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: qli@rsmas.miami.edu (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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