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www.elsevier.com/locate/marchem
Marine Chemistry 96
Continuous colorimetric determination of trace ammonium in
seawater with a long-path liquid waveguide capillary cell
Qian Perry Lia,b,*, Jia-Zhong Zhanga, Frank J. Millerob, Dennis A. Hansellb
aOcean Chemistry Division, Atlantic Oceanographic and Meteorological Laboratory, National Oceanic and Atmospheric Administration,
4301 Rickenbacker Causeway, Miami, FL 33149, USAbDivision of Marine and Atmospheric Chemistry, Rosenstiel School of Marine and Atmospheric Science, University of Miami,
4600 Rickenbacker Causeway, Miami, FL 33149, USA
Available online 16 March 2005
Abstract
An automated method for routine determination of nanomolar ammonium in seawater has been developed using segmented
flow analysis coupled with a 2-m-long liquid waveguide capillary cell. Conventional photometric detector and autosampler
were modified for this method. The optimal concentrations of the reagents and parameters for the development of indophenol
blue are discussed. The method has low detection limit (5 nM), high precision (5% at 10–100 nM) and the advantage of rapid
analysis of a large number of samples. The method has been used to examine the distribution of ammonium in Florida Bay and
Biscayne Bay.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Ammonium; Seawater; Automated analysis; Liquid waveguide
1. Introduction
Ammonia (NH3) is an important nitrogen species
in the natural environment. As a dominant gaseous
base in the air, it plays a very important role on the
acid–base chemistry of the atmosphere and greatly
influences the atmospheric sulfur cycle in the remote
0304-4203/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.marchem.2004.12.001
* Corresponding author. MAC/RSMAS/University of Miami,
4600 Rickenbacker Causeway, Miami, FL 33149, USA. Tel.: +1
305 4214019; fax: +1 305 4214689.
E-mail address: [email protected] (Q.P. Li).
marine boundary (Galloway, 1995; Quinn et al.,
1996). Being a gaseous compound, ammonia
exchanges at the air–sea interface although its flux
is not well quantified in a variety of environments
(Bouwman et al., 1997). Ammonia can easily dissolve
in water and become ammonium ion (NH4+). In the
ocean, ammonium is the dominant form, with
ammonia as a minor component. Ammonium is also
one of the most commonly used nutrients by marine
phytoplankton. Compared to nitrate, phytoplankton
generally prefer ammonium because additional energy
is required for them to reduce nitrate to ammonium
(D’Elia and DeBoer, 1978; Wheeler and Kokkinakis,
(2005) 73–85
Q.P. Li et al. / Marine Chemistry 96 (2005) 73–8574
1990; Harrison et al., 1996). Because ammonium is
consumed by phytoplankton in the surface waters of
the ocean, it is often well below micromolar concen-
trations and difficult to accurately quantify by conven-
tional analytical techniques.
To study the nutrient cycle in the oligotrophic
ocean, nanomolar-level nutrient analytical methods
are needed. Methods for nitrate and nitrite (Yao et
al., 1998; Zhang, 2000; Masserini and Fanning,
2001), phosphate (Karl and Tien, 1992; Zhang and
Chi, 2002), iron (Vink et al., 2000; Zhang et al.,
2001a,b) and ammonium (Brzezinski, 1987; Jones,
1991; Kerouel and Aminot, 1997) at nanomolar
concentrations have been developed. The applica-
tion of these methods to field studies (Law et al.,
2001; Woodward and Rees, 2001; Zhang et al.,
2001a,b) has greatly improved our understanding of
nutrient dynamics in the surface of the oligotrophic
ocean. However, low-level ammonium determina-
tion still suffers from low sensitivity and high
contamination (Aminot et al., 1997), particularly on
shipboard measurements (Harrison et al., 1996).
Therefore, it is desirable to develop a highly
sensitive method for shipboard automated measure-
ments of ammonium.
The most popular technique for the determination
of ammonium in aqueous samples is the colorimetric
method based on the formation of indophenol blue
(Solorzano, 1969; Hansen and Koroleff, 1999).
