The purpose of this paper is to analyze the dissolved ion concentrations of the anions; chloride, sulfate, and nitrate within a natural water sample using High Performance Liquid Chromatography, a specific application of Ion-Exchange Chromatography, as well as explain the mechanisms behind Ion Exchange Chromatography.

1. 1
Laboratory Report
Experiment # 7
Ionic Pollutant Analysis
Group #1
Jonathan Damora
Tuesday April 3, 2014

2. 2
Purpose
The purpose of thisexperimentisto analyze the dissolvedionconcentrationsof the anions;chloride,
sulfate,andnitrate withinanatural watersample using HighPerformance LiquidChromatography,a
specificapplication of Ion-Exchange Chromatography.
Introduction
Data from water analysis and chemical toxicity research throughout the world has
allowed government organizations to create standards of Maximum Contaminant Levels for
individual species in order to minimize health effects from water pollution. There are primary
and secondary standards for water treatment as well as wastewater treatment. Secondary
standards are those allocated to contaminants that currently do not pose a known significant
health risk but can affect the quality of the water, e.g. taste, odor, and appearance. The World
Health Organization uses Guideline Values and Provisional Guidelines. The Provisional
Guidelines indicate a possible long term health hazard,such as carcinogenic effects related to
long term nitrate ingestion, but the current data is limited. In this experiment we will be
analyzing an unknown sample for chlorides, sulfates, and nitrates.
Natural water samples often contain stable low levels of chloride from natural sources.
The relative stability, it rarely reacts with other compounds, makes it useful as a tracer for
groundwater analysis and loss determination, although other specialized compounds are now
preferred. Chloride concentrations above 250 mg/L will start to exhibit a salty taste that most
people find unpleasant to drink. Even the taste of coffee is affected when made with water
exceeding 400-500 mg/L of chloride. There is no data on the acute toxicity of chloride in
humans. Excessive consumption of water containing above 2500 mg/L of sodium chloride has
been shown to cause hypertension, although this might attributable to sodium. It is important

3. 3
to note that chlorination is commonly a necessary drinking water treatment step. The 4th
Edition of the WHO Guidelines sums up the competing factors in setting the chloride MCL; “In
all circumstances, disinfection efficiency should not be compromised in trying to meet
guidelines for DBPs, including chlorination by-products, or in trying to reduce concentrations
of these substances.”
Sulfates are currently not considered to be a toxic component of drinking water, but
many other sulfur compounds are very toxic. The simplest conversion from sulfate to a highly
toxic compound happens when waterhas very low dissolved oxygen concentration,such as
poorly aerated wastewater. Sulfates are reduced to sulfides by bacteria, which results in the
formation of hydrogen sulfide. Hydrogen Sulfide is a noxious chemical that can be lethal in
high doses, although no MCL specifically for sulfide has been established due to the extremely
apparent and unpleasanttaste/odor of water containing sulfides. When the water is well
chlorinated or dissolved oxygen is present, sulfides are rapidly oxidized to sulfates, meaning
controlling conditions can minimize this risk. Sulfates can affect taste at levels above 250 mg/L
of sodium sulfate or 1000mg/L of calcium sulfate, and extremely high levels can cause a
laxative/cathartic effect. The ratio of sulfate and chloride concentration to bicarbonate
concentration,known as the Larson Ratio, is used as an indicator of the corrosiveness of the
water to steel and cast iron pipes, which are common components of our drinking water
distribution infrastructure.
Nitrate is the only species analyzed in our experiment with a primary standard set by
the EPA. It is a common component of surface waters, as well as produced endogenously.
Unfortunately pollution from agriculture, poor/no wastewatermanagement,and industrial
factories increases the level significantly. Since 1980, there has been an increased use of

