Chemistry 370

Determination of Anions in Water by Ion Chromatography

Propose
The purpose of this experiment is to determine chloride, nitrite, nitrate, phosphate, and
sulfate in water samples.

Introduction
Nitrate and phosphate serve as the major anion nutrients in water. Increasing the nitrate
or particularly the phosphate levels can lead to stream eutrophication. Nitrite is not stable for
long periods in natural waters since it can be oxidized to nitrate. However, nitrite is toxic to fish
and therefore a buildup can be a cause of fish-kills. In the nutrient-poor water of western North
Carolina the levels are usually less that 1 ppm unless unusual human activity has raised the levels
(fertilization, waste water spills, etc.). Chloride is like sodium in that man’s activities have
moved it everywhere in our water systems. It is hard to find a natural water sample without
measurable levels of Cl-. Sulfate can come from mineral erosion but in our part of the country it
usually comes from man’s activities.
Ion chromatography is similar to HPLC in many ways: a high-pressure pump, an injector
(with injector loop), and a chromatographic column are used. The column is an ion-exchange
column with a fixed number of cationic sites that hold anions with varying tenacity. The force of
attraction is based on the charge on the anion as well as the size of the hydrated anion. The pH
of the eluting solution also plays a major role, since protonation of the anions of weak acids
changes their ionic charge. The eluting solvent is capable of displacing the anions of interest and
moving them along in the column. The anions elute from the column with the least attracted
coming off first. From the column the anions go to the suppressor and then on to the detector,
which is based on the conductivity of the solution. The suppressor is a unique system used to
remove most of the conductivity caused by the eluting solution while enhancing the conductivity
caused by the anions of interest.
The Dionex DX-120 is a computer-controlled instrument with software for peak
integration and calibration curve preparation. Once the calibration curve has been established,
quantitation of samples is automatic. In real samples the peaks corresponding to chloride, nitrite,
nitrate, phosphate, and sulfate are labeled, and the software determines the concentration. In real
world samples there are always peaks for components other than chloride, nitrite, nitrate,
phosphate, and sulfate. These components could also be quantified with appropriate calibration
data; however, they are of little interest in our study.

Procedure

Sample Collection
Obtain a water sample from a nearby stream and also one from the aquaculture lab on the
ground floor of the Natural Science Building. You will need only a few milliliters for the actual
analysis, so 25 mL should be plenty. If you cannot analyze the sample the same day you collect
it, the sample should be frozen until just before you begin your analysis. If you plan to freeze

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your samples you must use plastic collection containers. Half-full containers work better than
full containers.

Calibration and Analysis

Before you set up the values for the calibration you should run the Anion370 schedule to
see approximately where your unknowns fall. Place a blank (D.I. water) in vial 1, your unknown
in vial 2, and the fish lab water in vial 3. Place the auto-sampler cartridge in to auto-sampler and
press the hold/run button on it once. Start PeakNet and then select run. The run screen will
come up with HPLC on top and Anions & Cations under it. Click on the top bar of the Anions &
Cations box to put it on top. Under file select load schedule and then Anion370. This is a broad
range calibration curve but it does have the correct setting for operating the instrument. This will
make sure the pump and detector are running correctly. Don’t start the instrument for 20
minutes. This allows the instrument and column time to come to equilibrium. After 20 minutes
the instrument is ready for you to inject your sample. Click run to start the schedule. After you
obtain your results select the values for your calibration curve so you bracket both samples. For
example, if your results indicate 0.07 ppm PO43- for the creek and 2.5 ppm NO3- for the fish lab
your calibration curve should go from 0.05 to 3.00 for all five components. Prepare 5 standards
using the 1000 ppm stock solutions. All five components will be in the same solution and
therefore all five calibration curves will be prepared simultaneously. These standards are 1000
ppm N, not NO3-, etc.
To enter your standard values into the program close the run window and open the
methods window. Open the Anion370 method and immediately save it as Anion(your initials)
(i.e. AnionJRB). There are 8 boxes at the bottom of the run window. Select the 6th box, which
will open another window. Select Calibration Standards (middle tab) and yet another window
will open. This has a place for your new standards. You must replace the values in all 25
locations. Be sure to start with the lowest concentration and go up in concentration going right.
After you have entered all the standard values resave the method and exit the methods window.
You must prepare a schedule to run your calibration curve and your unknowns. Open the
schedule window and then open Anion370. Save it immediately as Anion(your initials). In the
schedule you need to insert 5 line between blank and your unknowns. Put in the values for your
five standards. You also need to change the method from Anion370.met to Anion(your
initials).met for every line except the last line. Make sure the stop-an.met is in the last line.
Return to the run part of the program. Click to put Anions & Cations on top. Load your
new schedule AnionInit.sch. The auto-sampler will inject your five standards starting with the
lowest concentration. Put in your initials for the Sample and under Sample Type select
Calibration Std. where it normally says sample, select level 1 for the first standard, level 2 for the
second standard, etc. You can ignore the Operator box or you can replace my initials with
yours. After you finish all of your standards open methods again and double click the 6th box.
Open the Calibrations Standards window and the click detail (lower right). You will be able to
see your calibration curves. If they look good print all of them on two sheets. If not, repeat any
"bad" samples. Return to the run part of the program. Run the samples and the printout should
have your results on it.

Waste Management
None of the anion solutions we are using are toxic at the levels used in this experiment.
All waste solutions may be poured into the sink.

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Data Analysis Include the unknown chromatograms, the calibration curves, and sample
calculations for your dilutions. What is the concentration of your samples in terms of ppm NO3 -,
NO2-, PO43-, and SO42- (Remember, the calibration standards were expressed in ppm N, P, or S)

Additional Problems

1. The eluting solvent is 1.0 millimolar in NaHCO3 and 3.5 millimolar in Na2CO3.
Calculate the pH of the solution.

2. At the pH calculated, what is the fraction of phosphate in the PO4-3 form? Hint:
remember the ? calculations. Which phosphate species H3PO4, H2PO4-, HPO4-2, or PO4-3 is the
dominant species at the pH of the analysis?

3. Of the ions we are studying, which is most affected by the pH of the eluting solvent
and why? Which anion is effected least and why?

4. Go to the Dionex web site (www.dionex.com) and find out how the ionization
suppressor works. Explain it in your own words.

NOTE: Since the computer does most of the work for you and this is a short report you
should pay close attention to the questions and the math associated with your dilutions.