Although this method is simple, economical and
easy for automation, it is not sensitive enough for
the determination of submicromolar concentrations
of ammonium (Aminot et al., 1997). A selective
electrode method was found easy to operate (Garside
et al., 1978), but requires long equilibration times.
Moreover, its detection limit of 0.2 AM is not
sufficient for routine work in oligotrophic waters. To
increase the sensitivity, a solvent extraction method
(Brzezinski, 1987) was developed, but the procedure
is time consuming and labor intensive and thus
impossible for shipboard automated measurements.
Although the fluorometric method (Jones, 1991;
Kerouel and Aminot, 1997) has a detection limit
of nanomolar concentrations for ammonium, the
method often suffers from high background fluo-
rescence and interference by methylamines. An ion
chromatography method coupled with a flow injec-
tion gas diffusion technique has a reported detection
limit of 20 nM (Gibb et al., 1995), but it requires
expensive chromatographic equipment and a long
diffusion time.
A liquid waveguide capillary cell made out of
AF-2400 Teflon has been applied to enhance the
sensitivity of spectrophotometric analysis of trace
concentrations of ferrous, chromate, nitrate and
phosphate ions in aqueous samples (Waterbury et
al., 1997; Yao et al., 1998; Zhang, 2000; Zhang and
Chi, 2002). This newly developed liquid waveguide
capillary cell (World Precision Instrument, Sarasota,
FL, USA) has the advantage of low light attenuation,
is easy to clean, and is, therefore, very suitable for
low-level photometric measurements. Here, we
incorporate a 2-m-long liquid waveguide capillary
cell to a modified gas-segmented continuous flow
auto-analyzer, thereby enhancing the sensitivity and
the precision of ammonium determination in sea-
water by the indophenol blue method.
2. Experiment
2.1. Liquid waveguide capillary cell and spectra
system UV–Vis detector
The liquid waveguide capillary cell (LWCC) is an
optical sample cell that uses the World Precision
Instruments’ patented Aqueous Waveguide Technol-
ogy (Liu, 1996). It offers an increased optical path
length compared to a standard cuvette and a small
sample volume for spectroscopy application. In this
study, a 2-m-long LWCC made of quartz capillary
tubing (550 Am ID) was used. A Spectra System
UV–Vis detector (UV1000) was modified to adapt
the LWCC to an auto-analyzer for continuous
analysis. The conventional flow cell assembly (0.55
cm path length) was removed and replaced with two
custom-made fiber optic connectors. The LWCC was
connected to the detector by two fiber optical cables
that transmit the source light from the lamp of the
detector through the LWCC and to the photodiode
detector. A detailed description of the coupling of an
LWCC with a detector is given by Zhang (2000). In
this study, before and after each run, the LWCC was
cleaned with 10% HCl, 1 M NaOH and deionized
water, respectively. To get a good signal, each step
should last at least 10 min.
Q.P. Li et al. / Marine Chemistry 96 (2005) 73–85 75
2.2. Automated analytical system
2.2.1. Autosampler
Contamination has been reported to be a major
problem that affects the precision and accuracy of
ammonium determination in submicromolar concen-
trations. Aminot et al. (1997) have pointed out that a
sample volume less than 50 ml is not suitable for
routine measurements of trace ammonium in natural
waters. However, most of the traditional high-speed
autosamplers for gas-segmented flow systems are
designed for small cups. Autosamplers that can
accommodate large volume samples are mostly
designed for FIA systems with relatively low sam-
pling speeds, which can introduce large intersample
bubbles. In FIA systems, intersample bubbles can be
avoided by the automated control of an injection
valve. In gas-segmented flow systems, bubbles are
usually removed to a waste line by a debubbler before
the stream flows into a detector. Extra large sizes of
intersample bubbles can escape from the debubblers
and get into the detector causing interferences. The
other reason to remove the intersample bubbles is to
reduce the air contamination. This will be further
discussed in the Section 3.1. In this study, a traditional
high-speed autosampler (WESTCO Scientific Instru-
ment) was modified for large cups (50 ml) and a
debubbler successfully removed the intersample bub-
bles generated by this sampler.
