RAPID AND ACCURATE TECHNIQUE FOR MEASURING N-15 OF NITRATE

Method Development, Calibration and Validation

A. I. M. Aly*, M. A. Ahmed*, H. E. Gomaa*a, N. Abdel Monem** and M. Hanafy**

* National Center for Nuclear Safety and Radiation Control, Atomic Energy Authority, Cairo, Egypt.** Chemical Engineering Department, Faculty of Engineering, Cairo University, Giza, Egypt.

ABSTRACT

A simplified ion exchange resin based procedure for measuring nitrogen-15 of nitrate in

water with moderate ionic strength and moderate to high organic loads is presented. The modified

analytical train consists of two main sections: a) Extraction and purification of nitrate from liquid

matrix and b) pyrolysis of purified potassium nitrate. The presented method was evaluated with

simulated solutions containing constant amounts of inter-laboratory nitrate standard salt (KNO3 ,

δ15N = -4.34±0.32‰Air N2) and potentially interfering anions (SO42-, Cl- and HCO3

-) as well as

Dissolved Organic Carbon (DOC) load. Dowex 1X8 anion exchange resin proved to be a suitable

mean and provide accurate and reproducible δ15N values (SD: ±0.48‰). It tolerates high

concentrations of chloride and sulphate ions for samples of small volumes (100 ml) of about four

folds of the amounts enough to fully saturate the resin column.

Rapid, accurate, precise, low–priced and less labor intensive activated graphite based

offline pyrolysis technique for measuring N-15 content of nitrate has been developed and

implemented. This involves combusting nitrate salt with activated graphite at 550oC for 30

minutes. Dramatic reduction in processing times needed for analysis of N-15 of nitrate at natural

abundance level was achieved. Performance characteristics of the modified analytical cascade

show that 1 to 2 hrs/sample/set of 5 samples processing time is required.

The pyrolysis step of the developed method was validated using internationally

distributed nitrate stable isotope reference materials. N-15 content of some selected nitrate

salts, namely, calcium nitrate, sodium nitrate, potassium nitrate and ammonium nitrate fertilizer

was measured using the modified technique, vacuum distillation method, modified ammonia

diffusion method, microbial dentrifier method and EA-CF IRMS method at two different

laboratories in Egypt and Germany. The results proved the validity and applicability of the

modified combustion procedure.

Keywords: 15N; Ion exchange; Dowex 1X8; nitrate; isotope analysis; Pyrolysis.

a. Corresponding Author, email address: Hassan_emgh@yahoo.com 1. INTRODUCTION

Nitrate (NO3-) contamination is an environmental problem worldwide (Dongmei Xue et

al., 2009). Most of the human activities responsible for the increase in global nitrogen are local in

scale, from the production and use of nitrogen fertilizers to the burning of fossil fuels in

automobiles, power generation plants, industries and nuclear fuel processing. Many other

industries also contribute to the problem such as fiber, plastics, petroleum and petrochemicals,

mining and detergents & surfactants industries. However, human activities have not only

increased the supply but enhanced the global movement of various forms of nitrogen through air

and water. Because of this increased mobility, excess nitrogen from human activities has serious

and long-term environmental consequences for large regions of the earth (http://

esa.sdsc.edu/tilman.htm, accessed at June, 2006).

Effective management practices directed towards preservation of water quality, and, if

necessary, involving adequate remediation measures require identification of major sources of

nitrates and their pathways to the affected groundwater systems (Wojciech et al., 2009). Nitrogen

isotope measurements constitute a powerful tool to better understand the nitrogen cycle in

terrestrial and aquatic ecosystems (Mathieu Sebilo et al., 2004). Since more than three decades,

nitrogen isotope ratio measurements have been used in countless case studies to identify nitrogen

sources and to describe nitrogen transformations in terrestrial and aquatic ecosystems (Kendall,

1998).

