Infrared spectroscopy

1
Infrared spectroscopy

• Principles and practice

2
Electromagnetic Spectrum
3
Infrared region of electromagnetic spectrum
4
Infrared spectroscopy

• In the infrared region of the spectrum photons do
  not excite electrons but may induce vibrational
  excitation of covalently bonded atoms and groups.
• Molecules experience a wide variety of
  vibrational motion.
• Virtually all organic compound will absorb
  radiation that corresponds to these vibrations.

5
Experimental approach

• In early experiments infrared light was passed
  through the sample to be studied and the
  absorption measured.
• This approach has been superseded by Fourier
  transform methods.
• A beam of light is split in two with only half of
  the light going through the sample.
• The difference in phase of the two waves creates
  constructive and/or destructive interference and
  is a measure of the sample absorbance.

6
Experimental approach

• The waves are rapidly scanned over a specific
  wavelength of the spectra and multiple scans are
  averaged to create the final spectrum.
• This method is much more sensitive than the
  earlier dispersion approach.

7
Infrared instrumentation

• Modern infrared spectrometers are very different
  from the early instruments that were introduced
  in the 1940s. Most instruments today use a
  Fourier Transform infrared (FT-IR) system.

8
Infrared instrumentation

• A Fourier transform is a mathematical operation
  used to translate a complex curve into its
  component curves. In a Fourier transform infrared
  instrument, the complex curve is an
  interferogram, or the sum of the constructive and
  destructive interferences generated by
  overlapping light waves, and the component curves
  are the infrared spectrum.

9
Infrared instrumentation

• The standard infrared spectrum is calculated from
  the Fourier-transformed interferogram, giving a
  spectrum in percent transmittance (T) vs. light
  frequency (cm-1).

10
Infrared instrumentation

• An interferogram is generated because of the
  unique optics of an FT-IR instrument. The key
  components are a moveable mirror and beam
  splitter. The moveable mirror is responsible for
  the quality of the interferogram, and it is very
  important to move the mirror at constant speed.
  For this reason, the moveable mirror is often the
  most expensive component of an FT-IR
  spectrometer.

11
Infrared instrumentation

• The beam splitter is just a piece of
  semi-reflective material, usually mylar film
  sandwiched between two pieces of IR-transparent
  material. The beam splitter splits the IR beam
  50/50 to the fixed and moveable mirrors, and then
  recombines the beams after being reflected at
  each mirror.

12
Infrared instrumentation
13
Infrared instrumentation
14
Michelson Interferometer
15
Michelson Interferometer

• An FT-IR is typically based on a Michelson
  Interferometer.
• The interferometer consists of a beam splitter, a
  fixed mirror, and a mirror that translates back
  and forth, very precisely.
• The beam splitter is made of a special material
  that transmits half of the radiation striking it
  and reflects the other half. Radiation from the
  source strikes the beam splitter and separates
  into two beams.

16
Michelson Interferometer

• One beam is transmitted through the beam splitter
  to the fixed mirror and the second is reflected
  off the beam splitter to the moving mirror.
• The fixed and moving mirrors reflect the
  radiation back to the beamsplitter. Half of this
  reflected radiation is transmitted and half is
  reflected at the beam splitter, resulting in one
  beam passing to the detector and the second back
  to the source.

17
Infrared instrumentation
18
Common sampling methods

• KBr pellet
• Good for powders a few milligrams of the sample
  power and an excess of KBr are finely ground and
  pressed under high pressure into a pellet. This
  is a useful and very general method for solids.
• Salt cells
• Good for organic liquids the liquid is placed
  into a reservoir milled in alkali salt windows.
• Nujol mull
• The material of interest is suspended in oil,
  such as mineral oil, and the resulting paste is
  spread thinly on a salt window to form a film.
  This is a good technique for oils and waxy solids
  that do not press well into pellets.

19
Infrared spectrum of vanillin
20
Infrared spectra

• The complexity of the spectrum is typical of most
  infrared spectra.
• The gap between 700 and 800 cm-1 is due to
  solvent (CCl4) absorption.
• The inverted display of absorption is
  characteristic.

21
Infrared spectra

• The frequency at the bottom of the spectrum is
  given in units of reciprocal centimetres (cm-1)
  rather than Hz to make the numbers more
  manageable.
• The reciprocal centimetre is the number of wave
  cycles in one centimetre.
• Hz expresses frequency as cycles per second or
  number of wave cycles in 3x1010 cm (the distance
  light travels in one second).
• Wavelength units are expressed in micrometers
  microns (µ) instead of nanometers, again for
  clarity.
• Infrared spectra are displayed using a linear
  frequency scale.

22
Infrared spectra

• Infrared spectra may be obtained from samples in
  all phases (liquid, solid and gaseous).
• Liquids are usually examined as a thin film
  sandwiched between two polished salt plates (note
  that glass absorbs infrared radiation, whereas
  NaCl is transparent).

23
Infrared spectra

• If solvents are used to dissolve solids, care
  must be taken to avoid obscuring important
  spectral regions by solvent absorption.
  Perchlorinated solvents such as carbon
  tetrachloride, chloroform and tetrachloroethene
  are commonly used.
• Alternatively, solids may either be incorporated
  in a thin KBr disk, prepared under high pressure,
  or mixed with a little non-volatile liquid and
  ground to a paste (or mull) that is smeared
  between salt plates.

