4- INFRARED (IR) SPECTROSCOPY

PRINCIPLE OF MID-IR SPECTROSCOPY

• Mid-infrared (mid-IR) spectroscopy involves measurement of the absorption of electromagnetic radiation over the wavenumber range of 4000-400 cm^-1 (which corresponds to the wavelength range of 2.5-25 μm) caused by the promotion of molecules from the ground state of their vibrational modes to an excited vibrational state 

Mid-infrared (mid-IR) spectroscopy involves measurement of the

absorption of electromagnetic radiation over the wavenumber range of 4000-400 cm^-1 (which corresponds to the wavelength range of 2.5-25 μm) caused by the promotion of molecules from the ground state of their vibrational modes to an excited vibrational state 

Different ways bonds would stretch or bend upon absorption of IR light

• symmetric stretching

• asymmetric stretching

• scissoring

• rocking

• wagging 

• twisting

STRETCHING

• symmetric stretching

• asymmetric stretching

BENDING

• scissoring

• rocking

• wagging 

• twisting

wavenumber

• number of waves per centimeter

• commonly used parameter to denote the energy of the transitions

the transition of certain functional group often occur in

narrow spectral ranges

Group frequencies:

The wavenumber at which these transitions (of certain functional groups) occur

Fingerprint Bands

• vibrations of the atoms in a molecule 

• occur over wider spectral ranges

• not characteristic of a particular functional group

• more characteristic of the molecules as a whole

Group Frequencies

• strong bands that absorb at wavenumbers above 1500 cm⁻¹

Either Group Frequencies or FIngerprint Bands

• strong bands that absorb below 1500 cm⁻¹

NIR

literally “near” Visible light

Mid IR:

4000-400 cm^-1 OR 2500-25000 nm

Far IR, Microwave

Far from visible light 

X-axis

is wave number;

Y-axis

is percent transmittance

↑ transmittance

= ↓ absorbed light

↓ transmittance

= ↑  absorbed light

Peaks (Inverted)

• low transmittance and high absorbance

area where there are peaks

= there are molecules or bonds that absorbed light at that frequency

Peak Height

• depends on the amount of light absorbed 

Peak Width

• influenced by the bond type or FG

Fingerprint bands can be seen at

500 - 1500 cm⁻¹

Group frequencies (or FGs) can be seen at

1500 - 4000 cm⁻¹

Group frequencies can be found in the fingerprint region, but

it would be difficult to identify.

SAMPLING PROCEDURES

1. Preparation of Alkali Halide

2. Preparation of Mulls

3. Preparation of Compression Cells

4. Preparation of Self-Supported Polymer Films

5. Preparation of Capillary Films

6. Liquids and Solutions in Transmission Cells

7. Gases

MODERN SAMPLING PROCEDURES /  INSTRUMENTS:

1. Attenuated Total Reflection (ATR) Spectroscopy

2. External Reflection Spectroscopy

3. Diffuse Reflection

 Preparation of Alkali Halide

• Alkali halide used is powdered, highly pure KBr, which is transparent to mid-IR radiation to approximately 400 cm −1 - referred to as KBr disks.

• Faulty, unsatisfactory, or poor-quality disks may be a consequence of inadequate or excessive grinding, moisture/humidity, or impurities (potassium nitrate) in the dispersion medium.

Faulty, unsatisfactory, or poor-quality disks may be a consequence of

inadequate or excessive grinding, moisture/humidity, or impurities (potassium nitrate) in the dispersion medium.

Alkali halide used is _

powdered,

KBr disks

• highly pure KBr

• transparent to mid-IR radiation to approximately 400 cm −1

Potassium bromide (KBr) is a good base for the sample; due to its transparency,

it won’t give a reading in the mid-IR region.

Preparation as KBr disks

used in pharmacopeial methods (official method)

Preparation as KBr disks procedures

1. Grind sample in KBr and transfer to die

2. Compress powder at 800 KPa under vacuum

3. The resulting KBr disc contains the dispersed sample

Preparation of Mulls

• Finely divided powder sample homogeneously distributed in a thin layer of a viscous liquid that is semi-transparent to mid-IR radiation and has a refractive index closely matched to that of the sample. 

• The mull should have the consistency of a paste.

• Most widely used mulling agent is saturated hydrocarbon mineral oil - AKA liquid paraffin, Nujol (brand name)

Finely divided powder sample homogeneously distributed in a thin layer of a viscous liquid that is

semi-transparent to mid-IR radiation and has a refractive index closely matched to that of the sample. 

viscous liquid:

SHOULD NOT dissolve the powder, but instead, disperse it to form a paste (final sample)

The mull should have the consistency of a

paste.

