Lindsey Tonge
Analytical Chemistry
Prof Hupp
December 7, 2009
Lab Report for Lab #9
The Separation of the Components in Tea using High Performance Liquid Chromatography
Abstract:
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.1 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.2,3,4 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
1
2
Eckert, R.; Crish, J.; Efimova, T.; Balasubramanian, S. Biochem. Pharmacology. 2004, 68, 6.
Srdjenovic, B.; Djordjevic-Millic, V.; Grujic, N.; Injac, R.; Lepojevic, Z. Journal of Chrom. Sci. 2008, 46,
2.
3
4
Sinija, V. R.; Mishra, H. N. LWT--Food Sci. & Tech. 2009, 42, 5.
Graham, A. Q.; Hathaway, C.; Geisberg, M. S. U.S. (2009). 2009, US 7569396, B1 20090804.
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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 chromatography2,5,6. 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 nm7 and
273 nm5 as does EGCG.8 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
5
Risner, C.H. Journal of Chrom. Sci. 2008, 46, 10.
Bispo, M.S.; Veloso, M. C. C.; Pinheiro, H. L. C.; De Oliveira, R. F. S.; Reis, J. O. N.; De Andrade, J. B.
Journal of Chrom. Sci. 2002, 40, 1.
7
Injac, R.; Srdjenovic, B.; Prijatelj, M.; Boskovic, M.; Karljikovic-Rajic, K.; Strukelj, B. Journal of
Chrom. Sci. 2008, 46, 2.
8
Saito, S. T.; Welzel, A.; Suyenaga, E. S.;Bueno, F. Campinas, 2006, 26, 2.
6
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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 Blum
Methods:
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,
3µ, 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,
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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 20µL 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
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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
factors (k) were also calculated for each compound as a comparison source. (Calculation1).
Table 1: Data from all separable elutions
Compound Tr (min) k 30%
Tr (min) k 20%
Tr(min) k 1st
Tr(min) k 2nd
st
30%
methanol 20%
methanol 1
gradient 2nd
gradient
methanol
methanol
gradient
gradient
NaNO3
1.184
NA
1.184
NA
1.184
NA
1.173
NA
EGCG
1.909
0.6123
1.887
0.5938
2.464
1.0811
2.475
1.1010
Caffeine
3.211
1.7120
3.179
1.6850
3.883
2.2796
4.245
2.6189
Figure 1: EGCG
Figure 2: Caffeine
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Calculation 1: Retention Factor
k = tr– tm
tm
k = 1.909-1.184 / 1.184
k = 0.6123
The 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
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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 separation
The 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
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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 Heights
Compound
Peak Height (mAu)
Concentration
EGCG by itself
90
125 ppm
Caffeine by itself
165
125 ppm
Calculation 2: Relative
Concentration of EGCG or
Caffeine in Tea
90 = 62
125 ppm x
x = 86 ppm
“EGCG” peak in
62
≈ 86 ppm
tea chromatogram
“Caffeine” peak in 175
≈ 133 ppm
tea chromatogram
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
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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
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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.