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Sreekanth Nama, M.Pharm, (Ph.

D)
ASSOCIATE PROFESSOR

INFRARED SPECTROSCOPY
Contents Introduction Infra red region and infrared spectrum Principle of IR Requirements for a molecule to absorb infra red radiation Theory of IR Fundamental vibrations Vibrational frequency Vibrational energy Number of fundamental vibrations Sampling techniques Instrumentation Pharmaceutical applications

Introduction It is the most important analytical tool for the chemist to gather the sufficient information about various organic molecules and as a technique to asses the purity of the compound. It is excellent technique to indentify the functional groups present in organic molecules. Spectral matching can be done by computer software and library spectra Since absorbance follows Beers Law, can do quantitative analysis Almost any compound having covalent bonds, whether organic or inorganic, absorbs various frequencies of electromagnetic radiation in the infrared region of the electromagnetic spectrum. Just as ultraviolet absorption spectra are strictly caused by changes in electronic, vibrational and rotational energy, so IR absorption spectra are due to changes in vibrational energy accompanied by changes in rotational energy. This is the technique example for molecular absorption spectroscopy (the energy changes that occurs at molecular level) When compared to ultraviolet, which comprising of relatively few peaks this spectrum containing large number of absorption bands.
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Sreekanth Nama, M.Pharm, (Ph.D)


ASSOCIATE PROFESSOR

From absorption band one can derive the wealthy of information about the structure of an organic compound. Infra red radiations are also called as thermal radiation and the extent absorption by the molecules in the form of vibrations are detected by thermal detectors.

In simple terms IR tells about Type of bonds are present Some structural information

Infrared region The most important region for an organic chemist is 2.5 to 15, in which molecular vibrations can be detected and measured in an infra red spectrum. Different regions of infrared radiation Near IR Mid IR Far IR Wavelength 0.8 2.5 15 200

Near IR

Mid IR

Far IR

12500

4000 Wave number

667

50 cm-1

The absorption of infrared radiations can be expressed either in terms of wavelength or wave number

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Infra red spectrum It is the plot in between transmittance against wave number. The spectrum is rich in numerous

absorption bands which give sufficient information about organic molecule. Relationship in between wavelength and wave number Wave number = Example Calculate the wave number for the wavelength 2.5 (2.5= 2.5X10-4 cm) Wave number = = 4000 cm-1

Wave number in cm-1=

The wavelength 15 corresponds to the wave number 667 cm-1 Principle of infrared spectroscopy The molecule absorbs IR radiation when the electric vector of IR radiation interacts with the molecule. Upon absorption of electromagnetic radiation in the infra red region causes an excitation of molecule from ground vibrational energy level to higher vibrational energy levels. We know that each vibrational level is associated with a number of closely spaced rotational levels. The wavelength or frequency of absorption of IR radiation depends on the relative masses of the atoms, the force constant of the bond and the geometry of the atoms. Requirements for a molecule to absorb infra red radiation Dipole momentum Center of symmetry

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Dipole momentum (Charge Distance) The concept is explained by taking simple example HCl. In HCl molecule the bonding electronic pair is not shared equally between the hydrogen atom and chlorine atom. The chlorine atom with its greater electro negativity pulls the electron pair close to it. This gives a slight positive charge (+q) to the hydrogen atom and a slight negative charge to the chlorine atom (-q). Such a molecule with a positive charge at one end and a negative charge at the other end is referred to as an electric dipole or simply dipole. The HCl molecule has dipole moment, its stretching vibration alters the bond distance and its dipole moment alters. Dipole moment and absorption of IR radiation All the bonds present in the molecule does not absorb infra red radiation. Those bonds which are accompanied by the change in the dipole momentum will absorb the infra red radiation. Vibrational transitions which are accompanied by change in the dipole momentum of the molecule are called infrared active transitions. Vibrational transitions which are not accompanied by a change in dipole momentum of the molecule are called as infrared inactive. Examples Vibrational transitions of C=O, N-H, O-H bands are accompanied by change in the dipole moment of the molecule and they absorb strongly in IR region. But the transitions in C-C bonds in symmetric alkenes and alkynes are not accompanied by change in the dipole moment and hence do not absorb in the IR region. Homo nuclear diatomic molecules such as Hydrogen (H 2), Nitrogen (N2) and Oxygen (O2) no infrared absorption is observed, as these molecules have zero dipole moment and stretching of the bonds will not produce one. For hetero nuclear diatomic molecules such Carbon Monoxide (CO) and Hydrogen Chloride (H Cl), which do possess a permanent dipole moment, infrared activity occurs because stretching of this bond leads to a change in dipole moment (since Dipole Moment = Charge Distance).