Most widely used mulling agent is

saturated hydrocarbon mineral oil - AKA liquid paraffin, Nujol (brand name)

Preparation of Mulls procedure

1. Grind sample in liquid paraffin

2. Sandwich between two NaCl discs

Preparation of Compression Cells

• used for small or limited-quantity solid sample e.g.:

  • a single particle of an API or excipient

  • a contaminant such as a short length of fiber, or 

  • a small fragment from a packaging material

• sample is placed between the diamond windows of the cell to compress the limited-quantity sample

Preparation of Compression Cells

used for 

• small or limited-quantity solid sample e.g.:

  • a single particle of an API or excipient

  • a contaminant such as a short length of fiber, or 

  • a small fragment from a packaging material

Preparation of Compression Cells
sample is placed between 

• diamond windows of the cell to compress the limited-quantity sample 

Preparation of Self-Supported Polymer Films

• polymers

• soft and low melting solids 

POLYMERS used as packaging materials can be recorded from

samples prepared as thin self-supporting films

Polymers (or plastic) used as packaging materials can be recorded from samples prepared as thin self-supporting films

• Hot compression or microtoming (slicing thinly to make a film) a thin section from a sample

soft and low-melting solids that do not crystallize when cooled can be prepared 

• either as a thin layer sandwiched between two mid-IR–transparent windows by gently warming the sample or from the melt

• thin films from some materials can be cast from solution

thin films from some materials can be

cast from solution

Preparation of Capillary Films

• nonvolatile liquids (prepared in a capillary film) can be examined neat in the form of a thin layer sandwiched between two matching windows that are transparent to IR radiation

nonvolatile liquids (prepared in a capillary film) can be examined neat in the form of a

thin layer sandwiched between two matching windows that are transparent to IR radiation

Liquids and Solutions in Transmission Cells

• transmission cell assemblies comprise a pair of windows constructed of mid-IR–transparent materials such as 

  • potassium bromide spacers

  • filling ports, and 

  • a holder

• optimum path length depends on: 

  • the liquid’s absorption characteristics and 

  • whether the application is qualitative or quantitative

transmission cell assemblies comprise a pair of windows constructed of mid-IR–transparent materials such as 

• potassium bromide spacers

• filling ports, and 

• a holder

optimum path length depends on: 

• the liquid’s absorption characteristics and 

• whether the application is qualitative or quantitative

 Gases

• traditional gas cell has been a 10-cm long cylinder made from borosilicate glass or stainless steel with an aperture of about 40 mm at each end 

• each open end is covered with an end cap that contains one of a pair of mid-IR–transparent windows constructed from

  • potassium bromide,

  • zinc selenide, or 

  • calcium fluoride

traditional gas cell

has been a 10-cm long cylinder made from borosilicate glass or stainless steel with an aperture of about 40 mm at each end 

each open end is covered with an end cap that contains one of a pair of mid-IR–transparent windows constructed from:

• potassium bromide,

• zinc selenide, or 

• calcium fluoride

Attenuated Total Reflection (ATR) Spectroscopy

aka

• internal reflection spectroscopy or evanescent wave spectroscopy

Attenuated Total Reflection (ATR) Spectroscopy

relies on the 

optical property that radiation passing through a medium of high refractive index at an angle of incidence greater than the critical angle will be totally internally reflected at a boundary in contact with a material of lower refractive index, the sample

Attenuated Total Reflection (ATR) Spectroscopy

• the refractive index of all molecules

is not constant and varies across absorption bands, this effect causes a shift in the measured wavenumber of a band with respect to transmission spectra

External Reflection Spectroscopy

• Fresnel reflection, transflection, reflection–absorption spectroscopy, and photoacoustic spectroscopy, but with the exception of diffuse reflection (commonly used), they are not widely used in pharmaceutical applications 

diffuse reflection (DR) spectra

• spectra recorded from powders or fairly fine granular samples 

diffuse reflection (DR) spectra

• the spectrum originates from

radiation that has penetrated through the surface of the sample and has been transmitted through multiple particles

INSTRUMENTATION: FT-IR

• Fourier-transform Infrared spectrophotometer

FT-IR

Light source 

• Incandescent silicon carbide (Globar®- type) source

Diffused reflection + FT-IR =

DRIFTS: Diffused Reflectance Infrared Fourier-transform Spectroscopy

Interferometer & Interferogram

• Beam emerging from the interferometer is then focused at the center of the sample compartment of the spectrophotometer by an off-axis paraboloidal mirror.

• After being transmitted through, or reflected from, the sample, the beam is focused onto the detector.

Beam emerging from the interferometer is then

focused at the center of the sample compartment of the spectrophotometer by an off-axis paraboloidal mirror.