Lamp
640 nm
Photodiode
2 m LWCC
UV/Vis Detector
Debubbler
Data Acquisition
System
Waste
Manifold
Wa
25 cm
25 cm
Fig. 1. Schematic flow diagram ofmanifold configuration for the ammonium
2.2.2. Manifold configuration
A gas-segmented continuous flow colorimetric
method was used for the analysis of ammonium in
seawater. The flow diagram is shown in Fig. 1. The
analytical method is based on the conventional
indophenol blue method (Solorzano, 1969) as modi-
fied to a gas-segmented continuous flow system
(Zhang et al., 1997). The chemical reaction takes
place in two steps. Firstly, the addition of hypo-
chlorite to the ammonium samples results in the
formation of mono-chloramines. Secondly, phenol
reacts with the mono-chloramines to produce an
indophenol blue dye. The maximum absorbance of
the indophenol blue is measured at 640 nm. To
increase the speed of this reaction, a catalyst (sodium
nitroferricyanide) and a heater were used in this
study.
2.2.3. Data acquisition system
Concentrations of ammonium in the samples are
calculated from the linear regression, obtained from
the standard curve in which the concentrations of the
calibration standards are entered as the independent
variable, and their corresponding peak heights are the
dependent variable. The operation of the autosampler
and the collection and analysis of data are simulta-
neously controlled by a computer based data acquis-
ition system (SOFTPAC, Measurement Microsystems
A-Z Inc.).
Nitroferricyanide
NaDTT
Complexing
reagent
Nitrogen
Sample/ wash
= 80s: 60s
Phenol
Pump (ml/min)
0.10
0.10
0.10
1.01
0.25
0.32
0.41
ste
Autosampler
Heater
0.41
25 cm
25 cm
150 cm
analysis with LWCC. The inner diameter of coils used here is 1.0mm.
Q.P. Li et al. / Marine Chemistry 96 (2005) 73–8576
2.3. Low ammonium seawater and standards
Low ammonium seawater was prepared by the
removal of background ammonium in low-nutrient
seawater (LNSW) collected from the surface of the
Gulf Stream. Several drops of 1 M NaOH were
added to the LNSW until a small amount of
precipitation was observed. After that, it was swirled
and heated to 60 8C. This solution was then sealed
and naturally cooled to room temperature and finally
filtered through a 0.45-Am filter. Ammonium stock
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.0 5.0 10.0 15
Pheno
Abs
orba
nce
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.0 5.0 10.0 1
Phen
Abs
orba
nce
Fig. 2. (a) Relationship between phenol and blank. (b) Relationship bet
solution. For (a) and (b), other reagents and temperature were fixed (NaD
standard solutions (10 mM) were prepared from
analytical reagent-grade pre-dried (105 8C for 2 h)
ammonium sulfate ((NH4)2SO4) and stored at 48C in
a refrigerator. Working standards were prepared from
serial dilutions of stock solutions with the low
ammonium seawater. Glass cups were found to be
subject to ambient ammonium contamination, which
might be caused by the adsorption of ammonium on
the glass walls. Therefore, plastic cups made of
polypropylene were used for both the samples and
standards.
.0 20.0 25.0 30.0
l (mM)
(a)
5.0 20.0 25.0 30.0
ol (mM)
(b)
ween phenol and the net signal of a 600 nM ammonium standard
TT: 1.6 mM, FSCN: 1.4 mM, T=80 8C).
Q.P. Li et al. / Marine Chemistry 96 (2005) 73–85 77
2.4. Reagents
All the chemicals used in this study were of
analytical reagent-grade. Deionized water (DIW) used
for preparing reagents was purified by a distilling unit
followed by a Millipore Super-Q Plus Water System
that produces water with 18 Mg resistance. To avoid
contamination in the analysis, the deionized water used
was purified daily. All the samples and reagents were
stored in high-density polypropylene bottles that were
ultrasonicated in 1 M NaOH for 6 h at room temper-
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.00 0.50 1.00 1.50 2.00NaDT
Abs
orba
nce
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.00 0.50 1.00 1.50 2.00
NaDT
Abs
orba
nce
Fig. 3. (a) Relationship between NaDTT and blank. (b) Relationship betw
solution. For (a) and (b) other reagents and temperature were fixed (Phen
ature and rinsed several times by deionized water prior
to their use. Concentrations of phenol, sodium dichlor-
oisocyanuric acid and sodium nitroferricyanide were
varied over a wide range to define the optimal reaction
concentration for each reagent. After optimization, the
following recipe was found suitable for routine
analyses of trace ammonium in seawater by LWCC.
