FTIT Spectroscopy- Dr. A. Amsavel

FTIR SPECTROSCOPY,
Principle, Theory, Instrumentation and Application in �Pharmaceutical Industry
IR Spectroscopy- Absorption Theory
Type of Vibrations & Vibration Energy level
FTIR Spectrophotometer-Instrumentation
Operation of the Spectrophotometer
Qualification & Calibration
IR Absorption by Organic compounds
Application
FDA citation in FTIR Analysis-Pharmaceutical Industries

1. Principle, Theory, Instrumentation and
FTIR SPECTROSCOPY
1
Principle, Theory, Instrumentation and
Application in
Pharmaceutical Industry
Dr. A. Amsavel, M.Sc., B.Ed., Ph.D.
[email protected]

2. An Overview
Introduction
IR Spectroscopy-Absorption Theory
Type of Vibrations & Vibration Energy level
2
FTIR Spectrophotometer-Instrumentation
Operation of the Spectrophotometer
Qualification & Calibration
Application
FDA citation in FTIR Analysis-Pharmaceutical Industries

3. History of FTIR
3
Chemical IR spectroscopy was evolved first in 1880s.
Interferometer was discovered by A. A. Michelson in 1890s
In late 1940s commercial optical null dispersive spectrophotometer
was developed
Peter Fellgettmeasured the light from celestial bodies by using an
interferometer. FTIR spectrum using interferometer was generated
in 1949.
Commercial FTIR spectrometers were made with micro-computers
in late 1960s.
Fast FTIR spectrum using algorithm was developed by Cooley-
Tukey1966

4. Electromagnetic Radiation
4
Vibration& Rotation

5. IR Spectroscopy
5
Spectroscopy is the study of interaction of electromagnetic
radiation with matter.
The Infrared region is classified as near, mid and far IR.
Infrared interacts with molecules rotational & vibrationalstructure
Molecule undergoes change in dipole moment when absorb IR
A diatomic molecule must have a permanent dipole (polar covalent
bond in which a pair of electrons is shared unequally) in order to
absorb, but larger molecules do not.
IR spectrum helps to identify the functional group of molecules.
Like a fingerprint no two unique molecular structures produce the
same infrared spectrum.

6. IR Spectroscopy
Mid-infrared (mid-IR) spectroscopy involves measurement of
the absorption of electromagnetic radiation over the wave
number range of 4000–400 cm
-1
(wavelength range of 2.5–25
µm) caused by the promotion of molecules from the ground
6
state of their vibrationalmodes to an excited vibrational
state.
Energy of the absorbed photon (E=hv)
Where h is Plank constant
vis wave number (number of waves per centimeter).
λ (µm) is wavelength (distance between the one cycle of wave).
Wave number = 1/ λ ie10000/ λ ṽ (cm-1)

7. IR Spectroscopy
7
When a vibrationalmode involves atomic motions of more than
just a few atoms, the frequencies occur over wider spectral ranges
and are not characteristic of a particular functional group.
Instead, they are more characteristic of the molecules as a whole.
Such bands are known as fingerprint bands. Such bands are known as fingerprint bands.
All strong bands that absorb at wavenumbersabove 1500 cm-1 are
group frequencies. Strong bands that absorb below 1500 cm-1 can
either be group frequencies or fingerprint bands
Polar groups, such as C–O, C=O, O–H, N–H, and C–F, typically give
rise to strong bands in the spectrum.

8. Absorption of IR
8
At temperatures above absolute zero, all the atoms in molecules are in
continuous vibration with respect to each other.
As a molecule vibrates, a regular fluctuation in the dipole moment
occurs.
A Dipole Moment = Charge Imbalance in the molecule
When the frequency of a specific vibration is equal to the frequency of
the IR radiation directed on the molecule, the molecule absorbs the
radiation and amplitude of the vibration increases.
The major types of molecular vibrations are Stretching and Bending
Stretching -along the line of the chemical bond
Bending -out of the line with the chemical bond.
Energy of Stretching > Bending

9. Hooke’s law
9
Total energy in IR absorption is sum of Energy of
rotation and energy of vibration
E tot= Erot+ Evib
Hooke’s lawHooke’s law
µ (kg) -the reduced mass of the system & k (N/m) force constant
V = 0, 1, 2 ….is called the vibrationalquantum number
0 -is strong band and 1, 2..are overtones
Evib = hν(V + ½)

