Brittany Hicks
IC & CZE Lab- Dr. Foy
Spring 2013
Ion Chromatography and Capillary Electrophoresis
Introduction
Ion chromatography is a specific type of liquid chromatography. The main purpose of this
chromatography is to analyze aqueous solutions for specific ion levels. Using a column, the IC separates
different anions. As seen before in our liquid chromatography lab, the IC uses a liquid mobile phase and
also a stationary phase which is either liquid or a solid. The plate height should be minimized and
number of theoretical plates maximized under analysis for best results.
Capillary Electrophoresis is able to separate ions because of their mobility within an electric
field. Charge to mass rations and the attraction to additives in the background both help in aiding in
determining the mobility of a specific substance. The most commonly used samples within the CZE are
proteins and enzymes.
Procedure – IC





Before operating the instrument, check the helium tank in the gas room
o If tank pressure is below 80 psi, let lab assistants and/or instructor know
 If pressure is below 40 psi, the tank must be replaced
Turn the helium gas on using the knob behind the instrument
o The pressure gauge should read 9 psi
 If the pressure is not near 9 psi, turn the black knob in front of the gauge in the
direction the needle needs to move
Check the Regenerant and Eluent bottles on the instrument
o If either bottle is less than half full, refill using containers near the instrument or make
more (regenerant should be replaced weekly)
 To make more eluent, use 10mL concentrated eluent and dilute to 1L with
distilled water
 To make more regenerant, use 5mL 6M sulfuric acid and dilute to 2L with
distilled water
Open the Chromeleon program from the icon on the desktop
Equilibrate the instrument
o In Chromeleon, choose File>Open
o Change the file type to Control Panel and the folder to MyPanels
o Open ICS-90 System
o Turn the pump off in the program
o Turn the small black knob on the pump to the left (one turn only)




Do not touch the knob that says DO NOT TOUCH
Let the system purge for 30 seconds
 Make sure that eluent is flowing into the waste jug below the counter
o Turn the pump on in the program, but leave the knob alone
 Let the system purge for another 30-45 seconds
 Make sure that eluent is dripping into the waste jug below the counter
o Turn the knob on the pump back to the right, but do not over-tighten
o Let the system equilibrate for 20-30 minutes
o Check the flow of the eluent by using a graduated cylinder to collect the waste
 There should be graduated cylinders below the counter with the waste jugs
 If the instrument is equilibrated, the flow should be ~1mL/1min
 If the flow is less than ~1mL/min, wait another 10-15 min and check
again
 If there is less than ~1mL/min, and the instrument has been
equilibrating for 40-60 min, find the experts, lab assistants, or Dr. Foy
Setting up the software
o Create a new sequence by clicking File>New>Sequence (From Wizard)
o Once the wizard opens, click Next twice
o Under the Specify Unknowns screen, adjust the number of vials (if you are not running
unknowns, this number is zero), the title format of the unknowns (i.e. Campbell Hall
Water), the number of injections per vial (typically one), the start position (if you are
running one standard, the unknown’s position would be 2), and injection volume
(should always be 10 μL)
o Click Apply, then Next
o Under the Specify Standards screen, adjust the number of vials (should always be
running one 7-anion standard), the title format (i.e. Seven Anion Standard), the number
of injections per vial (typically one), the start position (should always be 1), and injection
volume (should always be 10 μL)
o Click Apply, then Next
o Under the Methods and Reporting screen, change nothing and click Next
o Under the Saving the Sequence screen, choose a name for your sequence and a title for
it (top two boxes; ensure that these have your initials), but leave the bottom two boxes
unchanged)
o Click Finish
o The sequence should now be in your window
Running the Sequence
o Click Batch>Edit>Add Sequence and choose your sequence
o Click Start
o The software will prompt you to inject your first standard
o Fill a sterilized syringe halfway with your standard, then press it onto the injector
(ensure that it is fully on the injector)
o
o
o


Slowly inject the sample until you see 4-5 drops come out into the waste beaker
Click OK in Chromeleon
This scan should take 16 minutes
 If it does not, consult the experts, lab assistants, or Dr. Foy
 If there are no peaks after 16 minutes, your sample was not injected properly
 If there are no peaks after several injections and/or the conductivity is
very high, replace the regenerant
Naming Peaks
o Open your sequence
 File>Open
 File type should be Sequence
 Select your sequence
o Double click 7-anion standard in the dialogue box that lists the names of your injections
o Find the Peak Analysis tab
o Double click the first positive peak
o A dialogue box will appear
 Name each peak according to the sheet in the drawer which shows a spectrum
of the standard, then press Enter
 There is no peak for the acetate ion
Shutting Down
o In the Batch menu, select Stop
o Leave Chromeleon running (the system will go into low flow after 6 hours of inactivity)
o Fill the eluent to the top
o If the waste container is full, move the tube into a new container and do the following:
 Put a cap on the full waste container
 Bring the full container to the prep room using the elevator
 Neutralize the waste in a large beaker on a stir plate with magnetic stir bar using
20 mule team borax (located in storage area, consult lab assistants or Mrs.
Mowery if you need help)
 Use universal indicator to check the pH (the color should change from pink to
green)
 Flush the waste down the drain with plenty of water
o Be sure to turn off the helium knob behind the instrument
Lab Procedure:




