Experiment 8

Dan Palumbo
High performance liquid chromatography (HPLC) is considered to be the most widely used
analytical method for separation of many organic and inorganic substances. The UV-visible absorption
detector measures the absorption of eluents that flow through a small cell recording the retention time,
peak area, and height to allow for identificiation and concentration of a substance.
LC/MS which is a liquid chromatography mass spec instrument, detects additional properties of a
substance to accomplish a method like this is to utilize a low-flow rate atmospheric pressure ionization
technique. Retention time can be determined. This also determines the specific peak area corresponding to
the analyte of interest in an impure sample.
Shimadzu (HPLC)
1. Check if computer is on and verify A and B flasks contain enough degassed methanol and
distilled water.
2. Check pump lines for air bubbles
3. Turn on UV/Vis detector and LC-4B Amperometric Detector
4. “HPLC” icon on computer
5. Click Shimadzu and click methods button.
6. Select “TSW.met” and check parameters are correct.
7. Load/Inject knob is turned to Load
8. Select “Run Single” and enter a file name and click start
9. Inject a Blank sample (3 times) then caffeine sample (5 minutes)
10. Turn knob to inject and do not remove the syringe.
11. Click stop after correct time and then turn knob back to Load and remove syringe
12. Click analyze
13. Repeat for all other samples
Perkin Elmer (LC/MS)
1. Turn computer on and check nitrogen tank and slowly open small silver valve “builder Pressure:
on the tank. Then slowly open green valve on tank and verify pressure reads 40psi.
2. Turn on nitrogen at lab bench
3. Double click “Analyst Software” on screen, click “hardware configuration”, click “LCMS”, then
finally click “Activate Profile.”
4. After making samples, build an acquisition batch (More in dept instructions in lab notebook)
5. After running sample, double click “Open Data File”
6. Select previously entered “Set” file name and double click
7. Right click on graph and then left click on “List Data” and click “Peak List” tab.
8. Click empty box below and then right click the “Peak List” and left click “TurboChrom
9. Enter Bunching Factor and click OK. Then click “Print”
10. Repeat for all other samples.
HPLC Data:
Retention Time
2.57 seconds
2.63 seconds
2.64 seconds
Coffee – There were peaks with retention time at 1.62 and 2.33 seconds. The height of the peaks were
997623 and 360562, respectively. These peaks show the presence of caffeine in the sample.
<see graphs in lab notebook>
Due to the lack of helium, my partner and I ran LC/MS instead of GC for the first day. We
created our samples and put them in the vials and allowed the LC/MS to do its job. We obtained graphs
for each of our spectrums; however we failed to name the peaks. Our caffeine samples all showed similar
spectrum with a tall peak and then a slow decline but compared to the coffee it was more jagged and
irregular. With the HPLC, the caffeine samples showed similar spectra with the coffee spectrum showing
different peaks. The caffeine samples had a few small peaks which didn’t cover much area and then one
very large peak which took up over 99% of the area. The coffee spectrum showed a very large peak and
another smaller peak. Overall the procedure went well.