Microchim Acta 154, 31–38 (2006)

DOI 10.1007/s00604-006-0496-y

Original Paper

A Fast Sequential Injection Analysis System for the SimultaneousDetermination of Ammonia and Phosphate

Carsten Frank1;2;�, Friedhelm Schroeder1, Ralf Ebinghaus1;2, and Wolfgang Ruck2

1 GKSS, Institute for coastal research, Max-Planck-Strasse, D-21502 Geesthacht, Germany2 University of Lueneburg, D-21332 Lueneburg, Germany

Received August 6, 2005; accepted November 9, 2005; published online March 30, 2006

# Springer-Verlag 2006

Abstract. A flow system is described that is based

on sequential injection analysis (SIA) and is suitable

for the fast determination of ammonia and phosphate

in river and marine waters. It is applicable to nutri-

ent mapping in inhomogeneous coastal areas like the

Wadden Sea, and was optimised on several cruises on

the North Sea. The high sample throughput of 120

samples per analyte per hour and the low reagent con-

sumption (ammonia: 62.5 mL; phosphate: 60mL) were

achieved using a home made programme written in

the python programming language. The determination

of free reactive phosphate is based on the reaction of

phosphate with acidic molybdate to phosphomolybdate

which forms nonfluorescent ion pairs with rhodamine

6G. The remaining rhodamine fluorescence is detected

at 550 nm with an excitation at 470 nm. Ammonia

is determined with the help of o-phthaldialdehyde

and sodium sulfite. At 85 �C and a reaction time of

at least one minute a fluorescent species (exc. 365 nm,

em. 425 nm) is formed. The detection limits are

(3�) 0.3 mmol L�1 for phosphate and 1 mmol L�1 for

ammonia.

Key words: Phosphate; ammonia; sequential injection analysis;

marine waters.

Phosphorus as phosphate and nitrogen as nitrite,

nitrate and ammonia are the most important growth

limiting nutrients for phytoplankton in aquatic envi-

ronments [1]. In larger Seas like the North Sea phy-

toplankton accounts for more than 90% of the total

primary production [2]. Every change in the avail-

ability and composition of phosphorus and nitrogen

effects the phytoplankton population [3–5]. This again

has a direct or indirect effect on the phytoplankton

dependent food web and the whole ecosystem in the

affected area [6]. One example for a direct effect is the

increased amount and intensity of toxic and nontoxic

algal blooms [5].

To effectively emend past and prevent future errors

it is essential to determine and quantify the sources,

sinks and transport pathways of nutrients in the

problem areas (e.g. those defined by the OSPAR

Commission [7]). In areas with high hydrologic and

biologic dynamics like the Wadden Sea and the Elbe

estuary that quantification is difficult due to the in-

homogeneity of the water body. To succeed samples

have to be taken from a tight grid of sampling sta-

tions and out of different water depths. For larger

areas, like the Elbe estuary or parts of the North

Sea manual approaches for sampling and analysis

are too inefficient. An undulating towed vehicle like

the one developed and used by the BSH (Federal� Author for correspondence. E-mail: carsten.frank@gkss.de

Maritime and Hydrographic Agency, Germany) [8]

could be used as a carrier for a fast in situ nutrient

analyser.

The towfish (developed and operated by the BSH)

is designed to dive and surface in a sawtooth like

pattern (see Fig. 1). It is already used quarterly to

monitor physical, chemical and biological parameters

(like temperature, pH, salinity, algal fluorescence) in

the whole North Sea. A nutrient analyser attached to

or integrated into that towfish has to meet high

demands. These demands refer to technical and method-

ical demands. Technical demands are weight, size,

buoyancy and power consumption, which all have to

be accounted for. Even more difficult to accomplish

are the methodical demands, like high reliability,

low reagent consumption and high sample frequency.

While methods for the parallel determination of

ammonia and phosphate are already known (e.g.

[9, 10]), none of these methods fulfill all mentioned

requirements sufficiently.