After being transmitted through, or reflected from, the sample, the beam is focused onto

the detector.

INTERFEROGRAM

• analytical signal

• a record of the variation of the energy incident on the detector as a function of the optical path difference (OPD) (retardation) of the interferometer.

INTERFEROGRAM

• can be transformed using an equation called the

‘Fourier transform’ in order to extract the spectrum from a series of overlapping frequencies

Retardation

• or optical path difference 

• where variations of reading are recorded

• the dependent variable; reading depends on retardation

Laser

• Reference lights of FT-IR

• HeNe (Helium-Neon) laser (commonly used)

• A laser beam is superimposed to provide a reference for the operation of the instrument

A laser beam is __to provide a reference for the operation of the instrument

superimposed

Standard Detectors

• Room-temperature pyroelectric bolometer

• Most commonly used: Deuterated triglycine sulfate (DTGS) or deuterated L-alanine-doped triglycine sulfate

• MCT (mercury cadmium telluride) detector will provide more sensitivity

Standard Detectors (most commonly used)

• Deuterated triglycine sulfate (DTGS)

• deuterated L-alanine-doped triglycine sulfate

Standard Detectors (provide more sensitivity)

MCT (mercury cadmium telluride) detector

MEASUREMENT PERFORMANCE FACTORS

1. Spectral Resolution

2. Wavenumber Accuracy

3. Photometric Accuracy

4. Sensitivity

5. Beer’s Law Linearity

SPECTRAL RESOLUTION

• Main factor that affects the resolution is the maximum optical path difference of the interferogram

  • OPD or Retardation refers to the  difference in optical path length between the two arms to the interferometer

• Divergence angle of the beam passing through the interferometer may also degrade the resolution

Main factor that affects the resolution

is the maximum optical path difference of the interferogram

OPD or Retardation

refers to the  difference in optical path length between the two arms to the interferometer

Divergence angle of the beam passing through the interferometer

may also degrade the resolution

WAVENUMBER ACCURACY

• Main factors that affect wavenumber accuracy are the alignment of the laser and IR beams in the interferometer and the divergence of the beam passing through the interferometer as observed with the IR detector

Main factors that affect wavenumber accuracy are the

• alignment of the laser and IR beams in the interferometer

• divergence of the beam passing through the interferometer as observed with the IR detector

PHOTOMETRIC ACCURACY

• Main factor that affects the photometric accuracy of FT-IR spectrophotometers is the linearity of the detector response

Main factor that affects the photometric accuracy of FT-IR spectrophotometers is the

linearity of the detector response

SENSITIVITY

• can be determined by measuring two single-beam spectra under exactly the same conditions and calculating their ratio to produce what is commonly known as a 100% line

• measures signal-to-noise ratio to determine if the FTIR is sensitive enough for the amount of sample being tested

 BEER’S LAW LINEARITY

• for quantitative measurements

• the spectrum is measured (spectral bandwidth) at a resolution that is at least twice as narrow as the narrowest band in the spectrum

• for optimal photometric accuracy, the maximum absorbance of the analytical bands is no greater than 1.0 absorbance unit

 BEER’S LAW LINEARITY

• the spectrum is measured (spectral bandwidth) at a resolution that is

at least twice as narrow as the narrowest band in the spectrum

for optimal photometric accuracy, the maximum absorbance of the analytical bands is

no greater than 1.0 absorbance unit

APPLICATIONS

1. Qualitative fingerprint check

2. In synthetic chemistry

3. To characterize samples

4. Fingerprint test

5. Detect polymorphism

QUALITATIVE FINGERPRINT CHECK

• for the identity of raw material or drug product used in manufacture and for identifying drugs

IN SYNTHETIC CHEMISTRY

• as a preliminary check for compound identity, particularly for the presence or absence of a carbonyl group, which is difficult to check by any other method

TO CHARACTERIZE SAMPLES

• samples in the solid and semi-solid states, such as creams and tablets

FINGERPRINT TEST

• for films, coatings, and packaging plastics.

POLYMORPHISM

• IR is used to detect polymorphs of drugs

Polymorphs

are drugs with the same identity (i.e., functional group, structure) but different crystalline arrangements.

POLYMORPHISM

• an influential phenomenon, especially in pharmaceutical sciences

• can significantly influence variety of API properties and even biological performance

API properties:

flowability, tableting, dissolution rate, solubility, stability

biological performance:

efficacy and toxicity

STRENGTHS

• Provides a complex fingerprint which is unique to the compound being examined.

• Computer control of instruments means that matching of the spectrum of a compound to its standard fingerprint can now be readily carried out.