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Centre of symmetry If molecule has a centre of symmetry then the vibrations are Centro symmetric and are inactive in the IR. The vibrations which are not Centro symmetric are IR active. Since most of the organic compounds, the functional groups are not Centro symmetric, so IR spectroscopy is most informative. Theory of infrared spectroscopy The theory involved in the infra red spectroscopy explained by the following models. Classical model of a molecule Quantum mechanics model of molecule An harmonic model of molecule

Classical model of molecule

A diatomic molecule can be modeled by a spring with the force constant k attached to two balls of mass m. this is called the classical harmonic oscillator model. At the equilibrium length d the spring has zero potential energy. As spring is stretched or compressed the potential energy increases along a parabola. This curve is called harmonic potential curve.

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Vibrational frequency related to force constant k and mass m by the equation

The vibrational frequency of a molecule depends on two parameters Force constant (K) Reduced mass ()

The reduced mass of the atoms can be calculated by considering the masses of the atoms present in the molecule. If larger force constant (stronger spring), the spring has sufficient energy to pull the balls close to each other or to push the balls away from each other. So the vibrational frequency is increased. There fore smaller force constant results in a lower frequency.

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If larger mass of ball, the balls moves very slowly with respect to each other or moves very slowly away from each other. So the vibrational frequency decreased. Quantum mechanical model of molecule Quantum mechanics predicts the same relationships between vibrational frequencies, force constant and mass those are predicted by classical model. However quantum mechanics predicts that the molecule may vibrate only at energy levels that fit the formula

V= 0, 1, 2, 3, 4n h = Planks constant, 6.626X .

As per the theory of quantum mechanics model of molecule, the molecule can absorb or emit the amount of radiation of energy equal to h and the molecule breaks apart if it absorbs more energy. Transitions occur only from one level to the next higher or lower level.

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Transitions from the ground state (

to the first excited state (

absorb light strongly between

and give rise to intense bands called fundamental bands. The energy difference the lowest possible energy level of a bond and next higher energy level is given as

This gives the frequency of fundamental band in the infra red spectra. Effect of vibrational frequency on spacing between energy levels A higher frequency (due to larger force constant or small mass) results in larger spacing in between the energy levels. If the vibrational frequency is small, the spacing between energy levels also small.

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Anharmonic model of molecule

As per the theory of this model, a real molecule is not a harmonic oscillator. When the atoms are pushed very close together they repel more strongly than spring when they pulled apart far enough. This behavior can be modeled by an harmonic model or an harmonic potential curve. When they repelled more strongly than spring the bond existing between the atoms was disassociates. It is the realistic model for a molecule, the energy levels are equally spaced only in the region that is spaced like in the harmonic potential curve. The transitions which are allowed between one level and the next higher (or lower), it is not true. A transition from v=0 to v=2 called an over tone, corresponds to Molecular vibrations Absorption of infrared radiation is due to the changes in the vibrational and rotational energy levels. When radiations with frequency range less than 100 cm-1 are absorbed, molecular rotation takes place. The molecular vibrations are set in, when more energetic radiation in the region 104102 cm-1 are passed through the sample of the substance. The absorption causing molecular vibration also quantized. Clearly, a single vibrational energy change is accompanied by a large number of rotational energy changes. Thus the vibrational spectrum appears as vibrationalrotational bands. .

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In the infrared spectroscopy especially in the region of 2.5-15 the absorbed energy bring about predominant changes in the vibrational energy. The vibrational energy depends on Masses of the atoms present in the molecule Strength of the bonds (bond strength) The arrangement of atoms within the molecule

Fundamental vibrations

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Two kinds of fundamental vibrations observed in IR Stretching Bending

Stretching In this type of vibrations, the distance between the two atoms increases or decreases but the atoms remains in the same bond axis. Types of stretching vibrations Symmetric stretching Asymmetric stretching

Symmetric stretching In this type, the movement of the atoms with respect to a particular atom in a molecule is in the same direction. Asymmetric stretching In these vibrations, one atom approaches the central atom while other departs from it. Bending In this type of vibrations, the positions of the atoms change with respect to the original bond axis. Bending vibration accompanied by change in bond angle between the bonds, but the distance between the atoms remains same. Types of bending vibrations Scissoring Rocking Wagging Twisting