In this presentation a modified SIA system is intro-

duced which complies with all methodical as well as

some technical demands for the integration into a

towfish. The system consists of one syringe pump,

one valve, one thermostate two detectors and one

personal computer. Next to a low reagent consump-

tion (ammonia 62.5 mL and phosphate 60 mL per

determination) it reaches a sample throughput of up

to 180 samples per hour and per analyte. The soft-

ware controlling the SIA instrument is tolerant con-

cerning common errors. The validity of the gained

results is improved by continuously monitoring sys-

tem performance.

Experimental

Reagents

All reagents were prepared with fresh drawn degassed deionised

water. Sigma (www.sigmaaldrich.com) analytical grade chemicals

were used, unless otherwise stated.

Ammonia

O-phthaldialdehyde (OPA) stock solution was prepared by dissolv-

ing 2 g of o-phthaldialdehyde (Sigma P-1378) in 25 mL ethanol.

This solution has to be shaken for several minutes to achieve com-

plete dissolution. 2 g of sodium sulfite were dissolved in 250 mL to

prepare the sulfite stock solution.

Ammonia reagent (AR) 7.5 g of disodium tetraborate deca-

hydrate were diluted to 250 mL. The solution was stirred until

complete dissolution and then transferred into a dark glass bottle.

5 mL of OPA stock solution were added. After stirring 500mL of

sulfite stock solution and 0.1 mL of a 30% Brij (Merck 101894,

www.merck.de) solution were added. After stirring the solution was

left to stand for several hours [11].

Ammonia standard stock solution was 1 g L�1 from Merck.

Phosphate

Rhodamine stock solution was prepared by dissolving 0.20 g of

rhodamine 6G in 100 mL water. Molybdate stock solution was made

by dissolving 12.8 g of ammonium heptamolybdate tetrahydrate

(Merck, analytical grade) in 100 mL water. To prepare reagent 1

(PR1) 200mL rhodamine 6G stock solution was added to 90 mL

water. 500mL 5% IGEPAL (Polyoxyethylene(�)octylphenyl ether,

branched) was added and the solution diluted to 100 mL. Reagent 2

(PR2) was prepared by adding 8.45 mL of 30% (v=v) hydrochloric

acid to about 75 mL of water. Add 4 mL of molybdate stock solution

and dilute to 100 mL. The reagents PR1 and PR2 were derived from

Wei et al. [12].

Phosphate standard stock solution was 1 g L�1 from Merck.

Instrumentation

A schematic diagram of the flow system of the automated water

measurement system (ferrybox [13, 14]) with its connections to the

Fig. 1. The towfish of the BSH (Federal Maritime and Hydro-

graphic Agency, Germany) pulled by the research vessel GAUSS.

The towfish is able to dive and surface in a sawtooth like pattern.

This is used to continuously gain depth profiles without stopping

the ship

Fig. 2. The schematic of the flowsystem of the ship and the auto-

mated water measurement system (ferrybox), which was used as

source for the on-line samples. The tests and validations were

performed on the research vessel Ludwig Prandtl using the sam-

ples provided by the ferrybox. SWI Sea water intake; SWD sea

water discharge; CP centrifugal pump; PP peristaltic pump; TP1 &

TP2 T-pieces; F band-pass filter

32 C. Frank et al.

SIA-system and the ship (research vessel Ludwig Prandtl) is shown

in Fig. 2. The sample is drawn by the centrifugal pump out of about

1.20 m water depth at the bow of the ship. It is pumped through the

ship until it reaches the laboratory container which contains the

ferrybox and the SIA instrument. The T-piece (TP1) is used to split

the sample stream between ferrybox and waste using the high pump

rates to ensure sample freshness. After debubbling 4.5 L min�1 of

the sample stream are delivered to a band-pass filter (Metrohm

series 13, www.metrohm.de; Filter paper: Schleicher & Schuell type

1573; pore size 12–25mm, www.schleicher-schuell.de). The peri-

staltic pump (PP) of the band-pass filter is used to pump the filtrate

to the SIA system (Fig. 3). There T-piece TP2 is used as a bypass.

