Glucose_report_HD_3

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University of New South Wales *

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2041

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Chemistry

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Apr 25, 2024

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CHEM 2041 Laboratory Report 1 TITLE OF EXPERIMENT: Determining concentration of glucose in sports drinks with bioassays & biosensors INTRODUCTION Determining the concentration of glucose in sports drinks and determining concentrations of other compounds in drinks and foods is essential in the food and drink industry to verify claims companies make of their products. There are different methods which can be used to determine the glucose levels in a sports drink such as Gatorade, two of which are using a spectrophotometric enzyme bioassay and blood glucose biosensor, both of which can be used to create calibration curves to determine the concentration of an unknown solution of glucose. The LINEST function can then be used to determine uncertainties. Spectrophotometric enzyme bioassay The enzyme glucose oxidase (GOx) can be used to determine the concentration of glucose in a solution. GOx reacts almost exclusively with β-D-glucose in the following reaction: β − D − glucose + 0 2 → GOx β − D − gluconolactone + H 2 O 2 The H 2 O 2 produced in this reaction can be used to produce a UV-visible active species by reacting with ferrocyanide and the enzyme horseradish peroxidase (HRP) to produce water and ferricyanide in the following reaction. H 2 O 2 + ferrocyanide → HRP H 2 O + ferricyanide Ferricyanide is UV-visible active, so a solution prepared using these reagents can be used to create a calibration curve and determine an unknown glucose concentration. Blood glucose biosensor The blood glucose biosensor works using electrodes to host an oxidation reaction from the following equation. glucose + ferricyanide→ FAD − GDH gluconolactone + ferrocyanide The biosensor uses the enzyme flavin adenine dinucleotide glucose dehydrogenase (FAD-GDH) and an electrode, and the activity from the oxidation reaction can be measured to give a glucose concentration. In the same process as above, a calibration curve can be created from these measurements and LINEST can be used to calculate the uncertainties.

CHEM 2041 Laboratory Report 2 EXPERIMENTAL Concentrations referred to in method and associated information:  Phosphate buffer: 0.05 M from Na2HPO4 and NaH2PO4 adjusted to pH = 6.0  Glucose oxidase (GOx) and Horseradish peroxidise (HRP) stock: 3000 U GOx and 1000 U HRP  Glucose standard solution: 1 mM β-DGlucose in 0.05 M phosphate buffer, pH = 6.0. The Gatorade concentration is determined through two methods in this experiment. The first method uses a spectrophotometer enzyme assay with a UV-Vis absorption measurement, and the second a biosensor assay with a mmol/L measurement. Method A The concentration of glucose in Gatorade for this method was determined using a calibration curve created from measurements of absorbance from spectrophotometer enzyme assay, where the absorbance of a solution is measured after a certain time period and a calibration curve is created. Make up 6 5mL vials of standard solutions of glucose ranging from 0, 0.05, 0.1, 0.2, 0.4 and 0.6 mol/L of glucose. To make these solutions, use 1.25 mL of ferrocyanide, 0.5 mL of GOx and HRP enzyme, and the correct amount of phosphate buffer to achieve the desired glucose concentration. Add the glucose last in staggered intervals (~3 minutes), noting the time at which the glucose is added to each solution. Dilute the Gatorade by a factor of 20 with the phosphate buffer, and then repeat the previous steps, replacing the glucose with 0.5mL of the diluted solution. Make two vials of this solution. While the solutions are reacting, set up the UV-Vis spectrometer and open the SpectraSuite Software. Set the integration time to 100ms and scans to average is set to 9. Enable the light source by checking the Strobe/Lamp Enable box, place in a black dummy cuvette and record the dark spectrum. Then record the reference spectrum from a cuvette with the solution with no glucose in it. For each of the vials, record the absorbance exactly 20 minutes after the glucose or unknown solution is added at 420 nm using the spectrophotometer. Record these results and create a standard calibration curve from them, making sure to subtract the blank from the recorded values. Using the LINEST function on Excel and the Beer-Lambert Law, determine the concentration of the unknown solution and estimate the uncertainty associated with this calculated concentration. After these answers are calculated, account for the dilution factors performed when preparing the unknown solution to find the concentration of glucose in the Gatorade and the uncertainty in this concentration.

CHEM 2041 Laboratory Report 3 Method B The concentration of glucose in Gatorade for this method is determined by preparing a set of standard solutions and creating a calibration curve from concentration measurements from a blood glucose biosensor. Prepare a set of standard solutions of glucose of concentrations 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mg/mL using the given 4 mg/mL solution of glucose and diluting using the potassium chloride in phosphate buffer solution. Dilute the Gatorade by a factor of 10 using the potassium chloride in phosphate buffer solution. Measure the standard solutions using the biosensor and record the results (Note – the 0 mg/mL control solution should give an error when measured). Measure the unknown solution 4 times. Excluding the control solution, create a calibration curve from the results (make sure to convert the units of the results) Using the LINEST function on Excel, determine the concentration and uncertainty of the unknown solution. After these answers are calculated, account for the dilution factors performed when preparing the unknown solution to find the concentration of glucose in Gatorade.

