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Lindsey TongeAnalytical ChemistryProf HuppDecember 7, 2009Lab Report for Lab #9The Separation of the Components in Tea using High Performance Liquid ChromatographyAbstract:The presence of Epigallocatechin Gallate (EGCG) and caffeine in tea was determined using reversed-phase liquid chromatography using a mixture of water and methanol as the mobile phase and a C18 column as the stationary phase. The caffeine and EGCG were detected at 273 nm by UV-absorption. It was found that the EGCG eluted first, followed by the caffeine since reversed-phase liquid chromatography was used. The elution order proved that Bigelow’s Plantation Mint Tea contained both EGCG and caffeine. Introduction:It is commonly known that green tea promotes healthy bodies and prevents cancer and other malicious diseases. These benefits of green tea are attributed to its Epigallocatechin Gallate (EGCG), an antioxidant. Thus, many tea companies add “antioxidants” to their tea for health purposes. However, often the advertising on the box is wrong and there are not actually antioxidants in the infused teas. The purpose of this lab was to determine if there was in fact EGCG in Bigelow’s Plantation Mint Tea. Another component of tea that has been frequently researched is caffeine.,, Since caffeine is not always beneficial, many drinks such as tea, coffee, and soda are made both with and without caffeine. In this lab, the presence of caffeine in Bigelow’s Plantation Mint Tea was studied. Bigelow’s Plantation Mint Tea claimed to be a caffeinated tea, while most teas, like green tea claim to be decaffeinated. Therefore, often the presence of caffeine in decaffeinated teas is studied. Since the study of EGCG and caffeine in tea is so popular, many studies have been done using multiple different methods of analysis. The most popular methods of analysis on the tea components are infrared spectroscopy3 and liquid chromatography2,,. Liquid chromatography (LC), which was used in this lab, is popular for studying the components of tea, especially caffeine, because LC allows for simultaneous detection for all of the compounds, because the detection limits and recovery rates are good, and because the data generally creates good linear calibration curves.2 Since LC is such a popular and successful method of analysis it seems like a good method to analyze EGCG and caffeine. The optimal wavelength for both EGCG and caffeine was 273 nm. One of the reasons for this is because both samples have large peaks there. Caffeine has peaks at both 210 nm and 273 nm5 as does EGCG. Though both the EGCG and caffeine absorb well at 210 nm and 190 nm (most compounds absorb well at both 210 nm and 190 nm), 273 nm was chosen because at that wavelength the interference from the noise and other compounds is not as great.8 Since most things absorb at 190 nm and 210 nm, the spectra would be crowded and contain so many peaks that the resolution would be very bad. It turned out that there was indeed a great deal of caffeine in the caffeinated tea. Similarly, there was also a large peak associated with the EGCG, indicating that it too was present in the tea. In this case, the company was correct in advertising that this particular tea was caffeinated and infused with antioxidants. The methods section will outline the exact process used to determine the presence of EGCG and caffeine in the tea. The results and discussion section will provide the explanation and rationale behind the results obtained in the experiment. Finally the conclusion section will provide a quick summary of the experiment and propose a future direction for this experiment. Lab Partners: Mike Mandrioli and Brain BlumMethods:A Prominence Shiadzu LC-20AD pump and a Shimadzu Prominence Diode Array SPD: M20A were used. The column (stationary phase) was a nonpolar C18 column (EPS C18 100A, 3?, 7 mm ID, and 33 mm in length). The wavelength was set at 273 nm and the optimum flow rate was set as 1.5 mL/min. The run time was set for 12 minutes.The solvent (mobile phase) was made of methanol (Fisher Scientific, A452-4), distilled water, and 1% trifluoroacetic acid (from Sigma) which stabilized the EGCG. The percentage of water and the methanol was varied to change the mobile phase composition. First, we used 30% methanol and 70% water, then we used 100% methanol, then 40% methanol and finally 20% methanol.A regular store bought tea (Bigelow Plantation Mint) was brewed in 225 mL of distilled water on a hot plate in lab. The tea was filtered using a 0.45 micron glass filter. The mobile phase was set at 30% methanol and 70% water. A combination of 125 ppm EGCG (Epigallocatechin Gallate from Green Tea, minimum 80% HPLC from Sigma, 103K1121), the nonretained marker sodium nitrate (Sigma, minimum 99%, S5506-250g, 105K0681), and 125 ppm caffeine (Aldrich Chemical Company, 99% caffeine, C5-3) was used to determine the elution order. Every time the mobile phase was changed, the instrument was left to sit for 10 minutes so that the system could equilibrate. A series of isocratic elution (100% methanol, 40% methanol, and 20% methanol) were run to determine the effects of the mobile phase on separation. Each of the three components (EGCG, NaNO3, and caffeine) were injected in an equal mixture (1:1:1 ratio) through the 20?L injection loop. The retention