Into the T-piece a tubing (6.5 cm long, 0.8 mm i.d.) is inserted.

This tubing is used to draw fresh samples out of the filtrate stream.

The other end of the tubing is connected to the 17-port=1-channel

valve (V; Knauer Part-No. A1492, www.knauer.net). The SIA

system is composed of one syringe pump (CAVRO XL 3000,

www.tecansystems.com), one valve, two detectors (one fluorescence

detector exc. 365 nm, em. 425 nm build by ME Grisard GmbH,

www.me-grisard.de, and a Hitachi F1000 fluorescencespectrometer

exc. 470 nm, em. 550 nm, www.hitachi-hta.com) and four reaction

loops. The holding loop is 120 cm long (0.8 mm i.d.) and the reac-

tion loops are 60 cm long (0.8 mm i.d.). The Hitachi detector is

connected via an external multimeter with RS232 interface (HP

34401A, www.agilent.com) to the personal computer while the other

detector provides its own RS232 interface. Three reaction loops are

heated to 85 �C and are used to determine ammonia. The reaction

loop for the determination of phosphate is not temperature con-

trolled. A set of home made programmes written in python is used

to control the SIA instrument.

This programme set consists of three essential programmes and

four optional programmes. Two of the essential programmes are

used to control the detectors (one programme per detector). These

control programmes take care for the correct detector initialisation

and then switch into data server mode. The third essential pro-

gramme is the main programme which controls the SIA instrument

(syringe pump(s), valve(s), thermostate(s)). These three programmes

do only interact during peak determination. The main advantage of

this approach is the uninterrupted availability of the detector data.

Four more programmes can be used in conjunction with the three

essential programmes. Two are used to display the raw detector data

and two are used to display preliminary quantification results.

The structure of the main programme is shown in Fig. 4. The

programme starts with the initialisation of all used devices bringing

them into defined states. Due to the low reagent and sample con-

sumption the tubings (40 cm; 0.8 mm i.d.) connecting the reagents

and standards with the valve, have to be flushed with the respective

reagent or standard. The main loop starts with the ammonia readout

step. Directly after the startup none of the three reagent loops

is loaded with the sample-reagent-mix, so the actual reagent loop

is just flushed with carrier. Then the actual ammonia reagent loop is

loaded with the reagent-sample-mix (see Fig. 6). Phosphate is deter-

mined via phosphomolybdate and ion-pairing with rhodamine 6G in

the next step. Two factors limit the speed of the this step: The

dispersion of the reagents depend on the speed profile which is used

Fig. 3. Schematic of the sequential injection analysis (SIA) system

used for phosphate and ammonia analysis. A personal computer is

used to control syringe pump, valve and the detectors. SP Syringe

pump (2500 mL syringe); V 17 port=1 channel valve; C carrier

(degassed deionised water); hl holding loop; rl1–3 reaction loops

for the determination of ammonia; rl4 reaction loop for the deter-

mination of phosphate; D1 fluorescence detector (exc. 365, em.

425; for ammonia); D2 fluorescence detector (exc. 475, em. 550;

for phosphate); PR1 rhodamine reagent; PR2 acidic molybdate

reagent; AR OPA-reagent; Std standard; W waste

Fig. 4. Flow sheet of the home made software used to control the

SIA instrument. ‘x’ is the number of the ammonia reaction loop

used in the actual programme cycle. It is incremented prior to

every loop. The ‘wait’ times are calculated based on the time

needed for each step and the time left until the next ammonia

readout step has to occur

Sequential Injection Analysis of Ammonia and Phosphate 33

to push the reagent-sample-mix to the detector [15, 16]. This leads

to a compromise between speed and reaction efficiency. The limited

time resolution of the detector is the second speed limiting factor. At

this point the loop starts again with the ammonia readout step.

During the fourth run of the main loop the first reaction loop is

read out. This readout procedure starts with a ‘wait’ step which

ensures that the reaction time is reached before the actual determi-

nation starts. The ‘wait’ times are calculated based on the time

needed for each step and the time left until the next ammonia

readout step is due. The reaction time of the ammonia determination

was changed from 60 to 90 s (also decreasing the sample throughput

to 120 samples analyte and hour) while using the ME Grisard

detector to compensate the poor detector performance.