There are several methods in current use for collecting dissolved nitrate from water and

preparing for δ15N analysis. Reduction of nitrate to ammonium using Devard’s alloy and

collecting produced ammonium on suitable receiving acid media was the base of labor intensive

Kjeldahl-Rittenberg technique (Mulvaney, 1990), Steam distillation (Bremner & Keeny, 1965),

Vacuum distillation (Aly et al., 1982), diffusion methods (Mulvaney & Khan, 2001, Khan et al.,

2000 and Brooks et al., 1989) and modified ammonia diffusion method (Ahmed et al., 2008). The

extracted NH4+ is measured and/or dried to a salt which is subsequently converted to N 2 for

isotopic analysis by either hypobromite oxidation (Mulvaney, 1993, Hauck, 1982, Aly et al.,

1981, Sprinson & Rittenberg, 1948, 1949 and Rittenberg, 1948), Dumas combustion (Kendall &

Grim, 1990 and Fiedler & Proksch, 1972), or automated on-line combustion during analysis by

elemental analyzer-isotope ratio mass spectrometry (Stickrod and Marchall, 2000 and Kornexl et

al., 1999).

Downs et al., 1999 used 5 mL anion exchange resin (Dowex 1X-8), nitrate is eluted with

100 ml of 2 N KCl at a rate of 0.5 drops/sec and placed into 250 ml wide mouth polypropylene

bottle, when the sample was ready for diffusion, excess MgO (0.2 g) and Devarda's alloy (~ 0.4

g) was added. The bottles are quickly capped and swirled. The samples are diffused for 7 days at

room temperature. The packets are opened, the filter strips dried in a desiccator with an open

container of concentrated H2SO4. After drying the filter strips are put in tin boats and stored in

individual air-tight vials until analysis using EA-CF-IRMS configuration.

Another approach relying on collecting dissolved nitrate using anion exchange resin with

subsequent elution of collected nitrate using 15 ml of 3M HCl and neutralization using 6-8 g of

Ag2O for 2 ml resin columns of BioRad AG1X8 anion exchange resins was presented (Silva et

al., 2000, Chang et al., 1999 and Wassener et al., 1995) and finally combustion of freeze dried

AgNO3 using offline combustion method (Kendall and Grim, 1990) or online CF-IRMS. Each

method was developed to accommodate a particular type of sample. All of these improvements

have been driven by the widespread need for isotopic data in studies of contaminant transport and

identification (Scott et al., 2009, Van Dyke and Wasson, 2005, Ohte et al., 2004, Rock and

Mayer, 2004, Ostrom et al., 2002, Wassenaar, 1995, Durka et al., 1994, Aravana et al., 1993 and

Bottcher et al., 1990).

In this paper, precise, efficient and fast anion exchange resin based extraction method for

nitrate from water with moderate to high organic contents was described. More importantly,

simple, accurate, precise, robust, inexpensive and rapid offline as well as catalyst-free

combustion technique was devised. Moreover, validating the newly developed pyrloysis step with

both internationally distributed reference materials and comparison with well established methods

at two isotope analytical laboratories in Egypt and Germany was carried out.

2. Materials and Procedures

2.1 Preparation and Loading Anion Exchange Columns

Ion exchange resin columns were prepared as described in Aly et al., 2007. NO3-

concentrations are measured to determine how much water needs to be processed to retain 100–

200 μmol of nitrate on the anion exchange resin. Samples are filtered through a 0.45 μm pre-

combusted glass fiber filters to remove particles that might clog the resin. Samples are loaded

onto the resin columns by gravity with a flow rate of 6 ml/min., air pressing with a flow rate of 30

ml/minutes or vacuum-aided with a flow rate of 40 ml/minutes. After sequestering the sample-

NO3-, columns were rinsed with distilled water and stripped immediately or capped and

refrigerated until they are transported to the laboratory for analysis in case of nitrate collection in

the field or traveling abroad.

2.2 Stripping of Bound Nitrate

Sample-nitrate bound to the anion exchange resin is stripped by gravity dripping 30 ml

of 0.5 M HCl through the column for 2 ml resin columns and 40 ml of 1M HCl for 5 ml resin

columns. Positive air pressure, supplied by a one-way rubber bulb attached to the top of the anion

exchange resin tube, is applied to the columns after each increment to remove residual eluant and

sometimes is needed to start the subsequent aliquot dripping. The nitrate-bearing eluant is

collected in 100 ml glass beakers for the later processing steps.

2.3 Preparation of NO3-- Bearing Eluant

Eluted nitrate is in the form of nitric acid which is volatile. Neutralization of acid

nitrate-bearing eluent is made in two steps using both silver oxide (Ag2O, ReagentPlus®, 99%,

Aldrich, cat No. 221163-50G, lot# 05930KE.) and potassium hydroxide. This mixed

neutralization scheme results in lowering consumption of high-priced silver oxide and obtaining

sample nitrate in less sensitive potassium form.