10. Types of Vibrations & Rotations
10
Energy absorbed by the molecule due to vibration and rotation

11. IR Absorption Bands
11
Ref .: Vogel’s Quantitative Chemical Analysis

12. FTIR Spectrophotometer
12
Fundamental components of an FTIR system
1.The Source: A glowing black body emits the IR radiation. This beam is
passed to an aperture that limits amount of energy incident on sample.
2.Interferometer: Interferometer performs “spectral encoding” on
incoming radiation and exiting signal contains all the IR frequency incoming radiation and exiting signal contains all the IR frequency
components.
3.Sample: Beam is either transmitted through or reflected off of the
sample surface, depending on type. Sample absorbs energies at
frequencies characteristic to it.
4.The Detector: Detector measures the interferogramsignal.
5.The Computer: It performs Fourier transformation to obtain final IR
spectrum for examination. Spectrum obtained is %T Vs wave number.

13. FTIR Spectrophotometer
13

14. Source
14
Source:
In the mid-IR several type of sources are used. They are either
a lamp filament or a hollow rod, 1–3 mm in diameter and 2 to
4 cm long, made of fused mixtures of Zirconium oxide or
Yttrium and thorium oxides ( Nernst Globar) on heating to Yttrium and thorium oxides ( Nernst Globar) on heating to
1500°C, it emits IR radiation.
Helium–Neon (HeNe) laser is used in commercial FTIR
Other sources are Incandecentlamp or High pressure
mercury arc.

15. Interferometers
15
Interferometer: Itis heart of FTIR. Thisperforms “spectral encoding” the
radiation from source and exiting IR frequency signal to detector
Michelson Interferometer.
Albert Abraham Michelson
(1852–1931). Worked in the
study of light. He was awarded
Nobel Prize in Physics in 1907

16. Functioningof Michelson Interferometer
16
Light from the light source is directed to the beam splitter.
Half of the light isreflectedand half is transmitted.
The reflected light goes to the fixed mirror where it is reflected
back to the beam splitter.
The transmitted light is sent to the moving mirror and is also
reflected back towards the mirror.
At the beam splitter, each of the two beams (from the fixed and
moving mirrors) are split into two:
One goes back to the source and other goes towards the detector.
The resulting signal is called an interferogramwhich has the unique
property that every data point function

17. Functioningof Michelson Interferometer
17
The two beams reaching the detector come from the same source and
have an optical path difference determined by the positions of the two
mirrors,
That means they have a fixed phase difference and the two beams
interfere.
The two beams interfere constructively or destructively for a particular
frequency by positioning the moving mirror.
If the moving mirror is scanned over a range, a sinusoidal signal will be
detected for that frequency with its
maximum corresponding to constructive interference and
minimum corresponding to destructive interference.
This sinusoidal signal is called interferogram–detector signal (intensity)
against optical path difference.

18. Detectors in FTIR
18
Detectors: Two Popular detectors are used in FTIR
PyroelectricDetector: Deuteratedtriglycinesulphate(DTGS). The
pyroelectricdetector contains a mono-crystal of deuteratedtriglycine
sulfate (DTGS) or lithium tantalate(LiTaO3), sandwiched between two
electrodes, one of which is semi-transparent to radiation and receives the electrodes, one of which is semi-transparent to radiation and receives the
impact of the optical beam. It generates electric charges with small
temperature changes.
Photon diode Detectors contains semiconductors, such as Mercury
cadmium teluride(MCT) or indium antimonide(InSb). On effect of
infrared radiation, liberates electron/hole pairs which create a PD
measurable in an open circuit.

19. Spectrum from FTIR
19
The computer and software:
The measured interferogramsignal can not be interpreted directly
The measured signal is digitized and sent to the computer to get desired
Spectral information for analysis
Decoding ofindividual frequencies accomplished using mathematical Decoding ofindividual frequencies accomplished using mathematical
technique ieFourier transformation.
Background correction (nullify the blank / without sample)
Set the required scale (range) of absorption intensity or % transmittance
against wave number.
Compare the standard spectrum with sample and get the % matching (
confirmed by matching on min. 95%) thro sotware.
Standard spectrum is available in software library or run standard and
store as reference.

