Objectives

1. To determine the concentration an additive in beverages and an unknown sample

using UV-VIS spectroscopy.

Abstract
The purpose of this experiment was to determine the concentration of benzoic acid in
commercial soft drinks using high-performance liquid chromatography (HPLC). Standard
benzoic acid solutions (20–100 ppm) were prepared and analysed using an isocratic HPLC
system with a UV detector and a C18 column. A linear relationship between concentration and
peak area was observed (R² = 0.8858). The retention times for standards ranged from 5.890
to 6.009 minutes, indicating system stability. Mirinda Fruity and 7UP had retention times of
4.087 minutes and 6.420 minutes, respectively, with benzoic acid concentrations determined
as 40 ppm and 28 ppm. Both concentrations were below the regulatory limit of 0.1%. The
results highlight the method's effectiveness in quantifying benzoic acid but also suggest
interference from additional components in the samples. Recommendations for improved
chromatographic conditions are made to enhance precision across diverse sample matrices.

Procedure
Material

 Isocratic HPLC system with UV detector

 C18 column
 0.45 μm filter paper
 0.45 μm filter syringe
 100 μL syringe 60 mL syringe
 volumetric flask
 Pipette
 Dropper
 Beaker

Chemicals

 Benzoic acid
 Methanol (HPLC grade)
 Double distilled water (filtered with 0.45 μm filter paper)
 soft drink sample.

Equipment

 High Performance Liquid Chromatography

Procedures
Preparation of 1000 ppm Benzoic Acid
1. Approximately 0.1 g of benzoic acid was weighed and transferred to a 100 ml
volumetric flask.
2. The volumetric flask was filled to the mark level with distilled water.
3. The solution was mixed thoroughly by inverting the flask several times.

Preparation of 100 ppm Benzoic Acid

1. To prepare 100 ppm of benzoic acid, 5 ml of benzoic acid from the stock solution was
taken using a pipette and transferred to a 50 ml volumetric flask.
2. The volumetric flask was filled to the mark level with distilled water.
3. The solution was mixed thoroughly by inverting the flask several times.
4. Steps 1–3 were repeated to prepare serial standard solutions of benzoic acid with
concentrations of 20 ppm, 40 ppm, 60 ppm, and 80 ppm.

Preparation of Soda Samples

1. A soft drink sample was obtained.
2. The sample was degassed by placing it in a vacuum flask and connecting the flask to
a vacuum pump or water aspirator. It was left under vacuum until no more bubbles
appeared in the soda sample. (If no vacuum was available, the soda was allowed to
stand open overnight.)
3. The degassed soda was filtered through #42 filter paper.
4. After preparing the serial dilution and sample, the instructor briefed on the standard
operating procedure of HPLC.
RESULT /DATA ANALYSIS

CONCENTRATION (ppm) RETENTETION TIME (min) PEAK AREA (V.s)

20 5.900 193421

40 5.890 407698

60 5.920 1322515

80 5.935 1220029

100 6.009 245690

Sample 1: Mirinda fruity 4.087 90228

Sample 2: 7UP 6.420 58556

Calculation

 Formulation used: M1V1 = M2 V2

NO CONCENTRATION (ppm) CALCULATION

1 20 V1 = M2 x V2
V1 = (20 ppm) (50 ml)
V1 = 1000/1000
V1 = 1 ml
2 40 V1 = M2 x V2
V1 = (40 ppm) (50 ml)
V1 = 2000/1000
V1 = 2 ml
3 60 V1 = M2 x V2
V1 = (60 ppm) (50 ml)
V1 = 3000/1000
V1 = 3 ml
4 80 V1 = M2 x V2
V1 = (80 ppm) (50 ml)
V1 = 4000/1000
V1 = 4 ml
5 100 V1 = M2 x V2
V1 = (100 ppm) (50 ml)
V1 = 5000/1000
V1 = 5 ml
6 Concentration Benzoic Acid Concentration Benzoic Acid = 0.5mg/mL
x Volume soft drink = 1500mL
Volume soft drink 0.5mg/mL x 1500mL = 750mg
(Mirinda Fruity) Total Benzoic Acid = 0.75g
DATA ANALYSIS
The data shows a positive correlation between concentration and peak area, with peak areas
increasing up to 60 ppm but declining at higher concentrations, possibly due to system
saturation. Retention times for standards were consistent (5.890–6.009 minutes), indicating
system stability, while samples showed differing retention times (Mirinda Fruity: 4.087 minutes;
7UP: 6.420 minutes), suggesting different chemical compositions.

