LC-MS and HPLC
Purpose:
The purpose of this lab is to learn the basic functions of the HPLC, and the LC-MS
and to understand the separation techniques. Unknown and known samples will be
tested with both instruments.
Introduction:
The HPLC is known as high performance liquid chromatography, this is commonly
used to water-based sample. A liquid mobile phase and a solid or liquid mobile
phase are utilized. The instrument consists of a solvent pump, helium source, pump,
pulse damper, drain valve, pressure regular and an injector chamber and column.
The LC-MS is similar to the HPLC in parts however it uses a mass spectrometer as
the detector, which provides qualitative and quantitative data. Such as molar mass
and information about the compounds structure.
Procedure:
LC-MS
1. Prepare the samples by weighing out a range of concentrations in ppm of
caffeine
2. Once standards are created in a range fill the small vials up the 1.5 mL mark
3. Make sure all sections of the instrument are on
4. Turn the nitrogen gas on in the gas room to the about 40psi
5. Open the nitrogen on the bench top
6. On the desktop click the Analyst Software icon
7. In the vertical menu, click on the hardware configuration then LC-MS then
activate profile
8. In the vertical menu now click Build Acquisition Batch. In the window that
appears fill in the set name (file name)
9. Select the method “caffeine10”
10. Click add set then add sample the bottom of the window you select the
number of your samples
11. Under Vial position enter the numbers where the vials reside in the LC tray
12. Click on the submit tab then submit then view select “sample queue” then
acquire then ready then acquire then start
13. After a graph appears right click on the graph and click List Data
14. In the new window click on the peak list tab this is where needed data is
recorded
15. Print data
16. In the vertical menu, click on hardware configuration
17. The window from the startup will appear, click on LC-MS once more and then
click on deactivate profile this will shut down the instrument
18. Shut the nitrogen gas off at the desk and in the gas room
HPLC
1. Make sure to degas the methanol and water
2. Turn on the computer
3. Check the pump lines A and B for gas bubbles
4. If there are bubbles execute the purge with the pump
5. Purge the lines by opening the valve and hitting the purge button
6. Close the valve and hit the purge button again
7. Turn the pump back on
8. Turn on detector
9. Select method and run all the parameters
10. Run the standards and the unknowns
Data:
HPLC: The HPLC was down during our lab time
LC-MS: The data is inconclusive and no calibration curve can be drawn because of
problems with the MS detector during our lab time. However chromatograms are in
lab notebook.
Conclusion:
The HPLC was down during our lab thus we don’t have any data or experience on it.
However it is fixed and running now. So when I get a chance I plan on investigating
the operation of the HPLC by using the SOP and background information. The LC-MS
was easy to use in that setting up sample vials and running the program were
straightforward. However there was a problem with the MS and its ability to detect
the caffeine therefore our results had no identifiable use. After the LCMS I wish to
try to run samples on it again to see if we get better results.