Spectroscopy Letters

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Turbidimetric and Nephelometric Flow Analysis:

Concepts and Applications

Inês P. A. Morais , Ildikó V. Tóth & António O. S. S. Rangel

To cite this article: Inês P. A. Morais , Ildikó V. Tóth & António O. S. S. Rangel (2006)
Turbidimetric and Nephelometric Flow Analysis: Concepts and Applications, Spectroscopy Letters,
39:6, 547-579, DOI: 10.1080/00387010600824629

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Spectroscopy Letters, 39: 547–579, 2006
Copyright # Taylor & Francis Group, LLC
ISSN 0038-7010 print/1532-2289 online
DOI: 10.1080/00387010600824629

Turbidimetric and Nephelometric Flow

Analysis: Concepts and Applications

Inês P. A. Morais, Ildikó V. Tóth, and António O. S. S. Rangel

Escola Superior de Biotecnologia, Universidade Católica Portuguesa,
Porto, Portugal

Abstract: A review on flow analysis with turbidimetric and nephelometric detection is

presented. A brief discussion of the principles of turbidimetry and nephelometry is
given. Particular emphasis is devoted to coupling different flow techniques (flow
injection, sequential injection, multicommutation) to these detection techniques. Appli-
cations in environmental, pharmaceutical, biological, and food samples are sum-
marized and compared in terms of application range, flow configuration,
repeatability, and sampling rate.

Keywords: Flow analysis, nephelometry, turbidimetry

INTRODUCTION

Nephelometry and turbidimetry are closely related analytical techniques based

on the scattering of radiation by a solution containing dispersed particulate
matter. When a radiation passes through a transparent medium in which
solid particles are dispersed, part of the radiation is scattered in all directions,
giving a turbid appearance to the mixture. The decrease of the incident
radiation, as a result of scattering by particles, is the basis of turbidimetric
methods. Nephelometric methods, on the other hand, are based on the

Received 15 March 2006, Accepted 25 May 2006

The authors were invited to contribute this paper to a special issue of the journal
entitled “Spectroscopy and Automation”. This special issue was organized by
Miguel de la Guardia, Professor of Analytical Chemistry at Valencia University, Spain.
Address correspondence to António O. S. S. Rangel, Escola Superior de Biotecno-
logia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida,
4200-072 Porto, Portugal. Fax: þ351225090351; E-mail: [email protected]

547
548 I. P. A. Morais et al.

measurement of the scattered radiation, usually at a right angle to the incident

beam. The choice between a nephelometric and a turbidimetric measurement
depends upon the fraction of light scattered. When scattering is extensive,
owing to the presence of many particles, turbidimetry generally yields more
reliable results. Nephelometry is preferred at low concentrations because a
small scattered intensity against a black background is easier to measure than
a small change in intensity of intense transmitted radiation. It is important to
note that scattering associated with both nephelometry and turbidimetry does
not involve loss in radiant power; only the direction of propagation is affected.
The intensity of radiation appearing at any angle depends upon the number
of particles, their size and shape, as well as the wavelength of the radiation.

Effect of Concentration on Scattering

Turbidimetric analysis consists of the measurement of the decrease in the

intensity of the incident radiation that is caused by scattering and is
analogous to an absorptive measurement, although the reason for the
decrease in intensity is different.
When a beam of radiation of intensity I0 passes through a nonabsorbing
medium that scatters light, the transmitted intensity I is given by the expression:

I ¼ I0 e
tb
where t is the turbidity, or the turbidity coefficient, and b the pathlength in the
turbid medium. The turbidity t is often found to be linearly related to the concen-
tration C of the scattering particles. As a consequence, a relationship analogous
to Beer’s law is applied. That is,
S ¼
log I=I0 ¼ kbC
where
k ¼ 2:303 t=C
The equation is employed in turbidimetric analysis in exactly the same way as
Beer’s law is used in photometric analysis. The relationship between log I0/I
and C is established with the standard solutions, and the solvent is used as the
reference to determine I0. The resulting calibration curve is then used to
determine the concentration of the samples.[1]
Nephelometry is based on the measurement of scattered radiation by
sample particles at right angles to the beam. The detector is placed out of
the path of the incident radiation from the source. In most cases, the
detector is placed at 90 degrees relative to the path of the incident radiation.
It measures the intensity of that portion of the scattered radiation that is
emitted perpendicularly from the cell in the direction of the detector. For
nephelometric measurements, an equation describes the relationship
Turbidimetric and Nephelometric Flow Analysis 549

between the intensity of scattered radiation, the intensity of the incident

radiation, and the concentration of the particles that cause the scattering:
I ¼ KI0 C
The value K is constant only for a particular instrument and when experimental
conditions are carefully controlled. The intensity of the scattered radiation is
directly proportional to both the intensity of the incident radiation and to the con-
centration of the analyte. For assays of diluted solutions, it is advantageous to use
incident radiation that has a high intensity.[2]
The detected scattered signal may arise from the particles of interest but
also from dust, background scatter, or from other molecules (e.g., proteins and
lipids) in the sample. Reflection and scatter from optical components of the
instrument may also contribute to the background signal. Best performance
is obtained in dilute solutions where absorption and reflection are minimal.
Under these conditions, the relationship between concentration of scatter-
ing particles and scattered light intensity is almost linear over a very wide
range of concentration.

Effect of Particle Size on Scattering

Nephelometric and turbidimetric methods have advantages of being simple,

fast, and having high sensitivity. The difficulties arise not from the optical
measurement, which is simple, but from the preparation of the suspension. In
fact, the fraction of radiation scattered at any angle in colloidal systems
depends upon the size and the shape of the particles responsible for the
scattering. Because most analytical applications involve the generation of a col-
loidally dispersed phase in a solution, those variables that influence particle size
during precipitation also affect both turbidimetric and nephelometric measure-
ments. Thus, such factors that can affect the results and that must be controlled
include the concentrations of the reagents that are used to prepare the suspen-
sions, the rate and order of mixing, and the time after reagents have been
mixed and the time before the measurement is made. The pH, the total ionic
strength, and the temperature of the solution are other variables that are of
critical importance and must be carefully controlled. In order to stabilize the
suspensions and prevent the settling of the particles, a protective colloid is
usually added. The absence and presence of protective colloids in a suspension
also affect the size of the particles. Thus, during calibration and analysis, care
must be taken to reproduce all conditions likely to affect particle size.

Effect of Wavelength on Scattering

The wavelength selected for the measurements also has an important effect on
scattering. It has been shown, experimentally, that the turbidity coefficient t
550 I. P. A. Morais et al.

varies with wavelength according to the equation:

t ¼ sl
t

where s is a constant for a given system. The quantity t is dependent on particle

size and has a value of 4 when scattering particles are significantly smaller
than the wavelength of the radiation; for particles with dimensions similar
to the wavelength, t is found to be 2.[1] The latest situation is the usually
encountered in turbidimetric analysis.
The wavelength chosen for the turbidimetric or nephelometric assay is
also dependent upon the presence of other (interfering) absorbing or fluores-
cing species in solution. In this case, a wavelength where absorbance or fluor-
escence by the substances in solution does not occur has to be chosen. If the
scattering particles (those that are in the interest of the determination) also
absorb radiation, the sensitivity of turbidimetric, but not nephelometric, deter-
minations can be increased by choosing the wavelength at which absorbance
occurs. In that case, the instrument measures the sum of absorbance and tur-
bidance, which should also be proportional to concentration.