Run four different water samples
o Do not run salt water or flavored water as this will harm the instrument!
Run two samples per day
Run two trials of each sample
Follow the Standard Operating Procedure
o Day 1:

o
Unknown samples should be 2 vials and 2 injections per vial
 The first injection is the standard, followed by 2 injections of each
sample
Be sure to use the same standard for both days

Day 2:
 Same procedure as Day 1
 Make a calibration curve of your samples
 Plot ppm vs. height and area
Ask experts, assistants, or Dr. Foy if you have any questions
Procedure – CZE
Chemicals Required:






Capillary Performance Test Mixture B (mixture of p-hydroxybenzoic acid and phydroxyphenylacetic acid)
Capillary Performance Run Buffer A (pH 8.35)
Capillary Electrophoresis System Solution A (0.1 M NaOH)
Distilled water
1.0 M HCl Solution
All chemicals should be stored in the chemical refrigerator or under the instrument
Standard Operating Procedure:





Turn on the instrument
Let it warm up for 30 minutes
Read the Dr. Hu’s SOP while the instrument is warming up
Prepare chemicals
o Use five 1.5-mL vials
o Add 1.4 mL each of Run Buffer A (x2), Solution A, distilled water, and 1.0 M HCl
 Use P1000 micropipette and dispense two quantities of 700 μL for each solution
o Do not overfill the vials
o Cap these vials using the red caps from the drawer
o Cap 2-3 empty glass vials to be used for waste
o Use a plastic vial for your sample
 Fill with ~1mL of Test Mixture B
 Cap using blue cap from the drawer
Load buffer and sample vials according to Dr. Hu’s SOP
o

Direct Control>Load
 Wait until buffer trays are sent to the front, lift the cover, and insert vials as
follows:
 BI:A1 – 0.1 M NaOH
 BI:B1 – 1 M HCl
 BI:C1 – distilled water
 BI: D1 – Buffer A
 BO:A1 – Waste
 BO:B1 – Waste
 BO:C1 – Buffer A
 SI:A1 – Test Mix B
 SO:A1 – Waste
 Record the position of each vial in your lab notebook
Build Method
o Use Dr. Hu’s SOP for more details on how to build method
o Initial Condition: Leave all defaults
o PDA Detector Initial Conditions: Use Channel 1 (check both boxes) at 214 nm, uncheck
Acquisition Enabled (top left quadrant)
o Time Program:
 Rinse 1.0 min (20 psi, forward pressure) from 1.0 M HCl to Waste
 Rinse 1.0 min (20 psi, forward pressure) from distilled water to Waste
 Rinse 2.0 min (20 psi, forward pressure) from 0.1 M NaOH to Waste
 Rinse 1.0 min (20 psi, forward pressure) from distilled water to Waste
 Rinse 2.0 min (20 psi, forward pressure) from Buffer A to Waste





Inject (0.5 psi pressure, 10 seconds) from sample vial (Test Mixture B) to Waste
Separate at 0.0 min (Duration: 7 min, Voltage: 25 KV, Ramp: 0.17 min) from Run
Buffer A (BI) to Run Buffer A (BO)
 Autozero at time 1.50 min
 Stop data at 7.0 min
 Rinse 1.0 min at time 8.0 min (20 psi, forward pressure) from distilled water to
Waste
o Be sure to use the SO waste for the injection and be sure to use both of the BO waste
vials throughout the time program
Save and Run Method
o Use Dr. Hu’s SOP for more details on how to do this
Save and print electropherogram with report
o PRINTER MAY BE DOWN! SEE EXPERTS, LAB ASSISTANTS, OR DR. FOY
If you are the last group, exit the program, remove your vials and turn off the instrument
o FOR INSTRUMENT SHUT DOWN:
 Be sure to leave a buffer vial in the buffer inlet and buffer outlet trays
 Choose to inject the tray
 Allow the tray to inject, and turn off the instrument
 This ensures that the capillary will not dry out
Data Analysis:



Use peak area to calculate the percentage of each compound
We did not use standard to determine migration time of each benzoic acid derivative
Can you predict which is p-hydroxybenzoic acid and which is p-hydroxyphenylacetic acid?
Ask experts, assistants, or Dr. Foy if you have any questions
Data and Analysis
Unfortunately, this is the data results for the CE. As you can see, the mixture results are not very
good. We kept receiving error messages from the CE and they stated that the pressure was wrong. This
kept delaying the experiment and the instrument would sporadically stop running.
We got better results for the IC, which all the spectra containing the peaks, retention time, and
heights and relative areas. This data is all included within my lab notebook for further analysis. For all of
the spectra there are clear injection peaks along with distinguishable peaks.
Conclusion
This lab was not as fun as the other labs that have been done this year, however we did get
good results for the IC. The CE did not give as good as results due to the constant pressure error and
sporadic stopping of running the sample. The peaks showed good height, retention time and injection
points which showed that we were running the experiment right. I don’t know why the separation did
not work for mixture B that we did.