HPLC, LC-MS, & IC
Introduction
HPLC is High Pressure Liquid Chromatography. It is generally used for the
separation of compound mixtures, as well as for identification, quantity, and purity
measurements. A column separation technique is used, in which the analyte is separated
by mobile and stationary phases under extreme pressure. A detector gives a
chromatograph with retention times.
Procedure
Day 1: Investigate the operation of the HPLC and LC-MS by running standard samples of
caffeine and note retention times.
Day 2: Create calibration curve for caffeine. Run samples of caffeinated and decaf tea.
Preparation:
1) Ensure that there is enough degassed methanol and water, the lines coming from
the degassed species have no air bubbles, the computer is still on, and the injector
knob is towards the left on load
2) Turn on the detector
3) 1g of caffeine goes into 1000mL water to make 1000ppm solution
Software:
1) Click the HPLC icon on the desktop
2) Select Shimadzu
3) In the program window, click on the Methods icon at the top
4) In the Methods window, select the method: TSW.met
5) Click on the Parameters icon at the top
a) Flow rate A: .500 mL/min
b) Flow rate B: .500 mL/min
c) Flow rate C: 1.000 mL/min
d) Max pressure: 6000psi
e) Min pressure: 1 psi
f) Wavelength: 256nm
6) Click Download – Ok
Running Samples:
1) Prepare sample for injection by drawing it into a syringe
2) Insert into injection port but do not inject or turn the knob yet
3) Click on the Run Single icon at the top of program window
4) In the new window, assign file name with initials and click start
5) When the bottom of the window says “waiting for trigger,” the sample can be
injected
6) Inject until about 3-5 drops of sample fall from waste hose, switch knob to inject,
and leave syringe in
7) Program will run for about 5 minutes
Results:
1) Click red stop sign icon at top
2) Click analyze icon at top and label peaks on chromatogram
3) Click report icon at top and Area % in dropdown menu
Shutdown:
1) Click method icon and select: LOWFLOW.met
2) Click on parameters icon and ensure these values are assigned:
a. Flow rate A: .01 mL/min
b. Flow rate B: .01 mL/min
c. Flow rate C: 1.000 mL/min
3) Click download, then exit program
4) Ensure screens for A & B are at .01mL/min
5) Turn off detector
Results:
Retention Times of Samples in HPLC
Sample Number
Peak 1
Peak 2
Concentration
(ppm)
A
6.1 (cm)
7.4 (cm)
50
B
5.3 (cm)
6.3 (cm)
150
C
6.3 (cm)
7.5 (cm)
200
D
6.3 (cm)
7.5 (cm)
250
E
-----
-----
----
F
6.1 (cm)
-----
x
G
6.2 (cm)
-----
x
H
6.3 (cm)
-----
x
We did not get an area output so we could not make a calibration curve. Because of this,
we had to base our conclusions on retention times. All of the unknowns only had one
peak and the standards had two peaks. The standard peaks were at about 6.2cm and
7.4cm. The second peak was the characteristic caffeine peak.
Conclusion
Our unknown samples did not have caffeine peaks, most likely because our
samples were made ahead of time and stored so the caffeine may have degraded. Because
of this, we were not able to determine the amount of caffeine in the unknowns.
If we were to do the experiment again, we would make fresh samples instead of using the
same ones throughout the experiment.
Introduction
LC-MS stands for Liquid Chromatography-Mass Spectrometry. It uses a similar
column separation technique to the HPLC, but with mass analysis. After separation, the
solvent is removed and the sample is ionized. A mass detector scans the molecules by
mass and produces a spectrum that identifies compounds within samples.
Procedure
Sample Preparation:
Small vials are to be used that can be filled to a total volume of 1.5mL. These vials are to
be capped and placed into the numbered tray.
1) Make caffeine solution of 1000ppm
2) Make dilutions using M1V1=M2V2 equation: concentrations are 50ppm,
100ppm, 150ppm, 200ppm, 250ppm, 300ppm
3) Unknown samples made from 1mL tea in 100mL water
Sample Preparations of Standards and Unknown
Vial Number #
(mL) Of Stock
Dilution Flask
Desired
Retention
Solution
(mL)
Concen. (ppm)
Times
(min)
A
5 (mL)
100 (mL)
50 ppm
1.8480 (min)
B
10 (mL)
100 (mL)
100 ppm
1.8457 (min)
C
15(mL)
100 (mL)
150 ppm
1.8575 (min)
D
20(mL)
100 (mL)
200 ppm
1.8602 (min)
E
25(mL)
100 (mL)
250 ppm
1.8606 (min)
F
30 (mL)
100 (mL)
300 ppm
1.8540 (min)
G
1 (mL)
100 (mL)
(x)
1.7056 (min)
H
1 (mL)
100 (mL)
(x)
1.7579 (min)
L
1 (mL)
100 (mL)
(x)
1.6862 (min)
Preparation:
1) Ensure that all sections of instrument are powered on
2) Turn on nitrogen in gas room to about 40psi
3) Open nitrogen valve at bench top
4) Make sure flasks are full
Software:
1) Click analyst software icon on desktop
2) In vertical menu, click on hardware configuration – LCMS – activate profile
(green)
3) Click build acquisition batch and set file name
4) Select method “cafeine10”
5) Click add set – add sample and select number of samples
6) Under vial position, enter numbers where vials are
7) Click on submit tab – submit – view and select “sample queue” – ready – start
sample
8) Check LC pump to ensure it has pressure
Results:
1) In left panel, click on open data file and select set name
2) After graph appears, right click on it and click on list data
3) Click on peak list tab
Shutdown:
1) In left vertical menu, click on hardware configuration
2) Click on LC-MS
3) Click deactivate profile to shut down instrument
4) Shut off nitrogen gas valve at desk and in gas room
Calculations:
1) M1V1=M2V2
(1000ppm)(x) = (50ppm)(10mL)
X= 5mL
2) Y=mx+b
Sample G
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
Results:
LC-MS Results For Caffeine Standards (A-F) and Unknown Tea Samples (G-H)
Vial Number #
Concentration (ppm)
Area (Counts)
A
50 (ppm)
5495900
B
100 (ppm)
2853700
C
150 (ppm)
20375000
D
200 (ppm)
23934000
E
250 (ppm)
31307000
F
300 (ppm)
33924000
G
unknown
528110
H
unknown
480140
I
unknown
515490
A standard curve of Area vs. Concentration was made in order to analyze our results. The
standard curve could also be used to find concentration in unknown samples using the
equation y=mx+b
Conclusion:
The concentrations of the caffeine samples were determined to be about 8.4ppm, 8.8ppm,
and 8.7ppm. Sample H was decaffeinated and not supposed to have a caffeine peak so
there could have been some contamination. The retention times of the samples did not
exactly match the standards, but they were fairly close. The peaks, however, were
consistently found in the 1.7-1.8 area. The concentration of our unknowns may have been
too diluted, which could have affected retention time.
