14:00 29/12/2023 Appendix II A.

Infrared Spectrophotometry - British Pharmacopoeia

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Edition: BP 2023 (Ph. Eur. 11.2 update) View current BP

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Appendix II A. Infrared Spectrophotometry 

(Absorption Spectrophotometry, Infrared, Ph. Eur. method 2.2.24)

PRINCIPLE

Infrared absorption spectrophotometry (also known as infrared (IR) spectroscopy) is based on the
interaction of infrared radiation with matter. As a result of interaction between a molecule and IR
radiation, absorption of frequencies specific to that molecule can occur, and some intermolecular
and intramolecular vibrations can be excited to higher vibrational levels. This results in an infrared
absorption spectrum with characteristic bands that correspond to the functional groups of the
molecule.

The infrared wavelength region can be further divided into 3 subregions, namely near-infrared, mid-
infrared and far-infrared. These subregions have wavelength ranges that are generally accepted by
convention to be 0.8-2.5 µm, 2.5-25 µm and 25-1000 µm respectively. However, in IR spectroscopy,

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wavenumber is more commonly used than wavelength, and can be calculated using the following
equation:

1 4
ν̃ = ⋅ 10
λ

where ν̃ is the wavenumber in reciprocal centimetres (cm -1) and λ is the wavelength in micrometres.
-1 -1 -1
Thus 12 500-4000 cm is near-infrared, 4000-400 cm is mid-infrared and 400-10 cm is far-
infrared.

This chapter concerns only spectroscopy in the mid-infrared region, i.e. 4000-400 cm -1 (2.5-25 µm),
which hereafter is referred to as infrared for simplicity. This region is where the fundamental
molecular vibrations of functional groups appear in the spectrum as absorption bands. The region
-1
below 1500 cm is known as the ‘fingerprint region', a very complex and informative part of the
spectrum which characterises the molecule being investigated.

The mid-infrared region is flanked by the near-infrared region, where overtones and combinations of
fundamental vibrations, mainly C-H, N-H and O-H functional groups, are detected (2.2.40) and the
far-infrared region, where absorption bands associated with crystal lattice modes, hydrogen bonds,
angle deformation vibrations of heavy atoms and molecular rotations are observed.

APPLICATIONS

As the absorption bands in IR spectra are characteristic of the constituent functional groups of a
compound, IR spectroscopy is widely used to identify substances and provide information on their
structure. It can also be used for quantitative applications, which requires establishing a
mathematical relationship between the intensity of the radiation absorbed by the sample and the
concentration of the investigated component in the sample.

IR spectroscopy is widely used in the pharmaceutical field for chemical and physical analysis in the
laboratory, and has a wide variety of applications during the manufacturing process as outlined
below. IR spectroscopy thereby enables the application of Process Analytical Technology (PAT) as
part of an advanced control strategy.

Chemical analysis:

— identification of active substances, excipients, dosage forms, manufacturing intermediates,

chemicals and packaging materials;

— quality assessment of active substances, excipients, dosage forms, manufacturing

intermediates and packaging materials, including batch-to-batch spectral comparison and
supplier change assessment;

— quantification of active substances in a sample matrix, determination of water and solvent

content;

— quantification of impurities, e.g. in gases, inorganic materials;

— reaction monitoring, e.g. chemical synthesis.

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Physical analysis:

— determination of solid-state properties such as polymorphism.

LIMITATIONS

Notable limitations to the use of IR spectroscopy include the following:

— it may be necessary to use additional techniques to unambiguously identify a substance;

— pure enantiomers of a substance cannot be discriminated;

— it may not be a suitable method for trace analysis;

— sample preparation conditions (e.g. pressure, solvent) may change the crystalline form of a
substance that exhibits polymorphism;

— for heterogeneous samples, the limited sampling volume may be problematic.

MEASUREMENT MODES

IR measurements are based on passing radiation through or into a sample and measuring the
attenuation of the emerging beam at various wavelengths. This corresponds to 2 main measurement
modes, i.e. transmission and attenuated total reflection (ATR). However, other modes also exist for
specific applications (e.g. diffuse and specular reflection).

TRANSMISSION MODE

This mode is based on determination of the transmittance (T), namely the ability of the sample to
transmit IR radiation at a given wavelength (wavenumber). It is defined by the following ratio:

I
T =
I0

I0 = intensity of incident radiation;

I = intensity of transmitted radiation.

The resulting spectrum is presented in terms of transmittance (T) on the y-axis versus wavelength or
wavenumber on the x-axis. It can also be presented in terms of absorbance (A) on the y-axis, which
is related to transmittance (T) by the following equation:

1 I0
A = log10 ( ) = log10 ( ) = a ⋅ b ⋅ c
T I

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2
molar absorption coefficient of the sample, in square centimetres per mole (cm
a =
·mol -1);

b = sample thickness, in centimetres;

-3
c = sample concentration, in moles per cubic centimetre (mol·cm ).