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Scissoring In this type two atoms approach each other. Rocking In this type, the movement of the atoms takes place in the same direction. Wagging Two atoms move up and down the plane with respect to the central atom. Twisting In this type of vibration, one of the atoms moves up the plane while the other moves down the plane with respect to the central atom. Energies required for stretching and bending vibrations We know that more energy is required to stretch a spring than that required to bend it. Thus, we can clearly say that stretching absorptions of a bond appears at high frequencies (higher energy, less wave length) as compared to the bending vibrations or absorptions of the same bond. Vibrational frequency The value of stretching vibrational frequency of a bond can be approximated by the application of Hookes law. In this approximation, two atoms and connecting bond can be treated as a simple harmonic oscillator composed by two masses (atoms) joined by a spring (chemical bond). According to Hookes law, the frequency of the vibration of the spring is related to the mass and the force constant of the spring, k, by the following formula. The frequency of radiation absorbed in infrared region is expressed interns of wave number.

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Wave number =

m1, m2 are the masses of the atoms concerned in g (grams) in a particular bond. K= Force constant of the bond and related to the strength of the bond. For a single bond the value of K is 5X105 gm. sec-2 (it become double and triple for double and triple bonds respectively) C= velocity of radiation = 3X1010 cm/sec. Thus vibrational frequency or wave number depends on values of K, . How the k, values affect the vibrational frequency C=C stretching is expected to absorb at higher frequency than C-C stretching. It is due to the higher bond strength of the double bond compared to single bond. (Value of K) O-H stretching absorbs at higher frequency compared to C-C bond. It can be explained on the basis of the smaller value of reduced mass for O-H compared with C-C bond. (Value of ) Effect of electro negativity on vibrational frequency We can expect O-H absorbs at higher frequency as compared to F-H. But it is not true, actually, F-H absorbs at the higher frequency. This can be explained due to the higher electro negativity of fluorine compared that of oxygen. Vibrational energy

V= number of the vibrational energy level and have values 0, 1, 2, 3, 4, -------------------h = Planks constant v= vibrational frequency of the bond

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Number of fundamental vibrations Generally organic molecules are two types based on its arrangement of atoms with in the molecule. They are Linear molecule Non linear molecule

Linear molecule A molecule whose atoms are arranged so that the bond angle between each is 180, an example is carbon dioxide, CO2.

Degrees of freedom in a molecule Poly atomic molecules may exhibit more than one fundamental vibrational absorption bands. The number of these fundamental bands is related to the degrees of freedom in a molecule. The number of degrees of freedom is equal to the sum of the co-ordinates necessary to locate all the atoms in a molecule within the space. Each atom has three degrees of freedom corresponding to the three Cartesian co-ordinates (X, Y and Z) necessary to describe its position relative to other atoms in a molecule. An isolated atom which is considered as a point mass has only translational degrees of freedom and not has vibrational and rotational degrees of freedom. Total number of degrees of freedom of a molecule is equal to 3n where n is the total number of atoms in a molecule. A molecule of finite dimensions will, thus, be made up of rotational, vibrational and translational degrees of freedom, so 3n degrees of freedom= Translational+ Vibrational+ Rotational.

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Since in IR, we are concerned only with number fundamental vibrational modes of molecule. We calculate only the number of vibrational degrees of freedom of a molecule. We say that a molecule has always three translational degrees of freedom. Calculation of number of fundamental vibrations for linear molecule In case of linear molecule, there are only two degrees of freedom of rotation. It is due to the fact rotation of such molecule about its axis of linearity does not bring about any change in the position of the atoms while rotation about the other two axes change the position of atoms. Thus, for a linear molecule of n atoms Total degrees of freedom = 3n Translational degrees of freedom= 3 Rotational degrees of freedom= 2 Vibrational degrees of freedom= 3n- Translational degrees of freedom Rotational degrees of freedom = 3n-3-2 = 3n-5 Each vibrational degree of freedom corresponds to the fundamental mode of vibration and fundamental mode corresponds to a band. Hence theoretically there will be 3n-5 possible fundamental bands for linear molecule. Hence, for carbon dioxide molecule (CO2), the number of vibrational degrees of freedom can be calculated as follows. Number of atoms (n) = 3 (C, O, O) Total degrees of freedom = 3n = 3X3 = 9 Translational degrees of freedom = 3 Rotational degrees of freedom = 2 Vibrational degrees of freedom = 9-3-2 = 4

