Spectrophotometric Approach for the Analysis of Glucose - Lab Report Example

Name: Date: Experiment 5: Glucose Oxidase Enzyme Biosensor: Spectrophotometric Approach for the Analysis of Glucose Aim The main objective of this lab experiment was to determine the level of glucose concentration in three sport/soft drinks by use of spectrophotometric enzyme assay. INTRODUCTION Glucose is one of the most important compounds due to its chemical properties and physiological functions in the body cells. Analysis of this compound is of great importance because of its role in providing biological fuel and in diagnosis of a number of diseases. It can be an extremely difficult exercise to analyze complex mixtures such as foodstuffs and body fluids if the concentration of specific species in the sample cannot be determined due to interference from other species found in the same sample. Conventionally, such samples are analyzed by separation of the analyte of interest from the original sample. However, this traditional method is a tedious and convoluted process, which has called for the need of simple, quick and more accurate methods. One of the methods that has been developed to determine the concentration of glucose in a sample of a mixture is the spectrophotometric method. An important property of some enzymes is that they are substrate specific, which makes it easier to selectively recognize a specific species from a sample. For example, glucose oxidase is an enzyme that will almost exclusively oxidize E-D-glucose, even in the presence of compounds like D-D-glucose that is very much identical to E-D-glucose. Enzymes play the role of catalysts in conducting biochemical reactions. Their protein nature enables them to have dynamic molecules that can change conformation for proper functioning. When glucose solution undergoes a chemical reaction with the Glucose Assay Reagent, a coloured substance is produced. The amount of clouring in the substance is directly proportional to the quantity of glucose in the solution. Two enzymes are involved in the glucose enzyme assay, glucose oxidase (GOx) and horseradish peroxidase (HRP). In the first stage, E-D-glucose is oxidized by glucose oxidase to E-D-gluconolactone generating hydrogen peroxide (H2O2). HRP then oxidizes Ferrocyanide (colourless) in the assay solution to ferricyanide (coloured). The coloured species of ferricyanide generated in this biochemical reaction is UV Visible. In this lab experiment, a spectrophotometric enzyme assay was performed to determine the concentration of glucose in three samples of sports/soft drinks; Lemonade, Zero and Schweppes. PROCEDURE “Refer to the manual for “NANO3701/8701, Glucose oxidase enzyme biosensor: Spectrophotometric approach for the analysis of glucose”, 2015, pages (52-53). The initial step was the preparation of solutions: 1. Preparation of 100mL of 0.05M potassium ferrocyanide in phosphate buffer solution (0.05M, pH 7.0). From: Where: = no. of moles = volume (100mL) = concentration (0.05M) 2. Preparation of standard glucose solutions. From: c1 × v1 = c2 × v2 1 M× v1 = (1 × 10-3) M × 100 mL Therefore, v1 = 1 × 10-3× 100 /1 = 0.1 mL The figures below show pictures of glucose standard solutions and the sample solutions of sport/soft drinks that were prepared and used during the experiment. Figure 1(a): Glucose standard solution cuvettes Figure 1(b): Sample solutions of the three sport/soft drinks RESULTS AND DISCUSSION The UV-Visible spectrum of the standard solutions is as shown in the figure below: Figure 2: UV-Visible spectrum for the glucose standards The standard solution number six and eight have abnormal colour intensities. Solution is highly coloured than expected while solution number eight has a lighter colour than expected, according to the amount of glucose present in these solutions. The colour intensity is expected to increase as the concentration of glucose in the solutions increases progressively from low to high. In the results obtained in the first experiment, it was observed that solutions from number six through to eight had an abnormal trend of colour. A common observation in both experiments is that as the concentration of glucose tends to rise, the spectrum lines becomes closer. The calibration plot The calibration curve for glucose standard solutions was prepared from the absorbance results that were obtained. The standard curve translates absorbance values into different concentrations. The graph in figure 2 above clearly shows that there is a linear relationship between the concentration of glucose and the absorbance. The calibration plot is a straight line that can be described by Beer’s law. As the level of glucose increases in the standard solutions, the absorbance increases in a linear manner. The spectrophotometer measures the intensity of absorption at the maximum end-point level for every glucose standard solution. The concentration of the solutions were calculated using the relation: Where and are initial concentration and volume respectively while and are the final concentration and volume. For the sample with 0.25 ml of glucose solution, and final volume of 5 ml, the final concentration () is calculated as follows: = = = 0.00005 M = 0.05 mM In the same way, the concentrations of samples with 0.50, 1.00, 1.50, 2.00, 2.50 and 3.00 mL of the glucose solution were calculated