ATTENUATED TOTAL REFLECTION MODE

ATR mode is based on the phenomenon of total internal reflection. The sample, with a refractive
index n2, is brought into close contact with a crystal (diamond, germanium, zinc selenide or any
other suitable material), having a refractive index n1 which is greater than n2. A beam of IR light is
then passed through the crystal. When the angle α between the incident beam and the sample-
crystal interface exceeds a critical value αc, theoretically all of the radiation is reflected (total internal
reflection). However, an evanescent wave is produced which slightly penetrates the sample and part
of the energy is absorbed. The total reflection is attenuated, which makes it possible to generate an
absorption spectrum. In practice, multiple internal reflections are often used to amplify the
absorption intensity, although some accessories allow absorption measurements with a single
reflection.

The penetration depth dp is usually of the order of a few micrometres and is given for a wavelength λ
by the following equation:

λ/n1
dp =
2 2
2π√ sin α−(n2/n1)

where dp is the penetration depth, λ is the wavelength, α is the angle of incidence and n1, n2 are the
refractive indices of the reflection element and the sample, respectively.

Due to the relationship between these parameters, the absorption intensity in ATR is greater at
higher wavelengths (i.e. smaller wavenumbers) and slight band shifts occur compared to the
corresponding transmission spectrum. It is therefore not advisable to compare ATR spectra with
transmission spectra when identifying compounds.

EQUIPMENT

The most commonly used IR spectrometers are Fourier-transform (FT-IR) spectrometers which
typically consist of:

— a suitable polychromatic light source, e.g. a conducting ceramic rod;

— an interferometer;

— a sample presentation accessory, e.g. a sample holder;

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— a detector;

— appropriate software for controlling the spectrometer, and for spectral evaluation and data
processing.

Other spectrometers based on alternative principles may also be used if the requirements described
under Control of equipment performance are fulfilled.

IR spectrometers can also be used in association with a microscope for the study of a small part of
the sample or for chemical imaging.

IR spectroscopy can be coupled to other analytical techniques such as thermal analysis or

chromatography.

CONTROL OF EQUIPMENT PERFORMANCE

Accuracy of wavenumber scale and spectral resolution are critical parameters and must be verified.
The tests described below can be used for the control of instrument performance and for
qualification. They can also be used as system suitability tests.

These parameters are checked using suitable reference materials which are selected and presented
depending on the measurement mode (e.g. transmission or ATR).

For quantitative analysis, appropriate assessment criteria for the control of absorption intensity must
also be defined.

WAVENUMBER SCALE

The wavenumber scale is typically verified using a polystyrene film that exhibits IR absorption bands
at the wavenumbers shown in Table 2.2.24.-1.

Table 2.2.24.-.1 - Band positions and associated acceptable tolerances of the polystyrene film used
to verify wavenumber accuracy

-1
Band position (cm ) -1
Tolerance (cm )
Transmission ATR
906.6 906.1 ± 1.0
1028.3 1027.7 ± 1.0
1601.2 1601.0 ± 1.0
3060.0 3059.7 ± 1.0

For measurement modes other than transmission or ATR, reference materials must be defined by
the user.

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SPECTRAL RESOLUTION

Figure 2.2.24.-1. – Typical IR absorbance spectrum of polystyrene used to verify spectral resolution

Spectra recorded in transmission mode

The spectral resolution is typically verified using a polystyrene film approximately 35 µm thick.

Acceptance criteria (see Figure 2.2.24.-1) The difference between the absorbance values at the
absorption minimum at 2870 cm -1 (A) and the absorption maximum at 2849.5 cm -1 (B) is greater
-1
than 0.33; the difference between the absorbance values at the absorption minimum at 1589 cm
-1
(C) and the absorption maximum at 1583 cm (D) is greater than 0.08.

Spectra recorded in ATR mode

Appropriate assessment criteria for the control of spectral resolution according to the specifications
of each instrument need to be defined.

For measurement modes other than transmission or ATR, reference materials have to be defined by
the user.

PROCEDURE

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SAMPLE PREPARATION AND PRESENTATION

Sample preparation and presentation vary according to the physical state of the sample and the
measurement mode.

Transmission mode is applied to transparent samples, such as neat liquids, solutions, gases or
suitably prepared mulls and alkali halide discs. For liquids and gases, cells with fixed or variable
pathlength and IR transparent windows can be used. For alkali-halide disks, specific sample holders
are used. Reflection mode, e.g. ATR, is appropriate for the measurement of a wide range of samples
in the solid and liquid state.

Some preparation modes (e.g. for discs and mulls in transmission mode or for solids in ATR mode)
involve grinding and/or the application of pressure, which may induce unexpected crystal
modifications.

Transmission mode

Prepare the substance by one of the following methods depending on the sample state (solid, liquid
or gas). Sample bands in a spectrum have a minimum transmittance not lower than 5 per cent,
unless otherwise justified.

Liquids Examine liquids either in the form of a film between 2 plates transparent to infrared
radiation or in a cell of suitable pathlength with windows that are transparent to infrared radiation.

Liquids or solids in solution Prepare a solution of the substance to be examined in a suitable

solvent. Choose a concentration and a pathlength that give a satisfactory spectrum. Generally, good
results are obtained with concentrations of 10-100 g/L for a pathlength of 0.5-0.1 mm. The
absorption due to the solvent is usually compensated by successively recording the spectra of the
solvent and the sample solution and subtracting the solvent absorption bands from the spectrum of
the sample solution.