20. Sampling Techniques
20
Liquid Samples:Solid Samples:Gas Samples:
1.Neat sample
2.Dilute solution
1.Neat sample
2.Cast films
1.Short path cell
2.Long path cell2.Dilute solution
3.Liquid cell
2.Cast films
3.Pressed films
4.KBrpellets
5.Mull
2.Long path cell

21. Sampling Techniques
21
Alkali halides are transparent to mid-IR.
Sample cells are made from materials, such as NaCl
and KBr
Potassium Bromide (KBr) disks most commonly used Potassium Bromide (KBr) disks most commonly used
(above 400 cm-1).
Preparation KBrdisk dry; Use highly pure potassium
bromide powder (400mg) is ground with sample
(1mg) and pressed to make transparent disk of
13mm dia.

22. Preparation of KBrDisk / Pallet
22
Potassium nitrate absorption band at approximately 1378 cm-1 So KBr
must be pure
1 part of the sample to 100–400 parts,
Use clean Agate mortar.
Take 1mg sample add 10mg KBrand grind and add 20mg, 40mg… Take 1mg sample add 10mg KBrand grind and add 20mg, 40mg…
stepwise to 300mg
Spread mixture over die (13mm) and press to get disk
uniform transparency or exhibits good transmittance at about 2000 cm-1.
unsatisfactory, or poor-quality disks may be a consequence of inadequate
or excessive grinding, moisture/humidity, or impurities in the dispersion
medium.

23. Preparation of Mull Sample
23
Mull: Homogeneously distribute the finely divided powder sample in a thin layer
of a viscous liquid and should be semi-transparent to mid-IR.
Prepare a mull is to place 10–20 mg of the sample into an agate mortar, and then
grind the sample to a fine and add drop of the mulling agent (liquid paraffin or
Nujol) and grind into a uniform paste.
Transfer the paste to windows of sodium chloride and squeezed to form a thin, Transfer the paste to windows of sodium chloride and squeezed to form a thin,
translucent film and free from bubbles.
Mull agents (Mineral Oils) must have a refractive index (RI) close to RI of sample.
Chlorofluorosubstituted polymers mulling agent can be used if other mull
obscures.
Self support Film: Stretch the Primary packaging material LDPE or
Prepare thin self-supporting polymer films using hot compression molding or
microtoming

24. Liquid & Gas samples
24
Liquid: Neat non-volatile liquids spread between NaClwindows (sandwich)
Place a drop of the liquid between two NaClplates to get thin film of
about 0.01 mm thick.
Prepare a solutioninin highly volatile solvent and spread over the disk /
window and allow to evaporate, sample forms thin film and perform.window and allow to evaporate, sample forms thin film and perform.
Use spacers such as poly(tetrafluoroethylene), or poly(ethylene
terephthalate) as required path lengths of 0.015 to 1.0 mm
Transfer Liquids / solutions to commercially available cells ( neat)
Gases :
Use cells for static or flow-through gas and vapor sample.
10 cm long glass or stainless steel cells with ~40-mm aperture. Cover both
ends with Passiumbromide or zinc selenium or calcium fluoride windows.

25. ATR Sample
25
ATR sampler is an IR-transparent crystal of high Refractive index
than samples, such as ZnSe. Sample is surrounded by a crystal.
Radiation from the source enters the ATR crystal, where it
undergoes a series of total internal reflections before exiting the undergoes a series of total internal reflections before exiting the
crystal. During each reflection, the radiation penetrates into the
sample to a depth of a few microns.
By this action a selective attenuation of the radiation at those
wavelengths at which the sample absorbs and gives spectrum
Useful for polymers, fibers, fabrics,
powders &biological tissue samples.

26. Operation of the Spectrophotometer
Perform system suitability as required before test the sample
Ensure the calibration is performed and it is within validity date
Use reference polystyrene film (NIST traceable or certified) for calibration.
Use only spectroscopic reagents / solvents
26
Software used shall compliance with 21 CFR Part 11 complaince
The analysis of solution samples is limited by the solvent’s due to IR-
absorbing properties. Most commonly used solvents-CCl
4, CS
2, and CHCl
3
Ensure that % Relative humidity is not higher (RH <50%) and prevent to
use water near Instrument, since optics are water soluble egNaClwindow
Volatile liquids must kept in sealed cell to prevent evaporation.
Prepare sample and run the FTIR.