Nitrate blank associated with silver oxide reagent was removed following a modified

protocol to that described by Silva et al., 2000. Nitrate blank for the used lot was about 28

µgNO3-/g silver oxide. The benefit of using Ag2O as a neutralizing agent is that the resulting

silver chloride precipitate (AgCl) can be removed by filtration. To each column eluant, Ag2O is

added in successive increments to allow the heat of reaction to dissipate without producing vapor

till pH becomes ≈ 1. Each Ag2O addition is stirred and crushed with the flattened end of a glass

stirring rod to break the crust which tends to encapsulate the un-reacted silver oxide reagent. The

AgCl precipitate is removed by filtration through DI-rinsed, 0.2μm Nylon membrane filter (Cat

No. 7402-004, Whatman® Schleicher & Schuell). Additional DI water is used to rinse the

sample nitrate through the filter.

2.5 Preparation for Nitrogen-15 Isotope Analysis

Neutralized solution consisting of KNO3 and KCl is evaporated till complete dryness on

a hot plate. The dried salt mix is mixed with activated graphite, AG, (Graphite fine powder,

Riedel-deHäen, cat. No. 15553, lot No. 32180) and finally ground. Activated graphite amount

in 1:1 mass ratio with respect to potassium nitrate is added. The mixture is loaded onto a pre-

combusted 20 cm by 6 mm (OD) Pyrex glass tubes as depicted in Figure (1). About 50 mg pre-

combusted calcium oxide CaO is placed on the top of the reaction mix to absorb evolved carbon

dioxide and water. Combustion tubes are evacuated, Torch sealed and combusted at 550 oC for 30

minutes and then cooled at moderate cooling rate. Finally, sample tubes are ready for measuring

N-15 isotope by direct connection of the sample tubes to the vacuum manifold of IRMS in dual

inlet configuration without any further purification steps.

Activation of graphite was made by combusting graphite under continuous evacuation at

850oC until the vacuum stabilizes. Graphite to be activated is loaded onto quartz tube, 9 mm OD

and 50 cm in length between two wads of quartz wool (Alltech, Ca no. 4033, and lot 08N7) to

prevent graphite from going through the vacuum system.

Fig. (1): Portrayal vision of loading and arrangements of reagents in the Pyrolysis tube.

3. Experiments, Results and Discussion

3.1 Experimental Procedures, Materials, and Precision

The use of anion exchange resin columns to concentrate NO3- in the field allows the

collection of optimal sample sizes for maximum analytical precision, regardless of the nitrate

concentration of the water (Silva et al., 2000). Interlaboratory standard nitrate salt was used to

evaluate various aspects of sample collection and preservation. Interlaboratory standard solutions

made from KNO3 salt (99+%, A.C.S. reagent, Sigma-Aldrich, Cat. No. 221295-500G,

Batch# 12017AE) with δ15N = -4.34± 0.32‰. Isotope compositions for nitrogen are

reported in parts per thousand or per mil (‰) relative to atmospheric air and measured

using ThermoFinnigan DELTAplus XL Isotope Ratio Mass Spectrometer.

During sorption and desorption experiments, nitrate concentrations measured in sample

solutions after passage through the columns were determined using Ion Chromatograph (Dionex

DX 600). To determine the accuracy and precision of the method for δ 15N analyses, samples

solutions were prepared from KNO3 standard salt and average δ15N and 1s standard deviation of

dissolved nitrate after having been sorbed on and stripped from anion columns as described

above, was δ15N = -5.11± 0.48‰, n=4. Instrumental precision is ≈ 0.06‰.

3.2 Adsorption Efficiency and Flow Rate

Nitrate adsorption efficiency was tested using standard solutions containing 10 mg NO3-

(160 µmol) in 100, 250 and 500 ml samples volumes by comparing the NO3- content of samples

before and after passage through the anion exchange columns. The average percentage of NO3-

sorbed for the tested samples was 99.4 ± 0.35 %, n=9. There was no correlation (r2 = 0.13

between the extent of NO3- sorption and flow rate for rates between 6 and 40 ml/min. (350 to

2400 ml/h). Samples were loaded onto the resin columns by gravity with a flow rate of 6 ml/min.,

air pressing with a flow rate of 30 ml/min. and suction with a flow rate of 40 ml/min. No

measurable differences in nitrate recovery within the analytical range of uncertainty were

noticed using the three sample loading methods.