The contents of the main loop can be changed any time with the

changes taking effect in the next cycle. This is especially advanta-

geous during method development, but can also be of great help in

the field. This flexibility can, for example, be used to optimise a

speed profile during method development or to add an additional

standard to the sample sequence in the field. The sample sequences

(see Fig. 5) were introduced to improve the quality of the achieved

data by continuously monitoring system performance.

The sequences of reagents and sample are schematically pictured

in Fig. 6. The sequence for the phosphate determination contains

three reagent segments (PR1 and PR2) and the sample segment. For

the determination of ammonia only one reagent is used. However,

due to problems with the sensitivity of the detector, the amount and

volume of the reagent and sample segments were increased to reach

a satisfying sensitivity (a strategy proposed by Wang et al. [17]). So

three reagent and two sample segments were used (Fig. 6). Earlier

experiments using the Hitachi F1000 detector instead of the one

made by ME Grisard have shown significant better results with less

segments and lower volumes. However, only one of these detectors

was available and it had to be used for phosphate.

The methods for the determination of phosphate and ammonia are

both based on fluorescence determination. Phosphate is determined

via the formation of phosphomolybdate which quantitatively extin-

guishes the fluorescence of the rhodamine 6 g. The reaction of

ammonia with o-phthaldialdehyde (OPA) and sulfite is used to

measure the ammonia concentration.

Results

System Speed

Although the SIA is considered to be slower than the

FIA [18] it was accelerated to an at least similar

speed. This was achieved mainly using the self made

software which intensively uses the features integrated

into the syringe pump (CAVRO XL 3000). One of the

Fig. 5. An example for a sample sequence. At a rate of 120

samples per hour 6 minutes are needed to complete this se-

quence. The standard and blank values are used to monitor system

performance

Fig. 6. Sequence of reagents in the holding loop prior to injection

into the reagent loop. Volumes in mL. AR Ammonia reagent; PR1

phosphate reagent 1; PR2 phosphate reagent 2

Fig. 7. Schematic diagram of the readout step for the phosphate

determination. The speed ramps are used to prevent the formation

of air bubbles in the tubings due to vacuum conditions during

deceleration. (1) The reagents are pumped through the reaction

loop in direction to the detector. (2) The remaining fluorescence is

determined in the detector. (3) Reaction loop and detector are

flushed. (4) Syringe command

34 C. Frank et al.

most advantageous features are the free configurable

acceleration and deceleration slopes. These speed

ramps can be used to achieve high pump rates without

pressure peaks during acceleration and deceleration

phases. The speed ramp used for the phosphate deter-

mination is displayed schematically in Fig. 7.

A multichannel stopped flow approach was chosen

to successfully integrate a high throughput ammonia

determination. The system was tested for up to four

heated reaction loops reaching more than 200 samples

per hour. Since the reaction is interrupted before com-

pletion (70% of the final fluorescence intensity are

reached after 60 s reaction time) the timing has to

be very precise. A precision of about �0.2 seconds

was achieved using the timing described above.

Interferences Between Ammonia and Phosphate

Determination

The contemporaneous use of ammoniamolybdate as

reagent and ammonia as analyte in the same system

was not accompanied by any problems. This was

tested in the lab and during campaigns by deactivating

the phosphate or the ammonia determination for some

minutes and monitoring the effects on the preliminary

data. No difference in the signals of the standards and

the sample was observed.

System and Data Reliability

An improved system reliability was achieved by split-

ting the original large programme into a set of smaller

programmes. Each of these programmes has to ac-

complish only a small amount of tasks like gaining

the data from the detector, saving the data and deliver-

ing parts of these data on request. All non critical

tasks, like the visualisation of the data in a graph,

were moved to separate programmes which can be

used at will. This lead to a significant increase in

system stability and simplified debugging in case of

an error. Another improvement was the integration of

basic error correction features for comparatively com-

mon errors like erroneous data transmissions.

