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Pharmaceutical Analysis

INFRARED SPECTROSCOPY(Main Conceptes)

DR.WAEL ABU DAYYIH

Faculty of Pharmacy & Medical Science

Petra University2012

Infrared Spectroscopy

Gives information about the functional groups in a molecule

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Outline - What is spectroscopy ? - Electromagnetic spectrum. - IR active and inactive. - Infrared spectroscopy. - IR instrument. - Region of infrared spectrum. - Fingerprint molecule. - IR table. - Infrared energy mode.

IR CHART

42000200035003500 30003000 25002500 1000100015001500 500500

H—CH—COO

CHCH33CHCH22CHCH22CHCH22CCHCCH33

C=OC=O

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- Vibration modes leading to IR absorption.

- How to prepare IR sample.

- FT – IR spectrometer.

- References.

Outline

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What is Spectroscopy?

- Shine light ( energy ) on a sample to determine what is inside.

- Measure the amount of electromagnetic radiation absorbed and / or emitted by a compound.

- Different molecules absorb at different energies.

IR- SPECTROSCOPY

IR electromagnetic radiation ranging 600-4000 cm-1 is passed through a sample and is absorbed by the bonds of the molecules in the sample causing them to stretch or bend the wave length of the radiation absorbed is characteristics of the bond absorbing it

Theory of IR absorption : IR radiation doesn't have enough energy to induce electronic transition as seen with UV-VIS

Exploits the fact that molecules absorb specific frequencies that are characteristics of their structure

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IR- SPECTROSCOPY

The result of this energy absorption is an increased amplitude for the vibration

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IR Instrument

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DetectorDetectorSampleSample

CompartmentCompartment

IR SourceIR Source

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Electromagnetic Spectrum

Each form of spectroscopy uses a different part of the electromagnetic Spectrum

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IR Table

- Tables of Values

Wave number ( cm-1 (

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Region of the Infrared Spectrum IR region is lower in photon energy than visible light

(below red – produces heating as with a heat lamp( IR radiation – just outside visible light 400 –800 nm (infra

red( Wave length 2,500 nm – 16,700 nm Energy: 0.1 – 35 kcal/mol causes molecular vibrations IR energy in a spectrum is usually measured as

Wavenumber (cm-1(, the inverse of wavelength and proportional to frequency (Wavenumber (cm-1( = 1/ (

so IR Spectrum range: 600 – 4,000 cm-1

- Below 1500 cm-1 “ fingerprint “region

Region of the Infrared Spectrum

4000-2500 cm-1 N-H, C-H, O-H (stretching( 3300-3600 N-H, O-H 3000 C-H 2500-2000 cm-1 CºC and C º N (stretching( 2000-1500 cm-1 double bonds (stretching(

C=O 1680-1750 C=C 1640-1680 cm-1

- Below 1500 cm-1 “ fingerprint “region

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Uses of IR 1. Fingerprint.

2. Determination of structure information.

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Terminology * Fundamental absorption : V0 V1

* Overtones : V0 V1 or V1 V3 higher energy is required the absorption wavelength is shorter

*Combination band : when two vibration

Frequencies couple to give rise to a vibration of a new frequency ν

comb= ν

1 + ν

2

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*Difference band : the observed frequency is

a result of the difference between the two interacting bands

ν diff

= ν 2 – ν

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* Fermi resonance : occurs when a fundamental absorption is coupled with an overtone or combination band mostly in C=O

* The energy difference between the vibrational levels is modified by non- IR active rotational energy levels which add or subtract from the vibration energy levels

*This modifications leads to broad bands of spectra.

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IR ( Fingerprint ) * The frequency of the IR region of most uses

( vibrational IR ) is from 2.5 – 25 µm.

* While the whole region extends from 0.78 – 40 µm

* In IR charts it is normal to find wavelength expressed as wave number in units of cm-1

* 2.5-25 µm = 4000-400 cm-1 where E is directly proportional to wave number

/ λ where wavelength in µm * E = h ν

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* Using wave number provides easier interpretation of charts.

µm = ( 1 / cm-1) * 10000 µm * cm-1 = 10000

*So , Wavelength * Wave number( ) = 10000 ~

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Fingerprint of molecule

* Whole- molecule vibrations and bending vibrations are also quantitized.

* No two molecules will give exactly the same IR spectrum ( except enantiomers).

* Simple stretching : 1600-1400 cm-1.