3.3 Elution of Bound Nitrate

Potassium hydroxide was tested as eluant to replace the chloride load added from

hydrochloric acid which causes many problems in subsequent processing steps. Unfortunately,

potassium hydroxide gave lower stripping efficiencies (from 28 to 62 %) for concentration ranges

of 0.2, 3 and 6 N KOH and volume of 20 ml. Higher concentrations were not tested, since, higher

concentrations will be uneconomic. Transformation of Dowex 1X8 from chloride form to

hydroxide form using potassium hydroxide was adapted and it was found that 20 ml of 3N KOH

dripped through 2ml resin column, yield complete conversion of resin into OH- form. Resin color

changes from white to yellow when changed from Cl- to OH- form. Testing the effect of this

transformation on the capture-elution properties of Dowex 1X8 was conducted. Results showed

that capturing efficiency was not affected by this transformation. Although, elution characteristics

were improved, the improvement extent was low. Hence, conditioning of Dowex 1X8 using

potassium hydroxide is impractical.

Usage of hydrochloric acid as eluant was demonstrated. The effect of two factors was

tested, namely acid volume and acid concentration on the response variable, percentage nitrate

recovery. Acid concentrations of 0.2, 0.5 and 1 N of different volumes were applied in different

sequences for each column and the response variable was measured. At low concentration, 0.2 N

HCl, percentage nitrate recovery ranged between 65 to 85 ±13.44%, n=4 and did not improve

greatly with increasing acid volume. While, at 0.5N HCl, nitrate elution efficiency was enhanced

greatly and ranged between 96 and 100 % with dramatic reduction in variability between

individual columns (SD: ±1.03, n=6) and nitrate elution efficiency did not improve at higher

concentrations, 1 N HCl.

3.4 Competing Ions Interferences

The ability of anion exchange resin to adsorb and retain nitrate is a function of the

selectivity (affinity) of the anion, the exchange capacity of the resin, and the competition for sites

among other anions in solution (Francis, 1999). If the presence of other anions causes NO3- to be

incompletely sorbed during loading or incompletely eluted from the column during stripping,

isotope fractionation could result (Chang et al., 1999). The potential interference by chloride,

sulfate, bicarbonate and dissolved organic carbon was assessed.

3.4.1 Chloride Ion Interference

Standard solutions containing 160 µM NO3- were amended with NaCl to yield solutions

with Cl- ion concentrations ranging from 250 to 4000 mg/l (7.04 – 112.68 meq/l) for 100 ml

samples, 200 and 1600 mg/l (5.63 – 45.07 meq/l) for 250 ml samples and 100 to 800 mg/l (2.82 –

22.53 meq/l) for 500 ml samples. All tests were made in triplicates. Each column was eluted

using the adapted elution scheme, analyzed for nitrate recovery and processed to measure

nitrogen-15 isotopic content of eluted nitrate.

Figure (2) illustrates the results and indicates that nitrate recovery ranged from 75 to

100% for Cl- concentrations up to 4000 mg/l for 100 ml samples, 64 to 100 % for Cl-

concentrations up to 800 mg/l for 500 ml samples. The saturation concentration to exhaust 2 ml

of Dowex 1X8 (2.8 meq) should already be reached at Cl- concentrations higher than 1000 mg/l

in 100 ml of nitrate-free samples and 200 mg/l for 500 ml nitrate-free samples. It was found that

capturing efficiency decreases with increasing sample volume and amended chloride

concentration. This behavior of Dowex 1X8 for chloride ions agrees well with findings of Silva et

al., 2000 for anion exchange resin AG1X8, BIORAD.

Figure (3, A–C) represents the results from experiments with 100, 250, and 500 ml

solutions, respectively. Chloride ions as expected produced no measurable Nitrogen-15 isotope

fractionation, even if adsorption interference produced significant NO3- loss as seen in Fig. (3).

This behavior may be attributed to the same reasoning of Silva et al., 2000 that the loss of nitrate

is due to displacement not competition action, since the resin itself is in the chloride form.