Solids dispersed in a solid (disc) Grind the substance to be examined taking into consideration
any possible changes (e.g. crystalline form) and mix with a suitable amount of finely powdered and
dried potassium bromide R or potassium chloride R, unless otherwise specified. A mixture of a few
milligrams (e.g. 1-2 mg) of the substance to be examined in a few hundred milligrams (e.g. 300-
400 mg) of halide is normally sufficient to give a disc of 10-15 mm diameter and a spectrum of
suitable intensity. If the substance is a hydrochloride salt, it is recommended to use potassium
chloride R. Carefully grind the mixture, spread it uniformly in a suitable die and apply a suitable
pressure. A compacting force of about 800 MPa is generally sufficient to prepare a disc. For
substances that are unstable under normal atmospheric conditions or are hygroscopic, the disc may
be pressed under vacuum. Several factors may cause the formation of faulty discs, such as
insufficient or excessive grinding, humidity or impurities in the dispersion medium. For example, any
water in either the sample or the potassium bromide will cause clouding of the disc and produce a
low transmission spectrum. A disc is rejected if visual examination shows a lack of uniform
transparency or when, in the absence of a specific absorption band, the transmittance is less than
-1
60 per cent or the absorbance is more than 0.22 at about 2000 cm (5 µm) and without
compensation, unless otherwise prescribed.
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Solids dispersed in a liquid (mull) Triturate a small quantity of the substance to be examined
with the minimum quantity of liquid paraffin R or other suitable liquid. A mixture of a few milligrams
(e.g. 5-10 mg) of the substance to be examined in 1 drop of liquid paraffin R is generally sufficient to
make an adequate mull. Compress the mull between 2 plates transparent to infrared radiation. A
mull is rejected if a visual examination shows lack of uniform transparency or where the spectrum
shows features such as:
-1
— low transmission at 4000 cm ;
-1
— a strongly sloping baseline between 4000 and about 2500 cm ;

— a ratio of relative intensities of some absorption bands that is less than expected.

Molten solids If prescribed in the monograph, make a film of a molten mass and fix it on a suitable
mount.

Evaporated solution If prescribed in the monograph, dissolve the substance to be examined in a

suitable solvent. Prepare a film by evaporating the solvent on a suitable carrier and fix it on a
suitable mount.

Gases Use a suitable cell transparent to infrared radiation. Evacuate the air from the cell and fill to
the desired pressure through a stopcock or needle valve using a suitable gas transfer line between
the cell and the container of the gas to be examined. If necessary, adjust the pressure in the cell to
atmospheric pressure using a gas transparent to infrared radiation (e.g. nitrogen R or argon R), or
purge with carbon dioxide-free air. An appropriate measurement protocol must be followed to
compensate for water, carbon dioxide or other atmospheric gases.

ATR mode

ATR is suitable for liquid and solid samples, and requires no preparation apart from simple
treatments such as the grinding of large crystals and coarse material. Proceed as follows depending
on the sample state (liquid or solid).

Liquids Place the sample in contact with the crystal.

Solids Ensure close and uniform contact between the substance to be examined and the whole
crystal surface, either by applying pressure or by dissolving the substance in an appropriate solvent,
then covering the crystal with the resulting solution and evaporating to dryness.

METHODS

Infrared spectroscopy is mostly used to identify substances, but it may also be carried out for
quantitative applications. Quantitative analysis (based on the Beer-Lambert law, which relates the
absorbance of a sample to its concentration) will not be described in this chapter.

The measurement is performed on an appropriately prepared sample. The data is then processed
and evaluated, either to identify substances or quantify them (e.g. based on integration of IR-
absorption bands).

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Spectral quality may be enhanced by mathematical pretreatments. In practice, these are limited to
spectral normalisation and subtraction of bands caused by carbon-dioxide and water vapour. The
same pretreatments are performed on both the sample and the reference spectra.

Identification

Prepare the substance to be examined appropriately and record the spectra between 4000 and
-1
650 cm , unless otherwise prescribed.

Identification testing is performed by comparing the spectrum of the substance to be examined with
the spectrum obtained from a Ph. Eur. chemical reference substance (CRS) or with a Ph. Eur.
reference spectrum.

The spectrum of the current batch of the Ph. Eur. CRS may be recorded for immediate use or
stored, for example, in a spectral library for future consultation. A stored spectrum may be used,
provided traceability to the current batch of CRS is ensured.

In the case of substances that are not covered by individual monographs, a suitable reference
standard may be used.

In all cases, spectra must be recorded using the same operating conditions and procedure, and
especially the same measurement mode.

When comparison of the spectra recorded in the solid state show differences (see below), treat the
substance to be examined and the reference substance in the same manner so that they
recrystallise or are produced in the same crystalline form, or proceed as prescribed in the
monograph, then record the spectra again. However, this procedure must only be done for
substances where the monograph does not cover a particular form of a substance that exhibits
polymorphism.

