EUROPEAN PHARMACOPOEIA 11.0
Fluorimetry
Method. Place in the inner tube sufficient quantity of the
liquid or previously melted substance to be examined, to cover
the thermometer bulb and determine the approximate freezing
point by cooling rapidly. Place the inner tube in a bath about
5 °C above the approximate freezing point until all but the last
traces of crystals are melted. Fill the beaker with water or a
saturated solution of sodium chloride, at a temperature about
5 °C lower than the expected freezing point, insert the inner
tube into the outer tube, ensuring that some seed crystals are
present, and stir thoroughly until solidification takes place.
Note the highest temperature observed during solidification.
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2.2.19. AMPEROMETRIC TITRATION
In an amperometric titration, the end-point is determined
by following the variation of the current measured between
2 electrodes (either one indicator electrode and one reference curve can facilitate the determination of the end-point.
electrode or 2 indicator electrodes) immersed in the solution In potentiometric titrations of weak acids or bases using
to be examined and maintained at a constant potential
difference as a function of the quantity of titrant added.
The potential of the measuring electrode is sufficient to ensure it is impracticable to use potentiometric detection for this
a diffusion current for the electroactive substance.
Apparatus. The apparatus comprises an adjustable voltage
source and a sensitive microammeter ; the detection system
generally consists of an indicator electrode (for example, a
platinum electrode, a rotating-disc electrode or a carbon
electrode) and a reference electrode (for example, a silver-silver
chloride electrode).
A three-electrode apparatus is sometimes used, consisting of
an indicator electrode, a reference electrode and a polarised
auxiliary electrode.
Method. Set the potential of the indicator electrode as
prescribed and plot a graph of the initial current and the
values obtained during the titration as functions of the
quantity of titrant added. Add the titrant in not fewer than
3 successive quantities equal to a total of about 80 per cent
of the theoretical volume corresponding to the presumed
equivalence point. The 3 values must fall on a straight line.
Continue adding the titrant beyond the presumed equivalence
point in not fewer than 3 successive quantities. The values
obtained must fall on a straight line. The point of intersection
of the 2 lines represents the end-point of the titration.
For amperometric titrations with 2 indicator electrodes, the
whole titration curve is recorded and used to determine the
end-point.
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2.2.20. POTENTIOMETRIC TITRATION
In a potentiometric titration (volumetric titration with
potentiometric end-point determination) the end-point
is determined by recording the variation of the potential
difference between 2 electrodes (either 1 indicator electrode
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2.2.21.
and 1 reference electrode, or a combined electrode) immersed
in the solution to be examined as a function of the volume
of titrant added.
Apparatus. The apparatus used comprises a millivoltmeter.
Commercial autotitrator instruments may be used and are
operated in accordance with the manufacturer’s instructions,
using electrodes recommended for the type of titration
described.
The indicator electrode to be used depends on the substance
to be determined and may be a glass or metal electrode
(e.g. platinum, gold or silver).
For acid-base titrations, a glass-silver-silver chloride electrode
combination is generally used.
Method. Prepare the sample solution as described. Add
the titrant in suitable aliquots paying particular attention
to the rate of addition and the volume increments near the
end-point. Continue the titration beyond this point to allow a
clear detection of the end-point.
The end-point of the titration is reached when the maximum
change in potential occurs in a plot of potential versus
volume of titrant, and is expressed as the corresponding
volume of titrant. Recording the first or second derivative
non-aqueous solvents, if necessary, either carry out a blank
determination or pre-neutralise the solvent mixture. Where
purpose, the solvent mixture can be pre-neutralised by
titration using a suitable indicator. Some examples are given
below :
Titrant Indicator
Perchloric acid Crystal violet solution R
3 g/L solution of thymol blue R
Tetrabutylammonium hydroxide
in methanol R
Ethanolic sodium hydroxide Thymolphthalein solution R
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2.2.21. FLUORIMETRY
Fluorimetry is a procedure which uses the measurement of the
intensity of the fluorescent light emitted by the substance to
be examined in relation to that emitted by a given standard.
Method. Dissolve the substance to be examined in the solvent
or mixture of solvents prescribed in the monograph, transfer
the solution to the cell or the tube of the fluorimeter and
illuminate it with an excitant light beam of the wavelength
prescribed in the monograph and as near as possible
monochromatic.
Measure the intensity of the emitted light at an angle of 90°
to the excitant beam, after passing it through a filter which
transmits predominantly light of the wavelength of the
fluorescence. Other types of apparatus may be used provided
that the results obtained are identical.
41
2.2.22. Atomic emission spectrometry EUROPEAN PHARMACOPOEIA 11.0

For quantitative determinations, first introduce into the – a data-acquisition unit.