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CHEM 2041 Laboratory Report 4 RESULTS The following uses the data provided on the “Glucose Data” page on Moodle Volume of 1mM Glucose (mL) Absorbance Concentration of glucose (mol/L) Corrected absorbance (subtracting the blank) 0 0.099 0 0 0.25 0.110 0.05 0.011 0.5 0.198 0.1 0.099 1 0.347 0.2 0.248 2 0.526 0.4 0.427 3 0.620 0.6 0.521 Unknown replicate A 0.287 Unknown replicate A 0.188 Unknown Replicate B 0.298 Unknown Replicate B 0.199 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 0.1 0.2 0.3 0.4 0.5 0.6 f(x) = 0.95 x Absorbance vs. Concentration Concentration (mmol/L) Absorabnce

CHEM 2041 Laboratory Report 5 Using LINEST on the corrected absorbance and concentration, and setting the intercept to (0,0) slope (b) 0.949258 0 intercept (a) s(b) 0.057066 #N/A s(a) r^2 0.982251 0.043179 sy/x F 276.699 5 degree freedom SSreg 0.515874 0.009322 SSresidual From LINEST, y 0 = 0.1935 Using the Beer-Lambert law A = εcl Where εl is constant and equal to the slope of the graph and y 0 = A , 0.1935 = c ∗ 0.949258 c = 0.203843 mmol L = ^ x 0 Converting to mg mL , c = 0.203843 ∗ 180.156 ¿ 36.7236 g mL Accounting for dilution and conversion: c ( Gatorade )= 36.7236 g mL ∗ 200 1000 c ( Gatorade ) = 7.34473 g L Using the values from LINEST, the uncertainty U can be found. The unknown is measured n = 2 times and the graph has m = 6 data points. s ^ x 0 = s y x b √ 1 m + 1 n = 0.03714 U = s ^ x 0 ∗ t 0.05 , 5 = 0.09547 g mL Accounting for dilution, using the same steps as above:

CHEM 2041 Laboratory Report 6 U ( Gatorade ) = 0.09547 ∗ 200 1000 = 0.01909 g L Concentration of Glucose in Gatorade® ¿ 7.344 ± 0.019 g L

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CHEM 2041 Laboratory Report 7 Bioassay Glucose Standard (mg/mL) Measured conc (mmol/L) Measured conc (g/L) 2.5 10.6 1.9096536 2 9 1.621404 1.5 6.7 1.2070452 1 4.5 0.810702 0.5 2.2 0.3963432 Unknown replicate A 3.9 0.7026084 Unknown replicate B 4.1 0.7386396 Unknown replicate C 4.2 0.7566552 Unknown replicate D 4.2 0.7566552 0 0.5 1 1.5 2 2.5 3 0 0.5 1 1.5 2 2.5 f(x) = 0.79 x Expected concentration vs. measured glucose concentration Expected conc. (mg/mL) Measured conc. (mg/mL)

CHEM 2041 Laboratory Report 8 Using LINEST on the expected concentration vs. measured concentration, and setting the intercept to (0,0) slope (b) 0.748793847 0 intercept (a) s(b) 0.016183406 #N/A s(a) r^2 0.998135061 0.060009676 sy/x F 2140.842278 4 degree freedom SSreg 7.709518104 0.014404645 SSresidual y 0 = average of unknown replicates = 0.7386396 g L ^ x 0 = y 0 − a b = 0.986439 g L where ^ x 0 = c . Accounting for the dilution factor of 10, c ( Gatorade ) = 0.986439 ∗ 10 c ( Gatorade ) = 9.86439 g L Using the values from LINEST, the uncertainty U can be found. The unknown is measured n = 4 times and the graph has m = 6 data points. s ^ x 0 = s y x b √ 1 m + 1 n = 0.051731 U = s ^ x 0 ∗ t 0.05,4 = 0.143629 g L Accounting for the dilution factor of 10 U ( Gatorade ) = 10 ∗ U = 1.43629 g L Concentration of Glucose in Gatorade® ¿ 9.8 ± 1.4 g L

CHEM 2041 Laboratory Report 9 DISCUSSION The spectrophotometric enzyme assay will be referred to as Method A and the biosensor assay as Method B. Looking at the relative uncertainty in Method A (~0.26%) in comparison to Method B (~14%), Method B’s is approximately 50 times greater than Method A. This extreme disparity in uncertainties can be attributed to the methods used to prepare the solutions for the experiment as well as the instruments used for analysis. When preparing the unknown for Method A the unknown is diluted by a factor of 200 and unknown prepared for Method B is diluted by a factor of 10, meaning the spectrophotometric enzyme assay technique is more sensitive to glucose and more capable of giving a precise concentration because the concentration being measured is much smaller. Given this, these results indicate that Method A is more reliable than Method B as it has a smaller relative error. The LifeSmart biosensor is an appliance which is designed for convenience and portability and ease of use for measuring blood glucose levels, giving results in seconds. The measured values for Method B are within the measuring range given on the LifeSmart website is 20-600mg/dl (0.2-6 g/L). In comparison, the UV-Vis spectrometer is designed for precision and accuracy to determine absorbances of solutions, and with the method used gives results after 20 minutes. It is reasonable to assume that Method A would be more accurate and precise measurement since it is more sensitive and is designed for different applications than the biosensor, which most likely trades accuracy and precision for time and convenience. On the Sports Dietician Australia website, Gatorade is reported to have 5g of glucose per litre. This is within a reasonable range of the results from Method A (147% of the reported value), but almost half of the calculated result from Method B (196% of the reported value). This can likely be attributed to the inaccuracy in the biosensor and errors due to LINEST calculations. Compared to this result, Method A is once again more accurate than Method B. While the blood glucose biosensor gives quick and informative measurements for its intended use, using a spectrophotometric enzyme assay provides more accurate and precise results compared to literature values.

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CHEM 2041 Laboratory Report 10 REFERENCES: Sports Dieticians Australia. (2021). Sports drinks . https://www.sportsdietitians.com.au/factsheets/fuelling-recovery/sports-drinks/ LifeSmart. (2022). LifeSmart Blood Glucose Monitor . https://mylifesmart.net.au/products/ls-blood-glucose/