0

20

40

60

80

100

120

0 50 100 150 200 250 300 350 400 450

Amount of interfering chloride ion, mg

Rec

over

y of

nit

rate

, %

100 ml

250 ml

500 ml

Fig. (2): Effect of concentration of the competing ion (chloride) and the sample volume on the capture-elution characteristics of nitrate on Dowex 1X8 anion exchange resin. Nitrate recovery was that measured after elution.

Fig. (5): Relationship between the interfering calcium ion concentration and the resultant δ15N value of ammonium at 8 mg NH3-N /L using Dowex 50wX8

0

20

40

60

80

100

120

250 250 500 1000 2000 2000 3000 3000 4000

Cl - ion conc. mg/L

reco

very

of ni

trat

e , %

-12

-10

-8

-6

-4

-2

0

δ15N,

‰

Air N

2

RecoveryDeltaAssigned delta value

a. 100 ml sample volume

0

20

40

60

80

100

120

200 400 600 800 1200 1600

Cl - ion conc. mg/L

reco

very

of ni

trat

e , %

-12

-10

-8

-6

-4

-2

0

δ15N,

‰

Air N

2

RecoveryDeltaAssigned delta value

b. 250 ml sample volume

0.00

20.00

40.00

60.00

80.00

100.00

120.00

50 100 150 200 300 400 600 800

Cl - ion conc. mg/L

reco

very

of ni

trat

e , %

-12

-10

-8

-6

-4

-2

0

δ15N,

‰

Air N

2RecoveryDeltaAssigned delta value

c. 500 ml sample volume

Fig. (3): Relationship between the interfering chloride ion concentration and δ15N value of captured-eluted nitrate using Dowex 1X8 anion exchange resin.

3.4.2 Sulphate Ion Interference

To test the effect of SO42- ion, KNO3 standard solutions containing 160 µM NO3

- were

amended with Na2SO4 to yield solutions with SO42- ion concentrations ranging from 500 to 4000

mg/l (10.4 – 83.3 meq/l) for 100 ml samples, 200 and 1600 mg/l (4.16 – 33.3 meq/l) for 250 ml

samples and 100 to 800 mg/l (2.08 – 16.6 meq/l) for 500 ml samples. Twenty-one columns were

loaded with 100 ml each of solutions having SO42- concentrations between 500 and 4000 mg/l

(10.4 – 83.3 meq/l), 21 columns were loaded with 250 ml each of solutions having SO 42-

concentrations between 200 and 1600 mg/l (4.16–33.3 meq/l) and 18 columns were loaded with

500 ml each of solutions having SO42- concentrations between 100 and 600 mg/l (2.08 – 16.6

meq/l).

The exchange capacity of the columns (2.8 meq. per column) was exceeded by the

combined masses of NO3- and SO4

2- for solutions having sulphate amounts more than 134.4 mg.

For all columns, samples of the passed solutions were collected and tested for flooded nitrate

using. The columns were then eluted, prepared, and analyzed for their nitrate recovery. Results

obtained are shown in Fig. (4).

0

20

40

60

80

100

120

0 50 100 150 200 250 300 350 400 450

Amount of interfering sulphatee ion, mg

Rec

over

y of

nit

rate

, %

100 ml250 ml500 ml

Fig. (4): Relationship between concentration of the competing ion (sulphate) and capture-elution efficiency of nitrate using Dowex 1X8 anion exchange resin for different sample volumes.

Removal of sulphate ion before loading onto the anion exchange resin columns was tried.

Precipitation of SO42- ion using barium chloride will result in replacing SO4

2- ion load with an

equivalent Cl- ion load. Really, this will result in two benefits; one is to alleviate the double effect

(since SO42- can displace nitrate by both direct replacement or by producing Cl- rain via

exchange onto the resin) of SO42- and the other is to eliminate presence of sulpahte salt in the

combustion mixture. Another approach to remove sulphate ion load without adding an equivalent

chloride load was suggested and tested. In this approach, sulpahte ion is precipitated using barium

hydroxide monohydrate (Ba(OH)2.H2O). Barium hydroxide solution is added to the sample, left

for 30 minutes and filtered through DI pre-rinsed 0.2µm Nylon membrane filter to remove BaSO4

precipitate.