Equipment

Turbidimetric measurements are usually performed with simple filter photo-

meters, while instruments for nephelometric measurements are similar in
design to simple fluorometers. Both instruments comprise a light source that
emits in the visible region, a cell compartment, a detector, and a readout
device. In the apparatus used for nephelometric determinations the detector
is, in most cases, placed at 90 degrees relative to the path of the incident
radiation. Detectors that accurately and reliably respond to radiation in the
visible region are frequently used. Phototubes are usually used for turbidi-
metric measurements and phototubes or photomultiplier tubes for nephelo-
metric measurements. A wavelength selector may also be present between
the source and the cell compartment, and, for nephelometric measurements,
a second wavelength selector can also be placed between the cell compartment
and the detector. Laboratories that routinely use this technique for analysis
sometimes use instruments that have been specifically designed for
turbidimetric or nephelometric measurements, which are usually simpler in
design and less expensive than spectrophotometers or fluorometers. Often
they use the broad visible continuum emitted from a tungsten filament as
the incident radiation, have no monochromator, and apply a phototube or
the human eye as the detector. Cells that are used to hold the sample during
turbidimetric and nephelometric measurements are identical to the cuvets
used for measurements of absorbance and fluorescence. Nevertheless,
because scattered radiation from the walls can interfere with the assay, it is
sometimes advantageous to coat the exterior of the walls, except those
Turbidimetric and Nephelometric Flow Analysis 551

through which radiation must pass, with a nonreflective black paint. This is
particularly important for nephelometric measurements.

Applications of Scattering Methods

Turbidimetric or nephelometric methods are widely used in analysis of water,

for the determination of turbidity, and for the control of treatment processes.
In addition, the concentration of a variety of ions can be determined using
suitable precipitation reagents to form suspensions. Perhaps the best known
chemical turbidimetric analysis involves the precipitation of sulfate as
barium sulfate under controlled conditions that yield a stable monodisperse
suspension. Both techniques can also be used to locate the endpoint of some
titrations in which the titrand reacts with the titrant to form a suspension.
Generally, the turbidance or the intensity of the scattered radiation increases
before the end point and then remains constant. Turbidimetric or nephelo-
metric measurements have been used to locate potential precipitants in com-
mercially prepared soft drinks and alcoholic beverages, to measure potentially
equipment-clogging solids suspended in waters that are used in industrial
equipment, and as an environmental analytical tool to measure suspended
solids in waters.[3,4] Finally, they have also been used to measure suspended
particles in gases, like smog and fog.

FLOW ANALYSIS

Flow injection analysis (FIA), introduced in 1975 by Ruzicka and Hansen,[5] is

a simple and an alternative method to batch procedures. In a basic FIA
manifold, samples are introduced into the system through the injection
valve, dispersed in the carrier inside the tubes conduit. Most commonly, the
reagent is continuously added through a confluence point located after
the injection port and before a coil where reaction takes place. Finally, the
reaction product reaches the flow through detector where the detection
signal is acquired (Fig. 1A).
In 1990, Ruzicka and Marshall[6] proposed a new flow technique, sequen-
tial injection analysis (SIA), based on the same principles of FIA, and
conceived as a single pump, a single valve, and a single channel system.
The SIA is based on the sequential aspiration of well-defined sample and
reagent zones through a selection valve into a holding coil. The flow is then
reversed, to propel and mutually disperse these stacked zones through the
reaction coil and direct the reaction product to the detector (Fig. 1B).
Compared with FIA, these systems allow considerable saving of reagents
and a significant decrease on the chemical waste produced, because just the
required amounts are aspirated and carrier is not pumped continuously. In
addition, different analysis can be performed using the same manifold by
552 I. P. A. Morais et al.

Figure 1. Schematic diagram of flow systems with turbidimetric or nephelometric

detection: (A) flow injection analysis, (B) sequential injection analysis, (C) multicom-
muted flow-injection analysis. I, injection valve; SV, selection valve; V, individual
commutation devices (e.g., solenoid valves); S, sample; Ri, reagents; R1, surfactant,
washing solution; R2, precipitating agent; C, carrier; P, liquid drive; B, pistons bar;
D, detector; RC, reaction coil; HC, holding coil; W, waste.

simple reconfiguration of the sequence of events from the computer keyboard.

Besides this, the major difference between FIA and SIA methodologies
concerns the way that sample and carrier/reagent solutions are mixed inside
the tubes. While in FIA the solutions are most commonly mixed in confluence
points, giving rise to a concentration gradient of analyte in a constant back-
ground of reagent, in SIA efficient mixing is more difficult to achieve due
to the absence of confluence points. In fact, in SIA an initial sharp
boundary is formed between the adjacent sample/reagent zones stacked in
the holding coil. Even after the flow reversal, only a partial overlap of
analyte and reagent zones is achieved.[7]
In order to overcome this specific difficulty of SIA, and also to improve the
mixing between solutions in flow systems in general, various strategies were
Turbidimetric and Nephelometric Flow Analysis 553

published. In 1985, Pasquini and Oliveira proposed an approach, monosegmen-

ted flow (MSFA),[8] in which sample and reagent are introduced between two
air bubbles. The bubbles serve to limit the longitudinal sample dispersion and at
the same time to enhance the radial mixing. The bubbles are removed before
they enter the detection system using a gas-permeable membrane.
Another alternative to overcome mixing difficulties is the multicommuted
flow injection analysis (MCFIA), which was first described by Reis et al.[9]
associated with the binary sampling approach. This technique is characterized
by the use of individual commutation devices (solenoid valves) operating in a
simultaneous or a sequential way, where solutions can be accessed randomly.
In this approach, small plugs of sample and reagents are inserted in alternative
way in the flow system and mutually dispersed while directed to the detector
(Fig. 1C). Compared with other flow techniques, the main advantage intro-
duced by the multicommuted approach is versatility based on the use of
solenoid valves that can be arranged in multiple configurations. This
evidence was pointed out by Zagatto et al.,[10] when it mentioned that multi-
commutation can unify all concepts already proposed in flow analysis, consi-
dering the possibility of accommodating different flow modalities (FIA, SIA)
in a system with just solenoid valves.

Turbidimetric and Nephelometric Flow Analysis

Turbidimetry has been widely used as detection method in flow analysis.

Besides just automating batch turbidimetric methods, flow techniques such
as FIA, SIA, MCFIA, and MSFA, within others, even allowed improvement
of the analytical performance of these detectors. Undoubtedly, FIA is the
most widely used technique.
The use of a flow system does not affect any of the basic characteristics of
batch turbidimetric or nephelometric methods. The equations obtained are still
obeyed in the same range and with similar sensitivity.
Although any detector capable of flow-through detection can be inter-
faced with flow systems, to obtain reproducible signals the detector and the
readout device used must have a fast response.
The repeatability of the batch measurements are highly affected by the
skillfullness of the operator and, in some cases, by the time at which the
detection measurement is made. In fact, the time spent in each measurement
can be very high, because at the time of the detection the reaction has to be
in steady state. This is particularly observed when the reaction is relatively
slow, such as in turbidimetric determinations.
In turbidimetric analysis, the preparation of the standard suspensions is
particularly critical, and sample and standard suspensions must be prepared
using identical procedures. In fact, as pointed out by Brienza et al.[11] the
major problem of turbidimetry is related to processes of solution handling
rather than to quality and performance of the measurement instruments.
554 I. P. A. Morais et al.

The amount of light scattering in colloidal systems is a sensitive function of

the particle size, so any variation in the colloidal solution preparation may
result in a lack of particle size uniformity from one determination to the
next, altering significantly the turbidimetric or nephelometric measurement.
In this context, the flow systems are an attractive tool to improve the reprodu-
cibility and precision of turbidimetric determinations. The addition of colloid
protectors or surfactants is often required, which, in contrast with batch pro-
cedures, is efficiently accomplished in flow-based methodologies. The
presence of these agents guarantees the uniform nucleation and prevents the
settling of the precipitate, thereby improving the repeatability and reproduci-
bility of the analysis.[11,12] Carryover and memory effects can be lessened in
view of better uniformity of the particles, thus reducing washing time and
baseline drift. For this task, intermittent addition of a washing solution or a
fast washing stream has been exploited.[11]

TURBIDIMETRIC AND NEPHELOMETRIC FLOW ANALYSIS

APPLICATIONS

In the following sections, the description of turbidimetric and nephelometric

applications using flow methods is given. The applications are organized by
the type of analyte: inorganic ions, organic compounds, compounds with
immunological importance, and biomass.

