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The aim of this experiment was to determine the caffeine concentration in several commercially sold beverages such as Coca-Cola, Pepsi as well as Monster using High Performance Liquid Chromatography (H.P.L.C). High Liquid Performance Chromatography is a type of column chromatography. It uses differential migration techniques to separate various compounds, as well as, it’s used to find the concentration of said compounds. The objective of this experiment was accomplished by performing various steps. To start with, a stock solution of concentration 0,20mg/ml was prepared by using deionized water.
Consequently, standard concentrations of 0.01, 0.05, 0.10, 0.15, 0.20mg/ml were prepared in a 10ml solution.
For the Coca-Cola, 5ml was pipetted into a volumetric flask and a dropper was used to top up with the deionized water. Likewise, 5ml of Pepsi was pipetted into a different volumetric flask and a dropper was used to top up with the deionized water. 2.5ml of Monster was pipetted into another volumetric flask and was topped up with deionized water.
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For these samples, 2ml vials were filled with each standard and covered with screw caps. The samples were then loaded into the auto-sampler tray from the lowest to highest concentrations for standards and then in order of Coca-Cola, Pepsi and Monster for samples.
The stated value of caffeine concentration of Coca-Cola in a 12oz can was 34mg/ml, whereas the calculated value was 28.61mg/ml. Also, it was observed that the stated value of caffeine concentration of Pepsi in a 12oz can was 38mg/ml whilst the calculated value was 34,44mg/ml.
Moreover, the stated caffeine concentration value for Red Bull in a 16oz can was 160mg/ml while the calculated value was 156.98mg/ml. Based on these results, it indicates that the products had lower experimental values of caffeine concentration when compared to the respective stated values of the caffeine concentration in the products.
There are two known types of chromatography: reverse-phase and normal-phase. In reverse-phase, the stationary phase is non-polar (hydrophobic) while the mobile phase is polar (hydrophilic). This suggests that the hydrophobic molecule will be eluted last whereas the hydrophilic molecule will elute first. However, in normal-phase chromatography, the stationary phase is hydrophilic and the mobile phase is hydrophobic. This indicates that the hydrophilic molecule will be eluted last and the hydrophobic molecule will be eluted first. Reverse-phase chromatography was used in this experiment and furthermore, as caffeine is implied as being hydrophobic, it will be eluted last.
With respect to precautions, it would be noted to change the pipette tips when transferring samples to prevent contamination of samples. Additionally, other precautions would’ve been to decarbonize the samples prior to the start of the experiment as the carbon dioxide would affect the results as well as the volumetric flask should’ve been washed to avoid contamination. Possible sources of errors would be the loss of the solutions by splashing when using the pipette as well as adding excess deionized water to volumetric flasks when diluting the samples which would lead to incorrect concentration values being acquired.
The volume required for standard solutions to achieve a 100ml stock solution of various concentrations is presented in Table 1.0.
| Concentration of standard (mg/ml) | Amount of Stock Solution (ml) | Amount of Deionized Water (ml) |
|---|---|---|
| 0.01 | 0.50 | 9.50 |
| 0.05 | 2.50 | 7.50 |
| 0.10 | 5.00 | 5.00 |
| 0.15 | 7.50 | 2.50 |
| 0.20 | 10.00 | 0.00 |
The peak area values for caffeine standards and beverage samples were recorded, as shown in Table 1.1 and 1.2, respectively.
Table 1.1: Area Peak Values of Caffeine Standards
| Concentration of caffeine standard (mg/ml) | Average Peak Area/mAU*s |
|---|---|
| 0.01 | 127.78053 |
| 0.05 | 645.26920 |
| 0.10 | 1242.19660 |
| 0.15 | 1888.19770 |
| 0.20 | 2615.31958 |
Table 1.2: Area Peak Values for Beverage Samples
| Beverage Sample | Average Peak Area |
|---|---|
| Coca-Cola | 504.04105 |
| Pepsi | 609.80650 |
| Monster | 1055.57373 |
The calibration curve, shown in Figure 1, was derived from the peak area versus caffeine concentration, with the equation y=12961x – 18.272 and an R² value of 0.9987. Using this curve, caffeine concentration in beverage samples was calculated.
Table 1.3: Calculated Concentration of Caffeine in Samples
| Sample | Calculated Caffeine Concentration (mg/ml) |
|---|---|
| Coca-Cola | 0.0806 |
| Pepsi | 0.0970 |
| Monster | 0.3316 |
The calculated caffeine concentrations were compared to the stated values, revealing that the experimental values were lower. This discrepancy could be attributed to potential experimental errors such as solution loss or excessive dilution. To mitigate these issues, precautions like changing pipette tips and decarbonizing samples were suggested.
The experiment successfully determined the caffeine concentration in commercial beverages using H.P.L.C, illustrating the method's effectiveness in analyzing compound concentrations. Despite the observed discrepancies, the findings offer valuable insights into the caffeine content of popular beverages, underscoring the importance of precise measurement and preparation techniques in chemical analysis.
Determining Beverage Caffeine Content Using HPLC. (2024, Feb 17). Retrieved from https://studymoose.com/document/determining-beverage-caffeine-content-using-hplc
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