This experiment was performed to determine the glucose levels present in a Coke and Gatorade sample using Spectrophotometry. Due to an error in the method, the results of this experiment were inaccurate and the sample data was used. Using this data there was found to be a positive linear trend between glucose concentration and absorbance. From this the Coke and Gatorade glucose concentrations were calculated to be 0.439g/100ml and 0.944g/100ml respectively. However this was not consistent with previous research which actually found Coke to have the higher glucose levels. This suggests that the experiment could be improved to provide more accurate data.
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Enzymes are biological molecules that catalyse chemical reactions. This means they increase the rate of the reaction and they do this by lowering the activation energy required for the reaction to proceed. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Certain enzymes may require the presence of either co-factors (such as or ions) or co-enzymes (such as /NADH or /NADPH) to assist the reaction. Enzymes have no effect on free-energy changes associated with the reaction they catalyse nor do they influence the reaction equilibrium. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates sufficient for life. Since enzymes are selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.
Many enzyme-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 can be monitored spectrophotometrically. In this experiment, the concentration of sucrose and glucose in selected “sports drinks” will be determined via a reaction sequence involving the appearance of NADPH.In this experiment, a multi-step enzymatic reaction will be used to determine the concentration of glucose in selected drinks (Coke and Gatorade).
D-glucose + ATP à Glucose-6-phophate + ADP
Gluucose-6-phosphate + à 6-phosphogluconate + NADPH +
Enzyme: glucose-6-phospage dehydrogenase
A spectrophotometer will be used to measure the concentration of NADPH produced in this reaction, which is directly related to the concentration of glucose in the Coke and Gatorade. This will allow the determination of the naturally occurring D-glucose only in the sample. The aim of this experiment is to determine the glucose levels in both the Coke and the Gatorade and determine which of the two samples has the higher glucose content.
Sample of Coke (1.5mL – diluted 1/100)
TEA buffer pH 7.6 (containing NADP^+, ATP, enzymes (hexokinase, glucose-6-phosphate dehydrogenase)) Sample of Gatorade (1.5mL – diluted 1/100)
20 mM glucose
Each group of students was supplied with two samples (one coke and one Gatorade). One blank sample and 5 standards (5 glucose) needed to be prepared.
Distilled water was placed into the cuvette and the cuvette was placed into the spectrophotometer. The wavelength was set to 340nm and the absorbance reading was zeroed.
From the 20mM glucose standard, 1mL glucose standards were prepared of the following concentrations: 2 mM, 3 mM, 4 mM, 5 mM and 6 mM. These were labelled as 2G, 3G, 4G, 5G and 6G.
The calculated amounts for the glucose were added to the distilled water, mixed gently and incubated at 37ËšC for 5 minutes. The spectrophotometer was then used to read the absorbance at 340nm (A1).
The TEA buffer was then added to each glucose sample and mixed gently. After 15 minutes of incubation the absorbance was read from the spectrophotometer at 340nm and recorded (A2).
The readings from the spectrophotometer where recorded for all the standards and the two coke samples and placed into a data table.
Results and Calculations
Table of recorded and calculated Results
The data was collected and put into Table 1:
Using Excel, the concentration of the glucose in mM was plotted against the absorbance of each glucose sample (). Using these points a line of best fit and R^2 value was determined. Using the equation of the line of best fit, the concentration of the Coke and Gatorade was determined. The concentration of Coke was found to be 0.98g per 100mL and Gatorade was 1.156g per 100mL.
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Figure – Concentration vs. Absorbance for recorded data
However, the value for this figure of 0.47 is very low and shows a poor level of correlation. The data does not follow a linear trend as expected and there was very likely to be a lot of error in the data gathered, either through improper zeroing of the spectrophotometer or other errors. Therefore the data was taken into account but it was decided that use of the sample data will allow a more accurate calculation for the levels of glucose in the Coke and Gatorade. For sample data, refer to the lab manual page 67.
The sample data was plotted and the equation of the line of best fit was used to calculate the concentration of glucose in the Coke and Gatorade samples by substituting the values for absorbance into the equation and rearranging to find the concentration. There is a linear relationship between the data for absorbance and concentration.
