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2.1 INFRARED SPECTROPHOTOMETRY

APPENDIX 2 SPECTROSCOPY

      2.1 INFRARED SPECTROPHOTOMETRY

      Spectrophotometric measurements in the infrared region are used mainly as an identification test. The infrared spectrum is unique for any given chemical compound with the exception of optical isomers, which have identical spectra in solution. Polymorphism and other factors, such as variations in crystal size and orientation, the grinding procedure, and the possible formation of hydrates, may, however, be responsible for a difference in the infrared spectrum of a given compound in the solid state. The infrared spectrum is usually not greatly affected by the presence of small quantities of impurities in the tested substance. For identification purpose the spectrum may be compared with that of a reference substance, concurrently prepared or with a reference spectrum.

Apparatus

      Spectrophotometers suitable for use in the infrared region should operate in the range of 4000 to 625 cm–1 (2.5 to 16 μm). They should be checked frequently to ensure that they meet the standards of performance laid down by the manufacturer of the instrument, including the reliability of the wavelength scales, which should be checked by use of a polystyrene film.

      For the use of the attenuated total reflectance technique, the instrument should be equipped with a suitable attachment, which may be a single-reflection or a multi-reflection one. The attachment consists of a reflecting element and a suitable mounting permitting its alignment in the spectrophotometer for maximum transmission.

Use of Solvents

      The solvent used in infrared spectrophotometry must not affect the material, usually sodium chloride, of which the cell is made.

      No solvent in appreciable thickness is completely transparent throughout the infrared spectrum. Carbon tetrachloride is practically transparent (up to 1 mm in thickness) from 4000 to 1700 cm–1 (2.5 to 5.9 μm). Chloroformdichloromethane, and dibromomethane are other useful solvents. Carbon disulfide IR (up to 1 mm in thickness) is suitable as a solvent to 250 cm–1 (40 μm), except in the 2400 to 2000 cm–1 (4.2 to 5.0 μm) and the 1800 to 1300 cm–1 (5.6 to 7.7 -μm) regions, where it has strong absorption. Its weak absorption in the 875 to 845 cm–1 (11.4 to 11.8 μm) region should also be noted. Other solvents have relatively narrow regions of transparency.

Preparation of the Sample

      Substances are examined in one of the following forms, prepared as directed.

For measurement by transmission or absorption

      LIQUIDS Examine a liquid as a thin film held between two plates or in a cell of suitable path-length constructed of material transparent to infrared radiation in the region to be examined.

      LIQUIDS OR SOLIDS PREPARED AS SOLUTIONS Prepare a solution in a suitable solvent, and use a concentration and path-length to give a satisfactory spectrum over a sufficiently wide wavelength range. Absorption due to the solvent should be compensated for by placing in the reference beam a similar cell containing the solvent used; it should be noted that absorption bands due to the substance under examination that coincide with strong solvent absorption will not be recorded. Suitable concentrations of the solute will vary with the substance being examined but typical concentrations are 1 to 10 per cent at 0.5 to 0.1 mm path-length.

      SOLIDS Examine a solid after dispersion in a suitable liquid (mull) or solid (halide disc).

      Mull Triturate 1 to 5 mg of the substance with the minimum amount of liquid paraffin or other suitable liquid to give a smooth creamy paste. Compress a portion of the mull between two suitable plates.

      Disc Triturate about 1 mg of the substance with approximately 300 mg of dry, finely powdered potassium bromide IR or potassium chloride IR, as directed. These quantities are usually suitable for a disc 13 mm in diameter. Grind the mixture thoroughly, spread it uniformly in a suitable die and compress under vacuum at a high pressure. Commercial dies are available and the maker’s instructions should be followed. Mount the resultant disc in a suitable holder in the spectrophotometer. Several factors, such as inadequate or excessive grinding, moisture or other impurities in the halide carrier, may give rise to unsatisfactory discs. Unless its preparation presents particular difficulties, a disc should be rejected if visual inspection shows lack of uniformity or if the transmittance at about 2000 cm–1 (5 μm) in the absence of a specific absorption band is less than 75 per cent without compensation. If the other ingredients of tablets, injections, or other dosage forms are not completely removed from the substance being examined, they may contribute to the spectrum.

