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Effect of temperature on enzymic oxidation of chlorogenic acid in apples
What is the effect of varying temperature (0,10,15,20,25) °C on the average rate of oxidation of Chlorogenic Acid in red apples catalyzed by polyphenol oxidase when exposed to air for 30 minutes as determined by titration and UV spectroscopy?
Subject: Chemistry Extended Essay
Session: November 2020
Word Count: 4000
Contents
1.6 Chemicals and apparatus list
Control Experiment 1 (determination of equivalence point)
Control Experiment 2 (determination CGA present in gala apples)
3.2 Raw data collected for titration
3.3 Data processing for titration
3.4 Processed data for titration
3.7 Processed data for UV Vis spectroscopy
3.8 Comparison between UV Visible spectroscopy and titration
Effect of temperature on enzymic activity
Titration of NaOH against apple juice
Measuring absorbance of chlorogenic acid
1. Introduction
Apples are one of the most widely cultivated fruits in the world with over 70 million tons being produced and sold worldwide. Containing a wide variety of acids such as malic acid, ascorbic acid and chlorogenic acid, apples have become one of the most popular commercial fruits in the world. However when left out in the open, it is common to see apples quickly turning brown, this is often followed by it turning hard and dry and losing its flavor and appeal altogether. Since young, I was always curious about the scientific reasons behind this phenomenon and why it only happens to a select variety of fruits. Upon further reading, I discovered that this is caused by the oxidation of polyphenols in a process call enzymic browning, reducing the overall nutritional content of some fruits. A study done by A Kaanane on the maillard reaction in fruits revealed that certain fruits lose over 50% of its original nutritional content when kept at a temperature of 21oC over a period of one week. This has several negative implications in the food industry as food engineers seek to find better ways to preserve the freshness of fruits. Anecdotally, I have observed that keeping fruits and other foods in the fridge seems to keep them fresh for longer periods of time, and even slows down the browning. Current research into this area has shown that pH, temperature, oxygen availability and presence of enzyme inhibitors such as aromatic carboxylic acids and substituted resorcinol are all factors that affect the rate of enzymatic browning. Wageningen University revealed that a pH of 4 or below as well as temperatures below 7oC inhibits the action of enzyme polyphenol oxidase thus slowing down enzymatic browning. However, little research has been done on the effect of temperatures above 7oC on the rate of enzymatic browning. Chlorogenic acid and its derivatives is a type of polyphenol found in almost all fruits in varying concentrations and its oxidation is also catalyzed by polyphenol oxidase, therefore a measurement of the rate of oxidation could be an indirect measurement of the rate of enzymatic browning. Hence the motivation behind this research is to investigate the effects of temperature on the oxidation of chlorogenic acid of apples over an extended period of exposure to atmospheric oxygen.
1.1 Research Question
What is the effect of varying temperature (0,10,15,20,25) °C on the average rate of oxidation of Chlorogenic Acid in red apples catalyzed by polyphenol oxidase when exposed to air for 30 minutes as determined by titration and UV spectroscopy
1.2 Chlorogenic Acid
Chlorogenic acid is a widely found polyphenolic compound in fruits and vegetables as well as coffee beans. It is one of the major phenolic acids found in apples, giving apples and other fruits their characteristically sweet and bitter flavor depending on the concentration found. According to literature sources, the chlorogenic acid content of apples ranges from 10 to 80 mg per apple depending on species, most of which is found as free phenolic acids which can readily react with free radicals and other compounds. The species of apples used for this investigation would be the red gala apples, which has one of the highest concentrations of chlorogenic acid in apples at approximately 80mg; this makes it easier for the readings to be collected. Chlorogenic acid is the main ester of quinic acid with caffeic, ferulic, or coumaric acids and is one of the naturally occurring antioxidants found in apples.
1. Introduction
It contains an ester linkage between quinic acid and caffeic acid. As seen it is a polybasic acid with the Chemical formula: C16H18O9. Hence it’s able to donate 6 protons per molecule.