The calculated benzoic acid content in Mirinda Fruity was 0.75 g, equivalent to 0.05% by
weight, which is below the FDA’s safe limit of 0.1% for benzoic acid in soft drinks. The lower
peak areas for Mirinda Fruity (90228) and 7UP (58556) indicate lower analyte concentrations.
Overall, the chromatographic system is effective, but the differences in sample characteristics
suggest the presence of additional components requiring further analysis.

Discussion

Using chromatographic analysis, the experiment analyses the relationship between the
benzoic acid concentration (in ppm) and its related peak area (V.s). A linear relationship
between the two variables is demonstrated by the data, as indicated by the equation y =
2735.5x - 21058, which has a R2 value of 0.8858. Although small variances suggest minimal
experimental or instrumental differences, this R2 value shows a rather strong association. The
standard benzoic acid samples show stability and dependability in the chromatographic
system with retention times ranging from 5.890 to 6.009 minutes.

Two commercial samples were analysed, and the findings were different. The peak area for
Sample 1 (Mirinda Fruity) was 90228 V.s., and the retention time was 4.087 minutes. The
shorter retention period raises the possibility of interference or variations in the makeup of the
sample. The peak area for Sample 2 (7UP) was 58556 V.s. and the retention time was 6.420
minutes, both of which are in proximity with the typical benzoic acid retention duration and
suggest the probable existence of benzoic acid. Benzoic acid concentrations in the samples
were determined using the linear regression method. The concentration was about 40 parts
per million for Mirinda Fruity and about 28 parts per million for 7UP. These results offer a
numerical assessment of the amount of benzoic acid present in the commercial beverages.

According to Benzene in Soft Drinks (2018), preservatives such as benzoic acid and its salts
[INS 210-213] can be added to meals, especially soft drinks, to stop the growth of mould,
yeast, and bacteria. Benzoic acid and its salts are permitted to be used as preservatives in
certain foods, including soft drinks, in Hong Kong under the Preservatives in Food
Regulation. Cranberries, prunes, plums, and the majority of berries are among the foods that
naturally contain them.

According to the journal cited, excessive consumption of benzoic acid in beverages may have
harmful consequences on human health. Benzoic acid excess can cause negative side effects
such stomach lining inflammation and possible internal organ damage. Asthma, skin irritation,
and other hypersensitivity reactions are among the adverse reactions it can cause in sensitive
people. (View of Benzoic Acid Based Beverages: Health Implications, n.d.)

The data analysis presented gives significant new information about the connection between
peak area and concentration in benzoic acid analysis. It shows that concentration and peak
area are positively correlated, with the peak area rising to 60 ppm. The peak area does,
however, decrease with increasing concentrations, possibly as a result of system saturation.
This implies that even while the chromatographic technique is reliable, it might not be able to
precisely identify benzoic acid amounts above a particular level.

A stable system was observed throughout the investigation, as evidenced by the constant
retention periods for reference samples, which ranged from 5.890 to 6.009 minutes. However,
the two samples, 7UP (6.420 minutes) and Mirinda Fruity (4.087 minutes), had different
retention times, which suggests that their chemical contents changed and differ. These
variations might be explained by the existence of other substances in the samples that could
affect the retention period.

