injecting the sample into the continuously flowing mobile phase stream that
carries the sample into the HPLC column. The column contains the chromatographic packing
material needed to effect the separation. This packing material is called the stationary phase
because it is held in place by the column hardware. A detector is needed to see the separated
compound bands as they elute from the HPLC column. Most compounds have no color, so it is
impossible to see them with normal eyes. The mobile phase exits the detector and can be sent to
waste, or collected, as desired. When the mobile phase contains a separated compound band,
HPLC provides the ability to collect this fraction of the elute containing that purified compound
for further study. This is called preparative chromatography.
Before the solvent are prepared to test, it must go through the degasification process first
to make sure no more carbon dioxide is left in the soft drink sample. It disturbs the result of the
experiment if there is still carbon dioxide gas in the sample.
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2.1 Procedures
The following steps were carried out during the experiment.
2.1.1 Preparation of Benzoic acid/ caffeine standards
-The standard caffeine samples of 20 ppm, 40 ppm, 60 ppm, 80 ppm and 100 ppm
was prepared by diluting portions of the 1000 ppm solution with distilled water.
2.1.2 Preparation of soda samples
-The soft drink sample was obtained.
-The sample was degassed by placing it in a vacuum flask and connecting the
flask to a vacuum pump or water aspirator. It will left under vacuum until no more
bubbles appeared in the soda sample. (If no vacuum is available, allow the soda to
stand open overnight.)
-The degassed soda was filtered through filter paper.
2.2 Analysis of data
2.2.1 The standard benzoic acid /caffeine retention time was used to identify the
benzoic acid/caffeine peak and their retention time was recorded.
2.2.2 By using all information, the presence of benzoic acid/caffeine in the soda sample
was determined.
2.2.3 The different concentration of standards peaks area was recorded and a standard
calibration curve (concentration vs. peak area) was plotted.
2.2.4 The caffeine peak in the soda sample chromatograph was measured and standard
calibration curve (concentration vs. peak area) was used to determine the
concentration of Benzoic acid/ caffeine in the soda sample.
Note: All raw data must be record in table form.
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3.0 RESULT AND DISCUSSION
In this experiment, High Performance Liquid Chromatography (HPLC) was used to
determine caffeine in soft drink which was Coca-cola as the sample (Harris, 2005). It was also
used to find the amount of a chemical compound within a mixture of other chemicals. Before the
sample was run in the HPLC, standard of caffeine must be prepared. 1mL, 2mL, 3mL, 4mL and
5mL (see calculation in Appendix) of caffeine stock solution was taken out from 1000ppm
caffeine to dilute 20ppm, 40ppm, 60ppm, 80ppm, and 100ppm of caffeine standard respectively
in 50mL volumetric flask. Then, double distilled water was poured in the flask and the flask was
shacked until the mixture is well mixed.
To run the HPLC, Standard Caffeine sample was used to identify the Caffeine peak and
the retention time of the caffeine was being recorded. The peak was increased from 20ppm to
100ppm. The retention time was used to determine whether caffeine was present in the Coca-
cola sample or not. The amount of the caffeine in the sample was determined and the caffeine
peak of the standards was measured (see Table 3.1), and a standard curve was constructed (see
Figure 3.1). The caffeine in the Coca-cola sample chromatograph was measured and the
concentration to peak area relationship was used to determine the concentration of Caffeine in
the Coca-cola sample.
Table 3.1: Retention time of caffeine in standard
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Concentration
/ppm
Retention Time
[min]
Peak area / µv.s
20 1.122 87919.30
40 1.980 110641.08
60 2.068 156997.94
80 2.090 170023.59
100 2.105 216500.43
10 20 30 40 50 60 70 80 90 100 1100
50000
100000
150000
200000
250000
f(x) = 1582.72385 x + 53453.037R² = 0.9763453575622
Peak area / µv.s vs Concentration, ppm
Peak area / µv.sLinear (Peak area / µv.s)
Concentration, ppm
Peak
are
a, µ
v.s
Figure 3.1: Standard curve for peak area vs. concentration
Based on the result obtained, caffeine retention time is 2 minutes. The peak area for
20ppm is 87919.30 µv.s with time 1.122 minutes, 40ppm is 110641.08 µv.s with time 1.980
minutes, 60ppm is 156997.94 µv.s with time 2.068 minutes, 80ppm is 170023.59 µv.s with time
2.090 minutes, and 100ppm is 216500.43 µv.s with time 2.105 minutes. As expected from the
experiment, the highest peak area for this experiment is at 100ppm with 216500.43 µv.s. This
shows that the result obtained obeys the theory that states that the higher the concentration, the
higher the peak area whereas a higher concentration gives a greater effect towards the area
(Fernández, 2000). The data also shows a positive result when there is absence of caffeine in the
sample. Graph for this experiment is not a straight line and accurate because only got R 2 0.9763.
