Lab 5- HPLC, LC/MS and IC
Intro: Liquid chromatography can be used to separate liquid samples. The LC/MS and HPLC are very similar in their purpose, but perform them in different manners. The HPLC uses high pressure to separate compounds, while the LC/MS separates compounds by their mass. The IC can be used specifically when metals ions are involved.
Purpose: To become familiar with the operation of these instruments, learn their pros and cons, and learn which instrument is appropriate for which samples.
Procedure:
Day one: Run standard samples of caffeine for the LC/MS and HPLC. For the IC, create standard anion concentrations, and create a calibration curve for them.
Day two: Create a calibration curve for the caffeine samples. Run caffeinated and decaf samples of tea. For the IC, create calibration curves for various water samples.
HPLC Data:
Retention Times of Samples in HPLC
Sample Number Peak 1 Peak 2 Concentration
(ppm)
50 A
B
6.1 (cm)
5.3 (cm)
7.4 (cm)
6.3 (cm) 150
200 C
D
6.3 (cm)
6.3 (cm)
7.5 (cm)
7.5 (cm)
E
F
-----
6.1 (cm)
-----
-----
250
---- x
G
H
6.2 (cm)
6.3 (cm)
-----
----- x x

LC/MS data:
Sample Preparations of Standards and Unknown
Vial Number # (mL) Of Stock
Solution
Dilution Flask
(mL)
Desired Concen.
(ppm)
G
H
E
F
L
C
D
A
B
5 (mL)
10 (mL)
15(mL)
20(mL)
25(mL)
30 (mL)
1 (mL)
1 (mL)
1 (mL)
100 (mL)
100 (mL)
100 (mL)
100 (mL)
100 (mL)
100 (mL)
100 (mL)
100 (mL)
100 (mL)
50 ppm
100 ppm
150 ppm
200 ppm
250 ppm
300 ppm
(x)
(x)
(x)
Retention
Times(min)
1.8480 (min)
1.8457 (min)
1.8575 (min)
1.8602 (min)
1.8606 (min)
1.8540 (min)
1.7056 (min)
1.7579 (min)
1.6862 (min)

40000000
35000000
30000000
25000000
20000000
15000000
10000000
5000000
0
0 50 100 y = 117476x - 505400
R² = 0.9804
150 200
Concentration (ppm)
250 300 350
LC/MS calculations:
Sample concentrations were determined using the M1V1=M2V2 formula. For example:
(1000 ppm)( X ) = (50 ppm)(100 mL)
X = 5 mL
Sample G:
Y=mx+b
528110 = 117476 (x) – 505400
X = 8.7976ppm
Sample H:
480140 = 117476 (x) – 505400
X = 8.3989ppm
Sample I:
515490 = 117476 (x) – 505400
X = 8.6902ppm

IC data:
Peak 1
1
2
3
4
5
6
7
Seven Ion Standard Data
Peak Name
Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate
Retention Time (min)
3.01
3.60
3.93
4.45
4.75
5.24
5.77
Sample Name
Lab Water
Men’s BR
Water
Retention Times (min) of Water Sample Data
Peak 1
2.30 (min)
Peak 2
3.59 (min)
Peak 3
4.76 (min)
Peak 4
5.75 (min)
Peak 5
--
Peak 6
--
2.28 (min) 3.14 (min) 3.56 (min) 4.72 (min) 5.70 (min) 11.99
(min)
Conclusion:
Our HPLC data was mostly inconclusive. We made our caffeine samples ahead of time, and had not realized that they degraded in between lab meetings, despite storing them in dark glass reagent bottles. Our caffeine standards had two characteristic peaks, the first being the solvent, and the second being the caffeine peak. The caffeine samples all only had one peak, indicating that caffeine was not present. As a result, we could not create a calibration curve. In the future, we will know not to store caffeine samples. However, the LC/MS data was more successful. We ran these first, so the samples did not degrade. We determined the caffeine concentration to be 8.4, 8.7, and 8.8 ppm. However, sample H was supposed to be decaf, yet a caffeine peak was present. We believe this sample became contaminated, or was mixed up with another sample. We also think our retention time was effected by the dilution of our samples.
Next time, we won’t dilute them as much. For the IC, the different water samples were very similar. All of them contained chlorine, nitrate, and sulfate. However, the bathroom water also contained fluoride. We’re not sure why this was present, but we believe it may be due to fluoride build-up in the bathroom faucets. Perhaps the lab faucets did not accumulate fluoride, or were cleaned more recently. A few unknown peaks did show up in both spectra that did not match any retention times. We cannot account for these peaks. They may be contaminations, or trace elements.