4. 4
fertilizers in Northern China. According to one study this has resulted in over 50% of the 69
locations analyzed having nitrate concentrations above 50 mg/L (as NO3- - N). Concentrations
up to 300 mg/L (as NO3- - N) were found in groundwater below vegetable producing areas,
farmers’ yards, and population centers. Nitrate is currently being studied for its carcinogenic
qualities, including gastric cancers, although currently the data is inconclusive. Animal studies
have also correlated increased nitrate intake with hypertrophy including thyroid suppression
(goitrogenic effects). There is increased danger from nitrates, as it is reduced to nitrite by the
autotrophic bacteria within your body, from exposure to microbial contaminants or gastric
illness. Nitrite that gets into the blood will oxidize the Fe2+ in haemoglobin to Fe3+ which binds
with the remaining nitrite to form methaemoglobin. Methaemoglobin binds with oxygen too
strongly to release it, thus reducing the bloods ability to transport oxygen and suffocation
eventually occurs. Nitrates pose the largest health risk to bottle fed infants, with levels above
100 mg/L (as NO3- ) being associated with increased risk of Methaemoglobinaemia and
cyanosis, a.k.a. blue baby syndrome. There are also indications that Nitrates can contribute to
bladder cancer in women.
Laboratory techniques used to separate mixtures into their various constituents is
referred to collectively as chromatography. Ion-Exchange Chromatography (IC) is one of the
most sophisticated method available for dissolved ion analysis of both water and air samples.
The specific technique applied during this experiment is known as High Performance Liquid
Chromatography (HPLC), which is an application of Liquid Chromatography and Ion-
Exchange Chromatography. The difference between standard liquid chromatography and
HPLC is the pressure at which the sample is pumped through the column. HPLC uses high
pressure to pump the solution through the column, causing much faster adsorption of analyte

5. 5
ions compared to the lower pressure used in standard LC. HPLC equipment and techniques are
used to separate and individually analyze ion concentration of a water sample. The process by
which the ions are analyzed is complex and equipment varies according to analyte
characteristics, e.g. cation or anion.
HPLC is based on the adsorption of ions by an ion exchange resin contained within the
column of the HPLC instrument. A solution is pumped under high pressure through a column
(3-5 mm in diameter and 15 cm in length) containing a monolayer of small (100-300 nm in
diameter) polymeric beads electrostatically bonded to a neutral polymeric core (10 micrometers
in diameter). For this experiment, in order to determine anion concentration, the polymeric
beads are coated in a resin containing quaternary amines that retain the anions according to the
reaction below.
xRN(CH3)3+ OH- + Ax- ↔ [RN(CH3)3+]Ax- + xOH-
As the analyte passes through the column all anions are adsorbed completely by the
resin and held in place. The adsorption occurs rapidly, resulting in the ions being concentrated
near the head of the column. This completes the separation of the ions from the rest of the
solution, which is discarded. Desorption occurs when a strong base solution, referred to as
eluent, is pumped through the column, again at high pressure. Desorption is a result of an
excess of hydroxyl ions within the eluent, the resin preferentially desorbs analyte ions in
exchange for hydroxyl ions. The eluent, consisting of NaHCO3 and Na2CO3 in this experiment,
causes the above reaction to be reversed completely, releasing the ions back into solution.

6. 6
As the ions continue down the column they are subjected to continual adsorption and
desorption with the ion-exchange resin, which affects the velocity at which the ions move
through the column. The differences in adsorption affinity for each ion, as well as the
differences in diameter of the ions, results in a distinct separation between the species by the
end of the column. The concentration of each individual species can then be determined, since
the smaller diameter ions, and/or the ions with lower partition ratios, move down the column
faster than the larger diameter ions, and/or ones with higher partition ratios. For example, Cl-
appears on the chromatogramat approximately 1.25 minutes. Thus, Cl- will always appear at
approximately 1.25 minutes using the same equipment, and the only possible interference is
another anion appearing around the same time or if another anion has a large enough result to
combine parts of 2 separate peaks.
The ion concentration is usually determined by spectrophotometry or conductivity
analysis. The equipment used in this experiment determines analyte concentration using
conductivity measurements, the data is then presented as a graph showing peaks as each
species passes through the detector. This graph can be seen in Figure 1. The effect of the eluent
on conductivity is a source of interference, but the innovation of using a suppression column to
minimize conductivity of the eluent prior to analysis eliminates this interference. The small
amount of conductivity of the eluent alone, after suppression, is the baseline value shown on
the graph. The concentration of an analyte is directly related to the area under the curve of its
peak, therefore a calibration curve of standard solutions will allow you to convert the area given
into a concentration.