times were collected and recorded. Then the mobile phase was changed to 40% methanol. The same process was repeated. Finally, the mobile phase was changed to 20% methanol and the process was repeated.Then the mobile phase was changed so that the separations were collected using gradient elutions. The first gradient was set so that the percentage of the methanol changed from 25% to 35% over the first 5 minutes. After that, the percentage was held at 35% until the run was over. The three components were then injected in an equal mixture (with a 1:1:1 ratio) and their retention times were collected and recorded. The second gradient elution was set so that the percentage was held steady for the first 3 minutes at 25% methanol. Then the percentage methanol was increased from 25% to 35% over the next two minutes (until the fifth minute). Then the percentage was held steady at 35% until the run was over. The plantation mint tea was analyzed using the second gradient. Then the caffeine and EGCG were injected separately into the second gradient elution to determine their individual, unaffected retention times in the particular system. Results and Discussion:The purpose of this lab was to efficiently separate a complex mixture of tea so that its components could be identified. First, the elution order of the EGCG and caffeine was determined. Table 1 shows that the EGCG eluted before the caffeine did. Therefore, the caffeine must interact (temporarily bind) more with the stationary phase since the interactions would have slowed down its elution, thus resulting in a longer retention time. On the other hand, the EGCG, though larger, must have had fewer interactions with the stationary phase. The elution order identifies the EGCG as the second major peak (the nonretained marker being the first peak) and the caffeine was the third major peak. This makes sense when the polarity of both compounds are considered. EGCG has many very polar OH bonds (Figure 1). Caffeine, on the other hand, has no OH bonds and only a few weakly polar C-N bonds (Figure 2). Thus, EGCG is more polar than the caffeine is. This makes sense since this particular system is a reversed phase system with a nonpolar stationary phase (C18) and a polar mobile phase (water and methanol). In a reversed phase system, the first compound to elute is the most polar and the last is the least polar. Therefore, it makes sense that the more polar EGCG elutes first. Retention Table 1: Data from all separable elutionsfactors (k) were also calculated for each compound as a comparison source. (Calculation1).CompoundTr (min) 30% methanolk 30% methanolTr (min) 20% methanolk 20% methanolTr(min) 1st gradientk 1st gradient Tr(min) 2nd gradientk 2nd gradientNaNO31.184NA1.184NA1.184NA1.173NAEGCG1.9090.61231.8870.59382.4641.08112.4751.1010Caffeine3.2111.71203.1791.68503.8832.27964.2452.6189333375023018753238502235200 Figure 1: EGCGFigure 2: CaffeineCalculation 1: Retention Factork = tr– tm tmk = 1.909-1.184 / 1.184k = 0.6123The next thing done to help determine the best separation of the three compounds was to change the mobile phase. First different isocratic elutions were attempted. The first mobile phase composition that was tried was a mobile phase that was 100% methanol. The three peaks did not separate at all and the retention time for the large peak containing all three compounds was 1.419 min. The second mobile phase had a composition of 40% methanol and 60% water. While the caffeine did separate from the other two compounds, NaNO3 and EGCG did not separate but appeared as a large peak on the chromatogram. The retention times for the peaks were 1.355 min for the NaNO3/EGCG complex and 2.133 min for caffeine. The third mobile phase tried was 20% methanol and 80% water. Table 1 shows how all three peaks had good separation. Thus, it was determined that 20% methanol had the best separation with a small completion time (less than 4 minutes).The nonretained marker for the system, NaNO3, was a good nonretained marker for this particular system. At 30% methanol the NaNO3 had a retention time of 1.184 minutes as it did at 20% methanol. At any methanol percentage greater than 30%, the NaNO3 and the EGCG peaks did not separate enough to get a distinct retention time for the nonretained marker. However, at both 30% and 20% the NaNO3 gave the same retention time. Thus, the NaNO3 is a good and consistent nonretained marker. While the isocratic elutions produced fairly decent separations, the separation of the EGCG and the NaNO3 was not as good as it could be. However, decreasing the amount of methanol further would make the retention time of caffeine even greater. In an attempt to be time conscious and have good separations, the next separation was done using a gradient elution. Since the mobile phases that were 20% methanol and 30% methanol gave good separations, the gradient elution was started at 25% methanol. Table 1 shows that the spectra had good resolution and baseline separationThe resolution of the second gradient was pretty good since there was excellent baseline separation between all three peaks. Table 1 shows that the retention times for all three peaks were well spaced resulting in the best separation. As such, it was decided that this was the best separation for caffeine, EGCG, and NaNO3.It was decided that though the second gradient elution