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Theoretically carbon dioxide molecule is having four degrees of freedom. Non linear molecule It is a branched-chain molecule, that is, one whose atoms do not all lie along a straight line. In case of non linear molecule, there are three degrees of rotation as the rotation about three axes (X, Y and Z) will result in a change in the position of the atoms. So, for a non linear molecule of n atoms, the vibrational degrees of freedom can be calculated as follows Total degrees of freedom = 3n Translational degrees of freedom = 3 Rotational degrees of freedom = 3 Vibrational degrees of freedom = 3n-3-3 = 3n 6 Calculation of vibrational degrees of freedom for benzene Thus for a non linear molecule C6H6, the number of vibrational degrees of freedom can be calculated as follows Number of atoms (n) = 12 Total degrees of freedom = 3X12= 36 Translational degrees of freedom = 3 Rotational degrees of freedom = 3 Vibrational degrees of freedom = Total degrees of freedom Translational degrees of freedom Rotational degrees of freedom Vibrational degrees of freedom = 36- 3- 3 = 30. The fundamental vibrations of the molecule (Either linear or non linear) they should fall in the region under investigation, i.e, 2.5 to 15 . If those vibrations lying out side the range cannot be considered.

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Sampling techniques Various techniques employed for introducing the sample in the path of the infrared radiation depending upon whether sample is a gas, a liquid, a solid. Thus, sample of same substance shows shifts in the frequencies of absorption as we pass from solid to the gaseous state. In some cases, additional bands also observed with the change in the state of the molecule. Hence, it is always important to mention the state of the sample on the spectrum of IR. Different states of samples analysed by IR Solids Liquids Gasses Solutions

Sampling of solids Solid samples can be prepared for analysis by incorporating them in to a alkali halide. Alkali metal halides usually sodium chloride, which is transparent through out the infrared region and commonly used. Potassium bromide also serves the purpose well. The precautions has to be taken while preparing the discs The substance under study is absolutely dry i.e free from water ( if water is present in the substance it may absorb the IR radiation strongly at 3710 cm-1 and also near 1630 cm-1. Grinding is carried out under IR lamp to avoid the condensation of atmospheric moisture. The grinding sample with alkali halide is efficient. Discs obtained from poorly ground mixture scatter more light than disperse it. The optimum or desired particle size of mixture was prepared i.e. less than 2m.

Preparation of discs The sample is ground with KBr and it made into a disc after drying and then pressing it under elevated temperature at high pressures. In the same way a blank disc also prepared (with out sample) with pure potassium bromide which may placed in the path of the reference beam.

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Generally grinding was done by mortar and pestle. 1-2% of the solid sample mixed with potassium bromide and grinding it to the desired particle size. The IR spectrum of a solid can also recorded as a mull or paste. Some commonly used mulling agents are Nujol Hexachloro butadiene Chloro fluoro carbon oil

The most commonly used mulling agent is nujol, which is a mixture of liquid paraffinic hydrocarbons with high molecular weights. Solid film also be deposited over the alkali metal halide (Nacl or KBr) disc by putting a drop of solution of the solid sample (sample dissolved in a volatile solvent) and then evaporate the solvent. Solid samples also examined in solutions. Liquids It is the simplest technique where the liquid as a thin film (0.1 to 0.3 mm) squeezed between the two sodium chloride plates. The plates made of the material which is transparent to infrared light. A drop of the liquid sample is placed on the top of the sodium chloride plate and another sodium chloride plate is placed on it. The pair of sodium chloride plates enclosing the liquid film is placed in the path of sample beam. Gases The gaseous sample is introduced in to a gas cell. The walls of the cells are made of sodium chloride. Sodium chloride windows allow the cell to be placed directly in the path of sample beam. The gas cell is usually 10 cm long. Very few organic compounds can be examined as gases. Solutions It is the most convenient to record the spectrum in solutions. The solvents used in the IR technique are

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Chloroform Carbon tetra chloride Carbon disulphide

The sample under study is dissolved in a solvent. Its 1-5% of the solution is placed in a solution cell consisting of transparent windows made of alkali metal halide. A second cell containing the pure solvent is placed in the path of the reference beam to cancel out solvent interferences. In fact the solvent also having IR absorbing characteristics. From the spectra of the various pure solvents, it is observed that chloroform having good solvent properties and it was commonly used. Instrumentation The essential components of spectrometers A source of electromagnetic radiation A monochromator (To isolate a particular wavelength or range of wave lengths) A sample compartment A detector and associated electronics (To measure the intensity of electromagnetic radiation) Amplifier and read out device

