John Siller
Laboratory 3
LC, HPLC, and IC
Introduction
Liquid Chromatography is a separation technique that relies on the partitioning effect caused by
a liquid mobile phase and a solid-liquid stationary phase. Liquid chromatography is similar to gas
chromatography but differs in mobile phase and column designs. The objective of a good
chromatography technique is to maximize the number of theoretical plates and minimize the plate
height. These parameters are changed by varying the separation column and packing materials within
the column. Another key aspect of any chromatography technique is the detector used at the end of the
separation. Detectors range from mass analyzers to fluorometers, and can be used to identify separated
analytes using different techniques. High performance liquid chromatography (HPLC) is a liquid
chromatography technique that uses high pressure to quickly separate analytes in a mixture. In this lab
we used liquid chromatography mass spectroscopy (LCMS) and HPLC to separate and analyze caffeine in
an aqueous solution. In addition to liquid chromatography we also ran samples of water from various
sources around Campbell Hall using ion chromatography (IC). The IC results reported various metal ion
concentrations in water from two different water fountains. The water fountains were the one closest
to the men’s bathroom (A) and the one closest to the girl’s bathroom (B). Due to time constraints and
lack of nitrogen on the first day we were not able to run as many different samples as we would have
liked, but we were able to use all three instruments successfully.
Procedure
LCMS
 Turn on Nitrogen
 Open software and activate profile
 Build acquisition set to program automated sampling
 Place samples in auto sampling vial positions
 Run samples using computer program
 Analyze the data using list data, peak list, and data file
 Deactivate LCMS and shut down.
HPLC

Degas methanol and water if there is not enough
 Turn on computer

Check pump lines A and B to ensure no gas bubbles
 If gas bubbles execute purge with pump
Purge the lines by opening valve and hitting the purge button
 Close valve and quickly hit purge button again
 Turn pump back on
 Turn detector on
 Select the method and align parameters
 Run standards before running unknowns

IC
 Check helium pressure on tank in gas room and on instrument
 Make sure regenerate and eluent bottles are more than half full
 If bottles are less than half full they must be refilled
 Equilibrate system with pumps controlled by the software
 After 20 to 30 minutes check the hoses to insure proper flow rate
 Run program on the software by imputing proper parameters
 Run the sequence by injecting sample
 Each run takes 16 minutes
 After run is completed analyze the peaks
 Shut down computer program and IC
Data
All LCMS, HPLC, and IC chromatograms can be found in my lab notebook. Below are the
calibration curves created on Excel using the intensity of the strongest peak for each concentration.
The first graph reports the calibration curve for the LCMS with standard concentrations of 10 ppm,
20 ppm, 100 ppm, 200 ppm, and 400 ppm. Each standard was run twice therefore the peak heights
reported are the average of the two spectra collected.
Peak Height (cpsX10^6)
14
Calibration Curve LCMS
12
10
8
6
y = 0.0308x
R² = 0.7986
4
2
0
0
100
200
300
400
Concentration (ppm)
Figure 1: Calibration curve for LCMS using 5 calibration standards.
500
8000000
Calibration Curve HPLC
7000000
Peak Area
6000000
5000000
y = 47270x + 2E+06
R² = 0.9966
4000000
3000000
2000000
1000000
0
0
20
40
60
80
100
120
Concentration (ppm)
Figure 2: Calibration Curve for HPLC using 3 calibration standards. (10 ppm, 20 ppm, and 100 ppm)
Calculations
Dilution factors:
400 ppm
(V1)(1000ppm)=(100mL)(400ppm)
V1 =40 mL
200 ppm
(V1)(1000ppm)=(100mL)(200ppm)
V1 = 20 mL
100 ppm
(V1)(1000ppm)=(100mL)(100ppm)
V1 = 10 mL
20 ppm
(V1)(1000ppm)=(100mL)(20ppm)
V1 = 2 mL
10 ppm
(V1)(1000ppm)=(100mL)(10ppm)
V1 = 1 mL
IC Concentrations:
Standards
Ion
Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate
Peak Height (µS)
16.407
18.888
29.705
22.498
25.909
18.110
49.651
Concentration (mg/L)
30
30
100
100
100
150
150
Multiplication Factor
0.5469
0.629
0.29705
0.22498
0.25909
0.1207
0.331
Water Fountain A
Ion
Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate
Peak Height (μS)
4.432
17.906
8.150
5.918
11.329
4.722
16.057
Concentration (mg/L or ppm)
2.424
11.263
2.421
1.331
2.935
0.570
5.315
Peak Height (µS)
0.496
16.851
N/A
0.466
6.266
N/A
5.705
Concentration (mg/L or ppm)
0.271
10.600
N/A
0.105
1.623
N/A
1.888
Water Fountain B
Ion
Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate
Results
Ion Concentrations
Concentration (ppm)
12
Fluoride
10
Chloride
8
Nitrite
6
Bromide
4
Nitrate
2
Phosphate
Sulfate
0
Water Fountain A
Water Fountain B
Figure 3: Ion concentrations found in fountain water in Campbell Hall using IC
For both the LCMS and HPLC we unfortunately did not have enough time to prepare any
unknown caffeine solutions using different types of tea. Looking at our calibration curves however, this
was probably a blessing because any data would not be reportable due to lack of confidence. Our
calibration curves were not uniform enough to use to accurately report caffeine concentrations. We
were able to produce usable IC data however. The graph of ion concentrations in drinking fountain
water is reported above. Safe levels of these ions vary from state to state. For example in Pennsylvania
the nitrate concentration for public drinking water cannot exceed 45 mg/L (45 ppm). Both fountains
were significantly under this mark and therefore are safe to drink by this standard. Additionally the
chloride level, which appears high compared to other ions, is well within the Pennsylvania standard of
250 mg/L. It is interesting to note the difference between the ion concentrations of the two different
fountains. This could be due to different distances the water has to travel in pipes from the source to
the fountains. This could also be explained by experimental error and therefore more testing must be
done before any conclusions are made, but from these results it appears that our drinking water does
not contain dangerous levels of ion concentration. These results are important to me because I drink out
of fountain B almost every day, but now I am considering walking a little farther to fountain B.
Conclusion
Time constraints really hurt us during the process of this lab. Nitrogen is needed to run the LCMS,
and on the first day, when we planned to run the LCMS, the nitrogen tank was empty. This caused us to
make adjustments on the fly. We decided to instead work on the HPLC, but because we were unfamiliar
with the instrument and the programming used to control it we were only able to run four standard
solutions on Monday. This left us with a lot of work to do on Wednesday and as a result we were not
able to prepare and run unknown caffeine samples from tea. Therefore the only data we were able to
produce on the LCMS and HPLC were calibration standards. This was unfortunate, but if we did run any
unknown samples our calibration curves did not come out as organized as possible and would have led
to inconclusive results. Luckily the IC worked as it should, but again we were only able to analyze two
unknown samples due to time constraints. I would have preferred to run the water collected from each
fountain multiple times to ensure the results accuracy. Given that we were required to run three
instruments in two days and the nitrogen was empty on day one, I am content with the amount of data
collected. If this lab was repeated there would be many changes including being much more organized
and prepared to run the HPLC and IC on day one. The IC was originally included in another lab. This
would have allowed us to just focus on the liquid chromatography techniques, and we would have been
able to prepare unknowns and possibly correct our calibration standards. It would have also been nice
to spend more time with the IC to confirm our drinking water findings. Overall this lab was rushed and a
little sloppy, but we learned from our mistakes and future labs will benefit from these experiences.