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1. PURPOSE:

The purposes of this experiment are;

To illustrate the principles of volatilization and recovery of nitrogen compounds. To show the general analysis procedures applicable to solid samples and a way of

avoiding matrix effects.

REAGENTS:

1. Digestion Reagent: Dissolve 134 kg K2SO4 in 650 mL distilled water and 200 mL conc. H2SO4. Mix this with a solution prepared by dissolving 2 gram red HgO in 25 mL 6N H2SO4. Dilute this combination to 1 L.

2. Na2S2O3 - NaOH solution: Dissolve 100 gram NaOH and 5 gram Na2S2O3.5H2O in distilled water and make up to 200 mL.

3. Phenolphthalein indicator solution, 1 %

4. Mixed indicator solution: Dissolve 200 mg methyl red indicator in 100 mL 95 % ethyl alcohol. Mix this with a solution prepared by dissolving 100 mg methylene blue in 50 mL 95 % ethyl alcohol. This indicator is blue above pH 4.2 and pink below pH 4.2.

5. H3BO3 solution: Dissolve 20 gram H3BO3 in distilled water, add 10 mL mixed indicator solution and make up to 1 liter.

6. Standard sulfuric acid, 0.02 N: To 500 mL distilled water, add 0.56 mL concentrated H2SO4 and dilute to 1 liter. Standardize against sodium carbonate standard solution by using methyl orange indicator.

7. Sodium carbonate primary standard: Dry primary standard grade Na2CO3 AT 103oC. Weigh 1.060 g Na2CO3 and dissolve in 500 mL boiled and cooled distilled water and make up to 1 liter.

8. Borate Buffer: Add 88 mL 0.1 N NaOH solution to 500 mL 0.025 M sodiumtetraborate solution. (0.025 M Sodiumtetraborate solution: 5.0 gram Na2B4O7 or 9.5 gram Na2B4O7.10H2O complete to 1 L with distilled water.)

2. PROCEDURE:

For Ammonia Determination:

1. We take a soil sample from a pot or field and pass it through 0.15 mm (100 mesher/inch) sieve.

2. We weigh 1 gram of the soil (for sewage 100 mL is taken) and put into an 800 mL Kjeldahl digestion flask and add 300 mL distilled water with a few glass beads.

3. We add 15 mL borate buffer solution.

4. We adjust pH to 9.5 by NaOH solution.

5. We pipet 50 mL indicating boric acid solution into a 500 mL conical flask.

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6. We distill the contents of Kjeldahl flask into the indicating boric acid solution collecting 200 mL distillate below the surface of the boric acid.

7. We immediately disconnect the distillate collected in the conical flask.

8. Then we titrate the boric acid solution with standard sulfuric acid.

9. Blank solution is prepared similarly except the addition of the soil sample.

For The Determination of Total Nitrogen:

1. We again weigh 1 gram soil sample and put it into a 800 mL Kjeldahl flask.

2. We add 50 mL digestion reagent and 250 mL distilled water into the 800 mL Kjeldahl flask. Place the sample on digestion stand.

3. We prepare a blank by adding 50 mL digestion reagent and 250 mL distilled water. Place it on digestion stand along with the samples.

4. Digest for ½ hour by regulating the temperature to keep it between 360-400 Celsius.

5. We stop heating, cool the flasks. Then we add 250 mL distilled water and 0.5 mL phenolphthalein indicator solution and mix.

6. We make the solution alkaline by adding approximately 50 mL hydroxide thiosulfate solution. Color will be pink. Add extra hydroxide-thiosulfate solution if a pink phenolphthalein color fails to appear. Then mix thoroughly.

7. We mix the contents of the Kjeldahl flask and place the flasks on the distillation stand and connect it to the condenser. Start the cooling water flowing in the condenser. Light the burner.

8. We distill the contents of the Kjeldahl flask into 50 mL indicating boric acid solution collecting 200 mL distillate below the surface of the boric acid.

9. Once we have 200 mL collected, immediately disconnect the distillate collected in the conical flask.

10. We titrate the boric acid solution with standard sulfuric acid.

3. THEORY:

The compounds of nitrogen are of great importance in water resources, in the atmosphere and in the life processes of all plants and animals. The chemistry of nitrogen is complex because of the several oxidation states that nitrogen can assume and the fact that changes in oxidation state can be brought about by living organisms. (Sawyer et al., 2003, p.631)

Nitrogen Forms and Function

Forms of nitrogen available for plant uptake

Ammonium

Nitrate

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Functions of nitrogen in plants

Nitrogen is an essential element of all amino acids. Amino acids are the building

blocks of proteins.