Infrared light sources Characteristics of light source An ideal light source should emit a continuous spectrum of radiation without any fluctuations. The emission of light from a source varies exponentially with voltage of its power supply. Thus a regulated power source is often needed to provide required stability. The selected light source should be transparent in the given region and emit the radiation required for analysis. Generally the following light sources are widely used in infrared spectroscopy

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Nernst glower Globar source Incandescent lamp Mercury arc lamp Tungsten filament

Nernst glower It is composed of rare earth oxides formed in to a cylinder having a diameter of 1-2 mm and a length of 20 mm. platinum leads are sealed to the ends of the cylinder to permit electric connection. The current is passed through the device, temperatures between 1200 K and 2200K result. Globar source A Globar source a silicon carbide rod, usually about 50mm in length and 5 mm in diameter. It is also electrically heated to the temperatures of 1300 K to 1500 K. Globar source provides a greater output. Incandescent lamp A source, somewhat lower intensity but longer life than Globar and Nernst glower. It is a tightly wound spiral of a nichrome wire heated about 1100 K by an electrical current. A rhodium wire heater sealed in a ceramic cylinder had similar properties as a source. Mercury arc lamp This the lamp widely used in far infra red region. High energy mercury arc lamps are used. It is a quartz jacketed tube containing mercury at a pressure greater than one atmospheric pressure. Tungsten filament lamp An ordinary tungsten filament lamp is a convenient source for the near IR.

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Sample cells For the infrared instruments, various assorted cells are used. The most common is a cell of Nacl windows. Fixed thickness cells are available for these purposes and are the most commonly used. The solvent, of course, must not attack the windows of the cell. Nacl cells must be protected from atmospheric moisture and solvents. They require periodic polishing to remove fogging due to moisture contamination. Detectors used in infrared spectroscopy Infrared detectors are of three general types Thermal detectors Pyro electric detectors Photo conducting detector

The first two are commonly found in photometers and dispersive type spectrophotometers. Pyro electric detectors and Photoconductive detectors are found in FT-IR instruments. Thermal detectors Thermo couples In its simple form, a thermocouple of a pair of junctions formed when two pieces of metal such as bismuth are fused to either end of dissimilar metal such as antimony. A potential difference develops between the two junctions that vary with their difference in temperature. Among two junctions one junction absorbs the IR radiation (to improve its heat absorbing capacity the junction is usually blackened) and reference junction, which is usually shielded from the incident radiation. To enhance sensitivity, several thermocouples may be connected in series to give what is called thermopile. Bolometer It is a type of resistance thermometer constructed of strips of metals such as platinum or nickel or sometimes with semiconductor; the latter devices are sometimes called thermistors. These instruments are not widely used in mid IR.

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Pyro electric detectors These are constructed from pyro electric materials, which are with special thermal and electrical properties. Triglycine sulphate is most important pyro electric material. Photo conducting detectors It consists of a thin film of a semi conductor material, such as lead sulfide, mercury/cadmium telluride or indium antimonide deposited on a non conduction glass surface and sealed into an evacuated envelop to protect the material from atmosphere. Lead sulfide detector widely used in NIR. For mid and far IR mercury/ cadmium telluride was used due to its superior response characteristics. Types of instruments used in infrared spectroscopy Dispersive spectrophotometer Fourier transform spectrophotometers

Dispersive spectrophotometer (Classical, Older technology) The original infrared instruments were of the dispersive type. These instruments separated the individual frequencies of energy emitted from the infrared source. This was accomplished by the use of a prism or grating. An infrared prism works exactly the same as a visible prism which separates visible light into its colors (frequencies). A grating is a more modern dispersive element which better separates the frequencies of infrared energy. The detector measures the amount of energy at each frequency which has passed through the sample. This results in a spectrum which is a plot of intensity vs. frequency.

