Expert Instrument HPLC “2011”
Introduction
Two samples of tea (one caffeinated and one decaffeinated) were run through the HPLC to test for caffeine
concentrations. A provide 1000 ppm caffeine solution was diluted to make a 5ppm sample, 10ppm sample, 25ppm sample, 50ppm
sample, and 100 ppm sample. Each of the samples with known concentrations of caffeine was run in order to produce a calibration
curve.
Procedure
1. Dilutions were made using the provided stock caffeine solution and deionized water.
2. Dilutions were made using M1V1=M2V2, 5, 10, 25, 50, and 100ppm
3. All known samples were run through the HPLC 5 times
4. A Calibration curve was generated
5. The two tea samples were also run through the HPLC 5 times
6. Concentration of Caffeine for the two tea samples were found using the Calibration curve
Data
Caffeine Standards
Trial
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
Concentration (ppm)
100
100
100
100
100
50
50
50
50
50
25
25
25
25
25
10
10
10
10
10
5
5
5
5
5
Retention Time (min)
2.480
2.477
2.480
2.470
2.460
2.450
2.450
2.443
2.457
2.433
2.440
2.447
2.443
2.457
2.457
2.480
2.477
2.470
2.467
2.460
2.460
2.453
2.450
2.453
2.450
Area
4370644
4097123
4300269
3037130
1892232
1428354
333316
1636344
706452
435524
105199
42195
67707
390157
463076
701603
339450
248543
227375
196234
102380
55353
31105
75113
25630
Height
48762
71949
52187
52187
267352
165262
183523
212042
148920
172974
117265
105553
114856
74900
73415
1957
5882
61170
15024
19623
17683
20931
24408
20507
24367
Unknown Tea Concentrations
Trial
Tea
Concentrations Found (ppm)
Retention Time (min)
Area
Height
1
2
3
4
5
1
2
3
4
5
Black Tea
Black Tea
Black Tea
Black Tea
Black Tea
Raspberry Tea
Raspberry Tea
Raspberry Tea
Raspberry Tea
Raspberry Tea
105.55
101.9
97.91
91.4
96.55
20.97
22.18
19.40
18.54
18.55
2.457
2.430
2.433
2.430
2.433
2.210
2.210
2.210
2.210
2.210
3742515
3614982
3472162
3241712
3424114
748016
790836
692500
661928
662512
793461
40494
19823
37359
33294
64974
69240
60982
62866
63537
y= 35404x + 5521
R2=.9320
Caffeine Calibration Curve
4.010 0 6
Area
3.010 0 6
2.010 0 6
1000000
0
50
-1000000
100
150
Concentration of Caffeine (ppm)
Calculations
y=3540x + 5521
y=Area
x= Concentration of caffeine
M1V1=M2V2
Conclusion
Based off our results it was fond that Moring Thunder (the caffeinated tea) had an average caffeine
concentration of 98.6ppm and Raspberry Zinger (the decaffeinated tea) had an average caffeine concentration of 19.9
ppm. While running the samples consistency of data for the heights and the areas could not obtained. This may be
based on human error with preparing the dilutions and the tea samples.
The Data Collected in 2012
Procedure can be found in lab notebook.
Caffeine Standards
Concentration (ppm)
100
50
25
10
5
Retention Time (min)
2.367
2.358
2.450
2.383
2.392
Area
2591538
717679
547818
268653
162652
Height
384869
10868
105153
41657
25898
Unknown Caffeine Concentrations
Unknowns
Concentrations Found (ppm)
Retention Time (min)
Area
Height
Coffee
Tea
252.35
5.04
2.425
2.208
6164236
41784
573576
3461
The Y axis is Area
3000000
2500000
y = 24756x - 83044
R² = 0.9351
2000000
1500000
1000000
500000
0
0
50
100
150
The X axis is Concentration.
LC/MS Data
Some data was also obtained from the LC/MS but the graphs obtained were odd and sporadic. Also, do to user error a
portion of our results were lost due to printer issues.
Calculations
y = 24756x – 83044
y=Area
x= Concentration of caffeine
M1V1=M2V2
Conclusion
Based on information gathered from manipulating the data obtained from running samples on the HPLC, it was
determined that the Coffee sample had a caffeine concentration of 252.35ppm and the Tea sample had a caffeine
concentration of 2.208ppm. If time had permitted we would have reran the LC/MS for more constant results and would
have done more runs on the HPLC. In 2011 we had 3 days with the HPLC. In 2012 we had 2 days for the HPLC,
LC/MS, and IC.