Several comparison procedures may be used, and the analyst must document and justify the
method used and the specific acceptance criteria that allow a conclusion for identification. The
spectra can be compared either by overlaying the spectra (in the whole spectral range or in the
region of interest specified in the monograph) or by using mathematical calculations from the
software. It is possible for example to perform:

— visual comparison based on band positions and relative intensities unless otherwise specified
- the transmission minima (or absorption maxima) in the spectrum obtained with the substance
to be examined correspond in position and relative size to those of the reference;

— calculation of the correlation coefficient between the 2 spectra - this value is calculated by the
software and the identification threshold is defined by the user;

— evaluation by chemometric methods (e.g. Euclidean distance, Mahalanobis distance,

classification methods); these methods involve the set-up, assessment and validation of the
chemometric model by the analyst (see 5.21. Chemometric methods applied to analytical data).

Impurities in gases

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For the analysis of impurities, use a cell transparent to infrared radiation and of suitable optical
pathlength (e.g. 1-20 m). Fill the cell as prescribed under Gases. For detection and quantification of
the impurities, proceed as prescribed in the monograph.

Near-infrared Spectrophotometry
(Ph. Eur. method 2.2.40)

Near-infrared (NIR) spectroscopy is a technique with wide and varied applications in pharmaceutical
-1 -1
analysis. The NIR spectral range extends from 780 nm to 2500 nm (from 12 800 cm to 4000 cm ).
NIR spectra are dominated by C-H, N-H, O-H and S-H overtones and combinations of fundamental
mid-infrared (MIR) vibrations. They contain composite chemical and physical information and in
most cases this information can be extracted by suitable mathematical data treatment. NIR bands
are much weaker than the fundamental MIR vibrations from which they originate. Because
absorptivities in the NIR range are low, radiation can penetrate up to several millimetres into
materials, including solids. Furthermore, many materials such as glass are relatively transparent in
this region.

Measurements can be made directly in situ, in addition to standard sampling and testing procedures.
NIR measurements can be performed off-line, and also at-line or in-line, and on-line for process
analytical technology (PAT). Suitable chemometric methods may be required for identification.
However, when the specificity criteria for a qualitative method are met, chemical identification or
solid-state characterisation is possible by direct comparison of the untreated or pre-treated spectra
obtained with the chemical substance being examined with a spectrum of a reference substance.

NIR spectroscopy has a wide variety of applications for chemical, physical and process analysis, for
example:

Chemical analysis:

— identification of active substances, excipients, dosage forms, manufacturing intermediates,

chemical materials and packaging materials;

— qualification of active substances, excipients, dosage forms, manufacturing intermediates

and packaging materials, including batch-to-batch spectral comparison and supplier change
assessment;

— quantification of active substances in a sample matrix, determination of chemical values such

as hydroxyl value, determination of absolute water content, determination of degree of
hydroxylation and control of solvent content.

Physical analysis:

— crystalline form and crystallinity, polymorphism, solvates, particle size;

— disintegration, hardness;

— film properties.

Process analysis:

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— monitoring of unit operations such as synthesis, crystallisation, blending, drying, granulation

and coating, for the purpose of process control;

— control and endpoint detection.

Measurements in the NIR region are influenced by many chemical and physical factors as described
below; the reproducibility and relevance of results depend on control of these factors and
measurements are usually valid only for a defined calibration model.

APPARATUS

All NIR measurements are based on passing light through or into a sample and measuring the
attenuation of the emerging (transmitted or reflected) beam. Spectrometers for measurement in the
NIR region consist of a suitable light source (such as a highly-stable quartz-tungsten lamp), a
monochromator or interferometer, and a detector. Common monochromators are acousto-optic
tunable filters (AOTF), gratings or prisms. Traditionally, many NIR instruments have a single-beam
design, though some process instruments use internal referencing and can therefore be dual-beam
(for example in diode array instruments). Silicon, lead sulfide, and indium gallium arsenide are
examples of detector materials. Conventional cuvette sample holders, fibre-optic probes,
transmission dip cells, neutral borosilicate vials and spinning or traversing sample holders are a few
examples of sampling devices. The selection is based on the intended application, paying particular
attention to the suitability of the sampling system for the type of sample to be analysed. Suitable
data processing and evaluation units (e.g. software and computer) are usually part of the system.

It is common to express the wavelength (λ) in nanometres and the wavenumber (v) in reciprocal
centimetres (cm -1), depending on the measurement technique and apparatus. Conversion
between nm and cm -1 is performed according to the following expression:

7 1
vcm−1 = 10 ×
λnm

MEASUREMENT METHODS

Transmission mode

Transmittance (T) is a measure of the decrease in radiation intensity at given wavelengths when
radiation is passed through the sample. The sample is placed in the optical beam between the
source and the detector. The arrangement is analogous to that in many conventional spectrometers.
The resulting spectrum can be presented directly in terms of transmittance (T) and/or
absorbance (A) (y-axis) versus the wavelength or wavenumber (x-axis).

I
T =
I0

I0 = intensity of incident radiation;

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I = intensity of transmitted radiation;

1 I0
A = −log10T = log10 ( ) = log10 ( )
T I

Diffuse reflection mode

The diffuse reflection mode gives a measure of reflectance (R), the ratio of the intensity of light
reflected from the sample (I) to that reflected from a background or reference reflective surface (Ir).
Depending on the chemical composition and physical characteristics of the sample, NIR radiation
can penetrate a more or less substantial distance into the sample, where it can be absorbed by the
overtones and combinations of the fundamental vibrations of the analyte species present in the
sample. Non-absorbed radiation is partially reflected back from the sample to the detector. NIR
reflectance spectra are typically obtained by calculating and plotting log10 (1/R) (y-axis) versus the
wavelength or wavenumber (x-axis).