27. Qualification of FTIR
Ensure the Qualification is performed to ensure that Instrument meets
the intended purpose and documented.
Design Qualification: Ensure URS is suitable for the intended purpose
Installation Qualification: The IQ provides evidence that the hardware
and software are properly installed in the desired location
27
and software are properly installed in the desired location
Operational Qualification: Perform the series of test Wave number
accuracy , Resolution, Signal to Nose ratio, Zero test , contamination
test.
Performance Qualification : Determine that the instrument is capable
of meeting the user’s requirements for all the parameters that may
affect the quality of the measurement. Test the sample.
Ref: PA/PH/OMCL (07) 12 DEF CORR: Qualification Of Equipment: Annex 4: Qualification Of IR Spectrophotometers

28. Wave-Number Scale
28
The wave-number scale may be verified by recording the
spectrum of a polystyrene film,
Use Approx. 35 µm thick, matte polystyrene film (NIST).
Scan from 3800 to 650 cm
-1wave numbers.
Confirm the transmission minima (absorption maxima) at the
wave numbers (in cm
-1
) shown in the below table:

Transmission Minima (cm
-1
)
3060.0 1942.9 1583.0 1028.3
2849.5 1601.2 1154.5
Acceptable tolerance : ±1.0 (cm
-1
)

29. Detector Energy Ratio
29
Record the minimum energy ratio value for at least one of the
following measurement points and compare it to the vendor’s
specifications:
-Energy at 3990 cm
-1
/ energy at 2000 cm
-1
-Energy at 3990 cm/ energy at 2000 cm
-Energy at 4000 cm
-1
/ energy at 2000 cm
-1
-Energy at 3400 cm
-1
/ energy at 1300 cm
-1
-Energy at 2000 cm
-1
/ energy at 1000 cm
-1
Limits:
Energy ratio test specifications vary for each spectrometer
configuration. Refer manufacturer’s specification.

30. Signal / Noise Ratio
Peak-to-peak noise between:
4050 cm-1 and 3950 cm-1
2050 cm-1 and 1950 cm-1
RMS Noise between:
4050 cm-1 and 3950 cm-1
2050 cm-1 and 1950 cm-1
Record the maximum noise level for each of the following regions:
30
2050 cm-1 and 1950 cm-1
1050 cm-1 and 950 cm-1
550 cm-1 and 450 cm-1
(for DTGS detector only)
2050 cm-1 and 1950 cm-1
1050 cm-1 and 950 cm-1
550 cm-1 and 450 cm-1
(for DTGS detector only)
*RMS-root mean square
Limits (% T): Noise level test specifications vary for each
spectrometer configuration. Refer to the manufacturer’s
specifications.

31. Resolution
31
Use Certified polystyrene film of approximately 35 µm in
thickness.
Record the FTIR spectrum of the polystyrene film.
use suitable instrument resolution with the appropriate
apodisationprescribed by the manufacturer.
Limits:
Difference between the absorbancesat the absorption min. 2870 cm-1
and the absorption max. 2849.5 cm
-1
> 0.33.
Difference between the absorbancesat the absorption min 1589 cm
-1
and the absorption max at 1583 cm
-1
> 0.08.

32. Resolution
32
Typical spectrum of standard polystyrene used to verify the
resolution performance

33. Zero Test
33
When using a polystyrene film of approxi. 35 µm in thickness as
standard at the wavelength of 2925 cm
-1
and 700 cm
-1
, almost
complete absorption of the irradiated energy can be observed.
With this test, the remaining transmission is measured.
As the maximum absorption can be observed at 700 cm
-1
negative As the maximum absorption can be observed at 700 cmnegative
values may be observed.
The objective of the test is to evaluate if, despite the fact that
there is almost complete absorption, energy is still detectable.
Non-valid results are an indication of a non-linear behaviourof the
detector and the electronic system.
Limits (%T): Comply to manufacturer’s specification