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Working of dispersive type spectrophotometer The source of IR light is a ceramic tube electrically heated 12000c. This gives the radiation in the region of interest. Light from the source is reflected by a flat mirror and two toroidal mirrors to create focused sample and reference beams which pass through the sample chamber. The sample absorbs some of the infrared radiation from the sample beam. Only mirrors are used in the spectrophotometer, because lens material would absorb infra red light. The beams are reflected on to a rotating mirror, the mirror temporarily reflects the reference beam towards the rest of the optics, and blocks the sample beam. As the mirror rotates it alternates the passing the sample and reference beams to the rest of the optics, this alteration is important for proper detection of the absorption. The beam is refocused with another toroid mirror and reflected on to a diffraction grating, which disperses the light, similar to the action of prism. The dispersed light is refocused and sent to a thermocouple which acts as detector. The thermocouple generates an electrical signal proportional to the amount of light fall on it. By altering the reference and sample beams, an oscillating current is generated. The amplitude of generated oscillating signal indicates the amount of infrared absorption by the sample. While the beams alternate, the grating is slowly rotated passing different frequencies of the light to the detector. The amplitude is converted in to the %T by the sample relative to the reference %T.
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FT-IR spectrophotometer Why FT-IR Fourier Transform Infrared (FT-IR) spectrometry was developed in order to overcome the limitations encountered with dispersive instruments. A solution was developed which employed a very simple optical device called an interferometer. The main difficulty was the slow scanning process. A method for measuring all of the infrared frequencies simultaneously, rather than individually, was needed. The interferometer produces a unique type of signal which has all of the infrared frequencies encoded into it. The signal can be measured very quickly, usually on the order of one second or so. Thus, the time element per sample is reduced to a matter of a few seconds rather than several minutes. Fourier transform infrared spectroscopy is preferred over dispersive or filter methods of infrared Spectral analysis for several reasons: It is a non-destructive technique It provides a precise measurement method It can increase speed, collecting a scan every second It can increase sensitivity It has greater optical throughput It is mechanically simple with only one moving part

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The most commonly used interferometer is a Michelson interferometer. It consists of three active components A moving mirror A fixed mirror Beam splitter

Interference Consider two waves that are in phase, with amplitudes A1 and A2. Their troughs and peaks line up and the resultant wave will have amplitude A = A1 + A2. This is known as constructive interference. 180, out of phase, then one wave's crests will coincide with another wave's troughs and so will tend to cancel out. The resultant amplitude is A = |A1 A2|. This is known as destructive interference. Combined waveform wave 1 wave 2 Two waves in phase Optical path difference Optical Path Difference (OPD) is the optical path difference between the beams travelling through the two arms of an interferometer. OPD is equal to the product of the physical distance travelled by the moving mirror (multiplied by 2, 4, or other multiplier which is a function of the number of reflecting elements used. FT-IR has a natural reference point when the moving and fixed mirrors are the same distance from the beam splitter. This condition is called zero path difference or ZPD. The moving mirror displacement, , is measured from the ZPD. The beam reflected from the moving mirror travels 2 further than the beam reflected from the fixed mirror. Two waves 180 out of phase

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The relationship between optical path difference, and mirror displacement, , is OPD = 2 FT-IR instruments

At t=0, zero path difference between 2 beams for monochromatic radiation; constructive interference; maximum signal at detector. At a later time, path difference = l/2; destructive interference; minimum signal. Later on, l maximum signal. So interferogram is a cosine wave. Construction and working of FT-IR The two mirrors are perpendicular to each other. The beam splitter is a semi reflecting device and is often made by depositing a thin film of germanium onto a flat KBr substrate. Radiation from the broadband IR source is collimated and directed into the interferometer, and impinges on the beam splitter. At the beam splitter, half the IR beam is transmitted to the fixed mirror and the remaining half is reflected to the moving mirror. After the divided beams are reflected from the two mirrors, they are recombined at the beam splitter. Due to changes in the relative position of the moving mirror to the fixed mirror, an interference pattern is generated. The resulting beam then passes through the sample and is eventually focused on the detector. If the two arms of the interferometer are of equal length, the two split beams travel through the exact same path length.

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The two beams are totally in phase with each other; thus, they interfere constructively and lead to a maximum in the detector response. This position of the moving mirror is called the point of zero path difference (ZPD).

When the moving mirror travels in either direction by the distance l/4, the two beams are 180 out of phase with each other, and thus interfere destructively. As the moving mirror travels another l/4, the two beams are again in phase with each other and result in another constructive interference.

Schematic representation of waves and their phases, input, output, and the two arms of the interferometer as the scan goes from zero path difference condition to OPD= . (a) OPD=0 case. (b) /4 OPD case. (c) /2 OPD case. (d) 3/4 OPD case. (e) 1 OPD case.

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Fourier transformation A means of decoding the individual frequencies is required. This can be accomplished via a well-known mathematical technique called the Fourier transformation. This transformation is performed by the computer which then presents the user with the desired spectral information for analysis.