I
R =
Ir

I = intensity of light diffusively reflected from the sample;

Ir = intensity of light reflected from the background or reference reflective surface;

1 Ir
AR = log ( ) = log ( )
10 R 10 I

Transflection mode

This mode is a combination of transmittance and reflectance. In the measurement of

transflectance (T*), a mirror or a diffuse reflectance surface is used to reflect the transmitted
radiation back through the sample, thus doubling the pathlength. Non-absorbed radiation is reflected
back from the sample to the detector. The resulting spectrum can be presented directly in terms of
transflectance (T*) and/or absorbance (A*) (y-axis) versus the wavelength or wavenumber (x-axis).

* I
T =
IT

I = intensity of transflected radiation measured from the sample;

IT = intensity of transflected radiation of the reference material as background;

* 1 IT
A = log ( )= log ( )
10 T
* 10 I

SAMPLE PREPARATION/PRESENTATION

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Sample preparation and presentation may vary according to the measurement mode. The following
requirements are necessary for all sampling techniques:

— optimise the measuring time and number of scans to optimise the signal-to-noise ratio;

— find the best suitable measurement mode for the intended application (transmission, diffuse
reflection or transflection);

— find the best orientation of the sample (e.g. to minimise the impact of debossing on tablets);

— find the best suitable accessory (e.g. transmission cell or immersion probe);

— optimise pathlength in transmission and transflection modes;

— find a suitable spectroscopic background reference material;

— show that the background reference material is reliable over time and that the measurement
of the background is reproducible and stable over time;

— when measuring moving materials or samples (for process-related measurements) it is

important to obtain a representative spectrum (e.g. by adjusting the measuring time, the number
of scans, co-adding individual spectra, or increasing the beam size);

— ensure there is no fouling of the sensor, for example with build-up of material or
contamination;

— the measuring conditions (measuring time, beam size) in relation to the minimal sample size
should be justified.

In some process analysis situations it may be impossible to remove a probe for reference
background data collection; various options are therefore to be considered, including internal
nd
referencing, measurement of a background reference using a 2 detector, etc. Only spectra
measured against a background possessing the same optical properties can be directly compared
with one another.

Transmission mode

The measurement of transmittance (T) is dependent on a background transmittance spectrum for its
calculation. Examples of background references include air, a polymeric disc, an empty cell, a
solvent blank or in special cases a reference sample. The method generally applies to liquids
(diluted or undiluted), dispersions, solutions and solids (including tablets and capsules). For
transmittance measurements of solids, a suitable sample accessory is used. Liquid samples are
examined in a cell of suitable pathlength (typically 0.5-4 mm), transparent to NIR radiation, or
alternatively by immersion of a fibre-optic probe of a suitable configuration.

Diffuse reflection mode

This mode generally applies to solids. The sample is examined directly, or in a suitable device (for
example a sample holder), or in direct contact with a fibre-optic probe. For process monitoring,
material can be analysed through a polished window interface (e.g. sapphire), or using an in-line
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fibre-optic probe. Care must be taken to ensure that the measuring conditions are as reproducible
as possible from one sample to another. The reflected radiation of a background reference is
scanned to obtain the baseline, and then the reflectance of one or more analytical samples is
measured. Common reflectance references include ceramic, thermoplastic resins and gold. Other
suitable materials may be used.

Transflection mode

This mode generally applies to liquids, suspensions and clear plastic materials. A reflector is placed
behind the sample so as to double the pathlength. This configuration can be adopted to share the
same instrument geometry with reflectance and fibre-optic probe systems where the source and the
detector are on the same side of the sample. The sample is examined through a cell with a mirror or
a suitable diffusive reflector, made either of metal or of an inert substance (for example, dried
titanium dioxide) not absorbing in the NIR region. Liquids can also be measured using in-line
transflectance probes.

FACTORS AFFECTING SPECTRAL RESPONSE

Environment

The environment temperature and humidity must be taken into consideration before carrying out
measurements.

Sample presentation area

The sample presentation area or probe end must be clean of residue prior to measurement.
Similarly, the in-line or on-line interface to the sample should not have significant product or
contamination build-up, which would interfere with the desired measurement.

Sample temperature

This parameter is important for aqueous solutions and many liquids, where a difference of a few
degrees can result in measurable spectral changes which may have a significant effect on the
analysis. Temperature is also an important parameter for solids and powders containing water.

Moisture and solvent residues

Moisture and solvent residues present in the samples will contribute significant absorption bands in
the NIR region.

Sample thickness

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Sample thickness is a known source of spectral variability and must be assessed and/or controlled,
particularly for tablet and capsule analysis in transmittance mode. For the measurement of
compressed powders, an infinite thickness is typically reached after 5 mm of sample depth (e.g. in a
vial).

Sample optical properties

In solids, both surface and bulk scattering properties of samples must be taken into account.
Spectra of physically, chemically or optically heterogeneous samples may require increasing the
beam size, or examining multiple samples or spinning the sample to obtain a representative
spectrum of the sample. Certain factors such as differing degree of compaction or particle size in
powdered materials and surface finish can cause significant spectral differences.