Hence its average rate of oxidation can be indirectly measured using titration with a strong base, NaOH:
C16H18O9 (aq) + 6NaOH (aq) 6H2O (l) + C16H12O9Na6 (aq)
When the apple is exposed to atmospheric oxygen, a reaction catalyzed by Polyphenol oxidase turns fruits brown overtime. The browning effect is due to the oxidation of the polyphenols within the fruits when they react with oxygen forming Melanin as the end product. The oxidation of chlorogenic acid in open air is represented by figure 2:
1.3 Acid-base reaction
The acid-based reactions use the Bronsted-Lowry theory of acids and bases.
Since chlorogenic acid is an organic acid, it is a relatively weaker acid with a pKa of 3.33. Hence it chlorogenic acid undergoes partial dissociation in water, producing a low concentration of H3O+ ions.
The reaction of a chlorogenic acid with a NaOH results in the production of a basic salt. Since this salt contains an anion,C16H12O96+ which is the conjugate base of chlorogenic acid, it will undergo further reaction when dissolved in water to produce OH- ions, resulting in an alkaline solution. This reaction is known as salt hydrolysis. Hence this reveals that the end point of titration would be within the region of 7-11 pH
The basic salt dissociates in water to produce ions:
C16H12O9Na6C16H12O96++6Na+
Na+ is a very weak conjugate acid since it comes from a strong base, thus it does not undergo hydrolysis in an aqueous solution. On the other hand C16H12O96+ is a strong conjugate base, thus it undergoes hydrolysis in water to form OH- ions:
C16H12O96+aq+6H2Ol⇌C16H18O9aq+OH-(aq)
In addition, Chlorogenic acid (CGA) is soluble in polar solvents like ethanol since it is a phenolic acid, with solubility in ethanol to be approximately 25mg/ml. Thus liquid-liquid extraction of polyphenols is a viable method for isolating chlorogenic acid since it is isolated from other acids present in the apple such as malic acid and ascorbic acid. This opens the door to the use of UV VIS spectrophotometry as a method of quantifying the chlorogenic acid content of apples. Alternatively, a study done by China Agricultural University was able to isolate and quantify using complex reactions and polyacrylamide gel electrophoresis (PAGE) which gave accurate and replicable results. However due to the limited amount of laboratory equipment and chemical compounds, UV Vis spectrophotometry was used instead.
Figure 4 shows the absorption spectrum of Chlorogenic acid in ethanol between wavelengths 200 - 500 nm at room temperature taken from a research paper done by Belay A and Gholap AV. Hence for the purposes of this investigation, the wavelength of Chlorogenic acid will be taken to be 324nm.
Utilizing the Beer–Lambert law, the absorbance of chlorogenic acid is directly proportional to the concentration of chlorogenic acid. Thus, for a fixed path length of a cuvette, UV Vis spectroscopy can be used to determine the concentration of chlorogenic acid in a solution.
1.4 Polyphenol Oxidase
Enzyme polyphenol oxidase (PPO) is a naturally occurring compound in many fruits, including apples; they catalyze the oxidation of mono-, di-, and polyphenols to quinones. Polyphenol oxidases are a family of di-copper metalloenzymes that includes tyrosinase and catechol oxidase. Hence for this investigation we will be focusing more on the reactions catalyzed by Catechol Oxidase.
Catechol Oxidase is a copper oxidase found in a wide variety of fruits and fungi alike. O-quinones rapidly polymerize in the presence of oxygen to form melanin which gives fruits their dark brown coloration
Catechol oxidase is susceptible to changes in temperature. The optimum pH for catechol oxidase was found to be at 6.5 with an optimum temperature of 30oC. Lower temperatures and pH decrease the action of catechol oxidase, while extended exposure to high temperatures and pH causes a conformation change in the active site thereby reducing overall enzymic activity.
1.5 Hypothesis
H1: As the temperature of the apple increases, there will be a decrease in the amount of chlorogenic acid found.
H2: There is a directly proportional relationship between temperature of the apple and the rate of oxidation of chlorogenic acid (enzymatic activity of polyphenol oxidase)
1.6 Chemicals and apparatus list
2. Methodology
2.1 Preliminary trials
During preliminary trialing, the apples were cut into slices and blended without removing the peel. However, results collected from the first set of replicates were were slightly higher than literature sources as it showed a chlorogenic acid content of 90mg within the gala apples which was 10mg more than literature sources. Further analysis of the composition of apples revealed that the peel contains catechol and catechin which react with NaOH:
This causes the chlorogenic acid content found within apples to be higher than expected, thereby reducing the calculated rate of chlorogenic acid oxidation. Hence in realization of this after preliminary trials, the informed decision was made to peel the apples first to resolve the systematic error.
