Abstract
The aim of this experiment was to investigate the concentration of glucose in Coke and Gatorade samples via spectrophotometry at 340um wavelength. Soft drinks and electrolyte drinks often seem like the perfect way to quench your thirst when working out, and or intense physical activity as they replenish carbohydrates and electrolytes (Health, 2017). But they also have contributed to a negative metabolic health, causing obesity. A spectrophotometer was used to measure the absorbance difference in enzymatic reaction to calculate the glucose concentrations in coke and Gatorade. Data was collected and analysed and showed in table 3, coke (4.6404 g/100ml) has a higher concentration of glucose than that of Gatorade (2.9322 g/100ml). From this it can be concluded that consuming Coke is metabolically unhealthier to consume compared to Gatorade.
Introduction
Enzymes are biological molecules, usually protein, that significantly speed up the rate of virtually all of the chemical reactions that take place within cells (Castro, 2014). They achieve this by lowering the activation energy required for the reaction to proceed. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and each enzyme is specific to each substrate, for instance the enzyme that help breaks down fatty foods are not useful in breaking down carbohydrates (Kerr, 2019). The body must have the necessary enzymes in order to produce the correct chemical reaction for each specific bodily function, after substrates they’re then converted into different molecules, called products. Furthermore, some enzymes may require the presence of either co-factors or co-enzymes (such as NAD+/NADH or NADP+/NADPH) to assist the reaction. The protein in the enzymes are globular, the intra- and intermolecular bonds that hold proteins in their secondary and tertiary structures are disrupted by change in temperature and ph. Therefore, the catalytic activity of an enzyme is pH and temperature sensitive.
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Many enzymes-catalysed reactions followed by the use of spectrophotometry rely on differing absorbance characteristics of the oxidised and reduced forms of NADH or NADPH. Specifically, NADPH absorbs strongly at a wavelength of 340 nm whilst NADP+ does not. This then lets us see the appearance or disappearance of NADPH in reactions and is why we are using the spectrophotometry to monitor any change in the reaction. Specifically, in this experiment, we will be observing the concentration of glucose in selected drinks, this will consequently be determined by the reaction sequence involving the appearance of NADPH.
Reaction Sequence:
1. D-glucose + ATP -> Glucose-6-phosphate + ADP
Enzyme: Hexokinase
2. Glucose-6-phosphate + NADP+ -> 6-phosphogluconate + NADPH + H+
Enzyme: glucose-6-phosphate dehydrogenase
In this experiment a spectrophotometry will be used to measure the concentration of NAHPH produced in this reaction, which is directly related to the concentration of glucose in the Coke and Gatorade. This will allow the determination on naturally occurring D-glucose only in the sample. The aim of this experiment is to determine the glucose levels in both the Coke and Gatorade and determine which of the two samples had the higher glucose content.
Method/ Experiment
Each group of students was supplied with two samples; one with coke and one with Gatorade. One blank sample and 5 standard (glucose) needed to be prepared. Distilled water was placed into cuvette and the cuvette was placed into the spectrophotometer. The wavelength was set to 340nm and the absorbance reading was zeroed. From 20mM standard, 1ml glucose standards were prepared of the following concentrations: 1 mM, 2 mM, 3 mM, 4 mM, 6 mM, These were labelled 1G, 2G, 3G, 4G and 6G. the calculated amounts for glucose were added to distilled water, mixed gently and incubated for 2minutes, the spectrophotometer was then used to read the absorbance at 340nM (A1).
Results
Table 1: Data obtained from the experiment. The absorbance difference after 18 minutes.
Sample
A1 (0 mins)
A2 (+18 min)
Absorbance difference (A2 – A1)
0 mM
-0.08
-0.08
0.000
1 mM
0.004
0.192
0.188
2 mM
0.004
0.361
0.357
3 mM
0.011
0.616
0.605
4 mM
0.005
0.738
0.733
6 mM
0005
1.007
1.002
Coke
0.003
0.044
0.041
Gatorade
0.008
0.030
0.022
Sucrose
0.005
0.005
0.000
Figure 1: Table of nutritional information of coke (Coca-cola, 2017)
Figure 2: Table of nutritional information of Gatorade
(PepsiCO.Inc., 2019)
Figure 3: The Absorbance difference due to different concentrations of glucose, also displayed in the sucrose levels and the specific point of Cokes concentration and Gatorades concentration.
