Learn the essential steps for conducting FTIR spectrophotometry identification tests. Our guide provides clear instructions for accurate results.

FTIR Spectrophotometry identification tests

1.0     Objective

• • The objective of this General Test Procedure (GTP) is to describe the procedure to be used for performing Identification by Infrared / FTIR spectrophotometry.

2.0     Principle/Reaction

• • Every bond or functional group in a sample needs a different frequency of infrared photons to be absorbed in order for a spectrum to be produced in this spectroscopy.

• • As a result, each functional group or molecular component exhibits a distinctive peak.

• • Since FTIR spectra are a molecule’s fingerprint, we may quickly identify a sample by comparing its spectra with standard spectra.

3.0     Requirements for FTIR spectrophotometry

• • FTIR spectrophotometry as per IP, BP & USP

• • KBr press and accessories

• • IR spectrophotometer

• • Liquid paraffin

• • NaCl

4.0     Procedure for Identification Test by FTIR Spectrophotometer 

• FTIR Spectrophotometry as per Indian Pharmacopoeia (IP)

  • Apparatus:

• • An infra-red spectrophotometer for recording the spectra in the infra-red region consists of an optical system capable of providing the monochromatic light in the region of 4000 to 625 cm-1 (about 2.5 to 16 µm) and the means of measuring the quotient of the intensity of the transmitted light and the incident light.

• • Fourier transform infra-red spectrophotometers (FTIR Spectrophotometry) are replacing the conventional dispersive instruments.

• • For recording infra-red spectra, the instrument should comply with the following test for resolution.

• • Preparation of sample:

• • A sample of the substance being examined may be prepared in one of the following ways.

• • For normal recording: 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 infra-red 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 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 being examined 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 % at 0.5 to 0.1 mm path-length.

• • Solids – Examine a solid after dispersion in a suitable liquid (mull) or solid (halide disc), as appropriate.

• • Mulls – 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.

• • Discs – 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 pressure of about 10Ton. Commercial dies are available and the manufacture’s instruction should be strictly 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.

• • 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 % 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.

• • For recording by multiple reflections:

• • When directed in the individual STP and specification, prepare the sample by one of the following methods.

• • Solutions – Dissolve the substance in the appropriate solvent under the conditions described in the individual STP and specification.

• • Evaporate the solution on a thallium bromo iodide plate or any other suitable plate.

• • Solids: Place the substance on a thallium bromo iodide plate or any other suitable plate in a manner giving uniform contact.

• • Identification by reference substances: unless otherwise directed in the individual STP and specification,

• • prepare the substance being examined and the reference substance in the form of discs dispersed in potassium bromide.

• • IR or potassium chloride IR and record the spectra between 4000 cm-1 and 625 cm-1 (2.5 µm to 16 µm) under the same operational conditions.
  • The absorption maxima in the spectrum obtained with the substance being examined correspond in position and relative intensity to those in the spectrum obtained with the reference substance in the same manner so that they crystallize or are produced in the same form or otherwise proceed as directed in the STP and specification, and then record the spectra.

• • Identification by reference spectra:

• • Unless otherwise directed in the individual STP and specification or on the reference spectrum prepare the substance as a disc in a dispersion of potassium bromide IR and record the spectrum from 2000 to 625 cm-1 (5 to 16µm); in some cases the spectrum should be scanned from 4000 cm-1 (2.5 µm).

• • The spectrum should be scanned using the same instrumental conditions as were used to ascertain compliance with the requirement for resolution.

• • To allow for possible differences in wavelength calibration between the instruments on which the reference spectrum was obtained and that on which the spectrum of the substance is being recorded, suitable reference absorption maxima of a polystyrene spectrum are superimposed on the reference spectrum or the specimen should be checked with the specimen of polystyrene.

• • These will normally occur at about 2851 cm-1 (3.51µm), 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 are marked on the reference spectrum.

• • Similar reference maxima should be superimposed on the spectrum of the substance.