Determination of Inorganic Ions

Sulfate

Sulfate is undoubtedly the most popular analyte determined using turbidi-

metric flow methodologies (Table 1). The method that appears to be almost
universal is the barium sulfate turbidimetric procedure (Table 1), being the
measurement performed between 410 and 580 nm.[40] Turbidimetric flow pro-
cedures with barium chloride, as precipitating agent, have been successfully
applied to environmental,[12,13,15 – 20,22 – 37,39] clinical,[14,35] and wine[38]
samples. Krug et al.[12] were the first authors to adapt the turbidimetric
barium sulfate procedure to FIA for the determination of sulfate in natural
waters and plant digests, using various types of flow systems with more
than one reagent or carrier stream. This was also the first turbidimetric FIA
system reported, only a few years after the FIA concept been introduced,
which indicates the easy implementation of this reaction to flow systems.
Since then, several researchers have developed not only other FIA
systems,[13 – 30,35,36,39] but also SIA,[31,32,34,35,37,38] MCFIA,[33] and
MSFA[35] methodologies in order to obtain better precision and sensitivity,
shorter analytical cycles, and lower detection limits. Alternatively, a FI
 Turbidimetric and Nephelometric Flow Analysis
Table 1. Application of turbidimetric and nephelometric flow methods to sulfate determination

Flow RSD SR
Analyte method Sample Reagent Precipitate Surfactant Working range (%) (h21) Ref.

Sulfate FIA/tur Natural waters and BaCl2 BaSO4 PVA 10 – 200 mg L21 0.85 180 [12]
plant digests
Sulfate FIA/tur Natural waters and BaCl2 BaSO4 PVA 10 – 200 mg L21 250 [13]
predigested
plant material
Sulfate FIA/tur Urine BaCl2 BaSO4 Gelatin 4 – 15 mmol L21 ,1.2 120 [14]
Sulfate FIA/tur River and sea BaCl2 BaSO4 PVA 40 – 160 mg L21 1–2 [15]
water
Sulfate FIA/tur Natural waters BaCl2 BaSO4 Thymol and 20 – 500 mg L21 ,2.0 200 [16]
gelatin
Sulfate FIA/tur Surface, ground, BaCl2 BaSO4 Thymol and 50 – 200 mg L21 ,0.95 60 [17]
and domestic gelatin
waters
Sulfate FIA/tur Surface, ground, BaCl2 BaSO4 Thymol and Up to 200 mg L21 ,1 60 [18]
and domestic gelatin
waters
Sulfate FIA/tur Natural waters and BaCl2 BaSO4 PVA 1 – 30 mg L21 1 120 [19]
plant digests waters
5 – 200 mg L21
plants
Sulfate Reversed Effluent water BaCl2 BaSO4 Gelatin 50 – 200 mg L21 ,2.0 60 [20]
FIA/tur streams

555
(continued )
 556
Table 1. Continued

Flow RSD SR
Analyte method Sample Reagent Precipitate Surfactant Working range (%) (h21) Ref.

Sulfate FIA/tur 3% (m/v) cesium BaCl2 BaSO4 PVA 1 – 100 mg L21 [21]
iodide solution
Extracta- FIA/tur Plant material BaCl2 BaSO4 Arabic gum 0 – 35 mg L21 2 120 [22]
ble
sulfate
Total FIA/tur Plant material BaCl2 BaSO4 Arabic gum 0 – 200 mg L21 2.1 120 [23]
sulfur
Sulfate FIA/tur Petroleum indus- BaCl2 BaSO4 — 0 – 20 mmol L21 24 [24]
try –related
waters
Sulfate- FIA/tur Waters and plant BaCl2 BaSO4 Arabic gum 0 – 300 mg Kg21 0.01 plant 60 [25]
sulfur; materials digests
sulfur 0.3
waters
Sulfate FIA/tur Rain waters BaCl2 BaSO4 PVA 0.50 –2.00 mg L21 2 50 [26]
Sulfate FIA/tur Soil BaCl2 BaSO4 Arabic gum 0 – 180 mg L21 0.85 120 [27]
Sulfate FIA/tur Fresh and saline Pb(NO3)2 PbSO4 PVA 2 – 20 mg L21 ,3 35 [28]

I. P. A. Morais et al.
waters
Total FIA/tur Plants Pb(NO3)2 PbSO4 — 5.00 – 0.5 400 [29]
sulfur 25.00 mg S L21
Sulfate FIA/tur, Tap water BaCl2 BaSO4 — 20 – 2000 mg L21 4.0 [30]
neph tur
20 – 200 mg L21
neph
 Turbidimetric and Nephelometric Flow Analysis
Sulfate SIA/tur Natural waters and BaCl2 BaSO4 Thymol and 10 – 200 mg SO2-
4 ,3.9 26 [31]
industrial gelatin L21
effluents
Sulfate SIA/tur Industrial waters BaCl2 BaSO4 Thymol and 50 – 5000 mg SO2-4 ,4.5 20 – 24 [32]
gelatin L21
Sulfate MCFIA/ Plant materials BaCl2 BaSO4 Tween 80 10 – 500 mg SO2-
4 2 100 [33]
tur L21
Sulfate SIA/tur Waste waters BaCl2 BaSO4 Thymol and 5 – 200 mg SO2-
4 1.5 12 [34]
gelatin L21
Sulfate SIA, FIA, Plant, bovine liver, BaCl2 BaSO4 Tween 80 20 – 200 mg L21 ,3.2 30 – 40 [35]
MCFIA, and blood serum
MSFA/ digests
tur
Sulfate FIA/tur Natural and waste BaCl2 BaSO4 PVA 10 – 120 mg SO2-
4 ,3 40 [36]
waters L21
Sulfate SIA/tur Natural and waste BaCl2 BaSO4 PVA 10 – 100 mg SO2-
4 ,3.3 20 – 22 [37]
waters L21
Sulfate SIA/tur Wine BaCl2 BaSO4 PVA 300– 10 5 [38]
1500 mg K2SO4
L21
Sulfate FIA/neph Unknown waters BaCl2 BaSO4 PVA 10 – 80 mg L21 [39]

FIA, flow-injection analysis; MSFA, monosegmented flow analysis; SIA, sequential injection analysis; MCFIA, multicommuted flow injection
analysis; Tur, turbidimetry; Neph, nephelometry; RSD, relative standard deviation; SR, sampling rate; PVA, poly(vinyl alcohol).