Solid-state forms

Variations in solid-state forms (polymorphs, hydrates, solvates and amorphous forms) influence
vibrational spectra. Hence, different crystalline forms as well as the amorphous form of a solid may
be distinguished from one another on the basis of their NIR spectra. Where multiple crystalline forms
are present, care must be taken to ensure that the calibration samples have a distribution of forms
relevant to the intended application.

Age of samples

Samples may exhibit changes in their chemical, physical or optical properties over time. Depending
on the storage conditions, solid samples may either absorb or desorb water, and portions of
amorphous material may crystallise. Materials used for NIR calibration should be representative of
future samples and their matrix variables.

PRE-TREATMENT OF NIR SPECTRAL DATA

In many cases, and particularly for reflection mode spectra, some form of mathematical pre-
treatment of the spectrum may be useful prior to the development of a classification or calibration
model. The aim can be, for example, to reduce baseline variations, to reduce the impact of known
variations that are interfering in the subsequent mathematical models, or to simplify data before use.
In some cases spectra may also be normalised or corrected for scatter, for example using standard
normal variate (SNV) transformation. Spectral pre-treatment techniques may include, for example,
windowing and noise reduction and the numerical calculation of the first- or second-order derivative
of the spectrum. Higher-order derivatives are not recommended because of increased spectral
noise.

CONTROL OF INSTRUMENT PERFORMANCE

Use the apparatus according to the manufacturer’s instructions and carry out the prescribed
verification at regular intervals, according to the use of the apparatus and the application. For in-line
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and on-line applications, the use of alternative means of control of instrument performance must be
scientifically justified. For example, utilise the standards built into the instrument or separate
channels/probes to demonstrate instrument performance (pending practicality).

System suitability tests may be required prior to sample scanning, and the instrument attributes with
potential impact on suitability of the final measurement (typically photometric noise and wavelength
accuracy) must be tested. The frequency at which each performance test is carried out must be risk-
assessed depending on the instrument type and its environment. For example, instruments placed
in harsh environments with variations in temperature and humidity may need frequent performance
testing. Cases where the measurement system cannot be removed such as an in-line probe or flow
cell are also to be considered.

Some accessories are custom designed and therefore require adequate performance testing.

Verification and calibration of the wavelength or wavenumber scale (except for filter
apparatus) Verify the wavelength scale employed, generally in the region between 780 nm and
-1 -1
2500 nm ( 12 800 cm to 4000 cm ) or in the intended spectral range using one or more suitable
wavelength standards which have characteristic maxima or minima within the wavelength range to
be used. For example, methylene chloride R, talc R, wavelength reference lamps or a mixture of
rare-earth oxides are suitable reference materials. Other suitable standards may also be used.
Obtain a spectrum and measure the position of at least 3 peaks distributed over the range used. For
rare-earth oxides, the National Institute of Standards and Technology (NIST) provides suitable
reference materials. Fourier transform (FT) instruments have a linear frequency range, therefore
wavelength certification at a single frequency is sufficient.

Verification and calibration of photometric linearity The photometric linearity is demonstrated

with a set of transmittance or reflectance standards with known percentage values of transmittance
or reflectance. For reflectance measurements, carbon-doped polymer standards are available.
Ensure that the absorbance of the materials used is relevant to the intended linear working range of
the method. Subsequent verifications of photometric linearity can use the initial observed
absorbance values as the reference values. Non-linear calibration models and hence non-linear
responses are acceptable with understanding demonstrated by the user.

Spectra obtained from reflectance and transmittance standards are subject to variability due to the
differences between the experimental conditions under which they were factory-calibrated and those
under which they are subsequently put to use. Hence, the percentage reflectance values supplied
with a set of calibration standards may not be useful in the attempt to establish an ‘absolute’
calibration for a given instrument. As long as the standards do not change chemically or physically
and the same reference background is used as was used to obtain the certified values, subsequent
measurements of the same standards under identical conditions, including precise sample
positioning, give information on long-term stability of the photometric response. A tolerance of
± 2 per cent of the absorbance value is acceptable for long-term stability; this verification is only
necessary if the spectra are used without pre-treatment.

Recommendations for the conditions used to control instrument performance for the various
measurement modes are summarised in Table 2.2.40.-1.

Table 2.2.40.-1 – Control of instrument performance

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Measurement Reflection Transflection Transmission

mode
Verification of Typical tolerances for agreement with standard values are:
-1 -1
wavelength ± 1.0 nm at 780 nm (± 16 cm at 12 800 cm );
-1 -1
scale (except ± 1.0 nm at 1200 nm (± 8 cm at 8300 cm );
for filter -1
± 1.0 nm at 1600 nm (± 6 cm at 6250 cm );
-1

apparatus) -1
± 1.5 nm at 2000 nm (± 4 cm at 5000 cm );
-1

± 1.5 nm at 2500 nm (± 2 cm -1 at 4000 cm -1).