Table 2: Calculations
Working out
Coke
Working out
Gatorade
mM Diluted
0.2381 =
0.2381
0.1452 =
0.1452
mM Undiluted
0.2381 x 100 =
23.81
0.1452 x 100 =
14.52
Mol/L
23.81/ 100 =
0.2381
14.52/ 100 =
0.1452
g/mol
180
180
g/L
0.2381 x 180 =
42.871
0.1452 x 180 =
26.1327
g/100ml
42.871/ 10 =
4.2871
26.1327/ 10 =
2.61327
Discussion/Conclusion
The objective of this study was to conduct an objective experiment of sugar content and composition in popular sugar-sweetened beverage with a particular focus on glucose content. Soft drinks in Australia have sweetener in them with sugar cane-derived sucrose, sucrose is a disaccharide of 50% glucose and 50% fructose (Varsamis P, 2017), because there is high levels of sucrose in soft drinks and sport drinks there will also be glucose present in those drinks: Coke and Gatorade, despite their content labels not stating that, seen in figures 1 and figure 2. After comparing results among other experiments the trends became obvious that Coke has a higher concentration level than that of Gatorade also in comparison to the Article of “Sugar content of popular sweetened beverages based on objective laboratory analysis: Focus on Fructose content” (Emily Ventura, 2010) where there results were 3.9g/100ml of glucose in Coke and 2.4g/100ml of glucose in Gatorade. In supporting this from the experiment conducting in this report, the results can be seen in Table 2, where the glucose concentration in Coke was 4.2871g/100ml a lot higher than that of Gatorade which was 2.61327g/100ml.
Another trend in the results seen in figure 3, was that the higher the concentration of glucose in the dilutions, the more the enzymes are presents in the final product increasing the absorbance that the spectrophotometer measured. From this the trend line was draw in figure 3, where the results sat on and or close to that line, as well as having the glucose standards results plotted. the Coke and Gatorade results were also plotted individually. Interpreting this graph further the results from the experiment show that the glucose levels in Coke and Gatorade also follow the trend line of the glucose standards.
Additionally, after analysing the nutritional content labels and comparing them to the results from the experiment although they have the same trend of Coke having more glucose present then that of Gatorade however the labels have a much higher concentration then what the results showed from this experiment. This can be because of human error, when using the spectrophotometer, the process is typically done using a computer to increase the accuracy and decrease the liking hood of human error. Mostly likely the errors that occurred was improper zeroing of the spectrophotometer, miss use of the pipette, allowing for bubbles to occur causing there to be a difference in the amount.
Despite the results, from this experiment it can be concluded that enzymatic reaction can be used to determine the presence of glucose in drinks using a spectrophotometer. This experiment further supports the hypothesis that Coke has a higher glucose concentration than that of Gatorade because the spectrophotometer measured a higher glucose concentration than Gatorade because the spectrophotometer measured a higher absorbance difference in coke.
The objective of this experiment of detecting the presence of glucose in soft drinks and sports drinks i.e. Coke and Gatorade provide data that shows that there is more glucose present then assumed which raises concern about the accuracy of the sugar consumed by those who regular drink these beverages. In comparison to our data is can be supported by the Article (Emily Ventura, 2010) previously mentioned that further analyses should be conducted to examine the sugar content and composition of drinks particularly the glucose content. This is significant because the correlation between a negative metabolic health is related to a high consumption of sugars; including glucose.
References
Castro, J. (2014, April 26). How do Enzymes Work? Retrieved from Live Science: https://www.livescience.com/45145-how-do-enzymes-work.html
Coca-cola. (2017). Product facts. Retrieved from The Coca-cola co. : https://www.coca-colaproductfacts.com/en/products/coca-cola/original/12-oz/
Emily Ventura, J. D. (2010). Sugar Content of Popular Sweetened Beverages Based on Objective Laboratory Analysis: Focus on Fructose Content. Obesity Journal, 7.
Health, U. (2017, August 10). Water vs Sports Drink: Whats Best for our Bodies. Retrieved from Live Well with UnityPoint Health: https://www.unitypoint.org/livewell/article.aspx?id=9ab3d290-8767-4e22-8474-ab160992ae82
Kerr, S. (2019). How Do Cofactora & Coenzymes Affect Enzyme Activity. Retrieved from Seattlepi: https://education.seattlepi.com/cofactors-coenzymes-affect-enzyme-activity-6692.html
PepsiCO.Inc. (2019, September 26). Gatorade Lemon-Lime. Retrieved from Pepsico: the facts about your favourite beverages: http://www.pepsicobeveragefacts.com/Home/Product?formula=33877&form=RTD&size=11.6
Varsamis P, L. R. (2017). The sugar content of soft drinks in AUstralia, Europe and the United States. Unkown, 1-2.
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