* Complex vibrations : 600 - 1400 cm-1, called the “ fingerprint region.”

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Requirements for IR absorption : ( covalent bond involved (

1. Wave length of absorbed radiation : - Molecules absorb radiation when part of the molecule

vibrates at the same frequency as the incident radiant energy. - After absorbing radiation the molecules vibrate at an

increased amplitude.( see slide 8( - Molecules atoms can vibrate in several ways the rate at

which this atoms vibrate is quantized and only at will defined frequency that are characteristic of the atoms concerned.

- Since only radiation with certain frequency can produce this effect its wavelength is characteristic, and when measuring A

Those frequencies are recorded this record is the basis for IR spectrum of that molecule.

- The energy of the photon absorbed = energy difference between the vibrational energy states

- Vibration stretch and bending of the bonds.

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2 .Moment dipole: *Not all molecules can absorb in IR region► for absorption there must be

change in the dipole moment (polarity)of the molecule

• A change in dipole moment must occur when vibrational excitation occurs or IR absorption will not take place.

* The rate of change of the dipole during vibration is great absorbance is intense.

* If charge is large vibration is rapid rate of change of dipole is rapid absorption is intense.

* Symmetric molecules don’t absorb as H2 and Cl2

* Pseudo symmetric molecules don’t absorb Ex. symmetric alkenes and alkynes ( CH3 )2 –C = C- ( CH3 )2

CH3 – C ≡ C- CH3

* Ex. Pseudo symmetric alkenes and alkyne CH3 CH2 –C = C- (CH3)2

CH3

CH3 CH2 -C ≡C-CH3

H H

O

IR-Active and Inactive A polar bond is usually IR-active. A nonpolar bond in a symmetrical molecule will absorb

weakly or not at all

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H H

O

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Notes:* Triple bonds are stronger than double bonds which are stronger than

single bonds ≡ > = >- K3 >K2 >K1

K3 =15*105 dyne/cm, K2 = 10*105 dyne/cm, K1 = 5*105 dyne/cm is at lower energy levels

3 > 2 > 1 due to increasing K* As mass of the atom increases µ increases frequency decreases* Bending occur at lower energy levels than stretching* HybridizationSP > SP2 > SP3

KSP > KSP2 > KSP3

*

~ ~~

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Bond prosperities and absorption trends

* IR absorption frequency is affected by:

* bond strength

* masses of the bonded atoms

( Two masses connected with a spring since the avg distance is changing (

= 1/2П√k/ µ µ= mAmB )/mA+mB

* 1/2П = 4.12

* K= force constant dyne/cm

* µ = reduced mass dyne= 1.020*103 g *m-atomic weights

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Absorption trends

Factors that determining the absorption of IR:

1(The intensity: with which a bond absorb radiation which depends on its dipole moment and intensity depend on electro- negativity of the atoms involved in bond

2( Energy level Evib.

= √k/ µ

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Energy Absorption and Vibration1. IR electromagnetic radiation is just less energetic than visible light

2. This energy is sufficient to cause excitation of vibrational energy levels

3. Wavelength (( = 2.5-16.7 x 10-6 m

4. = wavenumbers. Larger = higher energy

5. Excitation depends on atomic mass and how tightly they are bound Hooke’s Law for 2 masses connected by a spring

k = constantf = force constant = bond strengthm-term = reduced mass

Many possible absorptions per molecule exist: stretching, bending,…

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kcal/mol 35– 0.1cm 4000-6001~ 1

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21 )(~mm

mmfk

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*Conclusion:

K is effected by : 1. Bond order 2. Mean distance 3. Electro negativity of the vibrating atoms Ex. Calculate for C = C K=10*105 dyne/cm µ = ( 12*12) / (12+12)=6 = 4.12 * ( 10*105/6) ½= 1682 cm-1 ( calculated) 1650 cm-1 (experimental) ~

~

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Infrared Spectroscopy

* Evaluates covalent bond stretching *Infrared ( IR ) spectroscopy measures the bond

vibration frequencies in a molecule and is used to determine the functional group.