For the reference material used, apply the tolerance for the nearest wavelength or
wavenumber for each peak used. For diode array instruments, most often the
pixel resolution (wavelength between pixels) can be as large as 10 nm. The pixel
resolution must be adapted to match the spectral resolution. The peak-finding
algorithms are critical to wavelength accuracy. Practically, ± 2 nm is appropriate
for peak wavelength accuracy using such instrumentation. Alternatively, refer to
manufacturer’s specifications for acceptance.
Bench/mobile Measure talc R via a A suspension of 1.2 g of Methylene chloride R may
instrument suitable medium or by dry titanium dioxide R in be used and has
fibre-optic probe. Talc R about 4 mL of methylene characteristic sharp bands
has characteristic peaks chloride R is used at 1155 nm, 1366 nm,
at 948 nm, 1391 nm and directly with a cell or a 1417 nm, 1690 nm,
2312 nm suitable for probe. Titanium dioxide 1838 nm, 1894 nm,
calibration. has no absorption in the 2068 nm and 2245 nm.
Alternatively, other NIR range. Spectra are Choose 3 peaks across
suitable standards may recorded with a the wavelength range for
also be used that ensure maximum nominal calibration. Other suitable
wavelength accuracy in instrument bandwidth of standards may also be
the region of working 10 nm at 2500 nm used.
-1 -1
methodology. For (16 cm at 4000 cm ).
example, measure an Methylene chloride has
internal polystyrene characteristic sharp
standard if built-in, or bands at 1155 nm,
measure a NIST standard 1366 nm, 1417 nm,
or other traceable 1690 nm, 1838 nm,
material, and assess 1894 nm, 2068 nm and
3 peaks across the 2245 nm. Choose
wavelength range for 3 peaks across the
calibration. wavelength range for
calibration. Other
suitable standards may
also be used, such as a
liquid transflection
standard mixed with
titanium dioxide or some
other reflective medium.
Process If it is not practically possible to measure a traceable reference material at the
instrument point of sample measurement, use internal material such as polystyrene,

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Measurement Reflection Transflection Transmission

mode
fibreglass or solvent and/or water vapour. Alternatively, adopt a second external
channel/probe.
For FT instruments, the calibration of the wavenumber scale may be performed
-1
using a narrow, isolated water-vapour line, for example, the line at 7306.74 cm ,
-1 -1
or 7299.45 cm , or 7299.81 cm or a narrow line from a certified reference
material.

Verification of The standard deviation of the wavelength is consistent with the specifications of
wavelength the instrument manufacturer, or otherwise scientifically justified.
repeatability
(except for
filter
apparatus)
Bench/mobile Verify the wavelength repeatability using a suitable external or internal standard.
instrument
Process Verify the wavelength repeatability using a suitable external or internal standard.
instrument

Verification of Measure 4 photometric standards across the working method absorbance range.
photometric
linearity and
response
(1)
stability

Bench/mobile Analyse 4 reflectance Transflection Analyse 4 transmittance

instrument standards, for example in measurements can use standards to cover the
the range of 10-99 per appropriate reflectance absorbance values over
cent, including 10 per or transmittance the working absorbance
cent, 20 per cent, 40 per standards and criteria. range of the modelled
cent and 80 per cent. In data. Evaluate the
some circumstances observed absorbance
2 per cent may be values against the
appropriate. Evaluate the reference absorbance
observed absorbance values, for example
values against the perform a linear
reference absorbance regression. Acceptable
values, for example tolerances are 1.00 ± 0.05
perform a linear for the slope and
regression. Acceptable 0.00 ± 0.05 for the
st
tolerances are 1.00 ± 0.05 intercept for the 1
for the slope and verification of photometric
0.00 ± 0.05 for the linearity of an instrument.
st
intercept for the 1 Subsequent verifications of
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Measurement Reflection Transflection Transmission

mode
verification of photometric photometric linearity can
linearity of an instrument. use the initial observed
Subsequent verifications absorbance values as the
of photometric linearity reference values.
can use the initial
observed absorbance
values as the reference
values.
Process If photometric reflectance and transmittance standards cannot be measured at
instrument the point of sample measurement, use the photometric standards built into the
instrument.
Process instruments can use internal photometric standards for photometric
linearity. Follow the manufacturer’s verified tolerances in such cases.

Verification of Determine the photometric noise at a relevant photometric region of the spectrum
photometric using a suitable reflectance standard, for example, white reflective ceramic tiles
(1)
noise or carbon-doped polymer standards. Follow the manufacturer’s methodology and
specifications.
Bench/mobile Scan the reflectance low flux standard (e.g. 5 or Scan the transmittance
instrument 10 per cent, carbon-doped polymer standard) over a high flux standard (e.g. 90
suitable wavelength range in accordance with the or 99 per cent, carbon-
manufacturer’s recommendation and calculate the doped polymer standard)
photometric noise as peak-to-peak noise. over a suitable
wavelength/wavenumber
range in accordance with
the manufacturer’s
recommendation and
calculate the photometric
noise as peak-to-peak
noise.
Process As above, or if not practically possible, use the As above, or if not
instrument standard built into the instrument for noise testing practically possible, use
and manufacturer specifications. the standard built into the
instrument for noise testing
and manufacturer
specifications.
(1)
Verification of photometric linearity and Verification of photometric noise are not required for
instruments using methods to perform simple identifications which do not use the photometric
absorbances as part of model strategy (for example, simple correlation with absorbing
wavelengths).