Applications of infrared spectroscopy Quantitative applications Qualitative applications

Quantitative applications The quantitative absorption methods differ some what from ultra-violet molecular spectroscopic methods because of the Greater complexity of the spectra The narrowness of absorption bands Instrumental limitations of the infrared instruments Non adherence to beers law

The quantitative data obtained from dispersive instruments are generally significantly inferior in quality to data obtained with ultra violet visible spectrophotometers. The precision and accuracy of measurements with fourier transform instruments is directly better than those with dispersive instruments.

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Sreekanth Nama, M.Pharm, (Ph.D)


ASSOCIATE PROFESSOR

Absorbance measurement Matched absorbance cells for solvent and solution are generally employed in the ultra violet and visible region and measure absorbance is then found from the relation

This technique seldom practical for measurements in the infrared region because of difficulty in obtaining cells whose transmission characteristics are identical. Most of the infrared cells have very short path lengths i.e. 0.025-0.1mm for assays and which are difficult to duplicate exactly. When compare to UV-Visible spectroscopy the concentrations of solutions used for analysis is differ and 10% solutions are more common in IR. In addition, the cell windows are readily attacked by contaminants in atmosphere and by the solvent.

Thus the transmission characteristics change continuously with the use. For these reason the reference absorber completely is often dispensed with entirely in infrared work. The intensity of radiation passing through the sample is simply compared with that of unobstructed beam. For quantitative work, it is necessary to correct for the scattering and absorption by the solvent and the cell. The so called cell in/cell out procedure, the spectra pure solvent and analyte solution are obtained successfully with reference to the unobstructed reference beam. The same cell used to both measurements.

This transmittance can be determined by

And

Where PT is the power of the unobstructed beam and and are the transmittances of the solvent and analyte solution, respectively. If the remains constant during the two measurements, then the transmittance of the sample with respect to the solvent can be obtained by division of two equations.

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Sreekanth Nama, M.Pharm, (Ph.D)


ASSOCIATE PROFESSOR

Base line method for quantification

An alternative way obtaining transmittance of the analyte with respect to reference is base line method. From obtained transmittance one can calculate the absorbance of the solution. A peak chosen that does not fall too close to others and a straight line is drawn at the base of the band, and P and Po are measured at the absorption peak. In this method transmittance recorded against the wavelength. From the P and Po values absorbance of the solution was measured and plotted against concentration. Qualitative applications Spectral matching Identification tests in the B.P monographs of many drug substances and their formulations are based upon a comparison of the sample spectrum with an authentic spectrum published in B.P. If the spectra are not identical and have been obtained by examination of the sample in the solid state, further tests may be carried out. Additional tests as per pharmacopoeia Recrystallize the sample from the solvent specified and repeat the determination Dissolve sample in a suitable solvent and measure the absorption spectrum against the solvent as blank. Then compare under identical conditions. Different crystalline forms of the same substance give rise to different spectra. Ex. Cortisone acetate, it exhibits five different crystalline forms.

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Sreekanth Nama, M.Pharm, (Ph.D)


ASSOCIATE PROFESSOR

Identification of organic compounds involved in two steps Determining what functional groups are most likely present by examining the group frequency region. Comparison of the spectrum of the unknown with the spectra of pure compound that contain all of the functional groups found in the first step. Finger print region The identity of organic compound can e established from its finger print region below 1500cm-1 and this region rich in many absorptions which are caused bending vibrations and those resulting from C-C, C-O and C-N bonds. Some of the molecules containing the same functional group show similar absorptions above 1500 cm-1 but their spectra differ in finger print region. It is particularly use full because small differences in the structure and the constitution of molecule results in significant changes in the appearance and distribution of absorption peaks in this region. Close match between the spectra in finger print region indicates evidence for identity. Structure determination All the functional groups absorb at their characteristic wave numbers. Based on those frequencies one can determine the structure. Some of group frequencies are
COMPOUND 1. Aromatic hydrocarbons TYPE OF VIBRATION CH stretching 3050-3000 C-H bending 900 700 FREQUENCY RANGE CM -1

1650 1450 C=C stretching C-H out of plane bending 900 690

Ar-H stretching

3050 3000

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Sreekanth Nama, M.Pharm, (Ph.D)


ASSOCIATE PROFESSOR
Mono substituted C H bending 710 690 & 770 730

Disubstituted meta supposition

C H bending

710 - 690 & 800 750

Disubstituted (ortho position)

C C bending

770 735

Disubstituted (para position )

