EUROPEAN PHARMACOPOEIA 9.2 2.2.1.

Clarity and degree of opalescence of liquids

07/2017:20201 MEASUREMENTS IN RATIO MODE

The determination of opalescence of coloured liquids is done
using instruments with ratio mode, since colour provides a
negative interference, attenuating both incident and scattered
light and lowering the turbidity value. The effect is so great,
2.2.1. CLARITY AND DEGREE OF even for moderately coloured samples, that conventional
nephelometers cannot be used.
OPALESCENCE OF LIQUIDS In turbidimetry or nephelometry with ratio mode, the ratio
Opalescence is the effect of light being absorbed or scattered by of the transmission measurement to the 90° scattered light
submicroscopic particles or optical density inhomogeneities. measurement is determined. This procedure compensates
The absence of any particles or inhomogeneities in a solution for the light that is diminished by the colour of the sample.
results in a clear solution. Instruments with ratio mode use as light source a tungsten
A liquid is considered clear if its clarity is the same as that lamp with spectral sensitivity at about 550 nm operating
of water R or of the solvent used, or if its opalescence is at a filament colour temperature of 2700 K. Other suitable
not more pronounced than that of reference suspension I light sources may also be used. Silicon photodiodes and
(see Table 2.2.1.-1), when examined under the conditions photomultipliers are commonly used as detectors and record
described below. changes in light scattered or transmitted by the sample. The
light scattered at 90 ± 2.5° is measured by the primary detector.
Requirements in monographs are expressed in terms of the Other detectors measure back and forward scatter (reflected
visual method by comparing with the defined reference light) as well as transmitted light. The results are obtained by
suspensions (see Table 2.2.1.-1). However, instrumental calculating the ratio of the 90° scattered light measured to the
methods may also be used for determining compliance sum of the components of forward scattered and transmitted
with monograph requirements once the suitability of the light values.
instrument has been established as described below and
calibration with reference suspensions I-IV and with water R The instruments used are calibrated against standards of
or the solvent used has been performed. known turbidity and are capable of automatic measurement
of turbidity. The test results are obtained directly from
VISUAL METHOD the instrument and compared to the specifications in the
Using identical test-tubes of colourless, transparent, neutral individual monograph.
glass with a flat base and an internal diameter of 15-25 mm, Alternatively, the influence of the colour of the sample
compare the liquid to be examined with a reference suspension may also be eliminated by using an infrared light-emitting
freshly prepared as described below. Ensure that the depths of diode (IR LED) having an emission maximum at 860 nm
the layers in the 2 test-tubes are the same (about 40 mm). with a 60 nm spectral bandwidth as the light source of the
Compare the liquids in diffused daylight 5 min after instrument.
preparation of the reference suspension, viewing vertically INSTRUMENT REQUIREMENTS
against a black background. Instruments complying with the following characteristics and
System suitability. The diffusion of light must be such that verified using reference suspensions as described below may
reference suspension I can readily be distinguished from be used instead of visual examination for determination of
water R, and that reference suspension II can readily be compliance with monograph requirements.
distinguished from reference suspension I (see Table 2.2.1.-1). – Measuring unit : NTU (nephelometric turbidity units).
NTU is based on the turbidity of a primary standard
INSTRUMENTAL METHOD of formazin. FTU (formazin turbidity units) or FNU
The instrumental assessment of clarity and opalescence (formazin nephelometric units) are also used, and are
provides a more discriminatory test that does not depend on equivalent to NTU in regions of low turbidity (up to
the visual acuity of the analyst. Numerical results are more 40 NTU). These units are used in all 3 instrumental
useful for process control and quality monitoring, especially methods (nephelometry, turbidimetry and in ratio mode).
in stability studies. For example, previous numerical data on – Measuring range : 0.01-1100 NTU.
stability can be extrapolated to determine whether a given – Resolution : 0.01 NTU within the range 0-9.99 NTU ;
batch of a preparation will exceed shelf-life limits prior to the 0.1 NTU within the range 10.0-99.9 NTU ; and 1 NTU for
expiry date. the range > 100 NTU.
TURBIDIMETRY AND NEPHELOMETRY – Accuracy : ± (10 per cent of reading + 0.01 NTU) within
When a suspension is viewed at right angles to the direction the range 0-20 NTU ; ± 7.5 per cent within the range
of the incident light, the system appears opalescent due to the 20-1100 NTU.
scattering of light by the particles of the suspension (Tyndall – Repeatability : ± 0.05 NTU within the range
effect). A certain portion of the light beam entering a turbid 0-20 NTU ; ± 2 per cent of the reading within the range
liquid is transmitted, another portion is absorbed and the 20-1100 NTU.
remaining portion is scattered by the suspended particles.
Instruments with measuring range or resolution, accuracy and
The light-scattering effect of suspended particles can be
repeatability capabilities other than those mentioned above
measured either indirectly by observation of the transmitted
may be used provided they are sufficiently validated and are
light (turbidimetry) or directly by measuring the scattered
capable for the intended use.
light (nephelometry). Turbidimetry and nephelometry are
more reliable in low turbidity ranges, where there is a linear CONTROL OF INSTRUMENT PERFORMANCE
relationship between turbidity values and detector signals. – Calibration : performed with at least 4 reference suspensions
As the degree of turbidity increases, not all the particles of formazin covering the measuring range of interest.
are exposed to the incident light and the scattered or the Reference suspensions described in this chapter or suitable
transmitted radiation of other particles is hindered on its way reference standards calibrated against the primary reference
to the detector. suspensions may be used.
For quantitative measurements, the construction of calibration – Stray light : < 0.15 NTU within the range 0-10 NTU ;
curves is essential. Linearity must be based on at least 4 < 0.5 NTU within the range 10-1100 NTU. Stray light is
levels of concentrations. Reference suspensions must show a defined as that light that reaches the nephelometric detector
sufficiently stable degree of turbidity and must be produced without being a result of scatter from the sample. Stray light
under well-defined conditions. is always a positive interference and is a significant source