34. Attenuated Total Reflection (ATR)
34
Contamination Test
This test checks the presence of peaks that
signal a contamination problem.
Use the automated function of the
instrument (if available) to perform this test.
Wave-number
(cm-1)
Upper limit
(A)
3100.0 –2800.0 0.1
1800.0 –1600.0 0.1
If not available, record a background
spectrum.
1800.0 –1600.0 0.1
1400.0 –1100.0 0.2
Throughput Check :
A background spectrum is recorded and the transmittance is measured at
3 wave numbers e.g. 4000, 2600 and 1000 cm-1.
Limits:
The lower limit of the transmittance for the 3 wave numbers must be 80%

35. Application
35
FT-IR used for various chemical analysis in Industries &Research
To identify unknown materials by comparing with Spectrum library
To determine the quality or consistency of a sample
Analysis of formulations: Amount of components in a mixture
With modern software algorithms, FTIRisused for quantitative With modern software algorithms, FTIRisused for quantitative
analysis
Identification of functional groups in unknown substances.
Eg. Ketones, Aldehydes, Alcohols,Amines, Carboxylic Acids etc.
Identification of polymers, plastics, and resins.
Concentrations gases such as carbon monoxide and dioxide,
ammonia, methanol, ethanol, methane at ppmlevel, etc

36. Application
Identification/ Characteristic of Organic compounds:
Functional groups have their characteristic fundamental vibrations which
give rise to absorption at certain frequency range in the spectrum.
However, several functional groups may absorb at the same frequency
range, and a functional group may have multiple-characteristic absorption
36
range, and a functional group may have multiple-characteristic absorption
peaks, especially for 1500 –650 cm-1, which is called the fingerprint
region. It is complex and more difficult to interpret.
Small structural differences results in significant in spectral differences
Complete interpretation may not be possible
Can be identified by using standard’s spectra and match the spectrum of
sample including finger print region

37. IR Absorption of Organic Compounds
Ref: Principles of Instrumental Anlalysis–D.A.Skoog
37

38. 38
Ref: Chemical Analysis: Francis and AnnickRouessac

39. Spectrum of Isopropyl alcohol
39

40. Spectrum of n-Butraldehyde
40

41. Application in Pharmaceuticals
Analysis of Pharmaceutical active & inactive ingredients
Identification test: to identify the Active Ingredient, primary
packing material, excipients, and Pharmaceutical dosage form
Semi quantitative (Limit test) and Quantitative analysis
To test the polymorph of certain APIs
41
To test the polymorph of certain APIs
Structure elucidation/ Characterisation
Detection of impurities in organic compound
In-process chemical reaction.
Study of Keto-Enoltautomerism, Conformational analysis.

42. IR Spectrum of Paracetamol
42
IR spectrum of Paracetamolin KBr

43. FTIR: Quantitative Analysis
Quantitative analysis:
FTIR Absorption spectra can be used for quantitative analysis.
Develop the Analytical Method and Validate the test method .
Lamberts Law: Absorbance (A) is proportional to the Path length (l) of the
absorbing medium.
43
absorbing medium.
Beers law: Absorbance (A) is proportional to the Concentration (C) of the
sample.
Beer-Lambert Law -Absorbance is (A) proportional to Concentration
and Path length of the sample.
A Cl; A = εCl
C-Concentration (Moles /litre) ; l-Path length (cm) & ε -Molar absorption coefficient
(molar absorptivity)

44. Analytical Method Validation-FTIR
44
Accuracy : Perform recovery studies with the appropriate matrix spiked with
known concentrations.
Validation criteria: Mean recovery for drug substances, 98.0%–102.0%; Drug
product assay, 95.0%–105.0%; for impurity analysis 70.0%–150.0%
Precision
Repeatability: Assess by measuring the concentrations of six preparations of
Precision
Repeatability: Assess by measuring the concentrations of six preparations of
sample (at 100% level) or 3 replicates of 3 preparations of concentrations (75%,
100% & 125%)
Validation criteria: RSD NMT 1.0% for drug substance, NMT 2.0% for drug
product, and NMT 20.0% for impurity analysis.
Intermediate precision: Assess the changes in variables such as different days,
using different instrumentation, or 2 or 3 analysts. At a minimum, any
combination of at least two of these factors totaling six experiments
Validation criteria: RSD NMT 1.0% for drug substance, NMT 3.0% for drug
product assay, and NMT 25.0% for impurity analysis.