Complexing reagent: Dissolve 80 g of sodium
citrate (Na3C6H5O7d 2H2O), 4.0 g of sodium
hydroxide, and 10.0 g of EDTA in 1000 ml distilled
water. The daily working solution is prepared by
2.50 3.00 3.50 4.00 4.50T (mM)
(a)
2.50 3.00 3.50 4.00 4.50
T (mM)
(b)
een NaDTT and the net signal of a 500 nM ammonium standard
ol: 10.0 mM, FSCN: 1.8 mM, T=80 8C).
Q.P. Li et al. / Marine Chemistry 96 (2005) 73–8578
adding 1 ml Brij-35 (ICI Americas) to 200 ml of
this complexing reagent.
Phenol reagent: Dissolve 1.0 g of solid phenol
(C6H5OH) in 1000 ml distilled water.
Hypochlorite reagent (NaDTT): Dissolve 0.35 g of
dichloroisocyanuric acid sodium salt (NaC3Cl2N3O3)
in 1000 ml distilled water.
Catalyst: Dissolve 0.55 g of sodium nitroferricya-
nide (Na2Fe(CN)5NOd 2H2O) in 1000 ml distilled
water.
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.00 0.50 1.00 1FSCN
Abs
orba
nce
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.00 0.50 1.00 1
FSCN
Abs
orba
nce
Fig. 4. (a) Relationship between nitroferricyanide (FSCN) and blank. (b) R
500 nM ammonium standard solution. For (a) and (b), other reagents and te
All the reagents were prepared fresh daily except
the complexing reagent that was prepared each week.
3. Results and discussion
3.1. The optimization of the flow configuration
For the gas segmented continuous flow analysis,
the injection of bubbles is critical to the final output
.50 2.00 2.50 3.00 (mM)
(a)
.50 2.00 2.50 3.00
(mM)
(b)
elationship between nitroferricyanide (FSCN) and the net signal of a
mperature were fixed (Phenol: 10.0 mM, NaDTT: 1.6 mM, T=80 8C).
Q.P. Li et al. / Marine Chemistry 96 (2005) 73–85 79
of the signal. The mixing of reagents and the
sample is achieved through the performance of the
bubble system, which contributes to the high
precision of gas-segmented flow analysis. Seg-
mented bubbles are usually injected from a pump
tube by pumping air to the flow stream. High
erratic peaks observed in trace measurements are
possibly due to ammonia contamination in the
ambient air. To minimize ammonia contamination,
pure nitrogen was used as the segmentation gas.
However, intersample bubbles will inevitably bring
ambient air into the flow system. Therefore, a
debubbler is introduced after the autosampler to
remove the intersample bubbles and to avoid the
potential contamination. Although the surfactant is
not involved in the chemical reaction, it does
influence the baseline signal. Therefore, the amount
of surfactant should be minimal. In this study, 1 ml
of Brij-35 in 200 ml of working complexing reagent
was found to be enough to keep a regular pattern
for the bubble stream. To get a smooth baseline, 1
M NaOH solution followed by deionized water was
pumped through the system before the experiment
to clean the trace amounts of ammonium left in the
system.
0.00
0.10
0.20
0.30
0.40
0.50
0.60
25 30 35 40 45 50 55
T(°C)
Abs
orba
nce
Fig. 5. Relationship of temperature and the net signal of 600 nM ammon
1.4 mM).