557
558 I. P. A. Morais et al.

procedure based on PbSO4 colloidal formation in ethanol – water was

proposed by Santelli el al. as a turbidimetric method for the determination
of sulfate in natural waters.[28] This reaction was also applied by
Brienza et al. in the determination of total sulfur in plants using crystal
seeding as an alternative approach for improving the rate of crystal growth
in turbidimetric flow analysis.[29]
The addition of colloid protectors or surfactants is often required and, in
contrast with batch procedures, is efficiently accomplished in flow-based
methodologies. These agents can be classified as aqueous solutions of
mono- and polyvalent alcohols, such as glycerol, and aqueous solutions of
macromolecular material, such as gelatine, various gums, or commercial pre-
parations of surface-active agents.[12] The presence of these agents guarantees
the uniform nucleation and prevents the settling of the precipitate, thereby
improving the repeatability and reproducibility of the analysis.[11,12,40] For
this purpose and for both reactions, different surfactants were used, namely
poly(vinyl alcohol) (PVA),[12,13,15,19,21,26,28,36 – 39] gelatine,[14,16 – 18,20,31,32,34]
thymol,[16 – 18,31,32,34] arabic gum,[22,23,25,27] and Tween 80.[33,35]
The nucleation of barium sulfate is strongly pH dependent.[11] The pH not
only affects the formation or dissolution of the barium sulfate precipitate but
also its structure. A precipitate obtained from a solution with a pH in the range
0– 1.5 consists of large, well-shaped crystals. At pH 1.5 – 3, uneven crystals of
medium particle size are obtained, whereas at pH 3– 7, the precipitate is
amorphous.[15] To obtain an acidic medium, hydrochloric acid was frequently
applied, and the samples were previously acidified or acidified in the flow
systems. Moreover, hydrochloric acid is added to prevent the formation of pre-
cipitates of carbonate, chromate, sulfite, phosphate, and oxalate of barium,
which may interfere.[40,41]
In turbidimetric flow methodologies, the build-up of precipitate can
occasionally occur, which leads to decrease of precision and finally can
even block the tubing.[15,41] To overcome this problem, the intermittent
addition of an alkaline buffer ethylenediaminetetraacetate (EDTA) washing
solution to dissolve the barium sulfate, and, consequently, to reduce the
accumulation of the precipitate on the conduit walls and/or on the windows
of the flow cell, has been widely exploited.[15,17 – 23,25,27,31 – 39]
Although very fast precipitation reactions are concerned, nucleation may
be a limiting factor in sample throughput.[11,29] In order to speed up nuclea-
tion, improvement of supersaturation conditions involving addition of a
nucleant species (often the same as the analyte) has been performed.[29]
Addition of sulfate ions into a carrier at a constant concentration or saturation
of the streams with barium sulphate result in an extension of the concentration
range to lower concentrations, better signal stability, and reduction of the
baseline drift.[40] In order to extend the range of the method to low concen-
trations, several FIA systems with continuous addition of sulfate to the
carrier stream[19,22,23,28] or addition of sulfate to the sample before it enters
the injection loop[25,26] have been reported. Brienza et al. proposed a
Turbidimetric and Nephelometric Flow Analysis 559

reproducible addition of in-line produced suspensions to improve supersaturation

conditions in flow turbidimetry. This crystal seeding leads to a simplification
in system design and an improvement in sampling rate and/or sensitivity in
procedures usually limited by rate of turbidity formation. The feasibility of
the approach was demonstrated in developing a turbidimetric FI procedure
for in the determination of total sulfur in plants based on lead sulfate precipi-
tation after adding a confluent stream with lead phosphate nucleant.[29]
Another problem related with the barium sulfate turbidimetric procedure
is the possible interference at the wavelength 420 nm caused by the suspended
solids, the presence of organic substances, and the intrinsic color of the
samples.[18] In order to minimize this difficulty, van Staden[18] proposed a
FI procedure with prevalve sample filtration. The interferences are automati-
cally removed by using an active carbon filter located between the sampler and
the sampling valve system.
Nephelometric flow injection systems for sulfate determination by pre-
cipitation as barium sulfate have also been reported. A liquid-drop windowless
optical cell with a reactor without walls for flow injection turbidimetric and
nephelometric determination of sulfate has been developed by Liu and
Dasgupta.[30] In this approach, problems arising from the deposition of pre-
cipitate on flow cell windows were avoided. Gradient dilution techniques
were conveniently implemented without precise external timing: with small
drops, a single FIA peak is spread over a multitude of drops. In 2003,
Jakmunee et al.[39] developed a simple and low-cost flow-through light-scat-
tering detection system for determining the particle mass concentration. The
methodology was based on nephelometric detection, using a laser pointer as
a light source and a photodiode as a light sensor.

Potassium

Although potassium quantification is generally carried out by flame emission

spectrometry, flow turbidimetric determination methodologies using sodium
tetraphenylboron (Na-TPB) have also been described (Table 2).
Torres and Tubino[42] proposed a turbidimetric flow injection system for
the determination of potassium after precipitation with Na-TPB in alkaline
medium. In order to determine low potassium concentrations, an additional
potassium solution was continuously added to the carrier. The methodology
was applied to the determination of potassium up to 20 mg K L21 in plant
leaves, bottle mineral waters, and serum rehydration solution.
A turbidimetric FI system was developed by Lima et al.[43] for the deter-
mination of total nitrogen and potassium in vegetable samples using a single
spectrophotometer as detector. A solution of Na-TPB prepared in PVA was
used as precipitating agent for the determination of potassium. A gas
diffusion process was included in the manifold to separate ammonium ions
from the rest of the sample and to allow paired analysis. Total potassium
 560
Table 2. Application of turbidimetric flow methods to potassium, nitrogen, phosphate, chloride, and total organic carbon determination

RSD SR
Analyte Flow method Sample Reagent Precipitate Surfactant Working range (%) (h21) Ref.

Potassium FIA Plant leaves, bottled Na-TPB K-TPB Glycerol Up to 1 60 [42]

mineral waters, and 20 mg K L21
serum rehydration
solutions
Potassium FIA Vegetables Na-TPB K-TPB PVA 78 – 1.6 70 [43]
390 mg K L21
Potassium MCFIA Fertilizers Na-TPB K-TPB PVA 6.00 – 1–3 240 [44]
60.0 mg K L21
Ammonia FIA Natural waters and Nessler NHn-1Hg2In — 0.5 – 6.0 mg 120 [46]
21
soil extracts N-NHþ 4 L
Total FIA Plant material Nessler NHn-1Hg2In — 0 – 5% N-NHþ 4 in ,3 100 [47]

I. P. A. Morais et al.
nitrogen plant material
Total FIA Vegetables Na-TPB NH4-TPB PVA 87 – 430 mg ,2.1 70 [44]
21
nitrogen N-NHþ 4 L
Phosphate FIA Serum samples; Molybdate Blue dye salt PVA Up to 1.25 mg 0.56 100 [48]
21
organic com- and crystal PO3-4 L
pounds; plant violet
materials
 Turbidimetric and Nephelometric Flow Analysis
Phosphate FIA Digested plant Zinc(II) Zn3(PO4)2 PVA 5 – 60 mg P L21 ,1.6 180 [49]
material
Phosphate SIA Urine CaCl2 Ca3(PO4)2 — 200– 1.1– 2.0 15 [50]
1500 mg L21
SIA Ca2þ / CO2-
3 CaCO3 — 0.1– 0.8 mg L21 0.97– 12
1.90
Chloride FIA River waters Agþ AgCl — 0 – 14 mg L21 15 [51]
Chloride FIA Natural waters (river) Agþ AgCl PVA Up to 10.00 mg 40 [52]
Cl2 L21
Chloride SIA Ground, surface, and Agþ AgCl PVA 2 – 400 mg Cl2 ,3.7 55– 57 [53]
waste waters L21
Chloride FIA Tap, river, deep Agþ AgCl — 3.0– 30 mg Cl2 [54]
ocean, and refer- L21
ence waters
Total FIA Industrial effluents Ba(OH)2 BaCO3 — 20 – 120 [55]
organic solution 800 mg C L21
carbon

TPB, tetraphenylboron; FIA, flow-injection analysis; MSFA, monosegmented flow analysis; SIA, sequential injection analysis; MCFIA, multicom-
muted flow injection analysis; Tur, turbidimetry; Neph, nephelometry; RSD, relative standard deviation; SR, sampling rate; PVA, poly(vinyl alcohol).