C H bending

840 800

2. Alkanes

C H stretching

2960 -2850

C H bending

1485 1340

C C bending

1300 800

C H bending in CH2 group

1485 1440

C H bending in CH3 group

1470 1430

3. Alkenes

C H stretching

3100 3000

C = C stretching

1680 1600

4. Alkynes

C H stretching

3300 3500

C C stretching

2250 2100

5. Aldehydes

C H stretching

2900 2800

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Sreekanth Nama, M.Pharm, (Ph.D)


ASSOCIATE PROFESSOR
C = O stretching 1750 1720

C O stretching 6. Ketones C O stretching 7. Carboxylic acids O H stretching

1725 1700

1725 1700

3000 2500

O H out and plane bond

930

C O stretching

1300 1000

C O stretching 8. Esters

1750 1735

3700 3500 9. Alcohols Free OH group Hydrogen bonded O H stretching

3400 3200

C O stretching

1300 1000

C O stretching 10. Phenols

1200 1410-1300

3650 3600 Free OH stretching 3400 3200 Hydrogen bonded OH stretching N H stretching 11. Amines N N stretching (2 Amine) 3500 3300 3500 3300

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Sreekanth Nama, M.Pharm, (Ph.D)


ASSOCIATE PROFESSOR

N H bending

1640 1550

C - N stretching 12. Nitriles C N stretching

1350 1000

2260 2290

C - O stretching 13. Amides 1 amides N - H stretching

1690

3500 3400

N - H stretching (Hydrogen bonded)

3350 3180

N - H bending

1620 1590

C = O stretching 2 amides N H stretching

1700 1670

3300 3140

Hydrogen bonded C = O stretching

1670 1630

1620 1535 N = O stretching (aliphatic) 14. Nitro compounds 1375 1275

1570 1500 N = O stretching (aromatic) 1370 1300

15. Sulfur compounds

S - H stretching

2550

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Sreekanth Nama, M.Pharm, (Ph.D)


ASSOCIATE PROFESSOR

S = O stretching 1050 Sulfones, sulfonyl Chloride, sulfates 1350 1140 1375 1300

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Sreekanth Nama, M.Pharm, (Ph.D)


ASSOCIATE PROFESSOR

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Sreekanth Nama, M.Pharm, (Ph.D)


ASSOCIATE PROFESSOR

Intermolecular vibrations and rotations Vibrations: Bonds can stretch, compress and bend like a spring. It is this vibration that is measured in IR-spec. Rotations: Molecules can rotate. Rotations produce waves mainly in the microwave region. However, part of the rotation spectra does overlap with the vibration spectra. Infrared spectra plots transmittance vs. wave numbers, cm-1

Transmittance increases as you go up the y-axis. Where transmittance dips down, that's a region of absorbance. Wave numbers decrease from left to right. Wave numbers are correlated to frequency. Peaks toward the left have higher frequency of vibration.

Recognizing common characteristic group absorptions, fingerprint region


Anything around 3000 cm-1 involves a hydrogen atom, be it O-H, N-H, or C-H. Anything around 2000 cm-1 and below does not involve hydrogen, be it C=O, C=C, C-C, or C-O.

With the same atoms, the higher the bond order, the faster it vibrates, and so the higher the wave number.

1700 cm-1 is for the carbonyl group. Remember this. 3300 cm-1 can be O-H, N-H, or alkyne C-H. OH is the broadest; N-H slightly sharper, alkyne C-H is very sharp.

Broad peaks are due to hydrogen bonding (OH and NH). Below 1300 cm-1 is called the fingerprint region.

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Sreekanth Nama, M.Pharm, (Ph.D)


ASSOCIATE PROFESSOR

Patterns in the fingerprint region are unique for each compound just like a fingerprint is unique for each person.

Key points to be remembered for the interpretation of IR spectrum 1. Look for evidence of alkane, alkenes, alkynes (or) aromatic ring systems by using C - H stretching, C - H bending, C C stretching, Ar - H stretching. 2. Look for OH stretching if it is present and check whether it is free OH stretching or it is associated with any hydrogen bonding. 3. Look for C = O stretching if it is present and check whether it belongs to aldehydes, ketones, carboxylic acids, ester. 4. Look for N - H stretching in amines and check whether it belongs to 1 amine or 2 amine. 5. Look for C = N stretching which is a characteristic peak for nitriles. 6. Look for C - O stretching in carboxylic acid. 7. Look for aromatic C H out of plane bending shows peak in region of 900 690.

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