and the results tabulated as shown in the table below: Table 1: Calculation of concentration of glucose standard solutions. Standard solution V(ml) V(L) Concentration(M) Concentration(mM) Absorbance (at 419.95nm) blank 0 0 0 0 0 2 0.25 0.00025 0.00005 0.05 0.081899 3 0.5 0.0005 0.0001 0.1 0.177589 4 1.0 0.001 0.0002 0.2 0.285232 5 1.5 0.0015 0.0003 0.3 0.494644 6 2.0 0.002 0.0004 0.4 0.712535 7 2.5 0.0025 0.0005 0.5 0.915249 8 3.0 0.003 0.0006 0.6 0.852894 The results in the table 1 above were used to draw a standard calibration curve, as in the figure 2 below: Figure 2: Calibration plot for standard solutions of glucose The absorbance values for the standard solutions do not indicate the concentration of glucose in the sports drinks, but by using a standard curve with known concentrations, the unknown concentration of glucose in these solutions can be determined. The slope of the straight line in figure 2 is higher than the slope of the straight line that was obtained in the first experiment. This may be due to different conditions in which the two experiments were carried out, and human errors involved. Determination of the Unknown glucose concentrations in the sport/soft drinks Using the calibration plot, the unknown concentration of glucose in three samples of sports/soft drinks (Lemonade, Zero and Schweppes) shown in figure 3 below was determined. This can be done by either using the equation of the straight line or by locating the absorbance of that sample solution on the vertical axis (y-axis) and drawing a horizontal line to the straight line, then drawing a vertical line from the point of intersection to the horizontal axis to determine the concentration. Figure 3: Bottles containing three different drinks (from the right: soft drink, sport blue and sport red) From the equation of the straight line on the calibration plot; Absorbance(y) = 1600.5[C] +0.0099, where C is the unknown concentration. Rearranging the equation, C= Below is a table that shows absorbance and wavelength of the three sport/soft drinks as obtained from the spectrophotometry device. Table 2: Absorbance of the three drinks at a wavelength of 419.95 Sample Absorbance(au) Average absorbance Wavelength(nm) Sport drink (red 1) Sport drink (red 2) Sport drink (red 3) 1.06807 0.870478 0.828278 0.922275 419.95 Sport drink (blue 1) Sport drink (blue 2) Sport drink (blue 3) 0.069955 0.068813 0.083959 0.074242 419.95 Soft drink 1 Soft drink 1 Soft drink 1 0.843667 0.79279 0.770264 0.80224 419.95 For every value of absorbance, there is a unique value of glucose concentration. The greater the value of absorbance, the higher the concentration of glucose in the samples. The red sport drink has a higher concentration of glucose, followed by the Schweppes soft drink while the Zero sport drink contains the lowest amount of glucose. The range of glucose concentration in all the three concentration is very narrow. Figure 4: The optical spectra of the sport/soft drinks from the spectrophotometric device As observed in the first experiment, all the samples of a specific drink show almost similar coloring, except for one sample of the red drink that shows abnormally higher intensity of colour (high concentration of glucose). Calculation of the unknown concentrations of glucose in the drinks and errors involved a) Sport drink (red) Concentration = Moles Standard error = = = 0.00008277 = 8 % Error = 14.5% b) Sport drink (blue) Concentration = Moles % Error = 20.6% c) Soft drink Concentration = Moles % Error = 16.7% Other Sources of errors in this experiment Other errors in this experiment might have arisen from inaccurate measurements of solutions and weighing of substances such as horseradish peroxidase enzyme and glucose oxidase enzyme. This would result in slightly more or less concentrated solutions than expected. Sometimes zeroing of the absorbance spectrophotometric device may not be very accurate. QUESTIONS Question 1 a) 20,000 units/g, b) 6,000 units/g and c) 35,600 units/g of GOx? a) g = 50mg b) g = 167mg c) g = 28.1mg Question 2 Glucose oxidase is an oxido-reductase enzyme that acts as a catalyst in the oxidation reaction of glucose to form D-gluconolactone and hydrogen peroxide. It is produced by certain species of insects and fungi and plays the role of an antibacterial in the presence of oxygen and glucose. The mechanism of action is through reduction of the underlying activation energy that will otherwise hinder the forward reaction. Question 3 As the reaction proceeds, more hydrogen peroxide is generated as displayed in the chemical equation below. -D-glucose + O2 + GOx (catalyst)  β –D – gluconolactone + H2O2 Question 4 In the GOx reaction, -D-glucose is oxidized while oxygen is reduced. In the HRP reaction Ferrocyanide is oxidized while hydrogen is reduced. CONCLUSION Testing of glucose concentration using the enzyme glucose oxidase is a two-step reaction process. The colour intensity in the solution is a measure of hydrogen peroxide generated, and thus the concentration of glucose present. Measurement of glucose concentration is a basic laboratory technique that can be utilized in both clinical and industrial applications. Accurate measurements glucose concentration is very significant in the diagnosis and management of diseases such as diabetes mellitus, hyperglycemia and hypoglycemia. References Read More

