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. NO 3-
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 (Ag 2O, 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

o
850 C 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 NO 3- 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 NO 3- 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
Fig. (5): Relationship between the
interfering solutions, respectively.
calcium ion Chloride ions as expected produced no measurable Nitrogen-15 isotope
concentration andfractionation,
the resultanteven if adsorption interference produced significant NO3- loss as seen in Fig. (3).
δ15N value of ammonium at 8 mg
NH3-N /L using DowexThis50wX8
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.

120

100
Recovery of nitrate, %

80

60

40
100 ml
250 ml
20
500 ml

0
0 50 100 150 200 250 300 350 400 450

Amount of interfering chloride ion, mg

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.
 120 -12 120 -12

100 -10 100 -10

80 -8 80 -8

Air N2

Air N2
recovery of nitrate , %

recovery of nitrate , %
60 -6 60 -6

δ 15 N, ‰

δ 15 N, ‰
40 -4 40 -4

R e c o ve ry R e c o ve ry
20 D e lta -2 20 D e lta -2
A s signe d d e lta va lue A s s igne d d e lta va lue
0 0 0 0
250 250 500 1000 2000 2000 3000 3000 4000 200 400 600 800 1200 1600
- -
C l i o n c o n c . m g /L C l i o n c o n c . m g /L

a. 100 ml sample volume b. 250 ml sample volume

1 2 0 .0 0 -12

1 0 0 .0 0 -10

8 0 .0 0 -8

Air N2
recovery of nitrate , %

6 0 .0 0 -6

δ 15 N, ‰
4 0 .0 0 -4

R e c o ve ry
2 0 .0 0 D e lta -2
A ssigne d d e lta va lue
0 .0 0 0
50 100 150 200 300 400 600 800
-
C l i o n c o n c . m g /L

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 SO 42- 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 SO42- 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).

120

100
Recovery of nitrate, %

80

60

40
100 ml
20 250 ml
500 ml
0
0 50 100 150 200 250 300 350 400 450

Amount of interfering sulphatee ion, mg

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 SO42- 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 BaSO 4
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 HCO 3-
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.
 120

100

Recovery of nitrate, %
80

60

40
100 ml

20 250 ml
500 ml
0
0 50 100 150 200 250 300 350 400 450

Amount of interfering bicarbonate ion, mg/L

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 NO 3- (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).
 2.5

2
y = 0.0048x + 0.7111

Passed DOC load, mg-C

R2 = 0.201
1.5

0.5

0
0 20 40 60 80 100 120 140 160

Inital amneded DOC amount, mg-C

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

120

100
Recovery of Nitrate, %

80

60

40

20

0
0 200 400 600 800 1000 1200 1400 1600

Conc. of interfering DOC, mg-C/L

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.
δ15N,‰Air N2
a
EAEA, Egypt UFZ, Germanyb
Material
Modified Vacuum Ammonia
Denitrifier EA-CF IRMS
combustion distillation diffusion
KNO3, Sigma- -4.57 -5.16 - -3.6 -4.11
Aldrich -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
17.7 - - 14.4 18.57
NaNO3,
18.6 - - 14.5 18.52
Average 18.15±0.64 14.45±0.07 18.54±0.04
-28.74 - - -24.3 -29.13
KNO3, Applichem
-27.72 - - -23.9 -28.81
Average -28.23±0.72 -24.1±0.28 -28.97±0.23
-1.08 -0.89 -1.34 - -
Ca(NO3)2, WINLAB
-0.422 -1.15 -1.8 - -
Average -0.75±0.47 -1.02±0.18 -1.57±0.32
Ammonium Nitrate 0.862 - - -0.4 0.57
fertilizer* 1.53 - - -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.
δ15N‰Air N2
Material
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 Ca. 30 – 40 minutes 90 – 100 %
• Cation exchange resin Ca. 5 to 120 minutes depending on sample
volume
Collection of NO3 - from water sample from 5 to 120 minutes, depending on the 98 – 100%
on anion exchange resin sample volume

NO3- stripping from anion exchange 10 to 15 minutes for 2 ml resin columns 95 – 100%
resins

Neutralization:
• Ag2O pre-neutralization step Ca. 20 minutes (Excluding pre-wash step)
• Filtration Ca. 5-10 minutes
• KOH final neutralization step Ca. 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