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QUALITATIVE ANALYSIS (IDENTIFICATION AND CHARACTERISATION)

Establishment of a spectral reference library

Record the spectra of a suitable number of representative samples of the substance which have
known, traceable identities, and that exhibit the variation typical for the substance to be analysed
(for example, solid-state form, particle size, etc.). Libraries are built using representative samples
under appropriate environmental conditions. The set of spectra obtained represents the information
which can be used for identification of the sample to be analysed.

The collection of spectra in the library may be represented in different ways defined by the
mathematical technique used for identification. These may be:

— all individual spectra representing the substance;

— a mean spectrum of the measured batches for each chemical substance;

— if necessary, a description of the variability within the substance spectra.

The number of substances in the library depends on the specific application. All spectra in the library
used have the same:

— spectral range and number of data points;

— technique of measurement;

— data pre-treatment.

If sub-groups (sub-libraries) are created, the above criteria are applied independently for each
group. Sub-libraries are individually validated. Original spectral data for the preparation of the
spectral library must be archived. Caution must be exercised when performing any mathematical
transformation, as artefacts can be introduced or essential information (important with qualification
methods) can be lost. The suitability of the algorithm used should be demonstrated by successful
method validation and in all cases the rationale for the use of transform must be documented.

Direct comparison of substance and reference spectra

Direct comparison of representative spectra of the substance to be examined and of a reference

substance for qualitative chemical or physical identification purposes may not require use of a
reference spectral library where specificity permits.

Data evaluation

Direct comparison of the representative spectrum of the substance to be examined is made with the
individual or mean reference spectra of all substances in the database on the basis of their
mathematical correlation or other suitable algorithms. A set of known reference mean spectra and
the variability around this mean can be used with an algorithm for classification; alternatively, this
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can be achieved visually by overlaying spectral data if specificity is inherent. There are different
techniques available, such as principal component analysis (PCA), cluster analysis, and soft
independent modelling by class analogy (SIMCA). The reliability of the technique chosen for a
particular application has to be validated according to the following:

Validation of the model

Identification methods using direct spectral comparison must be validated in accordance with
identification method validation procedures.

The validation parameters for qualitative methods are robustness and specificity.

LIMIT ANALYSIS

Relative comparison of spectra

A calibration is not required when comparing a set of spectra for limit analysis purposes, such as the
maximum or minimum absorbance at which an analyte absorbs. Also, dryer end point control may
use a qualitative approach around a specific absorbing wavelength. Appropriate spectral ranges and
pre-treatments (if used) must be shown to be fit for purpose.

Specificity

The relative discriminatory power for a limit test must be demonstrated. The extent of specificity
testing is dependent on the application and the risks being controlled. Variations in matrix
concentrations within the operating range of the method must not affect the measurement.

TREND ANALYSIS

Relative comparison of spectra

A calibration is not necessarily required when comparing a set of spectra for trend analysis
purposes, such as the moving block approach to estimate statistical parameters such as mean,
median and standard deviation. For example, blend uniformity monitoring using NIR spectroscopy
has adopted such data analysis approaches. Appropriate spectral ranges and algorithms must be
used for trend analyses.

Specificity

The relative discriminatory power for trend analysis must be demonstrated. The extent of specificity
testing is dependent on the application and the risks being controlled. Variations in matrix
concentrations within the operating range of the method must not affect the trend analysis.

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QUANTITATIVE ANALYSIS

Establishment of a spectral reference library for a calibration model

Calibration is the process of constructing a mathematical model to relate the response from a
sample scanned using an analytical instrument to the properties of the samples. Any calibration
model that can be clearly defined in a mathematical expression and gives suitable results can be
used. Record the spectra of a suitable number of representative samples with known or future-
established values of the attribute of interest throughout the range to be measured (for example,
content of water). The number of samples for calibration will depend on the complexity of the sample
matrix and interferences (e.g. temperature, particle size, etc.). All samples must give quantitative
results within a calibration interval as defined by the intended purpose of the method. Multiple linear
regression (MLR), principal component regression (PCR) and partial least squares regression (PLS)
are commonly used algorithms. For PLS or PCR calibrations, the regression coefficients and/or the
loadings should be plotted and the regions of large coefficients or loadings compared with the
spectrum of the analyte. Predicted residual error sum of squares (PRESS) plots (or similar) are
useful to facilitate the optimising of the number of PCR or PLS factors.

Pre-treatment of data

Wavelength selection or excluding certain wavelength ranges may enhance the accuracy and
robustness of calibration models. Wavelength compression (wavelength averaging) techniques may
be applied to the data.

Model validation parameters

Analytical performance characteristics to be considered for demonstrating the validation of NIR

methods are similar to those required for any analytical procedure. Specific acceptance criteria for
each validation parameter must be consistent with the intended use of the method. Validation
parameters for quantitative methods are accuracy, linearity, precision (repeatability and intermediate
precision), robustness and specificity.

ONGOING MODEL EVALUATION

NIR models validated for use are subjected to ongoing performance evaluation and monitoring of
validation parameters.

TRANSFER OF DATABASES

When databases are transferred to another instrument, spectral range, number of data points,
spectral resolution and other parameters have to be taken into consideration. Further procedures
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and criteria must be applied to demonstrate that the model remains valid with the new database or
new instrument.

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