561
562 I. P. A. Morais et al.

determination was carried out on the solutions remaining in the donor stream.
Analysis can be carried out within concentration range of 78– 390 mg K L21.
The turbidimetric determination of potassium in fertilizers using Na-TPB
in PVA was elected by Vicente et al.[44] to demonstrate the feasibility of
exploiting a tandem stream with large initial slugs in a MCFIA system.
Comparing with the other reported flow systems, sampling rate undergoes a
remarkable increase because three samples are simultaneously processed
inside the analytical path. Analysis can be carried out at a rate of 240
samples per hour between 6.0 and 60.0 mg K L21.

Nitrogen

In 1856, Nessler[45] introduced a reagent consisting of mercury (II) iodide and

potassium iodide in alkaline solution for the qualitative and quantitative deter-
mination of ammonia. Since then, Nessler’s reagent has been extensively
referred as the most sensitive test for ammonia; however, it is only accurate
if a number of conditions are carefully controlled. The turbidimetric FIA
systems applied to nitrogen determination are summarized in Table 2.
A turbidimetric FIA system for the determination of ammonia in low con-
centrations using Nessler’s reagent was first developed by Krug et al.[46] The
method was based on the reaction between ammonia and Nessler’s reagent
with the formation of a brown precipitate measured at 410 nm. The effects
of reagent composition, flow rate, temperature, and protective colloids in
the FI system are discussed in detail. Both natural waters and soil extracts
21
can be analyzed in the range 0.5 –6.0 mg N-NHþ 4 L .
In order to investigate the feasibility of isothermal distillation in flow
injection analysis, Zagatto et al.[47] proposed a turbidimetric FI system with
the Nessler reagent for the determination of total nitrogen in plant material.
The merging zones approach was employed to add Nessler’s reagent in a
discrete way so as to avoid baseline drift, which happens when this reagent
is added continuously,[48] and to diminish reagent consumption. The
influence of surfactant, flow rates, alkalinity, ionic strength, collector stream
pH, reagent concentration, and sample volume in ammonia distillation are
discussed.
In 1997, Lima et al.[43] developed a turbidimetric FI system for the deter-
mination of total nitrogen and potassium in vegetable samples using a single
spectrophotometer as detector. Sodium tetraphenylboron (Na-TPB) was used
as precipitating agent and poly(vinyl alcohol) (PVA) as surfactant.
Ammonium ions were withdrawn from the sample by diffusion of volatile
ammonia from the donor to the acceptor. Total nitrogen determination was
carried out on the solution in the acceptor stream after its injection into
the turbidimetric flow path where the ammonium tetraphenylboron preci-
pitation occurred. Analysis can be carried out within concentration range
21
87– 430 mg N-NHþ 4 L .
Turbidimetric and Nephelometric Flow Analysis 563

Phosphate

The majority of manual and automated methods for orthophosphate

determination, in a great variety of samples, are based on the spectrophoto-
metric determination of phosphorus as phosphomolybdenum blue.[49] Never-
theless, as an alternative to colorimetric procedures, different turbidimetric
methodologies have been proposed (Table 2).
Burns et al.[48] developed a FI manifold with a mixing chamber for the
determination of phosphate with molybdate and crystal violet. The insoluble
blue dye salt is kept in colloidal solution with PVA and measured at
560 nm. The system was applied to the determination of phosphate in
serum samples and after appropriate mineralization to organic compounds
and to plant materials.
A simple, fast, and low-cost FIA system was proposed by Diniz et al.[49] for
the turbidimetric determination of orthophosphate in digested plant material.
The determination was based on the precipitation of orthophosphate with zinc
in buffer medium (pH 6.0). PVA was added in all solutions as a colloidal
protector in order to increase both sensitivity and reproducibility and conse-
quently to reduce the washing time. Orthophosphate was determined in the con-
centration range from 5 to 60 mg P L21 with an analytical frequency of 180 h21.
In 2001, Simonet et al. proposed two SIA systems for the turbidimetric
determination of phosphate in urine samples.[50] One method was based on
the calcium phosphate crystallization, and the other on the inhibitory action
of phosphate on the calcium carbonate crystallization. As urine samples
with high calcium content (400 mg L21) can interfere in the method
based on the calcium phosphate crystallization, a cation exchange resin was
incorporated in the manifold. Phosphate could be determined within the
range of 0.2 –1.5 g L21 and 0.1– 1.8 mg L21 for calcium phosphate and for
the inhibitory method, respectively.

Chloride

The spectrophotometric mercury thiocyanate/iron (III) method has been

largely used for chloride determination.[52] However, because this methodo-
logy requires the use of a highly toxic reagent, an effort to replace it has
been recommended. As there are few other spectrophotometric methods for
chloride determination, the turbidimetric procedure involving silver nitrate
with the formation of silver chloride becomes attractive as it is environmen-
tally less harmful and it is easily implemented in flow analysis, requiring
similar instrumentation (Table 2).
Zaitsu et al.[51] were the first to propose a turbidimetric FI procedure for
the determination of chloride in river water. The method was based on the tur-
bidimetric measurement at 440 nm of a silver chloride suspension in nitric
acid medium. A prior separation step involving ion-exchange was required.
The method was applicable for chloride concentrations up to 14 mg L21.
564 I. P. A. Morais et al.

In 1997, Sartini et al.[52] also presented a FI procedure involving the silver

chloride precipitation for the automated turbidimetric determination of
chloride in river waters up to 10 mg L21. For accuracy improvement,
in-line cation exchange was accomplished by means of a resin minicolumn.
Studies aiming at the inclusion of the approaches of crystal seeding and the
addition of surfactants were also carried out.
Mesquita et al.[53] developed a SIA system using the silver chloride
reaction for the turbidimetric determination of chloride in different types of
water, where chloride concentration differs significantly. It was possible to
determine chloride between 2 and 400 mg L21 by simply changing the
sample aspiration time. The novelty of this work when comparing with the
previous FI applications is the possibility of the determination of chloride
over a wide range of concentration, with a single system. In addition, a con-
siderable saving of reagents is achieved due to noncontinuous consumption.
Zenki et al.[54] proposed a closed-loop FI system with turbidimetric
detection for a repetitive determination of chloride. The system of recycling
consists of a single manifold and is superior because of its simplicity, which
is an advisable feature for routine purposes. The method was applied to the
determination of chloride in tap, natural, and reference waters between 3.0
and 30 mg L21.

Total Carbon

The total organic carbon (TOC) is one of the most important parameters for
acquiring knowledge about water and waste water quality because it
concerns theoretically all organic compounds.[55] However, the determination
procedure is complex and time-consuming. In order to develop a simple,
robust methodology with higher analytical frequency, Paniz et al.[55]
proposed a FI turbidimetric system with a gas – liquid transfer microreactor
for the determination of TOC and its fractions in industrial effluent samples.
Samples were decomposed into glass vials in a microwave oven, and a
fraction of CO2 was injected into a carrier gas and pumped to a glass micro-
reactor. This device was specially developed to ensure a quantitative reaction
with a barium hydroxide solution. The resulting suspension was removed from
the microreactor, pumped to the flow cell, and the transient signal was
recorded. With minor modifications, the system allows the determination
of different carbon fractions. The dynamic range was 20– 800 mg C L21
and the maximum analytical frequency was 120 determinations per hour
(Table 2).

Determination of Organic Substances

Organic substances (Table 3) can be determined turbidimetrically either as ion

associates with voluminous organic dyes or metal chelates or as their chelates
 Turbidimetric and Nephelometric Flow Analysis
Table 3. Application of turbidimetric and nephelometric flow methods to the determination of organic compounds

Flow RSD SR
Analyte method Sample Reagent Precipitate Surfactant Working range (%) (h21) Ref.