3.2. The influence of reagent concentrations
According to the Lambert–Beer law, the absorbance
is proportional to the concentration of analyte and the
path length of the light in the sample solution. An
increase of the path length of the cell will directly
enhance the sensitivity of spectrophotometry, which
has been applied to improve the determination of
various ions in natural waters (Waterbury et al., 1997;
Yao et al., 1998; Zhang et al., 2001a,b; Zhang and Chi,
2002). However, the increase of light path proportion-
ally enlarges both the reagent blank and sample
signals. For measurements whose reagent blanks are
very low (e.g., nitrite and iron), the final blanks after
enhanced by the long flow cell still allow sufficient
source light to reach the detector. However, the reagent
blank for ammonium analysis is high even in the
conventional short-cell colorimetric measurement
(Aminot et al., 1997). In this case, the reagent blank
in the long flow cell absorbs so much source light that
there is not enough light to reach the detector. To
effectively reduce the reagent blanks in trace ammo-
nium analysis, a series of experiments were designed
to investigate the optimal concentrations for reagents
with minimal reagent blanks and maximal signals.
60 65 70 75 80 85 90
ium standard solution (Phenol: 10.0 mM, NaDTT: 1.6 mM, FSCN:
Q.P. Li et al. / Marine Chemistry 96 (2005) 73–8580
3.2.1. Complexing reagent
Citrate is the most common complexing reagent
used for the ammonium analysis in seawater to prevent
the precipitation of metal hydroxides such as Mg(OH)2and Ca(OH)2. Some authors use EDTA as the
complexing reagent (Gibb et al., 1995), while others
use EDTA and citrate together (Aminot et al., 1997;
Zhang et al., 1997). It has been argued that EDTA
should be excluded from reagents because it may
reduce available chlorine (Kempers and Kok, 1989).
Laboratory studies showed that the effective pH at
which citrate and EDTA work best is quite different
(Gibb et al., 1995). Citrate is effective only in pHb11
y = 0.0008x + 0.003
R2 = 0.9997
0.00
0.40
0.80
1.20
1.60
2.00
0 500 1000
Ammon
Abs
orba
nce
y = 0.0008x - 0.0004
R2 = 0.9979
0.000
0.020
0.040
0.060
0.080
0.100
0 10 20 30 40Ammo
Abs
orba
nce
Fig. 6. (a) The linear dynamic range of ammonium determination by gas-se
are data beyond the linearity. (b) The calibration curve for ammonia (10–
and EDTA works at pHN12. For ammonium analysis
by colorimetric methods, a pH range of 10.5–11.5 was
reported to give satisfactory results for the develop-
ment of indophenol blue (Patton and Crouch, 1977;
Hansen and Koroleff, 1999; Aminot et al., 1997). A
higher pH (N12) was used to increase the reaction rate,
especially in automated systems (Aminot et al., 1997;
Zhang et al., 1997). Therefore, for a pH change
between 11.0 and 12 or above, both citrate and EDTA
should be used. In this study, final concentrations of 54
mM of citrate (after dilution by reagents and sample),
together with 5.4 mM of EDTA, was enough to
prevent the precipitation of divalent metal ions in
3
1500 2000 2500
ia (nM)
(a)
50 60 70 80 90 100nia (nM)
(b)
gmented continuous-flow analysis with the LWCC. The open circles
100 nM).
Q.P. Li et al. / Marine Chemistry 96 (2005) 73–85 81
seawater in a pH range of 11–12. Large precipitates
were only observed when the citrate level was below
10 mM. The hydrolysis of the magnesium-citrate
complex was found to change the final pH of seawater
and interfere with the color formation (Pai et al., 2001),
and thus, an increased amount of NaOH in this study
can overcome the buffer capacity of this complex.
3.2.2. Phenol
Phenol, 2-methylphenol and 2-chlorophenol are
currently the most satisfactory reagents for the Berthe-
lot reaction (Patton and Crouch, 1977), for their high
sensitivity involved in the development of indophenol
blue. Due to the toxicity of phenol, some people used
salicylate as a substitute to measure ammonium in
seawater, but the sensitivity is significantly decreased
(Bower and Holm-Hansen, 1980; Kempers and Kok,
1989). The relationships of phenol concentrations with
blanks in low ammonia seawater and the net absorb-
ance of a 600 nM ammonium sample are shown in Fig.