Although the comparison of the calculated and literature values of the analyte concentrations
yielded large percent errors, the standard addition plots yielded R2 values close to 1, which
implies that the method of standard addition was successful (Leacock, et al., 2011). All the
objectives in this experiment were achieved as there was presence of caffeine in soft drink
sample and the amount of caffeine in soft drink sample can be determined.
As stated by Barone and Roberts (1996), caffeine is a pharmacological active substance
and depending on the dose, can be a mild central nervous system stimulant. It is noted that
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caffeine is not food but a drug working through nervous system. Excessive amount should be
avoided since caffeine consumed in large amounts has adverse health effects. In particular,
people suffering from high blood pressure should be advised to avoid use of caffeine containing
beverages since caffeine is known to increase the blood pressure. In addition those with coronary
heart disease should avoid such beverages as caffeine disrupts normal heart rhythm.
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4.0 CONCLUSION AND RECOMMENDATIONS
As the conclusion, the experiment has met the objectives which are to identify the present
of Benzoic acid/ Caffeine and determine the amount of caffeine in soft drink sample. The present
of Benzoic acid/ Caffeine was identified in the soft drink sample by using HPLC. A standard of
caffeine was prepared from caffeine stock solution to identify the caffeine peak and the retention
time. The retention time was used to determine whether caffeine was present in the soft drink
sample.The amount of the caffeine in the sample was determined and the caffeine peak of the
standards was measured as shown in Table 3.1 and a standard curve was constructed in Figure
3.1. Based on the result, the caffeine retention time is 2 minutes. A comparison of the caffeine
peak area in the soft drink sample compared to standard curve allows determination the amount
of caffeine. The result also shows that the result obtained obeyed the theory as the higher the
concentration the higher the peak area.The graph for this experiment is not a straight line and
accurate because only got R2 0.9763 the nearest the value of R2 to 1 is more accurate. All the
objectives in this experiment were achieved as there was presence of caffeine in soft drink
sample and the amount of caffeine in soft drink sample can be determined.
In order to get more accurate value, there are several recommendations to improve the
result obtained. Firstly, try to reduce as much of contamination as the procedures involve dealing
with the analysis of sample that contamination could affect the result obtained. Next, make sure
that avoid any errors while handling HPLC such as bubbles present in the syringe that injected
into HPLC.
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REFERENCES
Barone JJ, Roberts HR. (1996). Caffeine Consumption Food Chemical Toxicology, Volume 34,
p119, Coca-Cola Company, Atlanta
Harris, D.C., (2005). Quantitative Chemical Analysis; 5th Edition, W.H. Freeman and Company,
New York
Leacock, R. E., Stankus, J. J., Davis, J.M. (2011). Journal of Chemical Education. Simultaneous
Determination of Caffeine and Vitamin B6 in Energy Drinks by High-Performance Liquid
Chromatography. Volume 88, pp.2
Lindsay, S. (1997). High performance liquid chromatography. [Online]. Available from:
http://www.osti.gov/energycitations/product.biblio.jsp?osti_id=7013902 [Accessed by 26
October 2012].
P. L. Fernández, M. J. Martín, A. G. González and F. Pablos. (2000). Analyst, Volume 125,
pp.421-425, RSC Publishing
Union of European Soft Drink Association (UNESDA), (2010). Qualitative and quantitative
control of benzoic acid and caffeine in soft drinks. United States: UNESDA Publications.
Wikipedia (2011). Benzene in soft drinks. [Online]. Available from:
http://en.wikipedia.org/wiki/Soft_drink [Accessed by 27 October 2012].
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APPENDIX
Sample of calculation:
1) 20 ppm M1 = 1000 ppm M2 = 20 ppm
V1 = ? mL V2 = 50 mL
M1V1 = M2V2
(1000)(V1) = (20)(50)
V1 = 1 mL #
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