Levamisole FIA Pharmaceutical HgI2-

4 Ion-association — 7– 32 mg L21 0.9 80 [56]
hydrochloride samples complex
Chlorhexidine FIA Pharmaceutical Thymol blue Ion-association — 10.5– 63.0 mg L21 1.5 53 [57]
formulations complex
Diphenhydramine FIA Pharmaceutical Bromophenol Ion-association — 50 –230 mg L21 0.3 51 [58]
hydrochloride preparations blue complex
Amitriptyline FIA Pharmaceutical Bromocresol Ion-association — 30 –200 mg L21 1.4 39 [59]
formulations purple complex
Phenformin FIA Pharmaceutical Tungstate Tungstate poly-anion — 120– 122 mg L21 0.8 67 [60]
preparations
Thiamine FIA Pharmaceutical Silicotungstic [Thi]2[Si(W3O10)]4 PEG 5.0  1025 to ,1 90 [61]
formulations acid 3.0  1024 mol L21
Homatropine FIA Pharmaceutical Silicotungstic [Hom]4[Si(W3O10)]4 — 8.1  1025 to ,1.5 70 [62]
methylbromide preparations acid 2.2  1024 mol L21
Cyclamate FIA Low-calorie soft BaCl2 BaSO4 PVA 0.015– 0.120% (w/v) 5.9 45 [63]
drinks and artifi-
cial sweeteners
Dipyrone FIA Pharmaceutical Agþ Ag0 colloidal — 5.0  1024 to 1.8 45 [64]
formulations suspension 2.5  1023 mol L21
Dodecylbenzene FIA / Commercial o-tolidine Ion-association — 1.6– 300 mg L21 1.2 – 2.6 68/20 [65]
sulfonic acid SIA / sample complex
neph detergents

565
(continued )
 566
Table 3. Continued

Flow RSD SR
Analyte method Sample Reagent Precipitate Surfactant Working range (%) (h21) Ref.

L -lysine FIA Pharmaceutical L -glutamic L -glutamic acid (inhi- — 0.5– 20 mg L -lys L21 2.5 [66]
preparations acid bition assay)
L -arginine and L - FIA Pharmaceutical L -histidine L -histidine (inhibition — 0.2– 12 mg L -arg L21 2.3 7 [67]
ornithine preparations assay) 0.5– 20 mg L -orn L -arg
L21 2.6
L -orn
L and D -aspartic FIA Pharmaceutical L and D - L and D -histidine — 3 –40 mg L -asp L21 2.1 [68]
acid preparations; histidine (inhibition assay) 4 – 40 mg D -asp L21 L -asp
racemic sample 2.5
of L and D - D -asp
aspartic acid
D and L -glutamic FIA Pharmaceutical L and D - L and D -histidine — Up to 40 mg L21 2.6– 2.9 [69]
acid preparations; histidine (inhibition assay)
racemic sample
of L and D -glu-
tamic acid
L and D -histidine FIA Synthetic samples Land D -glu- L and D -histidine — 5 –100 mg L -his L21 3 [70]

I. P. A. Morais et al.
tamic acid (inhibition assay) 8 – 100 mg D -his L21
Phytic acid SIA Food samples Calcium Calcium oxalate — 0.05– 0.6 mg L21 2.0 20 [71]
oxalate (inhibition assay)

PEG, poly(ethyleneglycol); Thi, thiamine; Hom, homatropine; FIA, flow-injection analysis; MSFA, monosegmented flow analysis; SIA, sequential
injection analysis; MCFIA, multicommuted flow injection analysis; Tur, turbidimetry; Neph, nephelometry; RSD, relative standard deviation; SR,
sampling rate; PVA, poly(vinyl alcohol).
Turbidimetric and Nephelometric Flow Analysis 567

with metal ions. Turbidimetry, in most cases, avoids liquid/liquid extraction

procedures and application of organic solvents. The methods are faster and
simpler than the conventional methodologies.[40]

Nonkinetic Methods

Calatayud and Falco[56] developed a turbidimetric FI system for the determi-

nation of levamisole hydrochloride, a levo-isomer of tetramisole hydrochlo-
ride of the anthelmintic drug family, in pharmaceutical samples. The
method is based on ion-association compounds and deals with quantification
of levamisole with tetraiodomercurate (II) as precipitating agent. The usual
extraction into an organic phase is avoided.
Chlorhexidine, a bactericidal drug, is a member of the biguanide family,
several members of which are found in pharmaceutical formulations.
Calatayud et al.[57] proposed a FI methodology with turbidimetric detection
based on the formation of an ion pair between chlorhexidine and thymol
blue that avoided the extraction step. Studies of chlorhexidine – dye and chlor-
hexidine – Cu(II) were carried out to determine the best precipitate for this
determination.
Diphenylhydramine hydrochloride, usually found in many pharmaceutical
preparations, is a conventional antihistaminic of the H1 type (receptor anta-
gonists) with pronounced sedative properties. It also has antiemetic, anticholi-
nergetic, and local anesthetic properties. An ion associate of diphenylhydramine
hydrochloride with bromophenol blue has been employed for the FI turbidi-
metric determination of diphenylhydramine in pharmaceutical preparations
(tablets).[58] A single-channel manifold in which the sample solution was
injected into the carrier–reagent stream was used, with a monitoring
wavelength of 650 nm. In order to establish the most suitable precipitate for
this determination, several diphenylhydramine–dye systems were evaluated.
A number of interfering substances were also studied.
Amitriptyline is an odorless white powder with a bitter and burning taste. In
1990, Calatayud and Pastor proposed a FIA procedure with turbidimetric
detection for the determination of amitriptyline hydrochloride in pharmaceutical
preparations.[59] The method was based on the formation of an ion-association
compound with Bromocresol purple, and liquid–liquid extraction was required.
Phenformin is a hypoglycemic drug used in the treatment of diabetes
mellitus. Calatayud and Sampedro[60] developed a turbidimetric FI system
for the determination of phenformin in pharmaceutical preparations. After
studying some phenformin – counteranion compounds in order to determine
the suitable precipitate, tungstate was selected as reagent. The method is
based on the direct injection of the sample into a tungstate reagent stream
and the subsequent detection of the formed white precipitate at 700 nm.
Thiamine (vitamin B1) is a white crystalline powder, hygroscopic, and
with a nutlike taste used clinically in the treatment or prevention of
568 I. P. A. Morais et al.