2. The blank is very sensitive to the amount of phenol
used at low concentrations and reaches a stable value at
phenol concentrations greater than 10.0 mM (Fig. 2a).
The net signal of sample is calculated by the difference
between the absorbance of samples and the value of the
blank. The optimal concentration of phenol is 8.0–10.0
mM, at which the blank is low and the net signal of
sample is high (Fig. 2a and b).
3.2.3. Hypochlorite
Because sodium hypochlorite is sensitive to the
changes of light and temperature (Bower and Holm-
0
0.00
0.05
10Time (
Abs
orba
nce
Fig. 7. The typical output of signals of 10–50 nM of ammonium analy
concentrations of 0, 10, 20, 30, 40 and 50 nM, respectively.
Hansen, 1980), a more stable chlorine donor, sodium
dichloroisocyanurate (NaDTT), was used. However,
higher temperatures are required in order to liberate
its chlorine (Kempers and Kok, 1989). The concen-
tration of NaDTT was optimized by examining
signals of 500 nM ammonium samples after varying
the concentration levels of NaDTT, while keeping
the concentrations of all other reagents constant. A
plot of absorbance vs. concentration of NaDTT
shows the optimum of NaDTT to be between 1.0
mM and 2.0 mM (Fig. 3). The slight increase of the
sample signal with increasing chlorine concentration
is in agreement with the work of Kempers and Kok
(1989). Due to the reaction with seawater constitu-
ents, the available chlorine is lower than the chlorine
originally added to the system. In order to achieve
the same sensitivity, the chlorine concentration
required for the reaction in seawater is about four
times higher than that in pure water.
3.2.4. Catalyst
Without an appropriate catalyst, the reaction rate
for the formation of indophenol blue is very slow.
Generally, nitroprusside (NP) is used as a catalyst in
the IPB method, but in basic medium it becomes
nitroferricyanide (NF) and produces aquopentacyano-
ferrate (AqF). AqF was indeed the actual catalyst
(Patton and Crouch, 1977), but it usually needs
ultraviolet radiation to activate and is sensitive to
the change of pH. Therefore, sodium nitroferricyanide
is used as a catalyst in this study without radiation. To
study the effect of nitroferricyanide on the chemical
20minute)
sis by LWCC, Peaks are two replicates samples with ammonium
Q.P. Li et al. / Marine Chemistry 96 (2005) 73–8582
reaction, two experiments were carried out in which
deionized water was used as a wash. In one study, the
change of absorbance of reagent blank was examined
with a series of different concentrations of catalyst.
The other study examined the response of a 500 nM
ammonium sample to the same series of catalysts. The
net signal of the samples was calculated by subtract-
ing the blank from the value of sample. The best
-81.0 -80.8
24.8
25.0
25.2
25.4
25.6
25.8
45
6
78910
11
1213
14
15
16
17
2425
26272829
30
3132
33
3435
3637
383
Florida Bay and Biscayne(9/15~16/2004
Fig. 8. Spatial distribution of ammonium in Florida Bay and Biscayne Bay
stations in Florida Bay and Biscayne Bay, respectively.
concentration of nitroferricyanide is 1.6–2.0 mM, as
shown in Fig. 4.
The best temperature for the formation of indo-
phenol blue for this system is 808C (Fig. 5). It should
be noted that different systems might have a different
optimal temperature, because heat transfer efficiencies
may vary. These variations are related to the length of
the heating coil and the performance of the heater. We
-80.6 -80.4 -80.2
12
3
18
19
20
2122
23
9 40
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10B11
B12
B13
B14
B15
B16
0.2
0.4
0.6
0.8
1
2
4
6
8
10
Bay Survey)
Ammonium (uM)
during September 2004 survey. Numbers 1–40 and B1–B16 are the
y =1.0299x R2 = 0.9816
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40Colorimetric method (0.55cm)
Liq
uid
wav
egui
de m
etho
d
Fig. 9. Comparison of liquid waveguide method with the conven-
tional colorimetric method. The unit of ammonium concentration
used here is micromolar.