Tests showed that this is an effective and suitable way for removing sulphate before

pouring the sample through anion exchange resin column. Passage of the sample through cation

exchange resin column (in H+ form) is recommended to neutralize the alkalinity (OH -) added and

hence lower the interfering effect of carbonate ion formed. This approach results in lowering the

ionic strength load on the anion exchange resin by replacing Sulphate ions with hydroxyl ions

which are then neutralized by hydrogen counter ions liberated from cation exchange resin

column.

3.4.3 Bicarbonate Ion Interference

Selectivity of bicarbonate toward Dowex 1X8 anion exchange resin was unknown, and is

expected to be in the same order of magnitude as that for AG1X8 BioRad. Even if published

selectivity of bicarbonate ion for anion exchange resins is low in comparison to that for other

anions, the effect of bicarbonate ion is studied. This is because selectivity of certain ion is a

strong function on the concentration of this ion (Francis, 1999), and bicarbonate is an abundant

ion in freshwater and waste water.

Potassium nitrate KNO3 standard solutions with 160 µM NO3- were amended

with NaHCO3 to yield solutions having HCO3- concentrations ranging from 50 to 4000 mg/l

(0.82–65.6 meq/l). Tests were made in triplicates for samples volumes of 100, 250 and 500 ml.

The exchange capacity of each column (2.8 meq. per column) is exceeded by the combined

masses of NO3- and HCO3

- for solutions having bicarbonate amounts more than 170.8 mg.

Columns were immediately stripped, neutralized and analyzed for nitrate recovery. Fig. (5)

shows that, as expected, bicarbonate has a mild interfering impact on nitrate adsorption for

sample volumes of 250 and 500 ml. In contrary to Cl - and SO42-

ions, the highest interfering effect

of HCO3- ion was found for the 100 ml sample volumes. Maximum nitrate loss (≈ 15%) was

found for 100 ml sample volume with bicarbonate concentration of 4000 mg/l. Up to HCO3-

concentrations of 1500 mg/l, nitrate recovery was close to 100%. The reasoning of this

behavior is not straightforward. But it may be due to large interference effect of

bicarbonate ion on measuring nitrate concentration using ion chromatograph with

potassium hydroxide elution and not from the exchange process itself.

0

20

40

60

80

100

120

0 50 100 150 200 250 300 350 400 450

Amount of interfering bicarbonate ion, mg/L

Rec

over

y of

nit

rate

, %

100 ml

250 ml

500 ml

Fig. (5): Relationship between concentration of the competing ion (bicarbonate) and capture-elution efficiency of nitrate using Dowex 1X8 anion exchange resin for different sample volumes.

3.4.4 Dissolved Organic Carbon (DOC) Interference

DOC bounds to anion exchange resins by both ion exchange and hydrophobic interaction,

resulting in a much stronger bond than that of NO3- (Silva et al., 2000). In order to test the

potential interference caused by DOC on NO3- adsorption on anion exchange resin columns,

potassium hydrogen biphthalate (C8H5O4K), an organic acid typically used to prepare

concentration standards for analyses of dissolved carbon, was used as a proxy for DOC.

Potassium hydrogen phthalate was added to 160 µM NO3- standard solutions to produce solutions

with five DOC concentrations ranging from 100 to 1500 mg-C/l for 100 ml samples. Two

columns each were loaded with 100 ml of each of the five solutions. Escaping nitrate and DOC

load were monitored in the passed solutions. It is obvious that, Fig. (6), Dowex 1X8 has greater

tendency for DOC fouling and nearly almost initial DOC load bound to the resin (slope = 0.0048

and r2 = 0.2). Effect of DOC on capturing efficiency of nitrate was found to be low even at high

loads of DOC as indicated in Fig. (7).

y = 0.0048x + 0.7111

R2 = 0.201

0

0.5

1

1.5

2

2.5

0 20 40 60 80 100 120 140 160

Inital amneded DOC amount, mg-C

Pas

sed

DO

C lo

ad, m

g-C

Fig. (6): Relationship between Initial DOC and passed DOC amount after dripping through Dowex 1X8 anion exchange resin.

0

20

40

60

80

100

120

0 200 400 600 800 1000 1200 1400 1600

Conc. of interfering DOC, mg-C/L

Rec

over

y of

Nit

rate

, %

Fig. (7): Relationship between DOC concentration and capturing efficiency of nitrate on Dowex 1X8 anion exchange resin.