Mirinda Fruity had a calculated benzoic acid concentration of 0.75 g, or 0.05% by weight. This
indicates that the sample complies with regulatory requirements because it is less than the
FDA's safe limit of 0.1% for benzoic acid in soft drinks. Similarly, the lower peak areas for
Mirinda Fruity (90228) and 7UP (58556) indicate relatively low analyte concentrations, further
supporting compliance with safety limits.

The results obtained from the analysis indicate that the chromatographic technique is
applicable in analysing benzoic acid in beverages. However, variation in the features of studied
samples indicates that there might be other ingredients in the drinks that may require further
studies for full understanding of their effects on peak area and retention time. The present
study has highlighted the need to improve chromatographic conditions to increase the
system's capability of dealing with a wide range of concentrations with least interference from
other constituents of the sample.

The obtained results differ from the expected result due to issues that occurred during the
experiment, leading to variations in the data and the resulting graph. One frequent problem is
incorrect sample preparation, which includes handling contamination, incorrect dilution, and
weighing mistakes. Inappropriate solvent use or improper sample mixing can also cause
issues. Interference from other compounds in the sample is another factor. These substances,
such as benzoic acid, may overlap with the target peak or, in the case of impurities, may have
an impact on the outcomes. Finally, human errors may happen, such incorrectly handling the
equipment, entering data incorrectly, or utilizing the incorrect injection volume. It is crucial to
carefully follow preparation instructions, make sure operators are adequately trained, and
modify instrument settings to prevent these problems.

According to Trumentadmin (2020), the use of inaccurate measurement techniques,

inappropriate handling or operation of instruments, and malfunctions in the measuring
apparatus are the main causes of systematic sources of error in laboratory research. Errors
may also result from improper use of standard materials, variations in the experimental
environment, and the performance constraints of the testing instruments themselves.

PRE-LABORATORY QUESTION:

1. Explain the term food additive.

Any ingredient that is purposefully added to a food to improve its qualities but is not naturally
present is referred to as a food additive. Enhancing nutritional content, shelf life, texture,
flavour, and appearance are all examples of this. In order to guarantee the quality and safety
of food products, food additives which can be synthetic, natural, or derived from natural
sources are usually added during processing, packing, or storage.

2. Identify the regulatory limit for benzoic acid in soft drinks.

The regulatory limits for benzoic acid in soft drinks vary by region:

 European Union (EU): The maximum permitted level is 150 milligrams per liter (mg/L),
expressed as benzoic acid. (CNS Media. & CNS MEDIA, 2008)
 Hong Kong: The Centre for Food Safety specifies a maximum permitted concentration
of 160 mg/kg for benzoic acid in soft drinks. (Dietary Exposure to
Benzoic Acid from Prepackaged Non-alcoholic Beverages of Secondary School
Students, 2017)
 Canada: Benzoates are allowed in unstandardized foods, including soft drinks, at a
maximum use level of 0.1%, equivalent to 1,000 mg/L, expressed as benzoic acid.
(Darch et al., 2021)
These regulations are established to ensure consumer safety by limiting exposure to benzoic
acid, a common preservative in beverages.

3. Discuss the potential health effects of consuming benzoic acid in excess

Depending on the amount ingested and the sensitivity of the individual, excessive benzoic
acid consumption may result in a number of possible health problems.

 Irritation and Discomfort: Benzoic acid at high concentrations can irritate the digestive
tract, leading to nausea, vomiting, and stomach discomfort.
 Allergic Reactions: Benzoic acid sensitivity can cause allergic reactions in certain
people, including skin rashes, itching, and symptoms similar to asthma.
 Effect on the Nervous System: Overconsumption has been linked to neurological side
effects, such as headaches, feeling dizzy, or hyperactivity, especially in fragile people
like kids.

 Potential Toxicity: As the liver and kidneys metabolize and eliminate benzoic acid,
prolonged exposure to high concentrations of the chemical might put stress on these
organs.
 Carcinogenic Concerns: Benzoic acid is normally harmless at controlled levels, but
under some circumstances, it can combine with ascorbic acid (vitamin C) to create
benzene, which is known to cause cancer. Soft drinks and other liquids that contain
both preservatives may be at risk for this.

