David Millard
HPLC and LC/MS
Purpose:
The purpose of the lab was to run standards of caffeine on the HPLC and the LC/MS.
After the caffeine standards where run through the instrument, an unknown concentration was
determined from samples of caffeinated and decaffeinated tea.
Introduction:
Liquid chromatography is the separation of liquid solutions, which also, may have had
solids dissolved in it to try to determine the components in the solution. High performance
liquid chromatography, also known as HPLC, uses the peaks of standards to determine the
concentration of a specific molecule in a substance, for this lab, the concentration of caffeine in
tea. Another instrument that is similar to the HPLC is the LC/MS, liquid chromatography mass
spectroscopy. The components of this instrument are similar to that of the HPLC, but the
LC/MS has the addition of a mass spectrometer. With this addition, a more detailed analysis of
the samples being sent through can be obtained.
Procedure:
HPLC
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A 1000ppm stock solution of caffeine was made.
Varying concentrations of standards were made.
Degassed methanol and degassed water were checked to see if more had to be made.
The instrument was turned on.
Lines were checked for air bubbles.
o If bubbles were present, a purge of the line would be performed.
The method was loaded onto the instrument and pressure and flow rate was allowed to
stabilize.
Parameters were set to our specific instructions.
Standards were run.
Unknown samples were run.
Data:
The LC/MS was down during our instrumentation period. The standards for the HPLC were
made by diluting a 1000ppm solution of caffeine to 10, 20, 40, 60, 80, 100, 150, 200, and
300ppm.
+ 714250
Calibration CurveHPLC y = 31355x
R² = 0.99
12000000
10000000
Area
8000000
6000000
4000000
2000000
0
0
50
100
150
200
Concentration (ppm)
Dilution Calculations
(1000ppm)(V1)=(10mL)(10ppm)
V1= 0.1mL
(1000ppm)(V1)=(10mL)(20ppm)
V1= 0.2mL
(1000ppm)(V1)=(10mL)(40ppm)
V1= 0.4mL
(1000ppm)(V1)=(10mL)(60ppm)
V1= 0.6mL
(1000ppm)(V1)=(10mL)(80ppm)
V1= 0.8mL
(1000ppm)(V1)=(10mL)(100ppm)
V1= 1.0mL
250
300
350
(1000ppm)(V1)=(10mL)(150ppm)
V1= 1.5mL
(1000ppm)(V1)=(10mL)(200ppm)
V1= 2.0mL
(1000ppm)(V1)=(10mL)(300ppm)
V1= 3.0mL
Tea
Regular tea 1
Regular tea 2
Decaf tea 1
Decaf tea 2
Area
4619439
4577734
823233
829575
Concentration (ppm)
124.54757
123.21748
3.4757774
3.6780418
Results/Conclusion
During our lab time, the LC/MS was down so the only instrument used was the HPLC.
The retention times for the caffeine in the tea and the standards both showed on the HPLC at 2.5
minutes. The area for the regular tea and the decaf tea were both similar along with the
concentrations. The labeling of the decaf tea having less caffeine is true. The amount of
caffeine was significantly less than the regular tea.