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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)
1M
2M
3M
Regular Coffee
Decaf Coffee

Retention time (min)


2.425
2.375
2.375
2.367
2.408

% Area
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)
1M

Retention time (min)


1.89

Height (cps)
6.2348 x 106

2M

1.82

2.6125 x 106

3M

1.85

4.2459 x 106

1.86
1.88

5.0776 x 106

Decaf Tea
Regular Coffee

8.8857 x 106

6
Calibration Equation:y = .01256x + 1.94976 =y= .01256( 5.0776 x 10 + 1.94976 = 2.01M

decaf
6
y = .01256( 8.8857 x 10 + 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 shouldnt 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 didnt 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 wasnt 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.