Q.P. Li et al. / Marine Chemistry 96 (2005) 73–85 83
did observe a decrease in the optimal temperature
when the length of the heating coil was increased.
3.3. Linear dynamic range and detection limit
Using the manifold configuration shown in Fig. 1
and the above optimal recipe for reagents, the upper
limits of the linear dynamic range of ammonium
analysis is 1000 nM (Fig. 6). The calibration curve
was calculated from the average of six independent
runs with the standard deviation less than 5%. A
typical output signal of automated trace ammonium
analysis is shown in Fig. 7. A linear absorbance
response to ammonium concentrations below 1000
nM can be obtained as
Absorbance ¼ 0:0033F0:0013ð Þþ 0:000 8005F0:000 0065ð Þ� NHþ
4
� �nMð Þ
with r2=0.9997 (n=20). Above this concentration
range, the measured absorbance is lower than that
predicted from the linear relationship as shown in
open circles in Fig. 6. The linear dynamic range of the
ammonium analysis can be extended by either using a
shorter LWCC or by diluting the sample with low
ammonium seawater by adding a dilution line to the
sample flow. The detection limit of this method is 5
nM, which is estimated as three times the standard
deviation of measurement blanks.
For ammonium analysis in seawater, the correction
of refractive index interference is important (Aminot et
al., 1997). Because the refractive index signal is much
smaller than the analytical signal, it is usually qualified
by measuring the absorbance of samples with different
salinities relative to deionized water in the absence of
color formation. For ammonium analysis, this is
achieved by using a series of water samples with
different salinities as ammonium samples and deion-
ized water as the wash solution, with the exception of
the sodium nitroferricyanide being replaced by deion-
ized water. The resultant absorbance was converted to
ammonium concentrations. The relationship of meas-
ured refractive index with different salinities is:
NHþ4
� �ri¼ 0:233þ 0:376S; r2 ¼ 0:998; n ¼ 8
� �
Where [NH4+]ri is a correction for refractive index for
ammonium sample in nanomolar and S is the salinity
of sample. To avoid the significant refractive
interference for low-level ammonium samples, it is
necessary to match the salinity of the wash solution
with that of the sample. Low ammonium seawater is
recommended.
4. Field application
This method has been applied to water samples
from a Florida Bay and Biscayne Bay survey
conducted in September 2004. Samples were col-
lected from 40 stations in Florida Bay and 16 stations
in Biscayne Bay, as is shown in Fig. 8. The
ammonium samples were preserved by adding
several drops of chloroform and sent back to
laboratory for analyses at days end. These samples
were first measured by the conventional auto-ana-
lyzer with a 0.55-cm cell. Samples with ammonium
concentration around or below 1.0 AM were re-
determined by the above LWCC method. A compar-
ison of these two methods is shown in Fig. 9. The
agreement is quite good. Fig. 8 shows the spatial
distribution of ammonium in Florida Bay and
Biscayne Bay. The ammonium concentrations in
these two bays vary widely from above 10 AM to
several hundred nanomolar, which are agreeable with
the long-term observation of ammonium in the same
area (Boyer et al., 1999). We also found that there are
Q.P. Li et al. / Marine Chemistry 96 (2005) 73–8584
decreasing concentrations of ammonium from the
coast to open ocean both in Florida Bay and Biscayne
Bay.
Acknowledgements
We thank Christ Kelble for the collection of
samples and two anonymous reviewers for helpful
comments on the first draft of this paper. NOAA’s
South Florida Ecosystem Restoration Prediction and
Modeling Program under the Coastal Ocean Pro-
gram is acknowledged. Additional support comes
from the US National Science Foundation (OCE-
0241340) to DAH. This research was carried out
under the auspices of the Cooperative Institute of
Marine and Atmospheric Studies, a joint institute of
the University of Miami and the National Oceanic
and Atmospheric Administration (Contract no.
NA67RJ0149). Frank J. Millero also acknowledges
the support of the Oceanographic Section of the
National Science Foundation.
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