Another issue that occurred was the ambiguity of the end point. Since a universal indicator was used, the end point would be a green solution since it’s a reaction between a strong base and a weak acid. However, it was difficult to distinguish the exact end point by eye leading to variations in the end point. Thus, a pH probe was used to accurately and reliably determine the end point pH of titration which was calculated to be 8.05 using salt hydrolysis calculations.
Apart from this, the incubation time of apples was also revised. Initial trialing at 1 hour oxidation time prior to blending proved too slow as the rate of oxidation was limited by the small surface area in contact with atmospheric air. Thus blending the apples before oxidation allows all parts of the apple to oxidize simultaneously, rapidly speeding up the oxidation process.
Referencing research by M Murata on the properties of chlorogenic acid, chlorogenic acid content decreases at a decreasing rate with time, with the rate of oxidation plateauing after 30 minutes of incubation, thus the oxidation time was revised and reduced to 30 minutes.
2.2 Flowchart Methodology
Control Experiment 1 (determination of equivalence point)
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250cm3 of distilled water was measured out using a measuring cylinder
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8.8575g of chlorogenic acid powder was weighed with a electronic balance and dissolved in the distilled water to obtain 0.1 mol dm-3 of chlorogenic acid in a conical flask
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0.1 mol dm-3 NaOH solution was created by diluting 50cm3 of 2 mol dm-3 NaOH solution using the same process as titration
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50cm3 of 0.1 mol dm-3 of chlorogenic acid was titrated against 0.1 mol dm-3 NaOH solutions to determine the equivalence point.
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This point may be used as a reference point to indicate when the reaction has completed
Control Experiment 2 (determination CGA present in gala apples)
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Follow the steps 1-7 of the titration procedures
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Filter out 600cm3 of apple juice and titrate it against standardized NaOH
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The end point would be the same as the titration procedure.
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This would serve to indicate the initial amount of chlorogenic acid present in gala red apples.
Necessary safety precautions were taken throughout experimentation. Goggles and a lab coat were worn throughout the duration of experimentation for safety purposes.
3. Results
3.1 Qualitative Observations
Unoxidized apple juice
The apple juice was light yellow in colour. There were no noticeable chunks of apple in the juice, symbolizing that the apples had be properly blended into pulp. Upon filtering out the apple pulp, the apple juice turned light orange in colour.
Oxidized apple juice
After being exposed to the atmospheric air at 25 oC for 30 minutes, the apple juice gradually turned brown in colour. This is due to the oxidation of chlorogenic acid to melanin by the enzyme polyphenol oxidase. The brown coloration can be attributed to the increase in concentration of melanin in the apple juice.
1.3 Acid-base reaction
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3.2 Raw data collected for titration
After 30 minutes, the apple juice at 25°C was tested using a pH probe to ensure that there were no further ph changes occurring. Since Chlorogenic acid is acidic, its oxidation into melanin would lead to an increase in ph as the apple juice becomes less acidic due to the decrease in concentration of H+ ions. Hence the pH data reveals that after 30 minutes, most if not all the chlorogenic acid had been oxidized into melanin.
3.3 Data processing for titration
pH calculation
pH calculation
The initial and final pH substantiates the results collected during titration as well as the qualitative observations. The equivalence point corresponds to the theoretical point when equivalent amounts of acid and base have been reacted. Hence the pH derived at this point is taken to be the reference point for when reaction between chlorogenic acid and NaOH have ended for titration.
3.4 Processed data for titration
3.5 Calibration Curve
The final data point was taken to be an outlier hence it was omitted from the plotting of the maximum and minimum trend lines.
The beer-lambert equation states that:
Abs=εlc
Where Abs is absorbance measured in Arbitrary Units (AU), L is the path length and c is the concentration of chlorogenic acid.