      GASES Examine gases in a cell transparent to infrared radiation and having an optical path length of about 100 mm. Evacuate 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 being examined. If necessary, adjust the pressure in the cell to atmospheric pressure using a gas transparent to infrared radiation (for example, nitrogen or argon). To avoid absorption interferences due to water, carbon dioxide or other atmospheric gases, place in the reference beam an identical cell that is either evacuated or filled with the gas transparent to infrared radiation.

For measurement by diffuse reflectance

      SOLIDS Triturate a mixture of the test substance with finely powdered and dried potassium bromide or potassium chloride. Use a mixture containing approximately 5 per cent of the substance, unless otherwise specified. Grind the mixture, place it in a sample cup and examine the reflectance spectrum. The spectrum of the sample in absorbance mode may be obtained after mathematical treatment of the spectra by the Kubelka-Munk function.

For measurement by attenuated total reflection

      Attenuated total reflection (including multiple reflection) involves light being reflected internally by a transmitting medium, typically for a number of reflections. However, several accessories exist where only one reflection occurs. Prepare the substance as follows. Place the test substance in close contact with an internal reflection element (IRE) such as diamond, germanium, zinc selenide, thallium bromide-thallium iodide (KRS-5) or another suitable material of high refractive index. Ensure close and uniform contact between the substance and the whole crystal surface of the internal reflection element, either by applying pressure or by dissolving the substance in an appropriate solvent, then covering the IRE with the obtained solution and evaporating to dryness. Examine the attenuated total reflectance (ATR) spectrum.

Identification by Reference Substances

      Prepare the substance being examined and the Reference Substance under the same operational conditions. Some substances are known to exhibit polymorphism which could lead to differences between the two spectra. In such cases, the sample and the reference substance are therefore to be pretreated. The pretreatment is designed to ensure that the substance being examined and the reference substance are isomorphous. Record the spectrum of each from about 4000 to 600 cm–1 (2.5 to 16.5 μm) using the same instrumental conditions as were used to demonstrate compliance with the requirement for resolution.

Identification by Reference Spectra

      In certain special cases, it may be necessary to use a reference spectrum. The spectrum should be scanned using the same instrumental conditions as were used to demonstrate compliance with the requirement for resolution. To allow for possible differences in wavelength calibration between the instrument on which the reference spectrum was obtained and that on which the spectrum of the substance is to be recorded, suitable reference absorbance maxima of a polystyrene spectrum are superimposed on the reference spectrum. These will normally occur at about 2851 cm–1 (3.51 μm), 1601 cm–1 (6.25 μm) and 1028 cm–1 (9.73 μm), but when there is interference with any of these maxima by a band in the spectrum of the substance being examined, alternative reference maxima will be specified. Similar reference maxima should be superimposed on the spectrum of the substance. With reference to these polystyrene maxima, the positions and relative intensities of the absorbance bands of the substance should be concordant with those of the reference spectrum. When comparing the two spectra, care should be taken to allow for the possibility of differences in resolving power between the instrument on which the reference spectrum was prepared and the instrument being used to examine the substance. A reference spectrum of a polystyrene film recorded on the same instrument as the reference spectrum of the substance is included in the compendium of reference spectra for assessing these differences. It should be noted that the greatest variations due to differences in resolving power are likely to occur in the region of 4000 to 2000 cm–1 (2.5 to 5 μm).

NEAR-INFRARED SPECTROPHOTOMETRY

      Near-infrared spectrophotometry is a technique particularly useful for identifying organic substances. Although the spectra are restricted to C−H, N−H, O−H and S−H resonances, they usually have a high informative content. However, the spectra depend on a number of parameters such as particle size, polymorphism, residual solvents, humidity which cannot always be controlled.

      For this reason, direct comparison of the spectrum obtained with the preparation of sample with the reference spectrum is usually impossible and some suitable validated mathematical treatment of the data is required.

Apparatus

      Spectrophotometers for recording spectra in the near-infrared region consist of:

      (1) a filter, grating or interferometer system capable of providing the whole range of electromagnetic radiation in the region of about 780 nm to about 2500 nm (12821 cm–1 to 4000 cm–1);

      (2) a means of collecting and measuring the intensity of the transmitted or reflected radiation (transmission or reflection), such as an integration sphere, a fibre optic probe, etc, coupled to an appropriate detector;

      (3) a means of mathematical treatment of the spectral data obtained.