7. 7
Procedure
Firstwe prepared solutionsof 0.2,1, 3, 5, 10 mg/L of Chloride,Nitrate,andSulfate using astandard
solutionof 1000 mg/L of the saltsand an intermediate solutionof 100 mg/L.Thiswas preparedby
mixing1000 mL of DDI waterwithsaltsdriedtoconstantweightat 105 degreesCaccording tothe table
below.
Table 1. Standard Solution Preparation
Anoin Salt Amount (g/L)
Cl-
NaCl 1.6485
NO3
-
NaNO3 1.3707
SO4
2-
K2SO4 1.8141
These standard solutionswere analyzedbyHPLCinorderto developcalibrationcurvesrelatingthe area
underthe peakto concentration. Touse the HPLC we neededtofilterthe solutions,priortoinjecting
themintothe loop,inorderto eliminate anyparticulates whichcouldseverely damage the HPLC
equipment. We filtered20 mL of each solutionusinga0.22 micrometerfiltertip,thenflushedthe
sample loopwith5 mL of the solutiontwice. Finally, we injected2-4mL of solutionintothe loopand
programmedthe machine tobeginanalysisandrecordthe graph of the results. Thisprocesswas
repeatedforeachconcentrationof standardsolutionwe created,aswell asforthe unknownsolution. It
isimportantto ensure yourpeaksare distinctandseparate,astwo peaksoverlappingwillcause anerror
inthe analysisof bothspecies.

8. 8
Figure 1. Chromatogram
Results
Table 2. HPLC Data Table 3. Unknown Sample Species Concentrations
IC Sample
Concentration
(mg/L)
Cl-
NO3
-
SO4
2-
(Area Under Curve)
0.2 1,149,010 564,493 1,250,301
1 6,191,345 2,473,502 4,373,998
3 12,970,256 6,040,347 9,598,963
5 22,874,267 11,169,752 17,333,601
10 46,749,040 22,924,256 33,959,085
Unknown
Sample
13,475,992 7,242,783 10,452,095
IC ResultAnalysis
Cl-
NO3
–
(NO3
-
- N)
SO4
2-
UnkownSample
Concentration
(mg/L) 2.695* 3.62 3.48
Chloride 1.25 min
Nitrate 2.28

9. 9
y = 5E+06x
R² = 0.9972
0
5,000,000
10,000,000
15,000,000
20,000,000
25,000,000
30,000,000
35,000,000
40,000,000
45,000,000
50,000,000
0 2 4 6 8 10 12
AREAUNDERTHECURVE
CONCENTRATION (MG/L)
Cl- Calibration Curve
y = 2E+06x
R² = 0.9977
0
5,000,000
10,000,000
15,000,000
20,000,000
25,000,000
0 2 4 6 8 10 12
AREAUNDERTHECURVE
CONCENTRATION
NO3- Calibration Curve
Figure 2. Calibration Curve for Chloride
Figure 3. Calibration Curve for Nitrate
Figure 4. Calibration Curve for Sulfate

10. 10
Discussion
Using a linear regression of the data in Table 1 I was able to determine the concentration
of the three species within the unknown sample. The one anomaly present within the data is
the concentration of chloride within the sample, marked with an *. The reason this is an
unexpected, possibly erroneous, result is that at a concentration of 3 mg/L of Chloride the
HPLC gives a resulting area of 12,970,256. The unknown sample resulted in an area of
13,475,992 for chloride, thus the concentration of chloride should be above 3 mg/L. The linear
regression used to create the calibration curve gives a concentration of 2.7 mg/L, which is
below 3 mg/L. Using a 3rd order polynomial regression I obtained a concentration of 3.04 mg/L
for Chloride, even though the relation between area and concentration should be linear. It is
unclear exactly what this unexpected datum results from, but possibilities include human error
in standard solution preparation, errors in data collection,equipment malfunction, and
contamination. My recommendation would be to repeat the experiment to see if the anomaly
repeats itself.
y = 3E+06x
R² = 0.9974
0
5,000,000
10,000,000
15,000,000
20,000,000
25,000,000
30,000,000
35,000,000
40,000,000
0 2 4 6 8 10 12
AREAUNDERTHECURVE
CONCENTRATION
SO42- Calibration Curve