beriberi. Costa-Neto et al.[61] developed a FI merging zones system for the tur-
bidimetric determination of thiamine in pharmaceutical preparations. The
proposed method was based on the precipitation of thiamine with silicotung-
stic acid in acid medium to form a precipitate in suspension (thiamine silico-
tungstate) that is determined turbidimetrically at 420 nm. An improvement of
sensitivity, repeatability, and baseline stability of the FIA system was obtained
by adding poly(ethylene glycol) as colloidal protector.
Later on, the same research group proposed another system for the deter-
mination of homatropine.[62] Antimuscarinic compounds are drugs that play
an important role in the central nervous system. The most widely used are
areatropine, scopolamine, homatropine, and homatropine methylbromide
(HMB). A FI turbidimetric procedure exploiting merging zones for determin-
ing HMB in pharmaceutical preparations was proposed. The determination
was based on the precipitation reaction of HMB with silicotungstic acid in
acidic medium and the precipitate was measured at 410 nm.
Sodium and calcium cyclamates are additives widely used as non-
nutritive sweetener in many diet and medicinal products. They are no
longer permitted as a food additive in many countries including Canada, the
United States, and in European countries due to their conversion to cyclohexy-
lamine, which is a strong carcinogen. However, they are available in other
countries as a sweetener. In 2005, Llamas et al.[63] proposed a FI turbidimetric
in-direct method for determination of cyclamate in low-calorie soft drinks and
artificial sweeteners without pretreatment. It was based on the oxidation of the
sulfamic group, which is present in cyclamates, to sulfate by addition of
nitrite. Then, a precipitate of barium sulfate was obtained by reaction with
barium chloride, in presence of PVA in perchloric acid solution, at 308C.
The analytical signal was measured at 420 nm.
Dipyrone is a white crystalline powder, soluble in water and ethanol,
which presents anesthetic and antipyretic properties. A FI procedure using a
solid phase reactor with AgCl immobilized in a polyester resin was
developed by Marcolino-Jr et al.[64] in 2005 for determining dipyrone in
pharmaceutical formulations. The determination is based on the reduction
of Agþ ions of the solid phase reactor to Ag0 by dipyrone. A colloidal suspen-
sion of Ag0 produced is transported by carrier solution (0.01 mol L21 NaOH)
and turbidimetrically detected at 425 nm. The concentration of dipyrone
injected is proportional to the quantity of Ag0 produced.
Simple light scattering methods (batch, FI, SI) for the determination of
anionic active matter in detergents based on a novel reaction were reported
by March et al.[65] in 2005. The methods were based on formation of a
solid phase by association of anionic surfactants and protonated o-tolidine.
Measurements were carried out with a conventional spectrofluorimeter at
400 nm, and dodecylbenzene sulfonic acid (DBS) was selected as the
reference anionic surfactant. Influence of the main parameters affecting
the characteristics of the methods was studied by the univariate method.
The methods were applied to commercial samples and results successfully
compared with a volumetric recommended method.
Turbidimetric and Nephelometric Flow Analysis 569

Kinetic Methods

Some organic substances act as crystallization inhibitors for organic

molecules with similar chemical structures (or a slightly different bulk
component of molecular crystal). The inhibitory effect can be assigned to
selective interactions with the foreign molecule at specific points in the crys-
tallizing substances that induce marked changes in the crystallization rate at
very low inhibitor concentrations. These processes have found application
in analytical chemistry, mostly in the determination of amino acids.[66]
Several studies concerning the determination of different amino acids
using turbidimetric flow analysis methodology have been reported,[66 – 70] as
alternatives to spectrophotometric, liquid chromatographic, and chemilumino-
metric or electrochemical detection.[66] The high selectivity and sensitivity of
crystal growth inhibitory processes make these systems potentially useful for
the enantiomeric resolution of inhibitory substances.[70]
Ballesteros et al.[66] developed a FI turbidimetric method for the discrimi-
nation of L - and D -lysine enantiomers by the inhibitory action of L -lysine on
the crystallization of L -glutamic acid. A multidetection flow system
including an open-closed loop and a single detector permits the determination
of kinetic parameters for the crystallization of L -glutamic acid in the presence
of 2-propanol. L -lysine can thus be determined in the presence of D -lysine
concentration or other amino acids with no need for a prior separation. The
proposed method was applied to the determination of L -lysine in pharma-
ceutical preparations.
A FI method for the determination of L -arginine and L -ornithine based on
the inhibition of L -histidine crystallization was also presented by Ballesteros
et al.[67] The open-closed system permits turbidimetric multidetection of the
signal in the crystallization of L -histidine in the presence of an organic
solvent (2-propanol). The proposed method permits the selective determi-
nation of L -arginine and L -ornithine in pharmaceutical preparations in the
presence of their D -enantiomers and other L -amino acids without the need
for a prior separation.
Hosse et al.[68] proposed a FI system for the enantiomeric discrimination
of L - and D -aspartic acid that enables the turbidimetric multidetection of the
signal produced in the crystallization of histidine from a supersaturated
solution. The presence of L - and D -aspartic acid delays the growth of L - and
D -histidine crystals, respectively, the delay being proportional to the concen-
tration of aspartic acid. The method was applied to the determination of
L -aspartic acid in pharmaceutical preparations and the resolution of a
racemic sample of L ,D -aspartic acid.
A FI turbidimetric method for the indirect determination of D - and
L -glutamic acid by the inhibitory effect of these substances on the crystal
growth of D - and L -histidine, respectively, in the presence of an organic
solvent is proposed by Ballesteros et al.[69] This continuous method allowed
the sequential determination of D - and L -glutamic acid in a multidetection
flow system, including an open-closed loop and a single spectrophotometer.
570 I. P. A. Morais et al.

The methodology was applied to the determination of L -glutamic acid in

pharmaceutical preparations and the determination of D - and L -glutamic
acid in a racemate of DL -glutamic acid.
In 1998, Rodrı́guez et al.[70] developed a FI turbidimetric method for the
sequential determination of L - and D -histidine in synthetic samples, containing
both enantiomers in variable concentration ratios. The method was based in
the rate of crystal growth of L - and D -glutamic acid caused by the adsorption
of foreign species (of L - and D -histidine, respectively) at a specific point of the
crystal surface.
This kinetic-turbidimetric detection approach was also applied to the
determination of acid phytic in food samples using a SI system.[71] The
method was based on the diminution of the calcium oxalate crystallization
reaction rate in the presence of phytic acid. Such a crystallization rate has
been evaluated from the increase of turbidity with time.

Immunologic Reactions

The antigen – antibody interaction is a bimolecular association similar to an

enzyme –substrate interaction, with an important difference: it does not lead
to an irreversible chemical modification in either the antibody or in the
antigen. The association between both involves various nonconvalent inter-
actions. Antibody (precipitins) and the soluble antigen interacting in aqueous
solution form a lattice that eventually develops into a visible precipitate.[72]
The first quantitative determination of proteins based on an immuniprecipitin
reaction was reported by Heidelberger and Kendall in 1935. The current import-
ance of the immunoprecipitin technique for the analysis of proteins has been
emphasized by the development of an automated immunoprecipitin analyzer
and the subsequent use of laser nephelometry to increase the sensitivity of the
method. FIA provides an attractive high-speed, low-cost alternative to the
existing instrumentation for the study of immunoprecipitin reactions[72] (Table 4).
Immunoprecipitation reactions using FIA with merging zones was
applied to the determination of human serum immunoglobulin G (IgG) in
serum samples and human IgG antiserum.[73 – 78]
A stop-flow merging zones FI system for monitoring the precipitin inter-
action between yeast mannan (the model antigen) and concanavalin A (the
model antibody) was first developed by Worsfold[73] in 1983. In this paper,
the suitability of the FIA for the study of biochemically specific interactions
is also discussed.
In 1984, a study of a model immunoprecipitin reaction between concana-
valin A and yeast mannan using a microcomputer-controlled stop-flow
merging zones FIA manifold with turbidimetric detection was reported by
Worsfold and Hughes.[74] The system described could be used routinely for
immunoprecipitin analysis in clinical laboratories, IgG in human serum, and
also to study kinetic aspects of such reactions.
 Turbidimetric and Nephelometric Flow Analysis
Table 4. Application of turbidimetric and light-scattering flow methods to immunological methods and to the determination of biomass

Flow RSD SR
Analyte method Sample Reagent Surfactant Working range (%) (h21) Ref.

Concanavalin A FIA/tur Yeast mannan — Up to 10.0 mg mL21 ,5.3 [73]

Concanavalin A FIA/tur Yeast mannan — 0.1– 20.0 mg mL21 50 [74]
Antibody Ig G FIA/tur Human serum Goat anti-human Ig — 0 –3556 mg Ig G dL21 2.0 – 6.8 40 [75]
G antiserum
Antibody IgG FIA/tur Human serum Goat anti-human Ig PEG Up to 2844 mg dL21 ,6 40 [76]
G antiserum
Monoclonal anti- FIA/tur Fermentation of Anti-mouse IgG — 1 –1000 mg L21 2 [77]
bodies (mab) mouse– mouse
hybridoma cells
IgA FIA/tur Human serum Sheep anti-human — 0.09– 0.36 g L21 40 [78]
IgA
Pullulanase FIA/tur Fermentation of Clos- — 10 –1000 U L21 1.5 [79]
isoenzyme tridium
thermosulfurogenes
Antigen anti-A FIA/tur Mammalian cell culti- Solution of the anti- — [80]
Mab, a mono- vation processes bodies (anti-
clonal antibody mouse IgG)
of the IgG type

(continued )

571
 572
Table 4. Continued

Flow RSD SR
Analyte method Sample Reagent Surfactant Working range (%) (h21) Ref.

