Laboratory Report

Experiment # 7

Ionic Pollutant Analysis

Group #1

Jonathan Damora

Tuesday April 3, 2014

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Purpose

The purpose of this experiment 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.

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

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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 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 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,

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

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wastewater management, and industrial factories increases the level significantly.

Since 1980, there has been an increased use of 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

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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 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,

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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.

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 chromatogram at 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

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under the curve of its peak, therefore a calibration curve of standard solutions will

allow you to convert the area given into a concentration.

Procedure

First we prepared solutions of 0.2, 1, 3, 5, 10 mg/L of Chloride, Nitrate, and Sulfate using a standard

solution of 1000 mg/L of the salts and an intermediate solution of 100 mg/L. This was prepared by

mixing 1000 mL of DDI water with salts dried to constant weight at 105 degrees C according to the table

below.

Table 1. Standard Solution Preparation

Anoin Salt Amount (g/L)Cl- NaCl 1.6485NO3- NaNO3 1.3707SO42- K2SO4 1.8141

These standard solutions were analyzed by HPLC in order to develop calibration curves relating the area

under the peak to concentration. To use the HPLC we needed to filter the solutions, prior to injecting

them into the loop, in order to eliminate any particulates which could severely damage the HPLC

equipment. We filtered 20 mL of each solution using a 0.22 micrometer filter tip, then flushed the

sample loop with 5 mL of the solution twice. Finally, we injected 2-4 mL of solution into the loop and

programmed the machine to begin analysis and record the graph of the results. This process was

repeated for each concentration of standard solution we created, as well as for the unknown solution. It

is important to ensure your peaks are distinct and separate, as two peaks overlapping will cause an error

in the analysis of both species.

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8

Figure 1. Chromatogram

Results

Table 2. HPLC Data Table 3. Unknown Sample Species Concentrations

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Chloride 1.25 min

Nitrate 2.28 min Sulfate 3.95 min

IC Sample Concentration (mg/L)

Cl- NO3 - SO4 2-

(Area Under Curve)

0.2 1,149,010 564,493 1,250,3011 6,191,345 2,473,502 4,373,9983 12,970,256 6,040,347 9,598,9635 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 Result AnalysisCl- NO3 –

(NO3- - N)

SO4 2-

Unkown Sample Concentration (mg/L) 2.695* 3.62 3.48

0 2 4 6 8 10 120

5,000,00010,000,00015,000,00020,000,00025,000,00030,000,00035,000,00040,000,00045,000,00050,000,000

f(x) = 4644502.73992891 xR² = 0.998771603439141

Cl- Calibration Curve

Concentration (mg/L)

Area

Und

er th

e Cu

rve

0 2 4 6 8 10 120

5,000,000

10,000,000

15,000,000

20,000,000

25,000,000

f(x) = 2264505.04739337 xR² = 0.998954584152557

NO3- Calibration Curve

Concentration

Area

Und

er th

e Cu

rve

0 2 4 6 8 10 120

5,000,00010,000,00015,000,00020,000,00025,000,00030,000,00035,000,00040,000,000

f(x) = 3404026.97126777 xR² = 0.998885461053713

SO42- Calibration Curve

Concentration

Area

Und

er th

e Cu

rve

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Figure 2. Calibration Curve for Chloride

Figure 3. Calibration Curve for Nitrate

Figure 4. Calibration Curve for Sulfate

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.

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 at 50 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).

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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.

Discussion Questions

1. Discuss the significance of high chloride concentration in water supplies.

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 concentration in a water sample can

indicate toxic conditions. For this reason, it is used as an indicator for

contamination of water supplies. 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

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(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 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 water supplies and in

wastewater disposal?

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a. High sulfate concentrations in groundwater are 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.

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

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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 detect

concentrations 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 microbial

contaminants or gastric illness. Nitrite that gets into the blood will

15

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. 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.

[Nitrate ][50mg /L ]

+[Nitrite ][3mg /L ]

<1

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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

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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, cheaper to

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.

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Works Cited

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Drinking-water Quality. WHO/SDE/WSH, 03 Apr. 2003. Web. 4 Apr. 2014.

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"Disinfection By-Products." Centers for Disease Control and Prevention. Centers for Disease

Control and Prevention, 21 Mar. 2012. Web. 04 Apr. 2014.

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"Guidelines for Drinking-water Quality THIRD EDITION." Water Sanitation Health. WHO, 2008.

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L'Hirondel, J., and J-L L'hirondel. Nitrate and Man: Toxic, Harmless or Beneficial? Wallingford:

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"HPLC" Wikipedia. Wikimedia Foundation, 10 Mar. 2014. Web. 11 Mar. 2014.

"Ion-Exchange Chromatography" Wikipedia. Wikimedia Foundation, 10 Mar. 2014. Web. 11

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Nollet, Leo M. L. Handbook of Water Analysis. New York: Marcel Dekker, 2000. Print.

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Drinking-water Quality. WHO/SDE/WSH, 03 Apr. 2003. Web. 4 Apr. 2014.

<http://www.who.int/water_sanitation_health/dwq/chemicals/sulfate.pdf>.

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