Absorbance is directly related to concentration since the molar absorptivity is a constant for any given compound. By plotting an absorbance against concentration graph, the gradient of the curve obtained would be the molar absorptivity of chlorogenic acid. Hence referring to figure 10, the molar absorptivity of chlorogenic acid is 193.05 M-1cm3. Furthermore, the high R2 value indicates there are negligible differences between values of replicates, showing that there is a strong linear correlation of the data collected.
3.6 Raw data for Spectroscopy
3.7 Processed data for UV Vis spectroscopy
3.8 Comparison between UV Visible spectroscopy and titration
4. Discussion
Effect of temperature on enzymic activity
Polyphenol oxidases are biological catalysts that speed up the rate of reaction by offering an alternative pathway with a lower activation energy such that a greater portion of chlorogenic acid molecules have energy greater than or equal to the activation energy. A possible alternative reaction proposed by WS pierpoint on the oxidation of chlorogenic acid shows the formation chlorogenic acid quinones (CGA-Q) with the uptake of oxygen as the intermediate.
Quinones are formed through the uptake of half a mole of oxygen molecules by chlorogenic acid, losing 2 moles of H+ ions in the process, this reaction is catalyzed by polyphenol oxidase. Following this Non-enzymic reactions convert half of the CGA-Q into hydroxychlorogenic acid regenerating the rest back to chlorogenic acid and producing hydroxyquinones. The regenerated chlorogenic acid are fed back into the cycle to be converted into more quinones while the hydroxyquinones are polymerized through further reactions with amino acids to produce melanin resulting in the darkening of the apple:
For the enzymic portion of the reaction, decreasing the temperature causes the polyphenol oxidase to become inactive. Polyphenol oxidase and chlorogenic acid lose kinetic energy, decreasing the number of effective collisions. Hence the likelihood of chlorogenic acid forming a complex with polyphenol oxidase in the correct orientation with sufficient energy decreases. Therefore, at room temperature the average rate of oxidation is at its highest due to the increased enzymic activity at 3.33 X 10-6 mol-1 dm-3 min-1 from titration results and 4.6 X 10-6 mol-1 dm-3 min-1 from Spectrophotometry.
Equilibrium of reaction
As shown in the oxidation mechanism above, the oxidation of CGA is reversible as non-enzymic reactions convert a portion of the intermediate, CGA-Q, back to chlorogenic acid. Overall reaction:
2[C16H18O9]+ O2+H2O⇌ C16H18O10+ C16H16O9
As such the equilibrium constant of this reaction can be denoted as
Kc= [C16H18O10] [C16H16O9] / [C16H18O9]2[O2][H2O]
Following Le Chateliers Principle, a decrease in the temperature removes heat energy from the equilibrium mixture, hence the position of equilibrium shifts to favor the exothermic reaction and produce heat energy to maintain the temperature.
Referring to table 5, a decrease in temperature from 25 to 0 oC causes the concentration of chlorogenic acid in apples to increase from 0.0001258 to 0.0001885 mol contained within a single apple. As such the data shows that the backward reaction is exothermic, hence the position of the equilibrium shifts to the left to favor the backward reaction. Therefore lower temperatures also decrease the average rate of oxidation by causing a leftward shift in the equilibrium to favor the regeneration of CGA from the oxidation mechanism.
4.2 Analysis of results
Referencing figures 9 and 11, the average rate of oxidation of CGA increases as temperature decreases, leading to a negative correlation between concentration of CGA and temperature, corroborating with my initial hypotheses. From the graph in figure 11, there is an apparent deviation in average oxidative rates as the results collected from UV Vis Spectroscopy is observed to show an exponential trend line whilst results from titration revealed a more linear trend line. This can be explained by the formation of intermediates such as chlorogenic acid quinones during oxidation of CGA, as shown in a study done by W.S pierpoint (1966) :
In apples chlorogenic acid quinone (CGA Q) reacts further to regenerate CGA as well as produce hydrogen peroxide, H2O2. The production of H2O2 could have reacted with NaOH:
2 NaOH (aq) + H2O2 (aq) →Na2O2 (aq) + 2 H2O (l)
Furthermore, according to a research paper on the properties of CGA Q , equal amounts of H2O2 and CGA were formed in the regeneration of CGA. The formation of peroxidase has a significant impact on the enzymatic activity of polyphenol oxidase as research done by Richard-Forget, F. C., and Gauillard, F. A. revealed that the presence of peroxidase exponentiates the degradation of CGA into melanin. Therefore at higher temperatures, the increased function of polyphenol oxidase indicates an increase concentration of CGA Q and consequently, peroxidase. Therefore, the average rate of oxidation of CGA is seen to be exponentially faster due to the higher concentration of peroxidase as seen by the upward concavity of the UV Vis Spectrophotometry graph. The reason why titration values still indicated a linear trend line could be because of the reaction of NaOH with hydrogen peroxide as stated earlier. The bleaching effect of hydrogen peroxide on the brown colour of melanin was taken to be negligible as the time frame in which the melanin was exposed to hydrogen peroxide prior to spectrophotometry was short enough such that there was negligible impact on its colour.