References
1. Ahmed, M. A., Aly, A. I. M., Bastaweesy A. M. and Gomaa, H. E. (2008) Modified low-priced
ammonia diffusion method for the analysis of nitrogen isotopic composition of ammonium and
nitrate in different water matrices. Egypt. J. Rad. Sci. Applic., 21(1), 1.
2. Aly A. I. M., Bastaweesy, A. M., Ahmed, M. A., and Gomaa, H. E. (2007) Isolation of
Ammonium from Freshwaters for Nitrogen Isotope Analysis Using Amberlite IR120 and Dowex
50WX8 Ion Exchange Resins: A Comparative Study. Isotopes and Rad. Res. 38(4), 953.
3. Aly, A.I. M. and Mohamed, M. A. (1982) Separation of ammonium and nitrate for isotopic
analysis of Nitrogen-15 using vacuum distillation method. Isotopenpraxis, Bd. 19, H.3.
4. Aly, A.I. M.; Faust, H.; Hallaba, E. and Mohamed, M. A. (1981) Comparison between a
modified wet hypobromite method and a dry ampoule technique of sample preparation for precise
isotopic analysis of nitrogen-15. Arab journal of nuclear sciences and applications 15-1, 145.
5. Aravena R., Evans M. L. & Cherry J. A. (1993) Stable isotopes of oxygen and nitrogen in
source identification of nitrate from septic systems. Ground Water 31, 180.
6. Böttcher J., Strebel O., Voerkelius S. & Schmidt H.-L. (1990) Using isotope fractionation of
nitratenitrogen and nitrate-oxygen for evaluation of microbial denitrification in a sandy aquifer. J.
Hydrol. 114(3-4), 413.
7. Bremner, J. M. and Keeney, D. R. (1965) Steam distillation methods for determination of
ammonium, nitrate and nitrite. Anal. Chim. Acta, 485.
8. Brooks, P. D., Starks, J. M., McInteer, B. B. and Preston, T. (1989) Diffusion method to
prepare soil extracts for automated nitrogen-15 analysis. Soil Sci. Soc. Am. J. 53, 1707.
9. Casciotti K. L., Sigman D. M., Galanter Hastings M., Böhlke J. K., Hilkert A. (2002)
Measurement of the oxygen isotopic composition of nitrate in seawater and freshwater using the
denitrifier method. Anal. Chem. 74, 4905.
10. Chang, C.C.Y., Langston, J., Riggs, M., Campbell, D.H., Silva, S.R., Kendall, C. (1999) A
method for nitrate colleciton for δ15N and δ18O analysis from waters with low nitrate
concentrations. Can. J. Fish. Aquatic Sci. 56, 1856.
11. Dongmei Xue, Jorin Botte, Bernard De Baets, Frederik Accoe, Angelika Nestler, Philip
Taylor, Oswald Van Cleemput, Michael Berglund and Pascal Boeckx (2009) Present
limitations and future prospects of stable isotope methods for nitrate source identification in
surface- and groundwater. Water Research 43, 1159.
12. Downs, M., Michener, R., Fry, B. and Nadelhoffer, K. (1999) Routine measurement of
dissolved inorganic 15N in precipitation and stream water. Environmental Monitoring and
Assessment 55, 211.
13. Durka W., Schulze E. D., Gebauer G & Voerkelius S. (1994) Effects of forest decline on uptake
and leaching of deposited nitrate determined from 15N and 18O measurements. Nature, 372, 765.
14. Fiedler R. & Proksch G. (1972) The determination of the nitrogen content and nitrogen-15
abundance by means of the nitrogen gas generated from inorganic and organic materials. Anal.
Chim. Acta 60, 277.
15. Francis J. DeSilva (1999) Essentials of ion exchange, presented at the 25th annual WQA
conference.
16. Gomaa, H.E. (2007) Development and modification of some selected techniques for measuring
15
N isotopic content of ammonium and nitrate in water. M. Sc. Thesis, 2.
17. Hauck R. D. (1982) Nitrogen-isotope-ratio analysis. In: Methods of soil analysis, Part 2.
Chemical and microbiological properties. A. L. Page, R. H. Miller & D. R. Keeney (eds.).
American Society of Agronomy Inc. Soil Science Society of America Inc., Madison, Wisconsin.
Agronomy Series, 2nd edition, No. 9: 735-779.
18. Kendall C. (1998) Tracing nitrogen sources and cycling in catchments. In: Isotope tracers in
catchment hydrology. C. Kendall & J. J. McDonnell (eds.). Elsevier Science B.V, Amsterdam:
534-569.
19. Kendall C. and Grim E. (1990) Combustion tube method for measurement of nitrogen isotope
ratios using calcium oxide for total removal of carbon dioxide and water. Anal. Chem. 62, 526.
20. Khan, S. A., Mulvaney, R. L. and Hoeft, R. G. (2000) Direct diffusion methods for inorganic-
nitrogen analysis of soil. Soil Sci. Soc. Am J. 64, 1083.
21. Kornexl BE, Gehre M, Hofling R, Werner RA. (1999) On-line δ18O measurements of organic
and inorganic substances Rapid Commun. Mass Spectrom. 13, 1685.
22. Mathieu Sebilo, Bernhard Mayer, Micheline Grably, Daniel and Andre Mariotti (2004) The
use of the 'Ammonium diffusion' method for δ15N-NH4+ and δ15N-NO3- measurements: comparison
with other techniques. Environ. Chem. 1, 99 doi: 10.1071/EN04037.
23. Mulvaney R. L. (1993) Mass spectrometry. In: Nitrogen isotope techniques. R. Knowles & T. H.
Blackburn (eds.). Academic Press, San Diego, CA: 11-57.
24. Mulvaney R. L. and. Khan, S. A. (2001) Diffusion method to determine different forms of
nitrogen in soil hydrolysates. Soil Sci. Soc. Am J. 65, 1284.
25. Mulvaney R. L., Fohringer C. L., Bojan V. J., Michlik M. M. & Herzog L. F. (1990) A
commercial system for automated nitrogen isotope-ratio analysis by the Rittenberg technique.
Rev. Sci. Instrum. 61, 897.
26. Ohte, N., et al. (2004) Tracing sources of nitrate in snowmelt runoff using a high resolution
isotopic technique, Geophys. Res. Lett. 31, L21506, doi:10.1029/2004GL020908.
27. Ostrom, N. E., et al. (2002) Nitrogen transformations and NO3- removal at a soil-stream interface:
A stable isotope approach, Ecol. Appl., 12(4),1027.
28. Rittenberg D. (1948) The preparation of gas samples for mass spectrographic isotope analysis.
In:Preparation and measurement of isotopic tracers. D. W. Wilson, A. O. C. Nier & S. P. Reimann
(eds.). J.W. Edwards, Ann Arbor, Michigan: 31-42.
29. Rock, L., and B. Mayer (2004), Isotopic assessment of sources of surface water nitrate within the
Oldman River basin, Southern Alberta, Canada. Water Air Soil Pollut. 4, 545.
30. Scott D. Wanke, Carol Kendall, and Adina Paytan (2009) Using nitrate dual isotopic
composition (δ15N and δ18O) as a tool for exploring sources and cycling of nitrate in an estuarine
system: Elkhorn Slough, California. Journal of geophysical research 114, G01011, doi:
10.1029/2008JG000729.
31. Sigman, D. M., Casciotti, K. L., Andreani, M., Barford, C., Galanter, M. and Bo1hlke J. K.
(2001) A bacterial method for the nitrogen isotopic analysis of nitrate in seawater and freshwater.
Anal. Chem. 73, 4145.
32. Silva, S. R., Kendall, C., Wilkison, D. H., Ziegler, A. C., Chang, C. C. Y., Avanzino, R. J.
(2000) A new method for collection of nitrate from fresh water and the analysis of nitrogen and
oxygen isotope ratios. J. Hydrol. 228, 22.
33. Sprinson D. B. & Rittenberg D. (1948) Preparation of gas samples for mass spectrometric
analysis of isotope abundance. U. S. Naval Med. Bull., Supplement 115 (March-April): 82-93.
34. Sprinson D. B. & Rittenberg D. (1949) The rate of utilization of ammonia for protein synthesis. J.
Biol.Chem. 180, 707.
35. Stickrod R. D. & Marshall J. D. (2000) On-line nitrate-δ15N extracted from groundwater
determined by continuous-flow elemental analyzer/isotope ratio mass spectrometry. Rapid
Commun. Mass Spectrom. 14, 1266.
36. Van Dyke, E., and K. Wasson (2005) Historical ecology of a central California estuary: 150
years of habitat change. Estuaries 28(2), 173.
37. Wassenaar, L.I. (1995) Evaluation of the origin and fate of nitrate in the Abbotsford Aquifer
using the isotopes of 15N and 18O in NO3-. Appl. Geochem. 10, 391.
38. Wojciech M. Chmura, Kazimierz Różański, Tadeusz Kuc, Zbigniew Gorczyca (2009)
Comparison of two methods for the determination of nitrogen and oxygen isotope composition of
dissolved nitrates. NUKLEONIKA 54, 1.