11. 11
Drinking water standards are usually presented as Maximum Contaminant Level
(MCL), meaning any water with a concentration above the stated maximum level does not
comply with the regulation. California imposes a primary standard MCL of 10 mg/L (as NO3- -
N) on Nitrates due to the significant health risks of drinking water with high levels of nitrates.
The World Health Organization standards state the MCL for nitrate at50 mg/L (as NO3-) which
translates to approximately 11.4 mg/L (as NO3- - N). Additionally the WHO states a
Provisional Guideline of 0.2 mg/L of Nitrate (as NO3- - N).
The unknown sample complies with the nitrate MCL from US Drinking Water
Standards as well as WHO standards, including the Provisional Guideline of 0.2 mg/L
expressed as (as NO3- - N). The data shows the nitrate concentration of our unknown sample is
3.6 mg/L (as NO3-), which is equivalent to 0.82 mg/L (as NO3- - N). The MCL in California for
Chlorides (250 mg/L) and Sulfates (250 mg/L) are secondary standards, meant to provide a
guideline for water treatment but not to impose regulation or enforcement of these standards.
Levels higher than the MCL will begin to noticeably affect the cosmetic quality of the water.
Our sample was well under the stated MCLs for all three anions present. From these results,
the unknown sample is suitable for drinking water, although there might be cationic
contaminants.
DiscussionQuestions
1. Discuss the significance of high chloride concentration in watersupplies.
a. Natural water samples often contain stable low levels of chloride from natural
sources. The presence of unusually high chloride concentrations in water can
indicate fecal contamination. Chloride is excreted by humans in stable
concentrations above natural levels, thus, an unusually high chloride

12. 12
concentration in a water sample can indicate toxic conditions. For this reason, it
is used as an indicator for contamination of watersupplies. It is difficult to
notice concentrations below 250 mg/L, but any increase in chloride
concentration leads to an increase in corrosivity of the water as well as an
increase in concentration of metals in drinking water. Secondary effects include
faster galvanic corrosion of lead pipes, increased pitting corrosion of metal pipes,
deterioration of concrete, and scaling on water heaters (due to the large variation
in calcium chloride solubility depending on temperature).
2. Why has a secondary standard for chloride in drinking water been set by the U.S. EPA
and the WHO, and what is the recommended value?
a. A secondary standard has been set by the US EPA, as well as a provisional
guideline by the WHO, at 250 mg/L of chloride. The standard has been created
due to the significant health effects of some Disinfection By Products (DBP)
resulting from chlorination or other drinking water treatment options involving
chlorine. DBPs include Trihalomethanes and Haloacetic acids, such as the
carcinogens chloroform and bromoform. The reason the MCL is considered
secondary or provisional arises from the lack of data on the long term health
effects due to consumption of excess chloride. There are other sources of
chloride, and thus DBPs, according to the CDC, hot showers are responsible for
more DBP ingestion than drinking water. Swimming pools even contain DBPs,
e.g. Urea from sweat and urine react with the chlorine to create trichloramine
both in the water and in the air above the pool. The gaseous tricholoramine
causes the distinct smell of indoor pool rooms and also is attributed to an
increase in asthma among elite swimmers. To ensure quality control, treatment

13. 13
plants in the US distributing drinking water above the MCL must notify their
customers. In the US, treated water should contain at least 0.2 mg/L of chloride
throughout the distribution system.
3. What is the significance of high-sulfate concentration in watersupplies and in
wastewater disposal?
a. High sulfate concentrations in groundwaterare often naturally occurring,
although atmospheric deposition from industrial pollution and contamination
through industrial wastewater also occurs. Sulfate concentration is directly
related to the corrosivity of the water, meaning the rate of iron corrosion and
degradation of cement. The ratio of sulfate and chloride concentration to
bicarbonate concentration, known as the Larson Ratio, is used as an indicator of
the corrosiveness of the water to steel and cast iron pipes, which are common
components of our drinking water distribution infrastructure. When the water
has very low dissolved oxygen concentration,such as poorly aerated wastewater,
sulfates are reduced to sulfides by bacteria, which results in the formation of
hydrogen sulfide. Hydrogen Sulfide is a noxious chemical that can be lethal in
high doses, although no MCL specifically for sulfide has been established due to
the extremely apparent and unpleasant taste/odor of water containing sulfides.
When the water is well chlorinated or dissolved oxygen is present, sulfides are
rapidly oxidized to sulfates. Sulfates can affect taste at levels above 250 mg/L of
sodium sulfate or 1000mg/L of calcium sulfate, and extremely high levels can
cause a laxative/cathartic effect. In livestock, mainly ruminant animals, studies
have shown a correlation between high sulfate intake and various neurological
diseases.