3.5 Nitrogen-15 Measurements

This part of the analytical cascade involves presenting a new simple pyrolysis scheme for

measuring N-15 of nitrate. Most of published pyrolysis off line methods for measuring N-15 are

based on using Cu, CuO and CaO as the combustion reagents. The modified simple technique is

catalyst-free and is based on usage of activated graphite as the reducing agent. Placement and

loading of the combustion mixture was made as described in the experimental section. Prepared

samples were combusted at 550oC for 30 minutes and cooled with a moderate cooling rate (from

550oC to room temperature in ≈ 2 hrs). These conditions result in great reduction in the pyrolysis

time required to effect complete decomposition of nitrate salt and liberation of N2 gas.

Some selected nitrate salts and fertilizers, namely, potassium nitrate, sodium

nitrate, calcium nitrate and commercial ammonium nitrate fertilizer, are used for

evaluation and validation of the method. N-15 isotopic content of these salts was

measured using both modified and well established and already implemented techniques,

results are presented in table (1). Methods used in the validation process were vacuum

distillation (Aly et al., 1982) followed by hypobromite oxidation method (Aly et al.,

1981) and modified ammonia diffusion method (Ahmed et al., 2008) followed by

hypobromite oxidation method at the Central Laboratory for Environmental Isotopes

Hydrology, National Center for Nuclear Safety and Radiation Control, Atomic Energy

Authority Egypt. In addition, measuring N-15 isotopic content of these salts using

Elemental Analyzer Continuous Flow Isotope Ratio Mass Spectrometer EA-CF IRMS

and Denitrifeir method (Sigman et al., 2001 and Casciotti et al., 2002) at Isotope Hydrology

Department, Center for Environmental Research, Helmholtz-Zentrum für

Umweltforschung GmbH–UFZ, Germany was carried out.

Table (1): δ15N Values of some Nitrate salts and fertilizer processed by different techniques at EAEA, Egypt and UFZ, Germany labs.

Material

δ15N,‰Air N2 EAEA, Egypta UFZ, Germanyb

Modified combustion

Vacuum distillation

Ammonia diffusion

Denitrifier EA-CF IRMS

KNO3, Sigma-Aldrich

-4.57 -5.16 - -3.6 -4.11-5.3 -5.11 - -3.7 -4.56

Average -4.94±0.52 -5.14±0.04 -3.65±0.71 -4.34±0.32

NaNO3, 17.7 - - 14.4 18.5718.6 - - 14.5 18.52

Average 18.15±0.64 14.45±0.07 18.54±0.04

KNO3, Applichem-28.74 - - -24.3 -29.13-27.72 - - -23.9 -28.81

Average -28.23±0.72 -24.1±0.28 -28.97±0.23

Ca(NO3)2, WINLAB-1.08 -0.89 -1.34 - --0.422 -1.15 -1.8 - -

Average -0.75±0.47 -1.02±0.18 -1.57±0.32Ammonium Nitrate

fertilizer* 0.862 - - -0.4 0.571.53 - - -0.6 0.58

Average 1.2±0.47 -0.5±0.14 0.575±0.007* N-15 of total nitrogen. a. Central Laboratory for Environmental Isotopes Hydrology, National Center for Nuclear safety

and Radiation Control, Atomic Energy Authority.b. Isotope Hydrology Department, Center for Environmental Research, HelmHoltz-Zentrum fur

Umweltforschung GmbH–UFZ, Theodor-Leiser–Straβe 4, 06120 Halle-Saal, Germany.

Table (1) shows that, the modified combustion method is suitable for measuring N-15

isotopic content of nitrate salts. N-15 values of all salts were in good agreement with those

values measured using all other methods. In case of ammonium nitrate fertilizer, there was a good

agreement between both our modified combustion method (1.2±0.47 ‰) and EA-CF IRMS at

UFZ center 0.58±0.007 ‰). This agreement suggests that the method may be suitable for

measuring N-15 isotopic content of ammonium salts.

As a final validation stage, δ15N-NO3 of nitrate reference materials distributed by IAEA

with a wide range of δ15N values were measured using our modified Pyrolytic catalyst-free

method. Results revealed good agreement between measured and certified values, table (2).

Table (2): δ15N Values of Nitrate reference materials distributed by IAEA and our modified combustion method.