apparatus the solvent or mixture of solvents used to dissolve Oxygen, air and a combustible gas such as hydrogen, acetylene,
the substance to be examined and set the instrument to zero. propane or butane may be used in flames. The atomisation
Introduce the standard solution and adjust the sensitivity source is critical, since it must provide sufficient energy to
of the instrument so that the reading is greater than 50. If excite and atomise the atoms. The atomic spectra emitted
the second adjustment is made by altering the width of the from flames have the advantage of being simpler than those
slits, a new zero setting must be made and the intensity of emitted from other sources, the main limitation being that the
the standard must be measured again. Finally introduce the flames are not powerful enough to cause emission for many
solution of unknown concentration and read the result on the elements allowing their determination. Acidified water is the
instrument. Calculate the concentration cx of the substance in solvent of choice for preparing test and reference solutions,
the solution to be examined, using the formula : although organic solvents may also be used if precautions are
Ixcs taken to ensure that the solvent does not interfere with the
cx = stability of the flame.
Is
INTERFERENCES
cx = concentration of the solution to be examined,
Spectral interference is reduced or eliminated by choosing an
cs = concentration of the standard solution, appropriate emission line for measurement or by adjusting the
slit for spectral band-width. Physical interference is corrected
Ix = intensity of the light emitted by the solution to be by diluting the sample solution, by matching the matrix or by
examined, using the method of standard additions. Chemical interference
Is = intensity of the light emitted by the standard is reduced by using chemical modifiers or ionisation buffers.
solution.
MEMORY EFFECT
If the intensity of the fluorescence is not strictly proportional
to the concentration, the measurement may be effected using The memory effect caused by deposit of analyte in the
a calibration curve. apparatus may be limited by thoroughly rinsing between runs,
In some cases, measurement can be made with reference diluting the solutions to be measured if possible and thus
to a fixed standard (for example a fluorescent glass or a reducing their salt content, and by aspirating the solutions
solution of another fluorescent substance). In such cases, through as swiftly as possible.
the concentration of the substance to be examined must be METHOD
determined using a previously drawn calibration curve under
Use of plastic labware is recommended wherever possible.
the same conditions.
Operate an atomic emission spectrometer in accordance with
the manufacturer’s instructions at the prescribed wavelength.
01/2008:20222 Optimise the experimental conditions (flame temperature,
burner adjustment, use of an ionic buffer, concentration of
solutions) for the specific element to be analysed and in respect
of the sample matrix. Introduce a blank solution into the
atomic generator and adjust the instrument reading to zero or
to its blank value. Introduce the most concentrated reference
2.2.22. ATOMIC EMISSION solution and adjust the sensitivity to obtain a suitable reading.
SPECTROMETRY It is preferable to use concentrations which fall within the
linear part of the calibration curve. If this is not possible, the
GENERAL PRINCIPLE calibration plots may also be curved and are then to be applied
Atomic emission is a process that occurs when electromagnetic with appropriate calibration software.
radiation is emitted by excited atoms or ions. In atomic Determinations are made by comparison with reference
emission spectrometry the sample is subjected to temperatures solutions with known concentrations of the element to
high enough to cause not only dissociation into atoms, but be determined either by the method of direct calibration
also to cause significant amounts of collisional excitation (Method I) or the method of standard additions (Method II).
and ionisation of the sample atoms to take place. Once the
METHOD I - DIRECT CALIBRATION
atoms and ions are in the excited states, they can decay to
lower states through thermal or radiative (emission) energy For routine measurements 3 reference solutions of the element
transitions and electromagnetic radiation is emitted. An to be determined and a blank are prepared and examined.
emission spectrum of an element contains several more lines Prepare the solution of the substance to be examined (test
than the corresponding absorption spectrum. solution) as prescribed in the monograph. Prepare not fewer
Atomic emission spectrometry is a technique for determining than 3 reference solutions of the element to be determined,
the concentration of an element in a sample by measuring the the concentrations of which span the expected value in the
intensity of one of the emission lines of the atomic vapour of test solution. For assay purposes, optimal calibration levels
the element generated from the sample. The determination is are between 0.7 and 1.3 times the expected content of the
carried out at the wavelength corresponding to this emission element to be determined or the limit prescribed in the
line. monograph. For purity determination, calibration levels are
between the limit of detection and 1.2 times the limit specified
In this chapter only atomisation in flame is dealt with. The for the element to be determined. Any reagents used in the
method of inductively coupled plasma-atomic emission preparation of the test solution are added to the reference
spectrometry (ICP-AES) is described in a different general solutions and to the blank solution at the same concentration.
chapter.
Introduce each of the solutions into the instrument using the
APPARATUS same number of replicates for each solution, to obtain a steady
This consists essentially of : reading.
– a sample introduction and nebulisation system ; Calculation. Prepare a calibration curve from the mean of
the readings obtained with the reference solutions by plotting
– a flame to generate the atoms to be determined ;
the means as a function of concentration. Determine the
– a monochromator ; concentration of the element in the test solution from the
– a detector ; curve obtained.

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

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