14. 14
4. What analytical methods are available for the analysis of sulfate?
a. The most accurate method available is Ion Chromatography, this method is
described above and used in this experiment. IC has a variable minimum
detection limit depending on the instrument, the various components chosen,
and sample preparation but it is at least 0.003 mg/L or below. The other
methods available have much higher minimum detection limits, are usually
much more labor intensive, are subject to interferences thus require additional
analysis of the constituents of the sample, and the accuracy is highly dependent
on skill. Sulfate concentrations above 10 mg/L can be determined
gravimetrically by adding barium chloride to precipitate barium sulfate.
Turbidimetric detection of barium sulfate precipitate using a spectrophotometer
at 420 nm or turbidimeter which can detectconcentrations down to 1 mg/L.
Although interferences are possible, so other sources of turbidity must be filtered
out. The second most accurate method is colorimetric, with some papers
claiming to be able to detect concentrations down to 0.1 mg/L, although this is
highly dependent on skill and interference is possible depending on the color
compound chosen.
5. What is the health effect of nitrate in drinking water and what is the MCL set by the
EPA?
a. Nitrate itself is currently being studied for its carcinogenic qualities, including
gastric cancers, although currently the data is inconclusive. Animal studies have
also correlated increased nitrate intake with hypertrophy including thyroid
suppression (goitrogenic effects). There is increased danger from nitrates, as it is
reduced to nitrite by the autotrophic bacteria within your body, from exposure to

15. 15
microbial contaminants or gastric illness. Nitrite that gets into the blood will
oxidize the Fe2+ in haemoglobin to Fe3+ which binds with the remaining nitrite to
form methaemoglobin. Methaemoglobin binds with oxygen too strongly to
release it, thus reducing the bloods ability to transport oxygen and suffocation
eventually occurs. Nitrates pose the largest health risk to bottle fed infants, with
levels above 100 mg/L (as NO3- ) being associated with increased risk of
methaemoglobinaemia and cyanosis, a.k.a. blue baby syndrome. There are also
indications that Nitrates can contribute to bladder cancerin women. Nitrite is
the species that causes methaemoglobinaemia and the MCL is much lower at 3
mg/L, signifying the higher toxicity of nitrites compared to nitrates.
Nitrosamines, resulting from nitrites reacting with secondary amines, are known
carcinogens. The LD50 for nitrates in rats is 1072-6030 mg (of NO3-) /kg of body
weight, although ruminant animals are much more susceptible at 301.5 (of NO3-)
mg/kg of body weight. The EPA nitrate MCL is 10 mg/L (as NO3- - N), which
relates to our data this way; 4.4 mg/L (as NO3- ) = 1 mg/L (as NO3- - N). So the
EPA MCL for nitrates can also be stated as 44 mg/L (as NO3-). The EPA MCL for
Nitrites is 1 mg/L, but there is an additional requirement, the total nitrate-nitrite
concentration cannot be more than 10 mg/L. Thus if you have 1 mg/L of nitrite
you cannot have more than 9 mg/L of nitrate. The World Health Organization
standards state the MCL for nitrate at 50 mg/L (as NO3-) which translates to
approximately 11.4 mg/L (as NO3- - N). Additionally the ratio of actual value to
guideline value of nitrates and nitrites must be less than 1.
 