*IR spectra allow one to determine the presence or absence of certain functional groups ( i.e bond between O-H )

*IR easily detects- OH,-NH,-NO2

*Especially useful for detecting and distinguishing among C=O containing compounds

*Liquid, solid or gas samples *Rapid scanners linked to computers

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Absorption Spectra* Electronic energy ( UV/Vis ) - Absorption or emission in ultraviolet and visible - region (27.000 cm-1 to 13.000cm-1 )

* Rotational Energy ( microwave) - Absorption in far infrared region (400cm-1 to

50 cm-1 )

* Vibrational Energy ( infrared ) - Absorption in near , mid, and infrared region

( 12.800 cm-1 to 50 cm-1)

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Infrared Energy Modes

* IR energy absorption corresponds to specific modes, corresponds to combination of atomic movements, such as bending and stretching of bonds between groups of atoms called “normal modes “

* Energy is characteristic of the atoms of the group and their bonding

* Corresponding to vibrations and rotations

IR-Vibration modes

In general each atoms has 3 types of freedom(freedom of motion(

For linear molecules ,the number of vibrational mode equal (3N-5(

But, for non linear molecules the number of vibrational mode equal (3N-6(

Where N , No. of atoms

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IR-Vibration modes

Carbon dioxide has 3 x 3 - 5 = 4 vibrations

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Vibration modes

*The modes of IR stretching and bending

1- stretching: - vibration : Change in inter-atomic distance along bond axis and can e divided into two categories :

a . Symmetrical: Two atoms either move towards or away from the central carbon atom

b . Asymmetrical: In this case one H atom approaches the carbon atom while the other moves away from the carbon atom

- CH3 Symmetrical at 2872cm-1

- CH3 Asymmetrical at 2962 cm-1

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2- bending: vibration- change in angle between bonds and there are 4 types of bend-:

a . Wagging: The H atoms swing out of the plan weather back- forth or forth -back

b . Twisting: The structural unit rotates out of the plan about the bond

c . Rocking: The H atoms swing in plan weather forth- forth or back -back

d .Scissoring: The H atoms swing in plan weather forth and back or back and forth

a and b are out of plane which c and d are in plane

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Vibration modes

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An Infrared Spectrometer

An Infrared Spectrometer

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InfraredInfrared SourceSource

SampleSample CompartmentCompartment

DetectorDetector

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InstrumentationA. Radiation source

-Nernst glowers : zirconium, cerium, and thorium oxides ( 1200-2000K)

- Goobers: sintered silicon carbide - Nichrome wire

*They are electrically heated to 1000-1500C at such elevated temp the source emits IR radiation

*Advantages:1 .Continuous intensity

2 .Constant3 .Radiation extends over a wide wavelength range

*Dis- advantage: Intensity is not the same at all wavelength but rate of change is

gradualBut

1 .Double beam instruments compensate for that

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B.Monochromators: Function: to select radiation of a desired frequency from the

source and eliminate that of the other frequencies

1.Prism: ( NaCl, KBr, CaF2) - The choice among the materials is determined by the

wavelength range to be examined in addition , it has to be IR transparent and strong enough to be polished and shaped.

- The metal salts used to made the prisms are water soluble so if surface is wet it dissolves

- If dried itching “ its important to keep the prism dry” - Equipment use small heaters to keep temp of prism

above room temp at all times to prevent condensation of water vapor from the atmosphere on its surface

- If condensation occurs scattering of radiation - These are special tables to provide information about

the prism material including transmission range , solubility and R.I.

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2 .Gratings

* Increasingly popular * Made with materials that are stable in the

atmosphere and not affected by moisture * Can be used over a wide range of

wavelength in contrast to prisms * Problem: different orders of wavelength

travel the same light path upon leaving the face of the grating can be solved by using prisms or filters as order sorters

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C. Detectors

Slit Systems: * At longer wavelengths, the energy of each photon is decreased and the power from the source drops off and prisms if used lose their transparency

* So detectors will receive reduced light levels and thus the response of the detector will decrease

* To solve this problem use of programmed slit system

* As slit width increases, the intensity of radiation increases but this is accompanied by decrease in resolution

* This may lead to interference from unresolved bands in quantitative analysis

Lack of response from the detector is due to the above

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Response time: Time required for a detector to reach a steady signal when

radiation falls on it.

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How to Prepare IR liquid sample

IR transparent Salt Plates

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D. Sample cells: * Materials must be transparent to IR radiation ( NaCl and

KBr) * Choice depends on the wavelength range to be examined * NaCl 4000-650 cm-1 * KBr 4000-400 cm-1 Solid samples: 1. Solid samples may be grouped to a powder that can be

made into a thick slurry or mull by grinding it with a greasy liquid as Nujol ( paraffin oil) or chlorofluorocarbon greases. The thick suspension is placed between salt plates.