Nitrogen is also a component of nucleic acids, which form the DNA of all living

things and holds the genetic code.

Nitrogen is a component of chlorophyll, which is the site of carbohydrate formation

(photosynthesis). Chlorophyll is also the substance that gives plants their green

color.

o Photosynthesis occurs at high rates when there is sufficient nitrogen.

o A plant receiving sufficient nitrogen will typically exhibit vigorous plant

growth. Leaves will also develop a dark green color.

The Nitrogen Cycle

Figure : The nitrogen cycle

Source: http://www.physicalgeography.net

Gains of Nitrogen to the Soil

Biological and Atmospheric Fixation: Conversion of atmospheric nitrogen to

ammonium which is subsequently available for plant uptake

Direct additions of commercial and organic fertilizers

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Transformations in the Soil

Mineralization: Conversion of organic nitrogen to ammonium

Nitrification: Conversion of ammonium to nitrate

Losses of Nitrogen from the Soil

Denitrification: Conversion of nitrate to atmospheric forms of nitrogen

Volatilization: Loss of gaseous ammonia to the atmosphere

Run-off

Leaching

Consumption by plants and other organisms

Nitrogen is a very dynamic element. It not only exists on Earth in many forms, but also

undergoes many transformations in and out of the soil. The sum of these transformations is

known as the nitrogen cycle.

Though complex, the nitrogen cycle:

Helps us to understand the complex relationships that exist between the many

forms of nitrogen

Provides us with insight pertaining to the availability of ammonium and nitrate,

which are the only nitrogen forms usable by plants

To understand the many ways in which N may be lost from the soil

(Nitrogen,http://www.ctahr.hawaii.edu/mauisoil/c_nutrients01.aspx)

Most of the nitrogen is originally present in the form of organic nitrogen and ammonia

nitrogen.

Ammonia Nitrogen:

Ammonia-nitrogen is an inorganic, dissolved form of nitrogen that can be found in water and is the preferred form for algae and plant growth.  Ammonia is the most reduced form of nitrogen and is found in water where dissolved oxygen is lacking.  When dissolved oxygen is readily available, bacteria quickly oxidize ammonia to nitrate through a process known as nitrification.  Other types of bacteria produce ammonia as they decompose dead plant and animal matter.  Depending on temperature and pH (a measurement of acidity), high levels of ammonia can be toxic to aquatic life.  High pH and warmer temperatures increase the toxicity of a given ammonia concentration.  High ammonia concentrations can

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stimulate excessive aquatic production and indicate pollution.  Important sources of ammonia to lakes and streams can include: fertilizers, human and animal wastes, and byproducts from industrial manufacturing processes.  Techniques to prevent high ammonia concentrations involve filtration of runoff water especially from barnyards and other areas where animals may be kept in larger numbers, proper septic system maintenance, and not over-fertilizing yards or fields. ( Water and sustainability, http://www.unc.edu/~shashi/TablePages/ammonianitrogen.html )

Table :Various forms of nitrogen

Form of

Nitrogen

Formula Availability for plant uptake

Nitrogen gas N2 Although 78% of our atmosphere is nitrogen gas, this

form of nitrogen must be transformed to usable forms

before it is available for plant uptake.

Ammonia NH3 Ammonia is a gas. Ammonium can escape from the

surface of the soil under certain conditions and is

harmful to plants in high quantities. Ammonium is the

basic building block of commercial nitrogen fertilizers.

Ammonium NH4+ Soil particles attract and retain ammonium on cation

exchange complexes. This form may be directly taken

up by plants.

Nitrate NO3- Nitrate is the second form of nitrogen which is available

for plant uptake. In most soils, nitrate is highly mobile.

However, in the highly weathered soils of Hawaii,

nitrate is stored in soils with ‘anion exchange capacity’

and becomes less mobile.

Nitrite NO2- Nitrite is an intermediate product in the conversion of

ammonium to nitrate (nitrification). It is usually present

in low quantities, but is toxic to plants.

Organic

Nitrogen

Various

compounds

Organic nitrogen must be converted to ammonium

before it is used by plants. This conversion occurs with

time and is known as mineralization.

