Determining The Concentration Of Glucose In Drinks Using Spectrophotometry

Abstract

The purpose of this experiment was to determine the concentration of glucose in an assortment of drinks – Coke, Gatorade, and a sucrose solution. This was achieved through the use of a spectrophotometer to measure the absorbance of NADPH, which is directly linked to the glucose content, produced via a multistep enzymatic reaction. The results indicated a directly proportional relationship between glucose concentration and absorbance that followed a positive linear model, which was then used to determine the glucose content of Coke to be 2.89 g/100mL and Gatorade to be 0.72 g/100mL. This was consistent with research that suggested Coke would have higher sugar levels. The results of the sucrose solutions further corroborated this result as more acidic solutions (including Coke) yielded higher glucose concentrations.

Introduction

Consumption of sugar-sweetened drinks in Australia is extremely high; they constitute the primary source of the population’s sugar intake (Australian Bureau of Statistics, 2014) despite warnings from the World Health Organisation to limit consumption of such beverages (Lobstein, 2014). Coke reportedly contains 10.6 g/100mL of sugar and Gatorade contains 6 g/100mL (Coca Cola Australia, 2019; Cancer Council Victoria, n.d.). Based on this, it can be predicted that Coke has a higher concentration of glucose than Gatorade; however, exact concentrations can vary, and these differences can have a significant “biological impact” (Jameel, Phang, Wood & Garg, 2014). High levels of glucose have been positively associated with diabetes mellitus (Tuomilehto et al., 2001), chronic obstructive pulmonary disorder (Shi, et al., 2011), mental health problems (Shi et al., 2010) and other negative health effects. Therefore, although some research has been done into the sugar content of various drinks, it is beneficial from a public health perspective to further investigate glucose concentration in popular beverages such as Coke and Gatorade.

One common method for determining the concentration of glucose in various drinks is through the use of enzymes and spectrophotometry. Enzymes perform a catalytic function – they significantly increase the rate of a reaction by providing an alternative reaction pathway, thereby lowering the activation energy necessary for the reaction to proceed (Huang, Liu & Benkovic, 2016). As such, enzymes are not consumed within the catalysed reaction and therefore do not have an impact on the equilibrium or free-energy changes of said reaction (ref). Enzymes are highly selective due to the unique chemical composition of their active sites, where specific substrates (reactant molecules) bind to produce a given number of products (Rago, Saltzberg, Allen & Tolan, 2015). Some enzymes require additional entities to initiate functional activity; these may be co-factors such as metal ions or heme groups, or co-enzymes including ATP, NAD+/NADH, NADP+/NADPH, as well as a range of vitamins (Alexander, Mathie & Peters, 2011).

In this experiment, the enzyme hexokinase accelerated the phosphorylation of glucose to glucose-6-phosphate without effecting the reaction equilibrium (Stanton, 2012). Subsequently, the oxidation of glucose-6-phosphate into 6-phosphogluconate is catalysed by the enzyme glucose-6-phosphate dehydrogenase, which simultaneously facilitates the reduction of the coenzyme NADP+ to NADPH.

1. D-glucose + ATP Glucose-6-phosphate + ADP
2. Glucose-6-phosphate + NADP+ 6-phosphogluconate + NADPH + H+

Because the enzyme glucose-6-phosphate dehydrogenase is oxidising the glucose molecule at the same time as it reduces the co-enzyme in a 1:1 mole ratio between the reactants and products, the concentration of glucose can be directly linked with the concentration of NADPH. Therefore, since NADPH absorbs strongly at a wavelength of 340 nm whilst NADP+ does not, a spectrophotometer – which measures how much a chemical substance absorbs light (Dondelinger, 2011) – can be utilised to monitor the appearance or disappearance NADPH. This absorbance value can then be interpreted as equivalent to the concentration of glucose, as the reduction of each NADPH molecule would correspond with the oxidation of a D-glucose molecule.

Method

A spectrophotometer set to 340 nm was used to measure the absorbance of NADPH in 6 glucose standards, as well as Coke, Gatorade and Sucrose samples at the beginning and end of an 18-minute period. This absorbance difference was then used to calculate the glucose concentration of each drink. Refer to the 1015MSC Lab Manual for further details.

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Discussion & Conclusion

The results as displayed in Figure 1 established that glucose concentration increased as absorbance increased in a directly proportional relationship. Additionally, Table 1 showed that Coke had a significantly higher absorbance than Gatorade which therefore corresponded with a higher glucose concentration. The outcome of the sucrose samples demonstrated that increased temperature caused increased sucrose concentration, whilst increased pH (as in more basic solutions) decreased sucrose.

Although the majority of the data supported the overall trend that increased glucose concentration directly corresponded with increased absorbance, the results for Sample 6 deviated from this pattern and as such the sample was classified as an outlier. This can be attributed to an error in the time at which the sample was measured by the spectrophotometer, as it was placed in the machine too early and therefore did not have the full 18 minutes to absorb the co-enzyme like the other samples. This explains the comparatively lower glucose concentration and absorption recorded for sample 6.

Based on the absorbance values recorded by the spectrophotometer, the glucose concentration in Coke was found to be 2.89 g/100mL whilst for Gatorade it was 0.72 g/100mL; this supports the hypothesis that Coke would have a higher glucose content than Gatorade. However, these values are significantly lower than the reported overall sugar content in both drinks – 10.6 g/100mL for Coke and 6 g/100mL for Gatorade (Coca Cola Australia, 2019; Cancer Council Victoria, n.d.) – which could suggest that the composition of sugar in the beverages includes other molecules such as fructose (Varsamis et. al., 2017). Another study which used high-performance liquid chromatography found a total glucose concentration of ~5.5 g/100mL in Coke but did not test Gatorade. Given that this methodology and equipment is more sophisticated, it is likely that these results are more accurate than those produced through spectrophotometry and also used diluted samples, inhibiting accuracy.

