HPLC/LC-MS Laboratory
High Pressure Liquid Chromatography and Liquid Chromatography Mass Spectrometry
Purpose:
The objective of this lab was to become familiar with the HPLC and LC-MS by running
standard caffeine solutions and by running and analyzing the caffeine content in
caffeinated and decaffeinated teas.
Introduction:
Liquid chromatography is a method used to separate the components of a compound
that has been dissolved in a solvent. The sample is run through a liquid mobile phase
and a liquid or solid stationary phase in a pressurized column. The LC-MS uses a mass
spectrometer detector in order to separate the components by their mass to charge
ratio in order to analyze what is in the sample. The HPLC can be used for quantitative
analysis of a sample, showing the amount of substance in a sample.
Day 1: Run standard samples of caffeine through the LC-MS for comparison to the
samples and to gain an understanding of the instrument.
Day 2: Run caffeine standards through the HPLC and run tea samples through HPLC and
LC-MS.
(Note: IC was also supposed to be included in this laboratory, but due to time
constraints we were only able to run HPLC and LCMS)
Method:
Caffeine standards were made by dissolving caffeine in distilled water. Concentrations
of standards were 50ppm, 100ppm, 200ppm, 300ppm, 400ppm, and 500ppm.
Standards were run on HPLC and LCMS to create a calibration curve.
Two tea samples were made (Bigelow English Teatime, caffeinated and decaffeinated)
by boiling 150mL of distilled water for each tea bag, and letting the tea bag steep for
four minutes with regular stirring.
Tea samples were run through HPLC and LCMS for analysis of caffeine concentration.
All data and spectra are located in supplementary folder.
HPLC Data
Concentration
50 ppm
200 ppm
300 ppm
400 ppm
500 ppm
Caffeinated Tea
Decaffeinated Tea
Area
131119
2500
81616
199458
331786
31561624
46924160
Concentration vs. Area
y = 497.85x + 4918.5
350000
300000
Area
250000
200000
150000
100000
50000
0
0
100
200
300
400
500
600
Concentration (ppm)
Calculations:
Caffeinated
𝑦 = 497.85𝑥 + 4918.5
31561624 = 497.85𝑥 + 4918.5
𝑥 = 63385.97𝑝𝑝𝑚
Decaffeinated:
𝑦 = 497.85𝑥 + 4918.5
46924160 = 497.85𝑥 + 4918.5
𝑥 = 94243.73𝑝𝑝𝑚
LCMS Data
Concentration
50 ppm
100 ppm
200 ppm
300 ppm
400 ppm
500 ppm
Caffeinated Tea
Decaffeinated Tea
Intensity, cps
8.2e6
7.6e6
6.6e6
6.3e6
6.3e6
6.1e6
1.3e6
2.4e6
Concentration vs. Intensity
y = -4389x + 8E+06
9.00E+06
8.00E+06
Intensity (cps)
7.00E+06
6.00E+06
5.00E+06
4.00E+06
3.00E+06
2.00E+06
1.00E+06
0.00E+00
0
100
200
300
400
Concentration (ppm)
500
600
Calculations:
Caffeinated
𝑦 = −4389𝑥 + 8 × 106
1.3 × 106 = −4389𝑥 + 8 × 106
𝑥 = 1526.54𝑝𝑝𝑚
Decaffeinated:
𝑦 = −4389𝑥 + 8 × 106
2.4 × 106 = −4389𝑥 + 8 × 106
𝑥 = 1275.92𝑝𝑝𝑚
Conclusion:
In this lab, we were certainly able to learn how to use the HPLC and LCMS successfully.
Both instruments were very easy to learn how to run, and they did not give us any
problems during the experiment. The results, however, were certainly not what we
expected. After using the calibration curves we created with the standards, we
calculated the concentrations of the caffeine in the tea samples, but the calculations for
the HPLC and for the LCMS were vastly different from each other. The data acquired
from the LCMS is probably the closest to accurate, but the data and calculations we got
from this experiment definitely show some error.