Determination of the Unknown glucose concentrations in the sport/soft drinks Using the calibration plot, the unknown concentration of glucose in three samples of sports/soft drinks (Lemonade, Zero and Schweppes) shown in figure 3 below was determined. This can be done by either using the equation of the straight line or by locating the absorbance of that sample solution on the vertical axis (y-axis) and drawing a horizontal line to the straight line, then drawing a vertical line from the point of intersection to the horizontal axis to determine the concentration.

Figure 3: Bottles containing three different drinks (from the right: soft drink, sport blue and sport red) From the equation of the straight line on the calibration plot; Absorbance(y) = 1600.5[C] +0.0099, where C is the unknown concentration. Rearranging the equation, C= Below is a table that shows absorbance and wavelength of the three sport/soft drinks as obtained from the spectrophotometry device. Table 2: Absorbance of the three drinks at a wavelength of 419.95 Sample Absorbance(au) Average absorbance Wavelength(nm) Sport drink (red 1) Sport drink (red 2) Sport drink (red 3) 1.06807 0.870478 0.828278 0.922275 419.

95 Sport drink (blue 1) Sport drink (blue 2) Sport drink (blue 3) 0.069955 0.068813 0.083959 0.074242 419.95 Soft drink 1 Soft drink 1 Soft drink 1 0.843667 0.79279 0.770264 0.80224 419.95 For every value of absorbance, there is a unique value of glucose concentration. The greater the value of absorbance, the higher the concentration of glucose in the samples. The red sport drink has a higher concentration of glucose, followed by the Schweppes soft drink while the Zero sport drink contains the lowest amount of glucose.

The range of glucose concentration in all the three concentration is very narrow. Figure 4: The optical spectra of the sport/soft drinks from the spectrophotometric device As observed in the first experiment, all the samples of a specific drink show almost similar coloring, except for one sample of the red drink that shows abnormally higher intensity of colour (high concentration of glucose). Calculation of the unknown concentrations of glucose in the drinks and errors involved a) Sport drink (red) Concentration = Moles Standard error = = = 0.

00008277 = 8 % Error = 14.5% b) Sport drink (blue) Concentration = Moles % Error = 20.6% c) Soft drink Concentration = Moles % Error = 16.7% Other Sources of errors in this experiment Other errors in this experiment might have arisen from inaccurate measurements of solutions and weighing of substances such as horseradish peroxidase enzyme and glucose oxidase enzyme. This would result in slightly more or less concentrated solutions than expected. Sometimes zeroing of the absorbance spectrophotometric device may not be very accurate.

QUESTIONS Question 1 a) 20,000 units/g, b) 6,000 units/g and c) 35,600 units/g of GOx? a) g = 50mg b) g = 167mg c) g = 28.1mg Question 2 Glucose oxidase is an oxido-reductase enzyme that acts as a catalyst in the oxidation reaction of glucose to form D-gluconolactone and hydrogen peroxide. It is produced by certain species of insects and fungi and plays the role of an antibacterial in the presence of oxygen and glucose. The mechanism of action is through reduction of the underlying activation energy that will otherwise hinder the forward reaction.

Question 3 As the reaction proceeds, more hydrogen peroxide is generated as displayed in the chemical equation below. -D-glucose + O2 + GOx (catalyst)  β –D – gluconolactone + H2O2 Question 4 In the GOx reaction, -D-glucose is oxidized while oxygen is reduced. In the HRP reaction Ferrocyanide is oxidized while hydrogen is reduced. CONCLUSION Testing of glucose concentration using the enzyme glucose oxidase is a two-step reaction process. The colour intensity in the solution is a measure of hydrogen peroxide generated, and thus the concentration of glucose present.

Measurement of glucose concentration is a basic laboratory technique that can be utilized in both clinical and industrial applications.

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