POST-LABORATORY QUESTION:

1. Suggest the natural alternatives to benzoic acid for use in soft drink. ➢
Citric Acid: Acts as a preservative and enhances flavour.
 Ascorbic Acid (Vitamin C): Provides antioxidant properties and prevents spoilage.

 Rosemary Extract: A natural antioxidant and antimicrobial agent.

 Essential Oils (e.g., lemon or orange oil): Contain antimicrobial compounds.

 Vinegar (Acetic Acid): A natural preservative that inhibits microbial growth.

 2. How could you be identifying a peak in the soda was benzoic acid and not
another substance by using retention time?

Retention time is a crucial metric that can be used to determine whether a peak in soda is due
to benzoic acid and not another chemical. The retention period of the peak in the soda sample
can be compared to that of a reference solution of benzoic acid run under the same
chromatographic conditions. Benzoic acid is probably represented by the peak if the retention
times overlap. By adding a known quantity of benzoic acid to the soda sample and reanalysing
it, a spike test may be carried out to further validate its identity a steady rise in the peak at the
same retention time confirms it. The identity of the molecule can be further confirmed using
methods like mass spectrometry or UV-Vis spectroscopy.

3. Identify the potential sources of error in this analysis. Suggest the way to
minimize or eliminate.

During the analysis, a number of possible sources of mistake could appear. Inaccurate results
may arise from improper sample preparation, including contamination or dilution mistakes.
Accuracy may also be impacted by instrumental problems like detector drift or inadequate
column performance. Benzoic acid identification may be affected by the coelution of other
substances and misidentification may result from variations in retention time brought on by
irregular chromatographic conditions. Human error is another major source of disparities,
including errors in data interpretation or sample handling. Accurate sample preparation
utilizing calibrated equipment and appropriate handling practices is crucial to reducing or
eliminating these errors. Maintaining consistent performance requires routine calibration and
maintenance of the equipment. Peak separation can be enhanced by optimizing
chromatographic settings, such as modifying the composition and flow rate of the mobile
phase. Reliable results are ensured by include standards and blanks in the study, and human
error can be decreased by using automated systems or professional instruction.
Reference

Benzene in soft drinks. (2018, November 16).

https://www.cfs.gov.hk/english/multimedia/multimedia_pub/multimedia_pub_fsf_36_0
1.html

CNS Media. & CNS MEDIA. (2008, November 25). Levels of Benzoate and Sorbate in Soft
Drinks within Legal Limits: UK FSA. https://www.foodingredientsfirst.com.
https://www.foodingredientsfirst.com/news/levels-of-benzoate-and-sorbate-in-
softdrinks-within-legal-limits-uk-fsa.html?utm_source=chatgpt.com
Darch, M., Martyn, D., Ngo, K., & Jack, M. M. (2021). An updated estimate of benzoate
intakes from non-alcoholic beverages in Canada and the United States. Food
Additives & Contaminants Part A, 38(5), 701–717.
https://doi.org/10.1080/19440049.2020.1859624
Dietary Exposure to Benzoic Acid from Prepackaged Non-alcoholic Beverages of Secondary
School Students. (2017, June 16).
https://www.cfs.gov.hk/english/programme/programme_rafs/programme_rafs_fa_01_
05_ra.html?utm_source=chatgpt.com
Trumentadmin. (2020, May 10). Sources of error in lab experiments and laboratory tests |
Felixtrument. Felixtrument. https://felixtrument.ca/sources-error-
laboratory/?srsltid=AfmBOortYcSO1cK7yi1eaNHunqBZUX85LJSg38JeJ_KDdhsKXp
QLuSvj
View of Benzoic acid-based beverages: health implications. (n.d.).
https://journalafsj.com/index.php/AFSJ/article/view/383/765