 
 
 
1
/3/50

Lmg
Nitrite
Lmg
Nitrate

16. 16
6. What methods are available for analysis of nitrate?
a. UV Spectrophotometry is currently a standard method used to analyze nitrate
concentration using 220 nm light. The process is simple, fast, and does not use
any reagents but there are many interferences, such as nitrite, hexavalent
chromium, and various organic compounds. Techniques for direct
electrochemical detection of nitrates are split between Amperometric methods,
offering continuous detection (monitoring), and Potentiometric methods,
involving ion-selective electrodes. Amperometric methods can be applied to
nitrate but are mainly used for nitrite detection. Potentiometric techniques have
been highly developed over the last three decades, continually minimizing the
negative aspects. The problems associated with direct electrochemical methods,
e.g. ion-selective electrodes, include low selectivity of ions, poor stability, and
short instrument lifetime. In general, the standard potentiometric nitrate
electrode method has an MDL of 0.2 - 1 mg/L and gives results rapidly with little
to no pretreatment of the sample. Photometric methods detect only nitrite and
involve reducing nitrate to nitrite, allowing distinction between the two by
comparing results without reducing nitrate to nitrite. The method offers very
high selectivity, meaning minimal inferences from other compounds, and
extreme sensitivity, with one method developed by Motomizu et. al allowing
continuous nitrite detection in seawater with concentrations down to 1.4 ng/L
(as NO2- - N) or 0.0000014 mg/L

17. 17
Conclusion
Ionic analysis of water is of utmost importance for human health. The increased
efficiency of combined and consolidated water distribution systems also greatly increases the
risks from contamination and other toxic effects.It can be very difficult to determine whether
any of the many toxins have infiltrated a water supply, therefore water analysis has been
continually improved and refined throughout history. Ion-Exchange Chromatography has
recently emerged as a reliable as well as adaptable standard method for water analysis. The
other methods available to test ion concentration are, for the most part, cheaperto perform than
IC but subject to interferences. Also, many techniques only analyze one species in a longer
amount of time. This means that you must know your water contains a certain chemical prior
to testing for the level of it. IC has minimal interferences and allows you to individually
analyze the species of a complex solution in one process for anionic species and one for cationic
species. Since many common water pollutants are anionic, the process is simplified further.

18. 18
Works Cited
"Chloride in Drinking-water." Background Document for Development WHO Guidelines for
Drinking-water Quality. WHO/SDE/WSH, 03 Apr. 2003. Web. 4 Apr. 2014.
<http://www.who.int/water_sanitation_health/dwq/chloride.pdf>.
"Disinfection By-Products." Centers for Disease Control and Prevention. Centers for Disease
Control and Prevention, 21 Mar. 2012. Web. 04 Apr. 2014.
<http://www.cdc.gov/safewater/chlorination-byproducts.html>.
"Guidelines for Drinking-water Quality THIRD EDITION." Water Sanitation Health. WHO, 2008.
Web. <http://www.who.int/water_sanitation_health/dwq/fulltext.pdf>.
L'Hirondel, J., and J-L L'hirondel. Nitrate and Man: Toxic, Harmless or Beneficial? Wallingford:
CABI, 2001. Print.
"HPLC" Wikipedia. Wikimedia Foundation, 10 Mar. 2014. Web. 11 Mar. 2014.
"Ion-Exchange Chromatography" Wikipedia. Wikimedia Foundation, 10 Mar. 2014. Web. 11
Mar. 2014.
"National Primary Drinking Water Regulations." Drinking Water Contaminants. United States
Environmental Protection Agency, May 2009. Web. 04 Apr. 2014.
"Nitrate and Nitrite in Drinking-water." Background Document for Development of WHO
Guidelines for Drinking-water Quality. WHO/SDE/WSH, 16 Jan. 2007. Web.
<http://www.who.int/water_sanitation_health/dwq/chemicals/nitratenitrite2ndadd.pd
f>.

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Nollet, Leo M. L. Handbook of Water Analysis. New York: Marcel Dekker, 2000. Print.
Skipton, Sharon O., and Bruce I. Dvorak. "Nitrate-Nitrogen." Drinking Water:. University of
Nebraska–Lincoln Extension Publications, Dec. 2013. Web. 04 Apr. 2014.
<http://www.ianrpubs.unl.edu/pages/publicationD.jsp?publicationId=971>.
"Sulfate in Drinking-water." Background Document for Development WHO Guidelines for
Drinking-water Quality. WHO/SDE/WSH, 03 Apr. 2003. Web. 4 Apr. 2014.
<http://www.who.int/water_sanitation_health/dwq/chemicals/sulfate.pdf>.
Snoeyink, Vernon L., and David Jenkins. Water Chemistry. New York: Wiley, 1980. Print.