* Disadvantage: mineral oil obscures bands may be present in the analyzed compound

* Nujol bands appear at 2924 cm-1,1462 cm-1, 1377 cm-1

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2. KBr pellet: mixing finely ground solid powder with

powdered KBr and then the mixture is compressed to make a disc.

This pellet is inserted into a holder in the spectrometer.

* Disadvantages: KBr absorbs water 3. sample is placed on the surface of the solid to

dryness. Then IR radiation is passed through this thin layer deposited.

Useful in rapid qualitative analysis. 4. Dissolving the solid sample in a solvent

(CCl4) .Has a band at 785 cm-1 due to C-Cl stretch

Solid samples do not yield fine spectra as liquid samples so they are best avoided.

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Cells for gaseous samples: * Surface in the light path is made of KBr , NaCl,

….. * Large cells are used to compensate for the

small number of molecules of a sample * The gas must not react with the cell windows

or the reflecting surfaces * Multiple reflections can be used to make the

effective path length as long as 40 m so that constituents of the gas can be determined.

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*Diamond cells: - For longer λ- Advantages: transparent,

hard, stable, not affected by water.

- Uses to study metal oxides as Ag2O and TiO2

IR Handling Sample

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DessicatorDessicator

Water-freeWater-freeEnvironmentEnvironment

forfor Water-sensitiveWater-sensitive

Salt PlatesSalt Plates..

The plates must also be handled with The plates must also be handled with gloves to avoid contact of the plate gloves to avoid contact of the plate with moisture from one’s handswith moisture from one’s hands..

IR Handling Sample

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To run an IR To run an IR spectrum of a liquid spectrum of a liquid sample, a drop or sample, a drop or two of the liquid two of the liquid sample is applied to sample is applied to a salt platea salt plate..

IR Handling Sample

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The cell holder is then The cell holder is then placed in the beam of placed in the beam of the instrumentthe instrument..

IR Handling Sample

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Light PathLight Path))shown by red lineshown by red line((

The sample is then scanned The sample is then scanned by the instrument utilizing by the instrument utilizing predesignated parameterspredesignated parameters..

A relevant background scan A relevant background scan should already have been should already have been takentaken..

A sample of a printout of an IR spectrumA sample of a printout of an IR spectrum..

Click Here to Start ScanClick Here to Start Scan

IR Handling Sample

Well-definedWell-definedpeaks arepeaks are

labeled with the labeled with the WavenumbersWavenumbers of the Absorptionof the Absorption MaximaMaxima..

A satisfactory spectrum has A satisfactory spectrum has well defined peaks-but not so well defined peaks-but not so intense as to cause flattening intense as to cause flattening on the bottom of the peakson the bottom of the peaks..

Major peaks can be labeledMajor peaks can be labeled using the peak function of using the peak function of

the softwarethe software

The spectrum can then be The spectrum can then be printed using the print printed using the print function of the softwarefunction of the software..

DON’T FORGET!!!

The salt plates are cleaned by rinsing into The salt plates are cleaned by rinsing into a waste container with a suitable organic a waste container with a suitable organic solvent-commonly cyclohexane.solvent-commonly cyclohexane.

NEVER WATER!NEVER WATER!

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Info slide!!!……

Cloudy plates must be polished to return Cloudy plates must be polished to return them to a transparent condition.them to a transparent condition.

To polish cloudy windows, rotate salt plate To polish cloudy windows, rotate salt plate

on polishing cloth.on polishing cloth.

The clean plates and cell holder are stored The clean plates and cell holder are stored in the moisture free atmosphere of a in the moisture free atmosphere of a dessicator.dessicator.

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Solid samples

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Continued IR sample

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FT- IR Spectrometer

- Uses an interferometer. - Has better sensitivity. - Less energy is needed from source. Completes a scan in 1-2 seconds. - Takes severals scans and averages

them. - Has a laser beam that keeps the

instrument accurately calibrated.

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IR spectrophotometers: 1. Dispersive 2. Fourier transform IR ( FTIR) - They both provide information in the

common range of 4000 – 400 cm-1

- Both provide identical spectra for a given compound but FTIR provides the spectra much more rapidly than the dispersive instruments

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*FTIR - Involves interferogram: complex signal, but wave- like

pattern contains all the frequencies which make up the IR spectrum .It is plot of intensity vs. ( time domain spectrum) .