Figure 2 – Concentration vs. Absorbance for Sample Data
Calculation of glucose in Coke and Gatorade Sample
Using y = 0.1889x and absorbance = 0.046mM
0.046 = 0.1889x
x = 0.046/0.1889
x = 0.243515 mM
Using y = 0.1889x and absorbance = 0.099mM
0.099 = 0.1889x
x = 0.099/0.1889
x = 0.524087 mM
The values for the concentration of the diluted Coke and Gatorade were taken and converted into undiluted measurements by multiplying by 100.
0.243515*100 = 24.35151 mM
0.524087*100 = 52.40868 mM
Then this was converted from mM to mol/L by dividing by 1000.
24.35151/1000 = 0.024352 mol/L
52.40868/1000 = 0.052409 mol/L
The value of moles per litre was multiplied by the molecular weight of glucose which is 180.16 grams to get the value for grams per litre.
0.024352×180.16 = 4.387168 g/L
0.052409×180.16 = 9.441948 g/L
This was then converted from grams per litre into grams per 100mL by dividing by 10.
4.387168/10 = 0.438717 g/ 100mL
9.441948/10 = 0.944195 g/ 100mL
Therefore Coke was found to have 0.439g of glucose per 100mL and Gatorade was found to have 0.944g of glucose per 100mL.
The glucose concentration in Coke and Gatorade was found to be 0.98g per 100mL and 1.156g per 100mL respectively. However, on further analysis of the results it was found that these results did not show the expected linear relationship and were therefore deemed to not give an accurate determination of the glucose concentrations. There was certainly some kind of error in the experiment, most likely due to improper zeroing of the spectrophotometer. This ruined the results of our experiment so I decided to use the sample data provided in the lab manual to recalculate the glucose levels. Using excel to plot and analyse this data, it was found that the Gatorade sample had a glucose level of 0.944g per 100mL which was higher than that of the Coke sample which was 0.439g per 100mL.
This is not consistent with previous research  where the Glucose content in a Coca-Cola bottle (14 fluid ounces) was found to be 3.9g per 100ml. They also tested a bottle of Gatorade Lemon-Lime (20 fluid ounces) to have a glucose level of 2.4g per 100ml. This laboratory testing shows significantly higher levels of glucose in the coke and Gatorade then we calculated. However their results were calculated using high-performance liquid chromatography (HPLC) which means they are able to gather much more accurate data than what we have. Also, their tests were completed using drinks that were not diluted or modified in any way so this may lead to much more accurate results. Also, the drinks which the researchers used may be different varieties to the drinks we used, which would further increase differences in our results.
From our experiment we found that Gatorade had the higher glucose content. If we compare this to the sugar content from the article  we find that coke has much more fructose, 7.2g/100ml compared to 2.4g/100ml of Gatorade, and also a much higher overall sugar level. Coke had 11.1g/100ml to the 5.9g/100ml of Gatorade. This places Coke as the unhealthier drink because not only do sugar-sweetened beverages increase your risk of metabolic syndrome and type 2 diabetes, fructose is actually found to be unhealthier than glucose. Consuming fructose-sweetened, not glucose-sweetened, beverages increases visceral adiposity and lipids and decreases insulin sensitivity in overweight/ obese humans . So from this information we can conclude that Coke is the unhealthier drink.
From our recorded data and the sample data we were able to calculate that the Gatorade sample had the higher glucose levels than the Coke sample. From further research we are able to produce an alternate conclusion which is that Coke has both the higher glucose content and higher overall sugar content. This leads us to conclude that Coke is the unhealthier drink. This experiment is very significant to our daily lives as the glucose, fructose and the overall sugar content of the drinks we consume in our day to day lives is very important for us to know so that we can moderate our intake to ensure we live a healthy life and lower our risk of developing diseases.
When we participate in future experiments we need to make sure that we do not make any errors in the method or recording the results as this can greatly affect our final results and the conclusions we draw from it. We could undertake future experiments with higher quality and more accurate equipment as the equipment we use often places limitations to the accuracy of the data we can obtain. Future research can also be done to compare other sports drinks and soft drinks to find out their glucose content and their health implications for people in our society.
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