Experiment Nine: HPLC and LC
Introduction:
High Performance Liquid Chromatography (HPLC) is a type of instrument
that separates mixtures of compounds with a water base. Liquid Chromatography
Mass Spectrometry (LC-MS) which is very similar to the HPLC, it allows to
determine the mass-to-charge ratio of specific elements.
Purpose:
Within this experiment, the purpose was to test with various standards of
different concentrations of caffeine and test the decaffeinated and caffeinated tea.
The purpose of that was to determine the concentration of caffeine in the tea
samples with aide in the HPLC. Hence, the same experiment was conducted with the
LC-MS and to compare the results.
SOP for HPLC:
1. Make sure there is enough degassed methanol and water.
2. Turn computer on.
3. Check pump lines for air bubbles.
4. Turn detector on and oven.
5. From the desktop, select LabSolutions->Instruments->Type:LC->CH210SYSTEM1-Instrument1
6. Go to File->Open method file->Project 1->Taylor Test->Test Method 1
7. Select parameters
a. Organic/Aqueous ratio can be altered here.
b. A 10 minute run is standard.
c. Hit Download.
8. Hit Single Run
a. Give a Sample Name.
b. Give individual ID name.
c. Use first yellow folder button to make your own data folder.
d. Data File is what it will be saved as.
e. Hit Ok.
f. Hit Start.
9. Manually set to Load using the black dial next to column.
10. Syringe should be prepped by flushing at least once with deionized water and
once with sample.
11. Once pressures stabilize on screen, inject.
12. To start, turn knob quickly, leaving syringe in.
13. Hit start.
14. After run, turn back to Load and remove syringe.
15. Copy chromatogram in paint.
Troubleshooting
1. Problem: Where Ready should appear, Not Connected appears in gray box.
a. Go to Main->System Configuration->Reset->Auto Configuration->OK
Shut Down
1. Purge line by running closed line with 100% solvent from Pump B for 5 min
(Acquisition>Instrument Parameters>set parameters>Download). *Note:
You will have to time this because the system is closed. Repeat using 0%
solvent from Pump B for 5 min.
2. File-> Open method file->Low Flow Method->open->Download
3. Close program.
4. Computer and instrument stay on.
5. Ensure mobile flasks have adequate solvents.
6. Turn off detector and oven.
7. Check waste container.
Data:
HPLC:
Concentration (ppm)
Area (Caffeine Peak)
10
344338
20
571396
30
829879
40
986278
1200000
y = 21843x + 136897
R² = 0.9911
1000000
800000
Series1
600000
Linear (Series1)
400000
200000
0
0
10
20
Refer to spectra in notebook.
Results LC-MS:
30
40
50
25
20
y = 2.69x + 9.97
R² = 0.7555
15
Series1
Linear (Series1)
10
5
0
1
2
3
4
Conclusion:
Overall, the HPLC and LC-MS were not complicated instruments. However, the R
squared value for the LC-MS was terrible, having a value of 0.755. Hence, the
concentrations of the caffeinated and decaffeinated had skewed results.