• • With reference to these maxima of polystyrene, the position and relative intensities of the absorption bands of the substance should conform to 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 for assessing these differences.

• • The greatest variations due to differences in resolving power are likely to occur in the region between 4000 and 2000 cm-1 (2.5 to 5µm).

• • Reference spectra:

• • Suitable reference standard has been run and the spectrum is stored in software for overlay purpose.

• • In case reference standard is not available use reference working standard.

• • For standardization of spectra obtained by reference working standard, match the reference working standards spectra with reference spectra given in IP.

• FTIR spectrophotometry as per United States Pharmacopoeia (USP)

• • FTIR Spectrophotometric tests contribute meaningfully toward the identification of many chemical substances.

• • The test procedures that follow are applicable to substances that absorb infrared radiation.

• • The infrared absorption spectrum of a substance, compared with that obtained concomitantly for the corresponding USP reference standard or with working reference standard, provides perhaps the most conclusive evidence of the identity of the substance that can be realized from any single test.

• • Infrared Absorption –

• • Some methods are indicated for the preparation of previously dried test specimens and reference standard for analysis. These methods are;

• • • The substance under examination is mixed intimately with potassium bromide.

• • • The substance under examination is finely ground and dispersed in mineral oil.

• • • The substance under examination is suspended neat between suitable (for example, sodium chloride or potassium bromide) plates.

• • • A solution of designated concentration is prepared in the solvent specified in the individual STP and Specification.

• • • The solution is examined in 0.1-mm cells unless a different cell path length is specified in the individual STP and Specification.

• • • The substance under examination is intimately in contact with an internal reflection element for attenuated total reflectance (ATR) analysis.

• • • The substance under examination is pressed as a thin sample against a suitable plate for IR microscopic analysis.

• Sample preparation for above method is done as per the procedure given in IP for FTIR spectrophotometry.

  • Where testing is performed qualitatively and the reference standard spectra are similarly obtained.

• • Record the spectra of the test specimen and the corresponding USP reference standard over the range from about 5.0 µm to 16 µm (2000 to 625 cm-1) or where applicable scan between the range of 2.6 µm to 15µm (3800 cm-1 to 650 cm-1).

• • The infrared absorption spectrum of the preparation of the test specimen, previously dried under conditions specified for the corresponding reference standard unless otherwise specified, or unless the reference standard is to be used without drying, exhibits maxima only at the same wavelengths as that of a similar preparation of the corresponding USP reference standard.

• • Differences that may be observed in the spectra so obtained sometimes are attributing to the presence of polymorphs, which are not always acceptable unless otherwise directed in the individual STP and specification, therefore continue as follows.

• • If a difference appears in the infrared spectra of the analyte and the standard, dissolve equal portions of the test specimen and the reference standard in equal volumes of a suitable solvent, evaporate the solution to dryness in similar containers under identical conditions, and repeat the test on the residues.

• FTIR spectrophotometry As per British Pharmacopoeia (BP)/Ph.Eur.

  • Infrared spectrophotometers are used for recording spectra in the region of 4000 – 650 cm-1 (2.5 – 15.4 µm) or in some cases down to 200 cm-1 (50µm).

• • FTIR spectrophotometry Apparatus

  • Spectrophotometers for recording spectra consist of a suitable light source, monochromator or interferometer and detector.

• • Fourier transform spectrophotometers (FTIR spectrophotometry) use polychromatic radiation and calculate the spectrum in the frequency domain from the original data by Fourier transformation.

• • Spectrophotometers fitted with an optical system capable of producing monochromatic radiation in the measurement region may also be used.

• • Normally the spectrum is given as a function of transmittance, the quotient of the intensity of the transmitted radiation and the incident radiation.

• • It may also be given in absorbance.

• • The absorbance (A) is defined as the logarithm to base 10 of the reciprocal of the transmittance (T):

A = log₁₀ (1/T) = log₁₀ (I₀/I)

T = I/I₀

I₀ = intensity of incident radiation

I = intensity of transmitted radiation

• • Preparation of sample

• • For recording by transmission or absorption

• • Prepare the substance by one of the following methods.