Introduction
Ion chromatography is a form of liquid chromatography that is used to analyze
ion levels in aqueous samples. It contains a column that separates anions.
Procedure
Day 1: Investigate the operation of the IC by creating standard anion concentrations.
Create a calibration curve using an anion mixture and run the standards.
Day 2: Create a new calibration curve and run standards followed by a variety of water
samples
*Water samples were obtained from the lab, men’s bathroom, and women’s bathroom
Experimental
1. Check Helium tank in gas room. Pressure should be at 80 psi or above.
2. A pressure gauge on the instrument should be receiving helium and reading 9 psi.
Turn knob near the gauge to correct if needed.
3. Regenerant and Eluent bottles on the instrument should be full. If less than half
full they need to be refilled.
a. Gas must be turned off to refill either bottle.
b. Regenerant is made by diluting 1.7mL of sulfuric acid to 2 L volumetric
flask in distilled water.
c. Eluent is prepared by diluting 10mL of the eluent (in a bottle near the 7
ion standard) to 1 L volumetric flask in distilled water.
4. Open Chromeleon program from desktop.
5. From the open program, Click: File→ Open.
a. In the new menu change the file type to control panel. Click the
MyPannels folder and click to open the ICS-90 system.
6. Turn the pump off
7. Turn the small black knob on the pump counter clockwise for 30 sec to purge the
system
8. Turn the pump back on in the program window and continue to purge for 30 sec
9. Turn knob on pump clockwise
10. Stop and let the system equilibrate itself for 20 to 30 minutes
11. After the allotted time, check the hoses below. If eluent collects 1 mL over a full
minute, proceed with procedures.
12. A new sequence is started by clicking, File→ New→ Sequence.
13. Just click next in the box that appears.
14. Injection parameters:
a. Select number of vials to be used
b. State the number of injections of each vial
c. Start position is 2
d. Injection volume is 10μL
e. Click, Apply→ Next
f. Name your sequence with initials and click done.
15. Click Batch Menu→ Edit→ add. There should be a list of sequences, find yours
and click start.
a. A window will appear telling you to inject the sample, do so now till a
small amount drips out into a waste beaker
16. Click OK on the window. Each run will take 16 minutes
17. To open the sequence, click: File→ open→ select sequence→ your sequence
name (with initials)
18. Double click on the 7 anion standard in a window that has the names of your
injections listed.
19. In this new window click on the peak analysis tab
a. Double click on the first positive peak
b. After a box appears, pull the blue sheet from the drawer below and name
the peaks from the sheet.
20. Ensure the instrument has stopped running by opening the batch menu and
clicking stop.
21. Leave Chromeleon program running
22. Check eluent and regenerant levels, close the program by clicking the close button
at the top right of the program window.
Results
Seven Ion Standard Data
Peak 1
Peak Name
Retention Time (min)
1
Fluoride
3.01
2
Chloride
3.60
3
Nitrite
3.93
4
Bromide
4.45
5
Nitrate
4.75
6
Phosphate
5.24
7
Sulfate
5.77
Retention Times (min) of Water Sample Data
Sample Name
Peak 1
Peak 2
Peak 3
Peak 4
Peak 5
Peak 6
Lab Water
2.30 (min)
3.59 (min)
4.76 (min)
5.75 (min)
--
--
Men’s BR
2.28 (min)
3.14 (min)
3.56 (min)
4.72 (min)
5.70 (min)
11.99
Water
The lab water was shown to contain chloride, sulfate, and nitrate. The retention times of
chloride and sulfate appeared to match up with the seven ion standard samples. The
results of the bathroom sample were shown to contain fluoride, chloride, nitrate, and
sulfate.
(min)
Conclusion
Although the results seemed to show what each of the samples contained, we
cannot be sure that these were accurate because there were additional unknown peaks
present in the spectra. These unknown peaks did not match up with any of the known
retention times.