Naomi Bryner
CE and IC Lab
Week of 3/25/13
Introduction:
Ion chromatography (IC) is a separation process for aqueous solutions based on sample charge.
Ionic species of a sample interact differently within the stationary phase as the sample is forced through
a column. These differences between the sample and stationary phase’s level of interaction are due to
the species’ various charges and sizes. The concentration of analyte is detected by UV/Vis absorbance or
conductivity, and the retention time of the each ion is used for identification purposes.
Capillary electrophoresis (CE) operates based on the mass-to-charge ratio of each species within
a sample. This ratio coupled with the electrolyte which fills the capillary and the electric field applied to
the capillary allows for separation of species. Migration time of each species appear as peaks on the
produced electropherogram. Electrolyte flow can be replaced with a buffered gel matrix, allowing for
separation of tiny molecules such as DNA strands or proteins. CE improves significantly upon the speed
and resolution of traditional slab-style gel electrophoresis.
Purpose:
This lab is designed to become familiar with and run an experiment on the CE and IC. The IC will
be used to analyze a seven-anion sample as a standard and various tap water samples as unknowns. The
CE will be used to separate unknown mixtures of the known acids, p-hydroxybenzoic and phydroxyphenylacetic acid.
Procedure:
Ion Chromatography:
1. Turn on helium in gas room and behind instrument
2. Check helium tank pressure (80 psi) and pressure on instrument (9 psi)
3. Ensure that regenerant and eluent bottles are more than half full
a. If less than half full, they must be refilled
4. Equilibrate the instrument
a. After 20-30 min, test flow rate (1 mL/min)
5. Set up the software
6. Run the Sequence
a. Inject sample when prompted to do so
 Run four water samples
 salt water or flavored water can deteriorate the instrument
b. Each run should take 16 min
7. Analyze Peaks
8. Shut Down
a. Select Stop in Batch program
b. Leave Chromeleon running
c. Be sure to turn off the helium knob behind the instrument
Capillary Electrophoresis:
Solution Prep:
1. Capillary Performance Test Mixture B (mixture of p-hydroxybenzoic acid and phydroxyphenylacetic acid)
2. Capillary Performance Run Buffer A (pH 8.35)
3. Capillary Electrophoresis System Solution A (0.1 M NaOH)
4. Distilled water
5. 1.0 M HCl Solution
6. All chemicals should be stored in the chemical refrigerator or under the instrument
Instrumentation:
1. Turn on the instrument, allow for 30 minute warm-up
2. Read Dr. Hu’s SOP while the instrument is warming up
a. Prepare and label chemicals
b. Load buffer and sample vials according to Dr. Hu’s SOP
3. Build Method using Dr. Hu’s SOP
a. Time Program:
i. Rinse 1.0 min (20 psi, forward pressure) from distilled water to Waste
ii. Rinse 2.0 min (20 psi, forward pressure) from 0.1 M NaOH to Waste
iii. Rinse 1.0 min (20 psi, forward pressure) from distilled water to Waste
iv. Rinse 2.0 min (20 psi, forward pressure) from Buffer A to Waste
v. Inject (0.5 psi pressure, 10 seconds) from sample vial (Test Mixture B) to Waste
vi. 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)
vii. Autozero at time 1.50 min
viii. Stop data at 7.0 min
ix. Rinse 1.0 min at time 8.0 min (20 psi, forward pressure) from distilled water to
Waste
4. Save and Run Method
5. Save and print electropherogram with report
6. If you are the last group, exit the program and turn off the instrument
a. Remove all vials, except the buffer vials
7. Load the trays before turning off the instrument
Data:
Capillary Electrophoresis
Area % Report can be found on page 33
Prominent Peaks (min)
Area
Area %
5.308
110826 34.05
5.700
169710 52.15
Ion Chromatography
IC data can be found on page 33
1. Seven Anion Standard
Retention Time (min) Peak Name Amount (ppm)
4.22
Fluoride
1
4.76
Chloride
1
6.59
Nitrite
1
7.61
Bromide
1
9.41
Nitrate
1
10.40
Phosphate
1
13.59
Sulfate
1
2. Water Fountain
Retention Time (min) Peak Name Amount (ppm)
4.77
Chloride
0.779
6.60
Nitrite
0.925
7.62
Bromide
4.139
9.40
Nitrate
4.432
10.38
Phosphate
1.083
13.59
Sulfate
4.937
3. Lab Sink
Retention Time (min) Peak Name Amount (ppm)
4.76
Chloride
0.201
6.59
Nitrite
0.704
7.60
Bromide
0.800
9.37
Nitrate
1.285
10.35
Phosphate
0.719
13.57
Sulfate
1.069
4. Kenmore Refrigerator Filter
Retention Time (min) Peak Name Amount (ppm)
4.78
Chloride
0.692
6.60
Nitrite
0.635
7.61
Bromide
0.551
9.40
Nitrate
0.762
10.39
Phosphate
0.590
13.62
Sulfate
0.745
5. Bathroom Sink
Retention Time (min) Peak Name Amount (ppm)
6.62
Nitrite
0.802
10.41
Phosphate
0.758
6. Brita Filter
Retention Time (min) Peak Name Amount (ppm)
6.59
Nitrite
1.164
10.36
Phosphate
0.749
Conclusions:
On the CE, test mixture B was examined, which was known to be a mixture of p-hydroxybenzoic
acid and p-hydroxyphenylacetic acid. The first peak appeared at 5.308 min, and was the lightest
molecule, p-hydroxybenzoic acid. The second peak appeared at 5.700 min, and was the heavier
molecule, p-hydroxyphenylacetic acid. Percent area is equal to percent composition, so it is known that
there is 34.05% p-hydroxybenzoic acid and 52.15% p-hydroxyphenylacetic acid in text mixture B. On the
IC, a seven anion standard was used to calibrate the instrument. Unknown water samples came from
the water fountain in Campbell Hall, the sink in the Instrumental Lab, the spout of a Kenmore
refrigerator (filtered), the water from the bathroom, and a Brita water bottle filter. It was seen that the
Brita filter and bathroom water had the least contaminants.