45. Analytical Method Validation-FTIR
45
QuantitationLimit:
Measure six replicate blank preparation and calculate the standard deviation.
QL =SD X 10 /Slope of the calibration line
Prepare and measure the sample at calculated QL concentration and confirm accuracy.
Validation criteria: the measured concentration must be accurate and precise at a level
equal to or less than 50% of the specification.
Linearity
Prepare five standard preparations at concentrations encompassing the anticipated
concentration of the test preparation. Plot standard curve IR spectral response against
concentration and perform regression.
Validation criteria: Correlation coefficient (R), NLT 0.995 for assays and NLT 0.99 for Limit
tests.
Range:
This parameter is demonstrated by meeting linearity, precision, and accuracy requirements.
Validation criteria: Validation range for 100.0% is 80.0%–120.0%. For non-centered
acceptance criteria, the validation range ±10.0%. For content uniformity, the validation
range is 70.0%–130.0%.

46. Limitations of FTIR
46
Molecule must be active in the IR region. (alter the net dipole moment of the
molecule in order for absorption.)
Minimal elemental information is given for most samples.
Material under test must be transparency in the IR spectral region
Accuracy greater than 1% obtainable when analysis is done under favorable
conditioncondition
It is not possible to know purity of compound or a mixture of compound
Accuracy of FT-IR remains true if there is no change in atmospheric conditions
throughout the experiment.
Cannot detect atoms or monoatomicions –single atomic entities contain no
chemical bonds.
Cannot detect molecules of of two identical atoms symmetric-N
2or O
2.
Aqueous solutions are very difficult to analyze –water is a strong IR absorber.
Reagent used shall be highly pure

47. Examples of Form 483 in IR Analysis
47
Form 483 issued by USFDA is given below for understanding & action. This will help to
identify the regulatory gaps involving IR analysis at your firm. Review and take action on
the gap to overcome the inspectional Observations.
The identification test used for XX@-Na by FTIR compared the spectrum of XX@-Na against
the spectrum for USP XX@. These spectra are not equivalent and are in fact quite different.
 Idenficaon tesng of API *** batches …,…,…,…,… using IR was not actually done. The  Idenficaon tesng of API *** batches …,…,…,…,… using IR was not actually done. The
analyst simply used a previous spectrum and changed the batch number each time.
System audit trails are not available on the two FT-IR instruments, and quality control (QC)
operators have the option of not saving the IR data
Computerized system and instrument software ( Shimadzu IR solution 1.30) used in the
quality testing laboratory that are currently in use for routine testing is not validated (2015).
Your firm's laboratory analyst had modified printed raw data related to the IR Spectra test.
Your quality control unit failed to detect that IR spectra were being substituted by a
laboratory employee and detect the manipulation or alteration of laboratory documents. ….

48. FDA Citations-Classification
48

49. FDA Citations
49
Ref https://www.spectroscopyonline.com/view/analysis-fda-infrared-483-citations-have-you-data-integrity-problem

50. References
1.Quantitative Chemical Analysis. 7
th
Edition Daniel C. Harris
2.Pharmaceutical Analysis -David G. Watson
3.Chemical Analysis: Modern Instrumentation Methods and Techniques
Francis and AnnickRouessacand Steve Brooks, 2007,John Wiley & Sons Ltd.
4.Vogel’s –Quantitative Chemical Analysis-6
th
edition
50
4.Vogel’s –Quantitative Chemical Analysis-6edition
5.PA/PH/OMCL (07) 12 DEF CORR: Qualification Of Equipmen: Annex 4:
Qualification Of IR Spectrophotometers
6.USP General Chapter <854> and <1854>Mid-Infrared Spectroscopy &
Theory And Practice
7.European Pharmacopeia General Chapter 2.2.24. Absorption
Spectrophotometry, Infrared
8.Fundamentals of Analytical Chemistry-9
th
Edn. D.A Skoog, D.M West et al..

51. 51
About Author:
Dr. A. Amsavel, born at Begarahalli, Dharmapuri-Dist, Tamil Nadu, India.
Completed his M.Sc. in Dept of Analytical Chemistry, University of Madras. B.Ed. in
AnnamalaiUniversity and Ph.Din Anna University, Chennai.
Worked as Lecturer and also worked in various Chemical & Pharmaceutical
Industries for the past 34 years. Presently working as Assistant Vice President-
Quality at Malladi Drugs & Pharmaceuticals Ltd.