A Qualitative Analysis of Caffeine from Non-Alcoholic Beverages Using UV-Visible Spectrophotometer

Abstract

Caffeine is a widely consumed psychoactive compound found in various non-alcoholic beverages, including tea, coffee, and energy drinks. This study focuses on the qualitative analysis of caffeine using UV-Visible spectrophotometry, a rapid and reliable technique for detecting caffeine content. Samples were prepared using liquid-liquid separation method and analysed at their characteristic absorption wavelength, ensuring precise detection. The UV-Visible spectrophotometer, operating within a range of 200 nm to 400 nm, is sensitive to caffeine detection, providing precision and cost-effectiveness. Out of the 30 samples tested, 13 contained caffeine, despite all being labelled as non-alcoholic beverages without any indication of caffeine content. The research not only focuses on the toxicological analysis of caffeine but also highlights the importance of accurate labelling regarding its presence. The findings have significant implications for consumer awareness, regulatory bodies, and manufacturers, underscoring the need for accurate labelling of caffeine content.

Introduction

Overview

Beverages, excluding water, are consumed for taste and stimulation and are classified as alcoholic or non-alcoholic. Caffeine, a natural stimulant found in several plant sources (e.g., coffee, tea, cacao), is commonly added to energy drinks, soft drinks, and coffee products. It enhances alertness by blocking adenosine receptors in the brain but may cause health issues if consumed in excess. Regulatory bodies require labeling of added caffeine, especially due to its forensic and health significance.

Caffeine Detection and Extraction

Caffeine extraction is essential for analysis, with liquid-liquid extraction (LLE) being a popular method. Solvents like chloroform and ethyl acetate are used with additives like sodium carbonate to stabilize the process. More advanced methods include Microwave-Assisted Extraction (MAE) and Supercritical Fluid Extraction (SFE), though these are costlier.

For analysis, both chemical (e.g., murexide test) and instrumental techniques (e.g., UV-Visible spectrophotometry, HPLC, GC) are used. UV-Vis spectrophotometry is widely employed by measuring absorbance at around 273–275 nm. Caffeine quantification is crucial for consumer safety and legal compliance.

Review of Literature

Numerous studies have assessed caffeine in commercial beverages:

• Attipoe et al. (2016): Found discrepancies between labeled and actual caffeine content in energy drinks using HPLC and mass spectrometry.

• Goldberger et al. (2006): Used GC to analyze caffeine in energy drinks and sodas; noted content below maximum limits but raised concerns for pregnant women.

• Johnson et al. (2011): Used HPLC-DAD for caffeine detection in 8 popular brands; identified undeclared caffeine in some samples.

• Agatha Christie & Willfred (2021): Isolated caffeine from soft drinks using LLE and spectral analysis, confirming variance in content.

• Djapo et al. (2011): Applied reverse-phase HPLC to quantify caffeine in beverages from Bosnia, confirming high sensitivity of the method.

• Vuletic et al. (2021): Used UV/Vis spectrophotometry on teas, soft, and energy drinks in Croatia, highlighting labeling inconsistencies.

• Patil et al. (2018): Reviewed various extraction methods and caffeine content in tea and coffee, noting caffeine typically ranges from 1–5%.

• Saloni Desai (2020): Found caffeine in soft drinks exceeding FDA limits using UV and TLC, advocating better labeling.

• Gupta & Maurya (2023): Reviewed caffeine extraction methods and confirmed high caffeine content in coffee; discussed health risks of concentrated forms.

• Martin et al. (2014): Measured caffeine and pH levels in Nigerian soft drinks, noting all samples were acidic.

Experimental Methodology

• Samples: 30 non-alcoholic drinks (categorized as refreshers, nourishers, and stimulants) were collected without labeled caffeine content.

• Extraction Process:

  • 150 ml beverage boiled with sodium carbonate.

  • Mixture filtered and transferred to a separating funnel.

  • Caffeine extracted using chloroform, then dried.

• Detection Method: UV-Visible spectrophotometer used at ~273 nm to detect caffeine presence. Graphs plotted via Origin software.

Results

• Out of 30 samples, 13 showed presence of caffeine despite having no caffeine labeling.

• All caffeine-containing samples showed absorbance peaks in the 273–275 nm range.

• Caffeine was found in several milk-based, coffee-flavored, and cola-based drinks such as:

  • Nescafe, Dark Fantasy, Hershey’s shakes, Storia, Tilo Power Cola, and others.

• Some drinks, including fruit juices and lemon sodas, were caffeine-free.

Conclusion

This study aimed to extract and analyze caffeine from non-alcoholic beverages using liquid-liquid extraction followed by UV-Visible spectrophotometry. The method effectively confirmed caffeine presence through characteristic absorption spectra between 272–275 nm, demonstrating high specificity for a large number of samples. The results showed that beverages without caffeine labeling often contained significant amounts, sometimes exceeding those that did list caffeine. Among the 30 samples analyzed, 13 tested positive for caffeine, with most being coffee or chocolate-flavored drinks, except for two local stimulant beverages. The study highlighted concerns regarding the lack of proper caffeine labeling, which poses potential health risks, particularly for children and pregnant women. Additionally, products explicitly labeled as caffeine-free, such as 7Up Lemony Taste, were confirmed to contain no caffeine, reinforcing the importance of accurate labeling. The method employed was cost-effective, sensitive, and suitable for large-scale caffeine detection, making it an accessible option for routine use. Given the health risks associated with excessive caffeine consumption, including addiction and toxicity, regulatory bodies must enforce stricter labeling requirements. The FDA (Food and Drug Administration) has included caffeine in the list of substances that are mostly documented as safe and had set the maximum concentration of caffeine to be consumed.[8] Exceeding the pre-determined level is the cause of toxicity. Because of the above-mentioned health concerns arising from the consumption of caffeine, it seems appropriate that warning labels should be strictly accompany all the caffeinated beverages. Clearer labeling would ensure consumers make informed choices, ultimately promoting public health and safety.

References

[1] Arora, P., Ansari, S. H., & Arora, S. (n.d.). Nutritional beverages. American Journal of PharmTech Research, 10(03), 164–183. https://doi.org/10.46624/ajptr.2020.v10.i3.015 [2] Kumar, P. (2023, July 2). Classification: Non- Alcoholic Beverages - hmhub. hmhub. https://hmhub.in/1st-sem-f-b-service-notes/classification-non-alcoholic-beverages/ [3] Ahmad Bhawani, S., Fong, S. S., & Mohamad Ibrahim, M. N. (2015). Spectrophotometric Analysis of Caffeine. International journal of analytical chemistry, 2015, 170239. https://doi.org/10.1155/2015/170239 [4] National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 2519, Caffeine. Retrieved July 7, 2024 from https://pubchem.ncbi.nlm.nih.gov/compound/Caffeine [5] FSSAI. (n.d.). https://fssai.gov.in/ [6] Brobbey et.al. (2023). ‘caffeine content in energy drinks can be determined using a validated liquid-liquid extraction method coupled to UV-Visible spectrophotometry’. [7] Origin: Data analysis and graphing software. (n.d.). https://www.originlab.com/origin#:~:text=Origin%20is%20the%20data%20analysis,academia% 2C%20and% 20government%20laboratories%20worldwide. [8] FDA error. (n.d.). FDA. https://www.fda.gov/regulatory-information/fda-rules-and-regulations

Copyright

Copyright © 2025 Aruna B Ajikumar. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.