Especially during the first field tests the quality of

the achieved data is always questionable. Variations in

the overall system performance can be caused by tem-

perature differences, wind, vibrations and other fac-

tors. To improve the quality of the data gained with

this SIA system the system of sample sequences was

adopted. This was done using two or more additional

valve ports to attach two or more standards. During

the cruises performed to optimise and validate this

device the determination sequence usually comprises

of 50% standards and 50% samples as shown in Fig. 5.

Effect of Sample Salinity

Sample dilution and standard addition were used to

investigate the effect of the salinity of the sample on

the determined nutrient concentration. Both experi-

ments showed, that the salinity of the sample has

no distinguishable influence on the result of the

determination.

Detection Limits and Range Switching

In this SIA system two different fluorimetric methods

are used. While ammonia is determined via a fluores-

cent molecule which is formed by ammonia, sulfite

and o-phthaldialdehyde, the formation of phosphomo-

lybdate extinguishes quantitatively the fluorescence of

rhodamine 6 g, and therefore phosphate is determined

by the absence of fluorescence. Due to the strong

fluorescence of the rhodamine which is very effec-

tively quenched by phosphomolybdate, the linear

range of this system is quite small. This is compen-

sated using up to three different sample volumes for

different expected phosphate concentrations (50, 17

and 8 mL) [20].

Using 50mL as sample volume a detection limit

(3�) of 0.04 mmol L�1 can be reached for phosphate

in the laboratory. On the ship the situation is comple-

tely different. There the detection limit varies between

0.04 up to 0.3mmol L�1. This is most probably caused

by variations of environmental parameters like tem-

perature, wind, air humidity and incidence of light

which have an effect on the spectrometer, the reaction

loop and the reagents. These effects can most prob-

ably be reduced by exchanging the detector with one

more suitable for shipboard use. Up to 4 mmol L�1

phosphate can be measured using a sample volume

of 8mL.

Ammonia can be quantified directly using the fluo-

rescence intensity (peak area) and shows a calibration

graph with a good linearity up to 20mmol L�1. The

detection limit can be as low as 0.02mmol L�1 using

the Hitachi fluorescence detector in the laboratory.

Because that detector was already in use for the deter-

mination of phosphate a detector produced by ME

Grisard was used. That one has an inferior perfor-

Sequential Injection Analysis of Ammonia and Phosphate 35

mance compared to the Hitachi detector. By increas-

ing the reaction time, the sample volume and the

amount of reagent and sample segments a detection

limit of about 0.05mmol L�1 was achieved in the

laboratory. However, on the ship the average detection

limit reached values of about 1 mmol L�1 (range from

0.07 to 1.50mmol L�1) which was caused mainly by

failed or incorrect data transmissions between de-

tector and computer. Again a detector more suitable

for this environment seems to be the solution to this

problem.

Application and Validation

In order to optimise and validate the newly developed

system, several cruises to the German part of the

North Sea and Wadden Sea were performed. These

areas are characterised by strong nutrient concentra-

tion gradients (Wadden Sea) and low nutrient concen-

trations (North Sea). Therefore the chosen transects

perform an ideal validation scheme for the detection

of strong concentration gradients as well as for the

quantification of low nutrient concentrations directly

on the sea. One transect of a cruise performed in

August 2004 is described here in detail (map see

Fig. 8). The results of this experiment are shown in

the Figs. 9 and 10. The trend of the determined ammo-

nia and phosphate concentration was as expected from

earlier cruises and literature [21]. Starting with com-

paratively high values in Norddeich (the ship’s cen-

trifugal pump (CP see Fig. 2) was switched on about

2–4 km off the harbour after reaching 2–3 m water

depth) the concentrations decreased along the tideway

until the open sea was reached. The concentration of

about 0.5mmol L�1 for both, ammonia and phosphate,

remains stable until the area influenced by the Elbe

river is reached. Both values increase again during the

last part of that trip. About 7 km off Cuxhaven har-

bour the ammonia detector stopped to deliver data and

so the measurement was terminated.