- FT which is a mathematical operation can separate the individual absorption frequencies from the interferogram producing a spectrum identical to that obtained with the dispersive spectrometer

- Advantage: acquire interferogram in less than a second thus can correct dozens and store them in a computer.

When a FT is performed on the sum of accumulated interferogrames better S/N ratio , better sensitivity, and greater speed.

- FTIR operate on single beam mood.

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Advantages of FTIR

1. Multiplex advantage : scan the spectrum simultaneously2. Avoid narrow slits “ throughput advantage”3. The use of interferometer gives excellent wave number

reproducibility4. The use of computer facilitated data processing5. Single beam reference is stored in the computer memory 4 and 5 lead to : - Can be used on opaque or very diluted samples

- Improves S / N ratio - Spectral subtraction

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Multiple advantage: the sample is exposed to the entire radiation beam at the same time and absorption effects at all wavelengths which will modify the final signal

Figure showing a dispersive infrared spectrometer : a beam chopper is a rotating sector that passes two beams alternately to a diffraction grating.

* The spectrum is recorded as the frequency of IR radiation changes by the rotation of the diffraction grating.

* It is customary to plot frequency vs. light transmitted , not light absorbed.

* This is recorded as percent transmittance %T because the detector recorded the ratio of the intensities of the two beams

%T = Is / Ir * 100% * Max absorption is the minimal point i=on chart * Double beam record a spectrum in the frequency domain. Eliminates solvent and temperature effects since the use of

a reference.

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Comparison of dispersive and FTIR

Dispersive IRFTIR

1 -Many moving parts result in mechanical slippage.

2 -Calibration against reference spectra required to measure frequency.

3 -Star light within instrument causes suprious readings.

1 -Only mirrors move during an experiment.

2 -Use of laser provides high frequency accuracy (to 0.01cm-1).

3 -Stray light does not affect detector since all signals are modulated.

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Dispersive IRFTIR

4 -In order to improve resolution , only small amount of IR beam is allowed to pass through the slits.

5 -Only narrow frequency radiation falls on the detector at any time.

4 -Much larger beam may be used at all times ;data collection is easier.

5 -All frequencies of radiation fall on detector simultaneously ; improved S/N ratio obtained quickly ( Felget advantage (.

6 -Rapid scan speeds permit monitoring samples undergoing rapid change.

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Dispersive IRFTIR

6 -Slow scan speeds make dispersive instruments to slow for monitoring systems undergoing rapid change

) gas chromatographic effluents.(

7 -Sample subject to thermal effects from the focused beam.

8 -Any emission of IR radiation by sample will fall on detector.

9 -No multiplex advantage.

7 -Beam is not focused; hence sample is not subject to thermal effects.

8 -Any emission of IR radiation by sample will not be detected.

9 -Simultaneous interference by all wavelengths on all other wavelengths gives more complete spectra

(multiplex advantage.(

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Limitations for dispersive IR:

- Dealing with weak sources of radiation

- Relativity insensitive detector- Part of the spectrum is scanned

at any one moment - Use of narrow slit restrict the

light beam

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Attenuated total reflectance (ATR ) - Used for hard materials, flat surfaces, paint on a door or

wall, examining art objects - This system is an attachment that can be added to any

conventional IR system and is used for sampling - It is consists a material that is transparent to IR ( NaCl, KBr ,….) . Radiation is reflected frim the ligt source onto the thimble

and back out again into the IR light path. Then the thimble is immersed in the sample and scanned again. Loss of radiation will be observed

- Refractive index of the media > RI of the sample - Solid samples must be in actual contact with the thimble ART can also be used to monitor organic reactions , other

uses: 1- analysis of opaque material as inks , glues, stains 2- study of fossils

Strength of IR

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

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

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Limitations of IR

Rarely used as quantitative (determination( technique because of relative difficulty in sample preparation and the complexity of spectrum .

Usually can only detect gross impurities in sample. Sample preparation requires a degree of skills

particularly when KBr discus are being prepared. The technique is lacking in robustness since sample

handling can have an effect on the spectrum obtained and thus care has to be taken in sample processing

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Base Values for absorption of bonds

O-H3400C≡C2150

N-H3400C=O1750

C-H3000C=C1650

C ≡N2250C-O1100

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DR.WAEL ABU DAYYIH