Determining The Glucose Content Of And Orange Using GOD-PAP

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.

METHOD
The experiment was carried out as par the laboratory schedule (Coventry university schedule,2013)

RESULTS
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.

DISCUSSION

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 figure 1 which is a calibration curve shows that the graph is linear which means the Beer-lambert laws were obeyed. In table 1 the absorbance values of the solution were taken in duplicates toavoid any errors. The absorbance  values shown in the table have just a slight difference from one another which shows that the values were accurate. These values were taken using a spectrophotometry skills and a spectrophotometer. A spectrophotometer is a device that shines light through a solution measuring its absorbance of the light at certain wavelengths. In this experiment, the absorbance of standard and diluted glucose solutions were taken at 500nm. These absorbance values as shown in table 1 were used to plot a calibration curve to find an unknown concentration of glucose. From the table there were duplicate values for each solution or sample.

These samples were then plotted on a graph and a line of best fit was drawn through those points. To obtain the concentration of glucose based on the graph ,the value of one of the unknowns was chosen which was within the range of the graph was chosen and traced to meet the line of best fit and the concentration was derived. From the table, absorbance increased as the concentration increased showing precision and as for accuracy, the difference between the first and second trials were minimal. In this experiment and in other experiments like these, errors could be caused by improper handling of GOD-PAP assay and cuvettes .incorrect placement of cuvette into the spectrophotometer and poor calibration of the spectrophotometer. Errors could also be made when plotting a graph for the calibration curve. Weighing the wrong amount of orange. For such errors to be avoided in future experiments, the GOD-PAP assay should be handled with care and dispensed accordingly.

The cuvettes should also be handled with care and avoid touching the transparent side of the cuvette so as not to have fingerprints and inaccurate readings. Regarding the cuvette, they should be placed with the transparent side facing the light making it easy for the light to shine through. Proper scale should be used to plot the graphs to give a good graph and calibration curve. Without a good scale, there will be difficulty in drawing the line of best fit. Weighing the right mass of orange is very important so as to get accurate readings. There should always be a control or a blank solution which should also be in duplicates. These controls are used to zero the spectrophotometer for absorbance readings to be taken. An orange carries different types of sugars mainly sucrose, glucose and fructose. In an orange, there are 13g of natural sugars otherwise known as invert sugars. These sugars which are sucrose, glucose and fructose make up that 13g with sucrose being the most abundant sugar in an orange with 6.5g. Sucrose is a disaccharide made of two monosaccharides known as glucose and fructose which are also sugar contents of an orange.

From the calculations carried out in this experiment, it was gathered that an orange carries about 3g of glucose leaving fructose to be 3.5g in an orange which is the second highest amount (livestrong.com). Without experiments like these, people will always think glucose represents the total amount of sugar in an orange because it is believed that glucose makes everything sweet. In this experiment, the concentration of glucose was found to be 3g in an average orange. This was done with using the knowledge of spectrophotometry and Beer-lambert’s law. Also, the concentration of glucose was found using an enzymatic method known as the GOD-PAP assay and calibration curve. With this experiment, it has been made known that glucose does not represent the overall sugar content of an orange and in fact is the least most abundant sugar in an orange.

An orange also contains other types of sugars like sucrose and fructose with sucrose being the highest sugar component. The calibration curve obeyed Beer-lambert’s law because of its linear structure. Accuracy and precision of results were gotten because they were taken in duplicates. The difference between the absorbance values were minimal(results stayed in the same range) making the results accurate. An increasing absorbance was also recorded as the concentration increased. Overall, the main aim of the experiment was achieved which was determining concentration of glucose in an orange. The use of various equipments were understood and put into practice.

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