DETERMINING THE GLUCOSE CONTENT OF AND ORANGE USING GOD-PAP ASSAY Every fruit has a sweet taste only that some are sweeter than others. The sweetness of most fruits come from its sugar content and these sugars that the fruits contain are known as invert sugars. In this experiment, an orange was used. An orange which is an excellent source of vitamin C gets its sweetness from natural sugars which are sucrose, glucose and fructose (livestrong.com). In this experiment the concentration of glucose in an orange was measured. There are various methods which can be used to measure the glucose concentration but in this experiment a highly specific enzymatic method using the GOD-PAP assay (glucose oxidase peroxidase aminophenazone phenol) (schedule coventry,2013). This assay is based on 2 coupled enzyme reactions and a colorimetric end-point. D-glucose + O2 +H2O → H2O2 + gluconate aminophenazone + phenol + H2O2 → a red dye + H2O2 Under stable conditions, absorbance measured using a spectrophotometer will be proportional to the amount of glucose present (schedule Coventry,2013). Spectrophotometry is a method used to measure absorbance of light. This measurement is carried out using a spectrophotometer. A spectrophotometer is an equipment used to take accurate measurement of absorbance at various wavelengths. Electrons are usually present at different energy levels but are usually at ground state (stationary state) which is the lowest energy level. When electrons are exposed to energy such as heat or light they become excited. One quantum of energy is absorbed for an electron to become excited (from ground state to excited state) and for it to go back to its ground state one quantum of energy is released. There are two laws surrounding the absorbance of light in a solution known as the Beer-lambert relationship. These laws state that the absorption of light is exponentially related to the concentration of the solute. It also states that the absorption of light is exponentially related to the length of the light path through the absorbing solution. The Beer-lambert relationship is expressed as thus: A=el[C] which refers to the algorithm of the ration of the incident light to the emergent light expressed as (A=Log10(l0/l)) (Reed et al,2007). The purpose of this experiment was to determine the unknown concentration of glucose using the GOD-PAP assay and a calibration curve. The measurements were taken using a spectrophotometer.
The experiment was carried out as par the laboratory schedule (Coventry university schedule,2013)
Table 1 shows the calibration curve data which was used to find the concentration of glucose .This table shows the absorbance of standard glucose solutions and diluted glucose solutions which were measured at 500nm. These measurements were taken using a spectrophotometer. Figure 1 shows a calibration curve used to find an unknown concentration of glucose in 2.45g of orange with absorbance on the y-axis and concentration on the x-axis which are in duplicates. Table 1: Calibration curve data for glucose concentration at 500nm Tube
Absorbance a at 500nm
Absorbance b at 500nm
Average glucose in dilution B =0.815 mM
Dilution factor= 5
Weight of orange=2.45g
Glucose in orange= average glucose (mM) × dilution factor ×100 ml/weight of orange (g) ×180 ×100/106 g/100g orange = 0.815× 5×100/2.45 ×180 ×100/106 =0.815×204×0.018=2.993 ≈3g/100g orange
According to Beer-lambert’s law, absorbance is directly proportional to the concentration of a solution and the intensity of the light is proportional to the concentration of a solution under stable conditions. In this experiment the laws surrounding absorbance which is the Beer-lambert law was obeyed (Reed et al,2007). From...
References: 1. Reed,R.,Holmes,D.,Weyers,J.,Jones,A. (2007) Practical Skills in Biomolecular Sciences, 3rd edn., Essex: Pearson.
2. Ochs,C (2010) Natural Sugars in Oranges, Available at: www.livestrong.com/article/267094-natural-sugars-in-oranges/ (Accessed: 15th November 2013).
3. Coventry University. Schedule 116 BMS.(2013) Determination Of The Glucose Content O An Orange
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