(Nitrogen,http://www.ctahr.hawaii.edu/mauisoil/c_nutrients01.aspx)

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The most common method for determining organic nitrogen is the Kjeldahl method,which is based on a neutralization titration.The Kjeldahl method is a means of determining the nitrogen content of organic and inorganic substances. Although the technique and apparatus have been altered considerably over the past 100 years, the basic principles introduced by Johan Kjeldahl endure today. The Kjeldahl method may be broken down into three main steps:

Digestion - the decomposition of nitrogen in organic samples utilizing a concentrated acid solution. This is accomplished by boiling a homogeneous sample in concentrated sulfuric acid. The end result is an ammonium sulfate solution.

Distillation - adding excess base to the acid digestion mixture to convert NH4+ to NH3, followed by boiling and condensation of the NH3 gas in a receiving solution.

Titration - to quantify the amount of ammonia in the receiving solution.

The amount of nitrogen in a sample can be calculated from the quantified amount of ammonia ions in the receiving solution.

The Digestion Process :

A general equation for the digestion of an organic sample is shown below as one basic example:

Organic N + H2SO4 ➝(NH4)2SO4 + H2O + CO2 + other sample matrix by-products

A number of interrelated digestion conditions determine the rate of reaction and the completeness of the breakdown of nitrogen to ammonium sulfate. Among these are heat input to the acid digestion mixture, amount of inorganic salt added to elevate the acid boiling temperature, reflux rate of H2SO4 in the neck of the digestion flask, length of digestion, and catalyst addition. Adjusting any one of these factors has an influence on the others. Proper digestion conditions for a given sample matrix are achieved through establishing a balance of these factors in a controlled and repeatable fashion. In addition, if the sample contains nitrate or nitrite nitrogen, it is possible to chemically pretreat the digest to include or exclude this nitrogen source from the analysis as desired in a particular situation.

The Distillation Process :

The acid digestion mixture is diluted and made strongly alkaline with NaOH, liberating NH3 as follows:

(NH4)2SO4 + 2NaOH ➝ 2NH3↑ + Na2SO4 + 2H2O

Ammonium heat ammonia

sulfate gas

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The Kjeldahl flask is attached to a water condenser and is heated to boil off the NH3 gas from the digest. The tip of the condenser is submerged in a flask of acidic receiving solution, either standard acid or boric acid solution, to again trap the distilled NH3 in receiving solution.

The Titration Process :

There are two types of titration: back titration, commonly used in Macro Kjeldahl; and direct titration. Both methods indicate the ammonia present in the distillate with a color change and allow for calculation of unknown concentrations.

Nitrogen Determination by BACK TITRATION

The ammonia is captured by a carefully measured excess of a standardized acid solution in the receiving flask. The excess of acid in the receiving solution keeps the pH low, and the indicator does not change.

standard sulfuric ammonium excess sulfueic

ammonia acid sulfate acid

2 N H 3 + 2 H 2 S O 4 ➝ ( N H 4 ) 2 S O 4 + H 2 S O 4

(no color change)

The excess acid solution is exactly neutralized by a carefully measured standardized alkaline base solution such as sodium hydroxide. A color change is produced at the end point of the titration.

(NH4)2SO4 + H2SO4 + 2NaOH ➝ (Na)2SO4 + (NH4)2SO4 + 2H2O

(color change)

Nitrogen Determination by DIRECT TITRATION

If boric acid is used as the receiving solution instead of a standardized mineral acid, the chemical reaction is:

ammonia boric ammonium-borate excess

gas acid complex boric acid

N H 3 + H 3 B O 3 ➝ N H 4 +: H 2 B O 3- + H 3 B O 3

(color change)

The boric acid captures the ammonia gas, forming an ammonium-borate complex. As the ammonia collects, the color of the receiving solution changes.

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ammonium-borate sulfuric ammonium boric

complex acid sulfate acid

2 N H 4 H 2 B O 3- + H 2 S O 4 ➝ ( N H 4 ) 2 S O 4 + 2 H 3 B O 3

(color change in reverse)

The addition of sulfuric acid exactly neutralizes the ammonium borate complex, and a reverse color change is produced. The boric acid method has two advantages: only one standard solution is necessary for the determination and the solution has a long shelf life.