Additionally, it was found that one factor which significantly influenced glucose concentration in the Sucrose solution was pH. Lower pH yielded higher absorbance which corresponded with a higher concentration of sucrose; this increased the rate of acid hydrolysis in the sucrose, so more protons were available to form glucose. Ultimately, this resulted in an increased concentration of glucose in samples with lower pH. This supports the results of the Coke and Gatorade samples, which showed that Coke has a higher glucose concentration than Gatorade, as Coke is more acidic, with a pH of 2.52, in comparison with the pH of Gatorade which is 3.27 (Shelton, n.d.). This also aligns with other studies of sugar content in soft drinks, which found that “most glucose in Australian formulations is attributable to sucrose” (Varsamis et. al, 2017).

This experiment was limited in that it was only performed once and therefore there is no comparison to determine the consistency of the readings. Thus, a better outcome would be achieved if multiple trials were performed using the same equipment and conditions to prevent the results from being altered by random events and make it easier to identify systemic errors.

Overall, the data collected in this experimented supported both the reported values and other studies, which both asserted that Coke had a higher concentration of glucose than Gatorade. However, the exact values for each beverage differed significantly between all three results. Regardless, the information that Coke has a higher sugar content than Gatorade is extremely valuable in terms of public health as excess sugar is known to confer an array of negative health effects (Tuomilehto et al., 2001). Therefore, increased awareness about the content of sugar in popular beverages such as Coke and Gatorade will enable the population to make more educated, conscious decisions about their diet and help decrease the occurrence of preventable diseases due to excess sugar.

References

1. Alexander, S.P.H., Mathie, A., & Peters, J.A. (2011). Enzymes. British Journal of Pharmacology, 164, 279-324. https://dx-doi-org.libraryproxy.griffith.edu.au/10.1111%2Fj.1476-5381.2011.01649_9.x
2. Australian Bureau of Statistics. (2014). Australian Health Survey: Nutrition First Results – Food and Nutrients, 2011-12. Retrieved from https://www.abs.gov.au/AUSSTATS/abs@.nsf/DetailsPage/4364.0.55.0072011-12?OpenDocument
3. Cancer Council Victoria. (n.d.). Rethink Sugary Drink. Retrieved from http://www.rethinksugarydrink.org.au/how-much-sugar
4. Coca Cola Australia. (2019). How much sugar is in a can of Coke? Retrieved from https://www.coca-colacompany.com/au/faqs/how-much-glucose-is-in-a-can-of-coke.html
5. Dondelinger, R.M. (2011). Spectrophotometers. Biomedical Instrumentation & Technology, 45, 2, 139-143. https://dx.doi.org/10.2345/0899-8205-45.2.139
6. Huang, X., Liu, T., & Benkovic, S.J. (2016). Protein Conformation Motions: Enzyme Catalysis. In A. Svendsen (Ed.), Understanding Enzymes (pp. 19-44). New York: Jenny Stanford Publishing. https://doi-org.libraryproxy.griffith.edu.au/10.1201/b19951
7. Jameel, F., Phang, M., Wood, L.G., & Garg, M.L. (2014). Acute effects of feeding fructose, glucose and sucrose on blood lipid levels and systemic inflammation. Lipids in Health and Disease, 13, 195. doi:10.1186/1476-511X-13-195
8. Lobstein, T. (2014). Reducing consumption of sugar-sweetened beverages to reduce the risk of childhood overweight and obesity. Retrieved from https://www.who.int/elena/bbc/ssbs_childhood_obesity/en/
9. Rago, F., Saltzberg, D., Allen, K., & Tolan, D. (2015). Enzyme substrate specificity conferred by distinct conformational pathways. Journal of the American Chemical Society, 137, 43, 13876-13886. https://doi-org.libraryproxy.griffith.edu.au/10.1021/jacs.5b08149
10. Shelton, R.B. (n.d.). pH Values of Common Drinks. Retrieved from https://www.sheltondentistry.com/patient-information/ph-values-common-drinks/
11. Shi, Z., Taylor, A.W., Wittert, G., Goldney, R., & Gill, T.K. (2010). Soft drink consumption and mental health problems among adults in Australia. Cambridge University Press. https://doi.org/10.1017/S1368980009993132
12. Shi, Z., Grande, E.D., Taylor, A.W., Gill, T.K., Adams, R., & Wittert, G. (2011). Association between soft drink consumption and asthma and chronic obstructive pulmonary disease among adults in Australia. Respirology, 17, 2. https://doi.org/10.1111/j.1440-1843.2011.02115.x
13. Stanton, R.C. (2012). Glucose-6-phosphate, NADPH and cell survival. IUBMB Life, 64(5). https://doi-org.libraryproxy.griffith.edu.au/10.1002/iub.1017
14. Tuomilehto, J., Lindström, J., Eriksson, J., Valle, T., Hämäläinen, H., Ilanne-Parikka, P., Keinänen-Kiukaanniemi, S., Laakso, M., Louheranta, A., Rastas, M., Salminen, V., & Aunola, S. (2001). Prevention of Type 2 Diabetes Mellitus by Changes in Lifestyle among Subjects with Impaired Glucose Tolerance. New England Journal of Medicine, 344, 1343-1350. doi: 10.1056/NEJM200105033441801
15. Varsamis, P., Larsen, R.N., Dunstan, D.W., Jennings, G., Owen, N., & Kingwell, B. (2017). The sugar content of soft drinks in Australia, Europe and the United States. Medical Journal of Australia, 206 (10), 454-455. doi:10.5694/mja16.01316