HPLC/LC-MS Lab Report
Introduction: Liquid Chromatography similar to gas chromatography is the separation of compounds. There are several types of liquid chromatography but the main ones are High
Pressure Liquid Chromatography (HPLC) and liquid chromatography mass spec (LC-MS). The
HPLC separates compounds that are dissolved in a solution and they are separated by injecting a small amount of the sample into a column. The components pass through the column at different rates due to the partitioning of the samples. The HPLC is generally used for identifying, purifying, and quantifying components of a mixture. Some applications of HPLC include drug analysis, and protein levels in compounds. The LC-MS is mainly used for the detection and identification of chemicals in a mixture. Some applications of LC-MS include pharmacokinetic analysis and drug development.
Purpose: The purpose of this experiment was to determine the caffeine levels in various standard solutions of coffee, tea, and pure caffeine. The samples concentrated samples were run through both the HPLC and LC-MS.
Methods: Day 1: We worked on the HPLC for this lab period. First we made various concentrated standards of pure caffeine using 100mL volumetric flasks. We calculated the number of mols that would be needed for our standards. We made 1M, 2M, and 3M solution standards of caffeine each 10 mols with .52g, 20 mols with .10g, and 30 mols with .155g respectively and then made unknown concentrations of regular and decaf coffee. We first ran all our standards each by injecting a small amount into the column and letting it run for 10 minutes and obtained a chromatogram for each standard. Then we used the same method for the unknowns.
Day 2: This day we worked on the LC-MS. We first checked the degassed methanol and water levels. We ended up having to make more degassed methanol for the instrument but other than that everything was correct. We turned on the compressed nitrogen tank in the gas room as well as on the bench top. Loaded all the samples at once and started the software. Once the instrument was ready we let do its work. We used the same samples from the HPLC and this would give a mass spec of the samples. Each sample ran for 5 minutes and gave us a chromatogram and mass spec with the desired peaks for caffeine. We also made a calibration curve for these results to find the molar concentration of the unknown caffeine and tea samples.
Results: All of the chromatograms and mass spec data can be found in the lab notebook. The retention time, % area, and height were measured for each chromatogram on the HPLC and the
LC-MS only had retention time and height measured for results. A calibration curve was done for each instrument to test how well the instrument works and performs. We used the calibration curve equation to find the molar concentration of caffeine in the tea and coffee samples.
HPLC results:

Concentration(mol) Retention time (min) % Area
1M
2M
3M
Regular Coffee
Decaf Coffee
2.425
2.375
2.375
2.367
2.408
10.972
51.239
96.288
11.488
6.614
Height
5,098
20,737
93,552
102,617
16,400
Calibration equation: y = .00002x + 1.21019 = y = .00002(102,617) + 1.21019 = 3.26M regular y = .00002(16,400) + 1.21019 = 1.538M decaf
LC-MS results:
Concentration(mols) Retention time (min) Height (cps)
1M
2M
3M
Decaf Tea
Regular Coffee
1.89
1.82
1.85
1.86
1.88
6.2348 x 10 6
2.6125 x 10 6
4.2459 x 10 6
5.0776 x 10 6
8.8857 x 10 6
Calibration Equation:y = .01256x + 1.94976 =y= .01256( 5.0776x10
6 ) + 1.94976 = 2.01M decaf y = .01256( 8.8857x 10 6 ) + 1.94976 = 2.06M regular
Conclusion: The experiment was successful and we were able to figure out the molar concentrations of the unknowns from both the HPLC and LC-MS. Based on the tables with the retention times and heights, the higher the molar concentration the greater the height and peak width meaning that when the concentration was higher there was a higher caffeine level. Also the unknown concentrations were consistent with what we thought they should be, the regular coffee was 3.26M which was higher than our standards showing that coffee has high levels of caffeine in it. The decaf was 1.538M and based on our standards this is true because it shouldn’t have a lot of caffeine in it since its decaf. The same goes for the LC-MS, but the results from the calibration curve were unexpected. The decaf tea and regular coffee were almost the same molar concentration at 2.01M and 2.06M respectively. There are multiple reasons why this could be.
One reason is because we didn’t run any tea standards so there was nothing to base the unknown off of. Also there could have been possible contaminations in the tea because the tea sample was placed in between the coffee samples and maybe there were traces of coffee in the tea samples.
The other results were also unexpected because the 3M standard peak height wasn’t the highest as it should have been and the 1M standard should have been the smallest. Something went wrong somewhere during the experiment because the results should have been similar to that of the HPLC results, except for the tea since there was nothing to compare it to. To fix this error, more trials could have been done to ensure that it would yield the correct results and minimize errors and get a better concentration of caffeine.