Liquid Chromatography – Mass Spectrometry and High Pressure Liquid
Chromatography
Introduction: Liquid chromatography uses a liquid (usually H2O) to push the solution containing the
analyte onto the column. Depending on the polarity of the analyte relative to water determines the
retention time of the analyte on the column. The more water soluble the analyte, the longer it is
retained on the column. The converse is true as well, the less soluble in water the analyte is, the shorter
it is retained on the column.
Purpose: We will run standard samples of varying concentrations of caffeine on the LC-MS. We will
determine the retention time based on the chromatograms. After we determine the retention time of
caffeine, we will then run our unknown samples of Iced Tea. We will run regular Rutters Iced Tea as well
as decaffeinated Iced Tea. We will then use the data we obtained from our standard samples to
determine the presence of caffeine in both the regular and decaffeinated iced tea samples. We will be
running standard samples on the HPLC. Our standards are aqueous solutions of caffeine in varying
concentrations. Once we run the standards on the HPLC, we will determine retention time in order to
determine if caffeine is present in regular Rutters Iced Tea as well as in decaffeinated Rutters Iced Tea.
Procedure:
1. Make sure everything is turned on. Turn the nitrogen tank in the gas cylinder room on. The
gauge on the left should read about 40 psi. Then open the nitrogen valve on the bench, it should
also read approximately 40 psi.
2. Open ‘Analyst Software’ icon on the desktop. Then click on hardware configuration. Click LCMS
then click Activate Profile. Click build acquisition batch. Fill in the set name (filename), select
method caffeine 2011, click add set then click add sample.
3. Select the number of samples and then enter the numerical position of each vial. Then click
submit (you have to click it twice).
4. Click view then go to sample queue. Click ready (located next to the T button) then click start
sample.
5. To view the results, right click on the graph and select list data. Then open the Peak List tab and
click view. Record any required data.
6. To shut down the LC-MS, click hardware config, then LCMS, then deactivate profile.
7. Then turn off the nitrogen gas, first the valve on the lab bench, then the tank in the cylinder
storage room.
Data:
Concentration
Standards
Time (minutes)
10 ppm standard
1.7356
20 ppm standard
1.7055
30 ppm standard
1.6685
50 ppm standard
1.6884
100 ppm standard
1.6799
Area
% Area
9.3910e5 counts
1.7165e6 counts
7.0834e6 counts
1.3719e6 counts
1.1563e6 counts
5.87%
73.83%
6.33%
10.87%
11.49%
8000000
y = -14719x + 3E+06
R² = 0.0406
7000000
6000000
5000000
4000000
3000000
2000000
1000000
0
0
20
40
60
80
100
120
HPLC:
Caffeinated Tea
Peak
#
1
2
3
4
5
6
7
8
9
Retention
Time
0.884
1.478
1.741
2.307
2.533
3.675
4.141
5.24
8.071
Area
219290
9360053
11996103
4761067
2670732
6255841
23709668
2025015
2859
The peak at 2.307 matches up with all the peaks that we got for our caffeine standards on the HPLC.
From the intensity of our standard peaks compared to the intensity (area) of the caffeinated tea we
were able to determine approximate concentration of caffeine in the sample. This was determined to be
approximately 27 ppm.
We weren’t able to run a decaffeinated tea sample because of time constraints. We were having issues
getting the HPLC to run properly. Not really sure what happened, it was just not cooperative.
Conclusions: With the LCMS we were able to determine that decaffeinated tea has very trace amounts
of caffeine in it, <1 ppm.