General Notices (1) apply to all monographs and other texts 4285
2.2.46. Chromatographic separation techniques EUROPEAN PHARMACOPOEIA 9.2

of error in low-range turbidity measurements. Sources

of stray light include : imperfections in and scratches on
sample cells, internal reflections of the optical system,
contamination of the optics or sample cell chamber with
dust, and electronic noise. Instrument design can also
affect stray light. The influence of stray light becomes
negligible in ratio mode measurements. 07/2016:20246
The test methodology for the specific substance/product to be corrected 9.2
analysed must also be verified to demonstrate its analytical
capability. The instrument and methodology shall be
consistent with the attributes of the substance to be examined.
Measurements of standards and samples should be carried out
under the same temperature conditions, preferably between 2.2.46. CHROMATOGRAPHIC
20 °C and 25 °C.
SEPARATION TECHNIQUES
REFERENCE SUSPENSIONS
Chromatographic separation techniques are multi-stage
Formazin has several desirable characteristics that make it an
separation methods in which the components of a sample
excellent turbidity standard. It can be reproducibly prepared
are distributed between 2 phases, one of which is stationary,
from assayed raw materials. The physical characteristics
while the other is mobile. The stationary phase may be a
make it a desirable light-scatter calibration standard. The
solid or a liquid supported on a solid or a gel. The stationary
formazin polymer consists of chains of different lengths,
phase may be packed in a column, spread as a layer, or
which fold into random configurations. This results in a wide
distributed as a film, etc. The mobile phase may be gaseous
variety of particle shapes and sizes, which allows the analysis
or liquid or supercritical fluid. The separation may be based
of different particle sizes and shapes that are found in real
on adsorption, mass distribution (partition), ion-exchange,
samples. Stabilised formazin suspensions that can be used to
etc., or may be based on differences in the physico-chemical
prepare stable, diluted turbidity standards are commercially
properties of the molecules such as size, mass, volume, etc.
available and may be used after comparison with the standards
prepared as described. This chapter contains definitions and calculations of common
All steps of the preparation of reference suspensions as parameters and generally applicable requirements for system
described below are carried out at 25 ± 3 °C. suitability. Principles of separation, apparatus and methods
are given in the following general methods :
Hydrazine sulfate solution. Dissolve 1.0 g of hydrazine
sulfate R in water R and dilute to 100.0 mL with the same – paper chromatography (2.2.26) ;
solvent. Allow to stand for 4-6 h. – thin-layer chromatography (2.2.27) ;
Primary opalescent suspension (formazin suspension).
In a 100 mL ground-glass-stoppered flask, dissolve 2.5 g of – gas chromatography (2.2.28);
hexamethylenetetramine R in 25.0 mL of water R. Add 25.0 mL – liquid chromatography (2.2.29) ;
of the hydrazine sulfate solution. Mix and allow to stand for
24 h. This suspension is stable for 2 months, provided it is – size-exclusion chromatography (2.2.30) ;
stored in a glass container free from surface defects. The – supercritical fluid chromatography (2.2.45).
suspension must not adhere to the glass and must be mixed
thoroughly before use.
Standard of opalescence. Dilute 15.0 mL of the primary DEFINITIONS
opalescent suspension to 1000.0 mL with water R. This The system suitability and acceptance criteria in monographs
suspension is freshly prepared and may be stored for up to have been set using parameters as defined below. With some
24 h. equipment, certain parameters, such as the signal-to-noise ratio
Reference suspensions. Prepare the reference suspensions and resolution, can be calculated using software provided by
according to Table 2.2.1.-1. Mix and shake before use. the manufacturer. It is the responsibility of the user to ensure
that the calculation methods used in the software are equivalent
Table 2.2.1.-1 to the requirements of the European Pharmacopoeia and to
I II III IV
make any necessary corrections if this is not the case.
Chromatogram
Standard of opalescence 5.0 mL 10.0 mL 30.0 mL 50.0 mL
Water R 95.0 mL 90.0 mL 70.0 mL 50.0 mL
A graphical or other representation of detector response,
effluent concentration or other quantity used as a measure
Measurements of reference suspensions I-IV in ratio mode of effluent concentration, versus time or volume. Idealised
show a linear relationship between the concentrations and chromatograms are represented as a sequence of Gaussian
measured NTU values (see Table 2.2.1.-2). peaks on a baseline (Figure 2.2.46.-1).
Peak
Table 2.2.1.-2
The portion of a chromatogram recording the detector
Formazin suspensions Opalescent values (NTU) response when a single component (or 2 or more unresolved
Reference suspension I 3 components) is eluted from the column.
Reference suspension II 6 The peak may be defined by the peak area, or the peak
height (h) and the peak width at half-height (wh), or the
Reference suspension III 18
peak height (h) and the peak width between the points of
Reference suspension IV 30 inflection (wi). In Gaussian peaks (Figure 2.2.46.-1) there is
the following relationship :
Standard of opalescence 60

Primary opalescent suspension 4000

4286 See the information section on general monographs (cover pages)