( A Guide to Kjeldahl Nitrogen Determination Methods and Apparatus , http://www.expotechusa.com/Catalogs/Labconco/PDF/KJELDAHLguide.PDF )

Importance of nitrogen in the environment:

N2 comprises 80% of the atmosphere

N2 cannot be used most organisms N2 is not a problem until it is in a reactive form like NH3 or NO3 and is out of

balance in nature

N is the major component of proteins and nucleic acids

Often the most limiting nutrient for planr growth

When out of balance, N can have both direct and indirect negative impacts on the environment (Sawyer et al., 2003, pp.635-640)

4. DATA ANALYSIS AND CALCULATIONS:

The calculations for % nitrogen or % protein must take into account which type of receiving solution was used and any dilution factors used during the distillation process. The equations given here are in long form. They are often simplified in the published standard methods. In the equations below, “N” represents normality. “ml blank” refers to the milliliters of base needed to back titrate a reagent blank if standard acid is the receiving solution, or refers to milliliters of standard acid needed to titrate a reagent blank if boric acid is the receiving solution. When standard acid is used as the receiving solution, the equation is:

% Nitrogen = (ml standard acid - ml blank) x N of acid x 1.4007

weight of sample in grams

( A Guide to Kjeldahl Nitrogen Determination Methods and Apparatus , http://www.expotechusa.com/Catalogs/Labconco/PDF/KJELDAHLguide.PDF )

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NH2SO4=0.02 N

Table 1.

Sample weight(g) Volume of standard acid used(ml)

Sample 1 1.009 2.7Sample 2 1.040 2.8Sample blank Not used 0.6TKN 1 1.024 3.9TKN 2 1.006 3.4TKN blank Not used 0.3

Where TKN: Total Kjeldahl Nitrogen

1- ) % N in soil= (T-B)*N*1.4/S

Where:

T: Volume of standard acid used for the sample titration, mL.

B: Volume of standard acid used for the sample titration, mL.

N: Normality of standard acid.

S: Sample weight,g.

For Sample 1: % N= (2.7-0.6)*0.02*1.4/1.009

% N=0.058

For Sample 2: % N= (2.8-0.6)*0.02*1.4/1.040

% N=0.059

For TKN 1: % N= (3.9-0.3)*0.02*1.4/1.024

% N=0.098

For TKN 2: % N= (3.4-0.3)*0.02*1.4/1.006

% N=0.086

TKN= ORGANIC NITROGEN + AMMONIA NITROGEN

For organic nitrogen 1: 0.098-0.058 = 0.04

% N=0.04

For organic nitrogen 2: 0.086-0.059= 0.027

% N= 0.027

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5. DISCUSSION AND CONCLUSIONS:

1. We have to dry the soil sample until we leave moisture in the soil sample completely. If a small amount of moisture is still present in the soil sample, we cannot weigh the sample accurately because moisture in the soil increases the weight of the soil. Soil drying increases the release of ammonium. Moreover, we have to pulverize the soil sample in order to increase the surface area. Divided samples are more homogeneous, which means that a more representative sampling with a better accuracy and precision is possible. In addition smaller particles dissolve faster and are easier to extract because of a greater surface area. (Choromedia, Henk Lingeman, URL 1)

2. We have to distill the sample before titration because distillation removes from strange substances in our sample or makes purify the sample.

3. Digestion method is used for converting all organic form of nitrogen to NH3. In other words, this method provides us gain all nitrogen compounds in soil as NH3

4.

TABLO 25: ATIKSULARIN ATIKSU ALTYAPI TESİSLERİNE DEŞARJINDA ÖNGÖRÜLEN ATIKSU STANDARTLARI

PARAMETREKANALIZASYON SISTEMLERI TAM

ARITMA ILE SONUÇLANAN

ATIKSU ALTYAPI TESISLERINDE

KANALIZASYON SISTEMLERI

DERIN DENIZ DEŞARJI ILE

SONUÇLANAN ATIKSU

ALTYAPI TESISLERINDE

Sıcaklık (˚C) 40 40pH 6.5-10.0 6.0-10.0Askıda katı madde (mg/L) 500 350Yağ ve gres (mg/L) 250 50Katran ve petrol kökenli yağlar (mg/L)

50 10

Kimyasal oksijen ihtiyacı (KOİ) (mg/L)

4000 600

Sülfat (SO4=) (mg/L) 1700 1700

Toplam sülfür (S) (mg/L) 2 2Fenol (mg/L) 20 10Serbest klor (mg/L) 5 5Toplam azot (N) (mg/L) - (a) 40Toplam fosfor (P) (mg/L) - (a) 10Arsenik (As) (mg/L) 3 10Toplam siyanür (Toplam CNˉ) 10 10

