Experiment 3: HPLC and LC-MS
Introduction:
The instruments we will use are the HPLC and the LC-MS. The technique used for HPLC is analytical
separation. HPLC separates nonvolatile species, and runs industrial substances such as amino acids,
proteins, antibiotics, and other substances. The LC-MS is selective; it can isolate unresolved peaks by
monitoring their masses. The LC-MS can give the details about a sample instead of relying on its
retention time, like HPLC.
Purpose:
The purpose of the lab is to learn how to use the HPLC and the LC-MS to run standard samples of
caffeine, tea samples, and an Excedrin sample.
Procedure:
HP-LC
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Make sure there is enough degassed methanol and water in flasks
Turn on computer and check pump lines A and B, and make sure there are no air bubbles
between flasks and HPLC.
Purge the lines, by turning on pump button and detector power button.
Select HPLC method icon. Choose the Taylor Test method.
Set parameter according to Standard Operating Procedure and click Download button
Make sure HPLC knob is switched to load
Run standards and then unknowns
After sample is done running, select analyze
Record data, and print peaks
Shut down HPLC
LC-MS
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Make sure everything is turned on
Turn on nitrogen in gas room and on the bench
Click on Analyst Software icon, Hardware Configuration > LCMS > Activate Profile
Click Build Acquisition Batch
Fill in Set name
Select method: caffeine 2011
Click on Add Set > Add Sample, select number of samples
Enter numerical positions of vials in Vial Position
Click Submit
Click View > Sample Queue
Click Ready, start sample
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View results and record data
Deactivate LC-MS
Turn off nitrogen gas on bench and in gas room
Data:
The graphs and data for the HPLC are attached in the notebook.
The LC-MS was not working during the week that we were on these instruments, so we were not able to
use that instrument.
Calculations:
Stock Solution:
1 𝑚𝑔
0.001 𝑔
1𝑔
100 𝑚𝑔
=
=
=
1𝑔
1,000,000 𝑔 100 𝑚𝐿
1000𝑚𝐿(1 𝑚𝐿) 1000 𝑔
Dilutions:
M1V1=M2V2
1000(x)=(50)(50)
=
1000(x)=2500
=2.5mL
1000(x)=(40)(50)
=
1000(x)=2000
=2.0 mL
1000(x)=(30)(50)
=
1000(x)=1500
=1.5 mL
1000(x)=(20)(50)
=
1000(x)=1000
=1.0 mL
1000(x)=(10)(50)
=
1000(x)=500
Caffeinated tea
413654 = 20575(𝑥) + 271686
141968 = 20575(𝑥)
6.900 = 𝑥
6.900 𝑝𝑝𝑚
Decaffeinated tea
285692 = 20575(𝑥) + 271686
14006 = 20575(𝑥)
. 6807 = 𝑥
. 6807 𝑝𝑝𝑚
=0.5 mL
Excedrin
509424 = 20575(𝑥) + 271686
237738 = 20575(𝑥)
11.555 = 𝑥
11.555 𝑝𝑝𝑚
Concentration vs. Area
1400000
1200000
Area (nm)
1000000
800000
y = 20575x + 271686
R² = 0.9301
600000
400000
200000
0
0
10
20
30
40
50
60
Concentration (ppm)
Conclusion:
The LC-MS instrument was under repair during the week of working on the liquid chromatography
instruments, so we only worked on the HPLC. When first turning on the HPLC instrument we had an LK.S
error, all it took was a few tries of turning the instrument off and then back on for it to work. The HPLC
was an easy instrument to use the samples did not take long to run, the samples ran for about 10
minutes each. The R2 value was a little low and the 30ppm sample was not close to the line of best fit.
The decaffeinated tea sample seemed to have the least ppm value.