In addition to the on-line measurements off-line

samples were taken. These samples were sent to a

commercial lab which uses an SFA system to quantify

nutrients [22, 23]. Five of these off-line samples were

splitted and determined also off-line with the method

presented here. The results of these measurements are

displayed in the Figs. 9, 10 and in Table 1. The first

Table 1. Comparison of off-line data with data from the external

lab

Slope Intercept r2

Phosphate 0.95 0.010 0.996

Ammonia 1.35 �0.737 0.902

Fig. 8. Map of the transect from Norddeich to Cuxhaven. 5th of

August 2004

Fig. 9. Transect from Norddeich to Cuxhaven. Phosphate data.

Rectangles indicate lab samples

Fig. 10. Transect from Norddeich to Cuxhaven. Ammonia data.

Rectangles indicate lab samples

36 C. Frank et al.

two figures present the results of the on-line mea-

surements as described above and the results of the

samples sent to the lab. These graphs indicate that

the tendency of the on-line and lab results is the same

for phosphate (r2¼ 0.93) and similar for ammonia

(r2¼ 0.63). However the absolute values differ signif-

icantly. The difference between on-line ammonia

data and the lab results are caused by contamination,

inappropriate sample storage and by detector noise.

Furthermore the flow paths of the seawater differ for

the on-line and the lab samples. All lab samples were

drawn at TP1 (Fig. 2), filtered using Schleicher &

Schuell spartan 30=0.2 (pore size 0.2mm) filters

and directly stored in the freezer. Whatman GF=F

(pore size 0.7mm) filters were used for the five splitted

samples. The on-line samples however had to pass

the ferrybox with its integrated additional centrif-

ugal pump. This pump increases the number of

destroyed plankton cells whose contents are released

into the water. In the case of phosphate additional

problems were caused by the increased turbulence

in the pump and the ferrybox which leads to

an increased desorption of phosphate ions from

suspended particles. That water is then filtered

using Schleicher & Schuell type 1573 (pore size

12–25mm) filter paper in the band pass filter. The

increased amount of phosphate ions due to the turbu-

lence caused by centrifugal pump, the increased

amount of particles with adsorbent phosphate [24] in

the sample due to the coarse filter and the increased

number of destroyed plankton cells also lead to

higher phosphate concentrations in the on-line sample

stream.

Due to the significant differences between the on-

line data and the data provided by the external lab the

off-line results were also compared to the lab data.

The results of this comparison are given in Table 1.

The correlation between the off-line and lab results is

very good for phosphate and acceptable for ammonia.

While sample pretreatment and storage is difficult for

both analytes [25, 26], the procedure used in this case

is generally less optimal for ammonia due to the mate-

rial of the viles (PE) and the freezing and melting

processes.

A better approach to validate on-line methods like

the one presented here is to use another automated

method which also can be used on-line at a slower

pace. Another possibility would be a manual method

which can be carried on the ship directly after a sam-

ple is taken.

Discussion

A sequential injection analysis system is presented

which is suitable for the fast determination of ammo-

nia and phosphate in coastal waters. The system was

successfully tested on several cruises on the north sea.

One transact of these cruises was used to validate this

system. The methods used in this device were opti-

mised for the use in a towfish system.

The presented methods are especially useful for

three application fields. First it can be used for high

resolution surface mapping of ammonia and phos-

phate especially in coastal areas. Furthermore it is

useful for long term monitoring due to the low amount

of reagents used in this system, and the reliability

which comes with the integration of standards which

can be determined contemporaneous with the sam-

ples. A third field would be the integration of more

parameters like nitrate or silicate and use this system

as general purpose nutrient analyser.

In the near future the presented method will be

transferred to significant smaller components (e.g. a

Lee LPV pump instead of a CAVRO XL 3000, smaller

detectors with integrated analog to digital converters)

and then integrated into the towfish.

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38 Sequential Injection Analysis of Ammonia and Phosphate