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(mg/L)Toplam kurşun (Pb) (mg/L) 3 3Toplam kadmiyum (Cd) (mg/L) 2 2Toplam krom (Cr) (mg/L) 5 5Toplam civa (Hg) (mg/L) 0.2 0.2Toplam bakır (Cu) (mg/L) 2 2Toplam nikel (Ni) (mg/L) 5 5Toplam çinko (Zn) (mg/L) 10 10Toplam kalay (Sn) (mg/L) 5 5Toplam gümüş (Ag) (mg/L) 5 5Clˉ (Klorür) (mg/L) 10000 -Metilen mavisi ile reaksiyon veren yüzey aktif maddeleri(MBAS) (mg/L)

Biyolojik olarak parçalanması Türk Standartları Enstitüsü standartlarına uygun olmayan maddelerin boşaltımı prensip olarak yasaktır.

(a) Bu parametrelere atıksu değerlendirilmesinde bakılmıyacaktır.

( Su Kirlliği Kontrolü Yönetmeliği, www.ibb.gov.tr/tr- TR/kurumsal/Birimler/.../sukirliligiyonetmelik.doc )

Release of Nr to air, in both oxidized (NOy) and reduced (mostly ammonia, ammonium and urea)

forms, contributes to:

• Increases in Nr deposition to terrestrial and aquatic ecosystems, which generates a

“cascade” of direct and indirect effects on soil fertility, plant productivity, water quality

and estuarine productivity, and human structures. (Figure 1)

• Depletion of stratospheric ozone.

• Climate change attributable to greenhouse gas emissions, especially nitrous oxide (N2O).

• Fine particle formation and the resulting effects of fine particles on human health, air

clarity, visibility, and the radiative properties of the atmosphere.

• Formation of ozone in the troposphere, and subsequent human health effects associated

with ozone inhalation, as well as damage to plants that reduces crop and forest

production.

• Direct damage to plant foliage that reduces production and increases susceptibility to

insect and diseases.

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• Long-term loss of soil fertility through the soil acidification, depletion of base cations,

changes in element ratios, and increases in availability of harmful aluminum in soils.

• Shifts in plant community composition and loss of biodiversity.

In addition, increasing the loading of Nr to surface waters through direct inputs of sewage, and

diffuse non-point sources such as fertilizers, atmospheric deposition and animal wastes

contribute to:

• Nitrate contamination of drinking water supplies.

• Eutrophication-related algal and other vegetation blooms, loss of dissolved oxygen, fish

kills, loss of productivity, and loss of desirable habitat.

• Generation of periodic hypoxic zones in coastal waters associated with eutrophication.

• Acidification of lakes and streams.

• Reduced buffering capacity of estuarine and marine waters.

• Succession of wetland plant communities. ( The Significance of Reactive Nitrogen , http://www.epa.gov/wed/pages/research/nitrogen/ESRPNitrogenPlan082409.pdf)

These reasons show nitrogen must be regulated in standards.

REFERENCES:

1. Sawyer ,C.N., McCarty, P.L., Parkin , G.F., Chemistry for Environmental Engineering and Science 5th edition, The McGraw Hill Companies, New York, 2003 pp. 632-646

2. Nitrogen,retrieved 12 November 2012 from,

http://www.ctahr.hawaii.edu/mauisoil/c_nutrients01.aspx

3. Water and sustainability, retrieved 12 November 2012 from,

http://www.unc.edu/~shashi/TablePages/ammonianitrogen.html

4. A Guide to Kjeldahl Nitrogen Determination Methods and Apparatus, retrieved 12 November 2012 from, http://www.expotechusa.com/Catalogs/Labconco/PDF/KJELDAHLguide.PDF

5.Retrieved 12 November 2012 from,

http://www.chromedia.org/chromedia?waxtrapp=fwyqcDsHqnOxmOlIEcCzBY&subNav=tsmmyDsHqnOxmOlIEcCzBYK

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6. Su Kirlliği Kontrolü Yönetmeliği, retrieved 12 November 2012 from,

www.ibb.gov.tr/tr- TR/kurumsal/Birimler/.../ sukirliligi yonetmelik.doc

7. Skoog, D.A., West, D.M., Fundamentals of Analytical Chemistry 6th edition, Holt, Rinehart and Winston, Inc.,2004 pp. 456-458

8. The Significance of Reactive Nitrogen, retrieved 12 November 2012 from,

http://www.epa.gov/wed/pages/research/nitrogen/ESRPNitrogenPlan082409.pdf