• • Liquids

• • Examine a liquid either in the form of a film between 2 plates transparent to infrared radiation, or in a cell of suitable path length, also transparent to infrared radiation.

• • Liquids or solids in solution

• • Prepare a solution in a suitable solvent. Choose a concentration and a path length of the cell, which give a satisfactory spectrum.

• • Generally good results are obtained with concentrations of 10-100 g/l for a path length 0.5 – 0.1 mm. Absorption due to the solvent used.

• • If an FTIR spectrophotometry instrument is used, the absorption is compensated by recording the spectra for the solvent and the sample successively.

• • The solvent absorbance, corrected by a compensation factor, is subtracted using calculation software.

• • Solids

• • Examine solids dispersed in as suitable liquid (mull) or in a solid (halide disc), as appropriate.

• • If prescribed in the STP and specification, make a film of a molten mass between 2 plates transparent to infrared radiation.

1. • Mull

• • Triturate a small quantity of the substance to be examined with the minimum quantity of liquid paraffin R or other suitable liquid; 5-10 mg of the substance to be examined is usually sufficient to make an adequate mull using one drop of liquid paraffin R.

• • Compress the mull between 2 plates transparent to infrared radiation.

1. • Disc

• • Triturate 1-2 mg of the substance to be examined with 300-400mg, unless otherwise specified of finely powdered and dried potassium bromide R or potassium chloride R.

• • These quantities are usually sufficient to give a disc of 10-15 mm diameter and a spectrum of suitable intensity.

• • If the substance is a hydrochloride it is recommended to use potassium chloride R. carefully grind the mixture, spread it uniformly in a suitable die, and submit it to a pressure of about 10 Ton.

• • For substances that are unstable under normal atmospheric conditions or are hygroscopic, the disc is pressed in vacuo.

• • Several factors may cause the formation of faulty discs, such as insufficient or excessive grinding, humidity or other impurities in the dispersion medium or an insufficient reduction of particle size.

• • A disc is rejected if visual examination shows lack of uniform transparency or when transmittance at about 2000 cm^-1 (5µm) in the absence of a specific absorption band is less than 60 percent without compensation, unless otherwise prescribed.

• • 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 to be examined.

• • If necessary adjust the pressure in the cell to atmospheric pressure using a gas transparent to infrared radiation (for example nitrogen R and argon R).

• • To avoid absorption interferences due to water, carbon dioxide or other atmospheric gases, place in the reference beam, if possible an identical cell that is either evacuated or filled with the gas transparent to infrared radiation.

• • For recording by diffuse reflectance

• • Solids

• • Triturates a mixture of the substance to be examined with finely powdered and dried potassium bromide R or potassium chloride R.

• • Use a mixture containing approximately 5 % 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 recording by attenuated total reflection

• • Attenuated total reflection (including multiple reflections) 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 substance to be examined in close contact with an internal reelection 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 using reference substances

• • Prepare the substance to be examined and the reference substance by the same procedure and record the spectra between 4000-650 cm-1 (2.5- 15.4 µm) under the same operational conditions.

• • The transmission minima (absorption maxima) in the spectrum obtained with the substance to be examined correspond in position and relative size to those in the spectrum obtained with the reference substance (CRS).

• • When the spectra recorded in the solid state show differences in the positions of the transmission minima (absorption maxima), treat the substance to be examined and the reference substance in the same manner so that they crystalize or are produced in the same form, or proceed as prescribed in the STP and specification, then record the spectra.

• • Method prepares the substance to be examined according to the instructions accompanying the reference spectrum/reference substance.

• • Using the operating conditions that were to obtain the reference spectrum, which will usually be the same as those for verifying the resolution performance, record the spectrum of the substance to be examined.

• • The positions and the relative sizes of the bands in the spectrum of the substance to be examined and the reference spectrum are concordant in the 2 spectra.