had slightly longer retention times, the better of the two separations was obtained with the second gradient elution. Therefore, it was decided that the tea would be separated using the second gradient elution. The resolution of the tea spectra was not all that great though. The separation of the three peaks in the tea was pretty bad, probably because the tea did not simply contain EGCG and caffeine. There were a lot of other compounds in the tea that appeared in the chromatogram (Figure 3) making the chromatogram difficult to decipher. To be sure of their retention times in the second gradient, the caffeine (Figure 4) and the EGCG (Figure 5) were run and their retention times were recorded as 4.160 min and 2.432 min respectively. The caffeine peak was easy to find and identify in the tea chromatogram but the EGCG peak was harder to identify. The first peak was so large that might have contained EGCG since with the second gradient its retention time was 2.432 min and there is a peak there. However, it is hard to concretely say that the tea contains EGCG because the peak is so large and unseparated. Nonetheless, it is probably safe to say that there is some EGCG in there since there is a peak at the right place. Thus, the tea contained both caffeine and EGCG. This was expected for the tea because the tea did not claim to be decaffeinated tea and it also claimed to have antioxidants. To determine the relative concentration of EGCG and caffeine in the tea, the relative peak heights were looked at. Table 2 shows the relative peak heights for EGCG, caffeine and their correlating peaks in the components of tea spectrum. Calculation 2 shows how the ratio of the peak height to the known concentration (125 ppm) was used to calculate the relative concentration of either EGCG or caffeine in the tea. It was determined that the concentration of EGCG in tea was approximately 86 ppm and the concentration of caffeine in the tea was approximately 133 ppm. Table 2: Peak HeightsCompoundPeak Height (mAu)Calculation 2: Relative Concentration of EGCG or Caffeine in Tea 90 = 62125 ppm xx = 86 ppmConcentrationEGCG by itself90 125 ppmCaffeine by itself165125 ppm“EGCG” peak in tea chromatogram62≈ 86 ppm“Caffeine” peak in tea chromatogram175 ≈ 133 ppm As the mobile phase changes, so does the retention time and factor. The retention time when the percentage of methanol was high, was very small even for caffeine. The one large peak (100% methanol) had a retention time of 1.419 min. As the percentage of methanol was lowered to 40%, the separation of the peaks got better and two peaks appeared with retention times of 1.355 min (EGCG/NaNO3) and 2.133 min (caffeine). Without the caffeine contributing to the first peak, the first peak had a slightly smaller retention time. However, the general trend is that as the percentage of the methanol went down, the retention time went up, as Table 1 shows. This is because the caffeine and EGCG react more with water than with methanol. This is because methanol is less polar than water. Therefore, as the amount of methanol is decreased, the polarity of the mobile phase goes up and thus, the retention times increased. This can be seen most clearly in the caffeine peak as the methanol percentage was changed from 40% to 30%. The caffeine’s retention time at 40% methanol was 2.133 min while the retention time at 30% methanol was 3.211 min. The smaller the amount of methanol, the better the separation of the three peaks. However, as the percentage of methanol got smaller, the retention time continued to get longer. If a separation was attempted with 10% methanol, the retention time of caffeine would be impractically long. Therefore, the percentage of methanol was never lowered beyond 20% methanol. For the separations using the created gradient elutions, the same pattern applies. That is, that as the percentage of methanol decreases, the retention time increases. Since the first gradient elution was programmed so that the percentage of methanol was increased from 25% to 35% over the first five minutes, the percentage of methanol at the time of the EGCG peak was probably around 29%. The retention time of that peak was 2.464 min. On the other hand, the second gradient elution was programmed to stay consistent for the first three minutes at 25% methanol. The retention time at that peak was 2.475 min. Thus, the slightly lower methanol percentage resulted in a slightly higher retention time. Similarly, the retention time of the caffeine was also increased from 3.883 min to 4.245 min. Thus, with both the isocratic elutions and the gradient elutions, the pattern was that as the methanol percentage decreased, the retention time increased. Conclusions:In conclusion, it was determined that the best method of separation for this particular instrument and system was the developed second gradient. The components of Bigelow’s Plantation Mint Tea were separated. The chromatogram also contained peaks from other compounds but it is obvious that the tea contained both EGCG and caffeine. To develop better separations for the components of tea, it would be interesting to look at the interactions of caffeine and EGCG with other common components of tea (such as other antioxidants) and see how that would affect their separation. ................
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