      Preparation of the Sample

For measurement by transmission This method generally applies to liquids, diluted or undiluted, and to solids in solution. Examine the samples in a cell of suitable path-length (generally 0.5 mm to 4 mm), transparent to near-infrared radiation, or by immersion of a fibre optic probe of a suitable configuration, which yields a spectrum situated in a zone of transmittance compatible with the specifications of the apparatus and appropriate for the intended purpose. When recording the near-infrared spectrum of a liquid sample, the hazards of temperature dependent perturbations or any other effects of spectral disturbances must be taken into consideration. In all cases, compensation for background interferences must be made in a manner appropriate to the optical configuration of the apparatus, for example, a reference scan of air (for liquids) or solvent (for solutions) may be subtracted from the sample spectrum.

For measurement by diffuse reflection This method generally applies to solids.

      Examine the samples in a suitable device.

      When immersing a fibre optic probe in the sample, care must be taken in the positioning of the probe to ensure that it remains stationary during the acquisition of the spectra and that the measuring conditions are as reproducible as possible from one sample to another. In all cases, compensation for background interferences must be made in a manner appropriate to the optical configuration of the instrument, for example, a reference scan of an internal or external reflection standard must be subtracted from the sample spectrum. The particle size and the state of hydration or of solvation must also be taken into consideration.

For measurement by transflection This method generally applies to liquids, diluted or undiluted, and to solids in solution or in suspension. Examine the sample in a cell with a suitable diffuse reflector, made of either metal or of an inert substance (for example titanium oxide), not exhibiting a spectrum in the near-infrared region and introduced at a suitable concentration into the sample. The samples are examined as described above under For measurement by transmission or For measurement by diffuse reflection. 

Control of Instrument Performance

      Use the apparatus according to the manufacturer’s instructions and carry out the prescribed verifications at regular intervals, according to the use of the apparatus and the substances to be tested.

VERIFICATION OF THE WAVELENGTH SCALE (EXCEPT FOR FILTER APPARATUS) Verify the wavelength scale employed, generally in the region between 780 nm and 2500 nm using (a) suitable wavelength standard(s) which has characteristic maxima at the wavelengths under investigation, for example polystyrene or rareearth oxides.

VERIFICATION OF THE WAVELENGTH REPEATABILITY (EXCEPT FOR FILTER APPARATUS) Verify the wavelength repeatability using (a) suitable standard(s), for example polystyrene or rare-earth oxides. The standard deviation of the wavelengths is consistent with the spectrophotometer specification.

VERIFICATION OF RESPONSE REPEATABILITY Verify the response repeatability using (a) suitable standard(s), for example reflective thermoplastic resins doped with carbon black. The standard deviation of the maxima response is consistent with the spectrophotometer specification.

VERIFICATION OF PHOTOMETRIC NOISE Determine the photometric noise using a suitable reflectance standard, for example white reflective ceramic tiles or reflective thermoplastic resins. Scan the reflection standard in accordance with the spectrophotometer manufacturer’s recommendation and calculate the photometric noise, either peak to peak, or for a given wavelength. In the latter case, the photometric noise is represented by the standard deviation of the responses. The photometric noise is consistent with the spectrophotometer specification.

Establishment of a Spectral Reference Library

      Record the spectra of a suitable number of batches of the substance which have been fully tested as prescribed in the monograph and which exhibit the variation typical (e.g., manufacturer, particle size,...) of the substance being analyzed. The set of spectra represents the information that defines the similarity border for that substance and is the entry for that substance in the spectral database used to identify the substance. The number of substances in the database depends on the specific application.

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

      (1) individual spectra representing the substance;

      (2) mean spectra of each substance and a description of the variability.

      The selectivity of the database to identify positively a given material and discriminate adequately against other materials in the database is to be established during the validation procedure. This selectivity must be challenged on a regular basis to ensure ongoing validity of the database; this is especially necessary after any major change in a substance, for example: a change of supplier or in the manufacturing process of the material.

      This database is then valid for use only on the originating instrument or on a similar instrument provided the transferred database has been demonstrated to remain valid.

Method

      Prepare the sample being examined in the same manner as for the establishment of the database. A suitable mathematical transformation of the log (1/T) or log (1/R) spectrum may be calculated for both the sample and the spectral reference library, for example second derivative or multiplicative scatter correction, to facilitate spectral comparison.

      Comparison of the transforms of the sample and the spectral reference library involves the use of a suitable chemometric classification technique. 

APPENDICES • 2.1 INFRARED SPECTROPHOTOMETRY
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หมายเหตุ / Note : TP II 2011 PAGE 372-374