Total prothrom- FIA/tur Human plasma (venous Calcium — 10– 100% of total clot- ,2.8 50 [81]
binase complex blood) thromboplastine ting activity
(prothrombin,
factor V, factor
Xa, Ca2þ,
phospholipids)
Fibrinogen FIA/LS Human plasma Ammonium sulfate — 1– 20 mg L21 ,1.33 80 [82]
and guanidine
hydrochloride
Biomass FIA/tur Bacterial and yeast — 15– 4000 mg L21 0.95 90 [83]
fermentation broth
Total biomass SIA/tur Unfiltered yeast fer- — 0.2– 80 g L21 3 [85]
mentation broth
Biomass FIA/tur Microalga bioreactor — 5.9 [86]

I. P. A. Morais et al.
PEG, poly(ethyleneglycol); LS, light scattering; FIA, flow-injection analysis; MSFA, monosegmented flow analysis; SIA, sequential injection
analysis; MCFIA, multicommuted flow injection analysis; Tur, turbidimetry; Neph, nephelometry; RSD, relative standard deviation; SR,
sampling rate; PVA, poly(vinyl alcohol).
Turbidimetric and Nephelometric Flow Analysis 573

An immunological reaction between human serum immunoglobulin

G (IgG) and goat anti-human IgG was developed using automated stop-
flow merging zones FIA manifolds by Worsfold et al. Turbidimetric
detection was used to monitor the rate of reaction.[74,75] Serum samples and
human reference serum were analyzed and their IgG concentrations inter-
polated from a second-order fit[75] or from the linear[76] calibration data. In
order to enhance the formation of large molecular aggregates and to
increase the sensitivity, polyethylene glycol was introduced to the carrier
stream.[76]
Freitag et al.[77] proposed a stop-flow merging zones FI system for
real-time monitoring of specific proteins in fermentation processes. The
method is based on the formation of aggregates between the proteins to be
determined and their antibodies, with the subsequent turbidimetric
determination. The analyzer was used to measure monoclonal antibodies
produced in fermentations of mouse– mouse hybridoma cells and to
quantify pullulanase isoenzymes produced in a fermentation of Clostridium
thermosulfurogenes.
An automated merging zones FIA procedure for the determination of
IgA in human serum via its interaction with sheep anti-human IgA was
developed by Wang et al.[78] The FIA coupled with turbidimetric detection
provided a precise, rapid, and simple system for the study of immunoprecipitin
interaction.
An online assay for a thermostable pullulanase and antithrombin III is
described by Freitag et al.[79] The assay is based on the formation of aggre-
gates between the protein to be measured and the antibodies raised against
the protein. A stop-flow merging zones FIA manifold was used to monitor
pollulanase activity of Clostridium thermosulfurogenes cultures.
Hitzman et al.[80] used an assay with turbidimetric detection for the online
or offline monitoring of mammalian cell cultivation. A FI system with
merging zones and stop-flow approach was applied. Reference channel was
also incorporated where no immunoreactant was supplied so that medium
blank absorption could be assessed. The difference of peak high within the
two channels was used to establish linear regression model and to calculate
the sample concentrations.
Romero et al.[81] developed an automatic FI method for the evaluation of
the hemostasy process based on the estimation of the extrinsic coagulation
pathway (prothrombin, factor V, factor Xa, Ca2þ, phospholipids). A stop-
flow merging zones manifold was proposed, and the clotting reaction rate
was monitored at 340 nm.
A light-scattering method for the determination of fibrinogen in human
plasma is presented by Silva et al.[82] The method is based on the analyte pre-
cipitation in the presence of ammonium sulfate in glycine hydrochloride
buffer. The approach was developed by using a flow-injection manifold
where the light scattered by the solid suspension formed was monitored in
spectrofluorimeter with an incident wavelength of 340 nm.
574 I. P. A. Morais et al.

Determination of Biomass

In order to make microbial processes most efficient, several parameters that

give information about physical and chemical environment, as well as about
growth and production, have to be determined continuously.[83,84] FIA is a
very promising method for online process control, due to its versatility, the
simplicity of experimental setup, low cost, and good reproducibility. The com-
bination of suitable sampling devices with FIA systems is a prerequisite
toward online control of bioreactor processes. It includes problem-orientated
pretreatments of the sample and allows the application of FIA to the control of
almost all kinds of bioreactors.[84] Biomass is a basic parameter in bioreactor
operation that is often used as an indirect measure of product formation,
subtract consumption, and process disturbances.[85,86] Traditional direct deter-
minations by counting the cell number under the microscope or determining
cell dry weight are both tedious and time-consuming and are not suitable
for online bioprocess control.[84,85] The use of turbidity of the fermentation
broth as analytical signal for bacterial and yeast fermentations biomass
measurement is the usual method of noninvasive biomass estimation. The tur-
bidimetric FI methods applied to biomass determination are summarized in
Table 4.
An automated FI analyzer for measuring the concentration of biomass,
glucose, and lactate during lactic acid fermentations was described by
Benthin et al.[83] Biomass concentrations were determined by absorbance
(turbidity) measurements. Traditionally, the absorbance of the broth is
measured by continuously diluting the broth to the range of linear response.
Despite automatic washing procedures, these analyzers are more or less liable
to clogging and forming deposits on the optical surfaces. Applying the FI prin-
ciples, these problems can be minimized. The sample was injected into a small
stirred mixing chamber (MC) with subsequent detection at 565 nm. In the MC,
rapid and reproducible dilution of the sample occurs, and consequently potential
matrix effects from the viscosity of the fermentation broth are reduced. The
analyzer is calibrated by injection of potassium permanganate standard
solution and the absorbance values converted to biomass concentration
(g cell dry mass L21) by a linear relationship between the measured absorbance
and measured biomass concentration during batch fermentation.
In 1994, Baxter et al.[85] developed a SI system for the determination of
total biomass from yeast (Saccharomyces cerevisiae) fermentation. The
assay uses both turbidimetric (absorbance) and nephelometric measurements
at a wavelength that is not absorbed by the liquid medium. In contrast with
the FI system previously described, the biomass is determined without pretreat-
ment or dilution of the original sample. The assay uses a SIA system to sample
a precise volume of biomass obtained from the bioreactor and to deliver it to a
flow cell where it is quickly mixed and the analytical signal detected.
A FI system for the online determination of biomass in a microalga
(Pavlova lutheri) bioreactor was developed by Meireles et al.[86] The device
Turbidimetric and Nephelometric Flow Analysis 575

was fully computerized and was based on diluting small aliquots of the culture
followed by measuring optical density (turbidity); this figure was then accu-
rately correlated with biomass, in terms of both cell number and ash-free
dry weight, during the entire culture time. The growth rate and biomass pro-
ductivity of P. lutheri, cultivated under batch and semicontinuous modes,
were monitored as experimental testing model.

ACKNOWLEDGMENTS

Inês Morais and Ildikó Tóth thank Fundação para a Ciência e a Tecnologia
(FCT) and FSE (III Quadro Comunitário) for the grants SFRH/BPD/
26127/2005 and SFRH/BPD/5631/2001, respectively.

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