Materialδ15N‰Air N2

Modified combustion Certified Values*USGS32, KNO3 176.76 ± 1.5 ‰, n=3 +180 ±1 ‰USGS34, KNO3 -2.11±0.38 ‰, n=3 -1.8 ±0.2 ‰USGS35, NaNO3 +2.36± 0.42 ‰, n=3 +2.7 ±0.2 ‰

IAEA-NO-3, KNO3 + 4.2± 0.28 ‰, n=3 +4.7 ±0.2 ‰*full details can be found in IAEA Reference materials and catalogue–Stable Isotopes (http://curem.iaea.org/catalogue/SI/SI_004320000.html).

3.7 Simulated Samples

Cumulative effects of the whole analytical train on produced N-15 values of nitrate were

addressed using simulated samples. Samples of hypothetical composition of 100 mg/l NO 3

(KNO3, δ15N= - 4.34 ± 0.32‰Air N2 ), 400 mg/l Cl- (KCl), 200mg/l SO42- (Na2SO4), 400 mg/l

HCO3- (NaHCO3) and 10 mg-C/l (potassium hydrogen phthalate) were prepared and processed

according to the modified analytical procedure. Nitrogen-15 isotope values of processed

samples through the whole analytical train ensure suitability and applicability of the

developed method (Average δ15N = - 4.86 ± 0.72, n=5).

4. Method Characteristics

The basic characteristics of modified sample preparation procedure are summarized in

Table (3). It is clear that, there was a great reduction in time required for samples processing. One

sample requires 1 to 2 hrs for complete processing from field to IRMS in a set of 5 samples.

Previously developed ion exchange resin based method of Silva et al., 2000, requires 20 to 68 hrs

for a set of 8 samples and 28 days for a set of 12 samples in case of the denitrifier method of

Casciotti et al., 2002 (Wojciech et al., 2009).

Table (2): Characteristics of the modified method of nitrate conversion to N2 for isotope analysis.Step Duration, minutes Efficiency, %

Removal of SO4-

Precipitation and filtration Cation exchange resin

Ca. 30 – 40 minutesCa. 5 to 120 minutes depending on sample volume

90 – 100 %

Collection of NO3 - from water sample on anion exchange resin

from 5 to 120 minutes, depending on the sample volume

98 – 100%

NO3- stripping from anion exchange

resins10 to 15 minutes for 2 ml resin columns 95 – 100%

Neutralization: Ag2O pre-neutralization step Filtration KOH final neutralization step

Ca. 20 minutes (Excluding pre-wash step)Ca. 5-10 minutesCa. 3-10 minutes

Drying, grinding and reagents mixing Ca. 60 – 75 minutes

Thermal reduction Ca. 30 minutes at 550oC, 2h for cooling

Total 1to 2hrs/sample (set of 5 samples > 90%

Conclusions

Concentrating nitrate on anion exchange resins provides an efficient, rapid and reliable

means for extracting nitrate from low to moderate ionic strength water samples. Although,

reduced nitrate recovery showed no measurable isotopic fractionations at high chloride contents,

it is strongly recommended to collect all nitrate dissolved in the sample. Through this work we

have devised a new, simple, rapid, precise, accurate, reliable, catalyst-free, less time consuming

and robust pyrolysis method for measuring N-15 of nitrate. The new devised pyrolytic technique

offers greater flexibility in comparison with the previously published offline combustion methods

for N-15 isotope measurements. The modified method presented has the following advantages:

1. Eliminating the need for cryogenic purification before IRMS measurements.2. Allowing collection of sufficient nitrate amount, one analysis needs about 5 mg NO3 or ≈

15 μmol NO3-N, from nitrate-diluted waters.3. Implementing catalyst-free Pyrolysis procedure lowers consumables price.

4. Shortening the time cycle required for the whole analytical train from sampling to isotope measurement, can be considered one-day technique.

5. Requires no fixed installations (e.g. Kjeldahl technique or vacuum distillation).6. Eliminating the need to hazardous preservatives. 7. Combining neutralization using both silver oxide and potassium hydroxide which lowers

the chemicals cost (1g of Ag2O costs about 4-5 USD).8. Offering a robust, low labor intensive and rapid offline combustion based method.9. Low temperature allows usage of Pyrex instead of more expensive quartz as a combustion

container. 10. No previous evaporation is necessary to reduce the sample volume

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