Another possible reasoning behind the higher results for titration is the presence of other forms of phenolic acids such as caffeic acid and quinic acid which are also soluble in ethanol. During experimentation however, the presence of these acids were considered to be negligible since chlorogenic acid is the primary phenolic acid found in apples, accounting for more than 85% of all phenolic acids according the paper on Composition of phenolic acids content in apple by Soares, M. C., and Ribeiro, É. T.
Analyzing the Spectrophotometry graph, the rate of change in average oxidation rate between 0oC and 10oC was significantly smaller compared to other temperatures, represented by the relatively lower gradient between those two points. This indicates that there was negligible change in activity of polyphenol oxidase between these temperatures, suggesting that enzymic activity is inhibited at 10oC and below. This corroborates with sources such as Food-info.net which states that the temperature at which polyphenol oxidase is inhibited is at 7 oC. Furthermore, the calibration line obtained had relatively small error bars and presented a good fit to the linear trend line. This adds impetus to the accuracy results collected for Spectrophotometry as it allows for fewer errors.
Furthermore the data collected from both titration and spectrophotometry showed a reasonably low standard of deviation of approximately 0.05 for titration and 0.015 for UV Vis spectroscopy. A low percentage uncertainty was also calculated, with 4% for UV Vis spectroscopy and 1.7% for titration which adds impetus to the reliability, consistency and accuracy of the results collected. The low standard deviation and percentage uncertainty are reflective of the relatively small random errors encountered during preparation and the replicability of the results and experiment. Comparing the results collected for Titration and Spectrophotometry, it can be concluded that UV Vis Spectrophotometry is the more reliable method for quantifying the chlorogenic acid content in apples. This is because the standard deviation of results for UV Vis spectrophotometry was consistently lower for all data sets compared to titration values, indicative that the results were closer to the mean trend line. This shows that the methodology applied contains fewer systematic and random errors compared to titration. Thus, the results and trend line obtained from spectrophotometry more accurately represents the oxidation pathway of chlorogenic acid as compared to titration.
5. Evaluation
5.1 Strengths & limitations
The methodology utilizes the conventional liquid-liquid extraction of polyphenols using an alcohol. This is the simplest method of extraction as highly polar phenolic acids such as benzoic acid are only partially soluble in alcohol, hence are not extracted. This reduces the uncertainty caused by the presence of other phenolic acids to the absorbance readings. Hence this isolates chlorogenic acid from other compounds in apples such as malic and ascorbic acid, thereby reducing the standard of error. This is supported by the relatively high R2 values of 0.9941 and 0.9915 respectively which reveals that the methodology applied is reliable and is representative of the actual oxidative rates.
Furthermore, the results of this investigation would be commercially beneficial to the food industry as it provides firms insight to the ideal temperatures to store their products to inhibit enzymic activity, keeping them fresh. This extends shelf life and increases profits.
The slow oxidation of apples in the open air prior to data collection is a limitation as some time is needed for the preparation of the apple juice for titration. This unrecorded time frame is not taken in account as it is seen as negligible relative to the incubation time of 30 minutes. However, CGA is still being oxidized during this period, hence the results collected for the Number of mole of chlorogenic acid present after oxidation would be lower than expected. One possible improvement on this would be to use lids to cover the beakers and flasks to limit the amount of oxygen available and slow down the oxidation of chlorogenic acid prior to incubation.
Another limiting factor would be the temperature of the apple juice. The timer for incubation started when the apple juice was place in the water basin and not when the temperature of the apple juice reached the desired temperature. This was done because a significant amount of chlorogenic acid would have oxidized in the time required for the apple juice to cool to the desired temperature. Thus the rate of oxidation of chlorogenic acid initially would be higher than expected as the temperature of the apple juice is higher at the beginning.
5.2 Conclusion
In conclusion, the initial hypothesis that concentration of chlorogenic acid decreases as temperature increases proved to be true. The effect of temperature on the enzymic activity of polyphenol oxidase impacts the production of intermediates and other compounds hence affecting the production of melanin which served as an indicator of oxidation for this investigation.
The average rate of oxidation could be seen to increase as temperature increased due to increased enzymic activity. The relatively small drop in oxidative rate between the temperatures 10oC and 0oC indicate that the ideal temperature to store fruit would be within this region.
Naturally, some questions arised that remains unanswered over the course of this investigation such as:
Were some enzymes lost when the apple pulp was removed from apple juice?
To what extent does the surface area in contact with atmospheric air affect rate of oxidation?
Are there possibly other compounds present in apple juice that affect the average rate of oxidation?
5.3 Extensions
A possible extension would be to investigate the chlorogenic acid content against time to obtain how the rate of chlorogenic acid oxidation changes with time for a respective temperature. This has applications to the food industry as it provides insight as to how long a particular food can remain fresh at a particular temperature.
Another extension would be to quantify the relative levels of chlorogenic acid in each portion of the apple ie the peel, flesh, stalk and seeds, allowing us to examine the nutritional benefits of each part of the apple.
6. Appendix
Full Methodology
Titration of NaOH against apple juice
The apples were skinned before they were cut into small cubes. An electronic balanced was used to weigh out approximately 400g of apple cubes. The apples cubes were then added to a blended and mixed with 240 cm3 of ethanol. After blending continuously for 1 minute, no apple noticeable apple chunks can be seen. The 600cm3 of ethanol-apple solution “apple juice” was filtered out using a strainer. The apple juice was left in a basin of cold water at 0 °C for 30 minutes. Ice cubes were added into the basin at regular intervals to control the temperature of the water, keeping it consistent. After 30 minutes had elapsed, the apple juice was removed from the water bath and 50cm3 of apple juice was measured and poured into a conical flask using a measuring cylinder. 50cm3 of apple juice was titrated against the standardized 0.02mol/dm3 NaOH solution. 5 drops universal indicator was used to show the colour change as the acidic apple juice is neutralized by the base NaOH. The end point will be when the solution turns dark green in colour. When the end point is reached, a pH probe was used to measure the pH of the solution. This process was repeated with the varying temperatures of apples (0, 5, 10, 20, and 25) °C.
Plotting calibration curve
3.543g of chlorogenic acid powder was added to 100 cm3 of ethanol in a beaker to produce a solution of 0.1 concentrations labeling it as solution A. Using a 20 cm3 pipette, 40 cm3 of A is transferred to a 100cm3 volumetric flask and diluted to 50cm3 with ethanol to produce a 0.08 solution labeled as B. This dilution process is repeated to obtain solutions of 0.06, 0.04, 0.02 and 0.01 concentrations, labeling them in alphabetical order.
Measuring absorbance of chlorogenic acid
Apple juice was obtained using the same method for titration; however the ethanol was replaced by water. 9cm3 of ethanol was measured out using a 10cm3 measuring cylinder. 1cm3 of apple juice was measured out using another 10cm3 measuring cylinder. The two solution were mixed together to obtain a 9:1 ratio of ethanol-apple juice solution. Continuous shaking for 1 minute to allows the chlorogenic acid to dissolve into the ethanol as chlorogenic acid is a phenolic compound and an organic acid. This separates chlorogenic acid from the other compounds. This was repeated to obtain 5 replicates of 10cm3 ethanol-apple juice solution. They were centrifuged at 40 X 103 RPM for 15 minutes. These were then added to cuvettes and using for Uv-Vis Spectroscopy. The entire process was repeated to obtain 5 sets of data for apple juice that has been oxidized at varying temperatures (0, 10, 15, 20, and 25) oC
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