24
Infrared spectra

• Water has a dipole moment and absorbs light in
  the infrared very strongly.
• It is difficult to measure the infrared spectrum
  of biological materials in water due to the
  spectrum of water obscuring the information.
• A combination of H2O and D2O is often used to
  overcome this problem.
• Hydrated films may also be used.

25
Infrared spectrum of formaldehyde
26
Infrared spectrum of formaldehyde

• A molecule composed of n-atoms has 3n degrees of
  freedom, six of which are translations and
  rotations of the molecule itself.
• This leaves 3n-6 degrees of vibrational freedom
  (3n-5 if the molecule is linear).
• Vibrational modes are often given descriptive
  names, such as stretching, bending, scissoring,
  rocking and twisting.

27
Infrared spectrum of formaldehyde

• The four-atom molecule of formaldehyde, the gas
  phase spectrum of which is shown, provides an
  example of these terms.
• We expect six fundamental vibrations (12 minus
  6), and these have been assigned to the spectrum
  absorptions.

28
General features of infrared spectra

• The exact frequency at which a given vibration
  occurs is determined by the strengths of the
  bonds involved and the mass of the component
  atoms.
• In practice, infrared spectra do not normally
  display separate absorption signals for each of
  the 3n-6 fundamental vibrational modes of a
  molecule.
• The number of observed absorptions may be
  increased by additive and subtractive
  interactions leading to combination tones and
  overtones of the fundamental vibrations, in much
  the same way that sound vibrations from a musical
  instrument interact.

29
General features of infrared spectra

• The number of observed absorptions may be
  decreased by molecular symmetry, spectrometer
  limitations, and spectroscopic selection rules.
• One selection rule that influences the intensity
  of infrared absorptions, is that a change in
  dipole moment should occur for a vibration to
  absorb infrared energy.
• Absorption bands associated with CO bond
  stretching are usually very strong because a
  large change in the dipole takes place in that
  mode.

30
General features of infrared spectra

• Stretching frequencies are higher than
  corresponding bending frequencies. (It is easier
  to bend a bond than to stretch or compress it.)
• Bonds to hydrogen have higher stretching
  frequencies than those to heavier atoms.
• Triple bonds have higher stretching frequencies
  than corresponding double bonds, which in turn
  have higher frequencies than single
  bonds.(Except for bonds to hydrogen).

31
General regions of an infrared spectrum
32
General regions of an infrared spectrum

• Note that the blue coloured sections above the
  dashed line refer to stretching vibrations, and
  the green coloured band below the line
  encompasses bending vibrations.
• The complexity of infrared spectra in the 1450 to
  600 cm-1 region makes it difficult to assign all
  the absorption bands, and because of the unique
  patterns found there, it is often called the
  fingerprint region.
• Absorption bands in the 4000 to 1450 cm-1 region
  are usually due to stretching vibrations of
  diatomic units, and this is sometimes called the
  group frequency region.

33
Features of infrared spectroscopy

• It is rarely, if ever, possible to identify an
  unknown compound by using IR spectroscopy alone.
• The principal strengths are
• It is a quick and relatively cheap spectroscopic
  technique,
• It is useful for identifying certain functional
  groups in molecules and
• An IR spectrum of a given compound is unique and
  can therefore serve as a fingerprint for this
  compound.

34
Infrared spectroscopy

• Basic interpretation

35
Infrared spectroscopy group frequencies
3700 2500 cm-1 Single bonds to hydrogen 2300
2000 cm-1 Triple bonds 1900 - 1500 cm-1 Double
bonds 1400 - 650 cm-1 Single bonds (other than
hydrogen)
36
Infrared group frequencies
37
Infrared group frequencies
38
Infrared group frequencies
39
Infrared group frequencies

• Alcohols and amines display strong broad O-H and
  N-H stretching bands in the region 3400-3100
  cm-1. The bands are broadened due to hydrogen
  bonding and a sharp 'non-bonded' peak can often
  be seen at around 3400 cm-1.
• Alkene and alkyne C-H bonds display sharp
  stretching absorptions in the region 3100-3000
  cm-1. The bands are of medium intensity and are
  often obscured by other absorbances in the region
  (i.e., OH).

40
Infrared group frequencies

• Triple bond stretching absorptions occur in the
  region 2400-2200 cm-1. Absorptions from nitriles
  are generally of medium intensity and are clearly
  defined. Alkynes absorb weakly in this region
  unless they are highly asymmetric symmetrical
  alkynes do not show absorption bands.

41
Infrared group frequencies

• Carbonyl stretching bands occur in the region
  1800-1700 cm-1. The bands are generally very
  strong and broad. Carbonyl compounds which are
  more reactive in nucleophilic addition reactions
  (acyl halides, esters) are generally at higher
  wave number than simple ketones and aldehydes,
  and amides are the lowest, absorbing in the
  region 1700-1650 cm-1.

42
Infrared group frequencies

• Carbon-carbon double bond stretching occurs in
  the region around 1650-1600 cm-1. The bands are
  generally sharp and of medium intensity. Aromatic
  compounds will typically display a series of
  sharp bands in this region.
• Carbon-oxygen single bonds display stretching
  bands in the region 1200-1100 cm-1. The bands are
  generally strong and broad. You should note that
  many other functional groups have bands in this
  region which appear similar.

43
Infrared spectrum of vanillin
44
Examples of infrared spectra
45
Examples of infrared spectra

• The infrared spectrum of benzyl alcohol displays
  a broad, hydrogen-bonded -OH stretching band in
  the region 3400 cm-1, a sharp unsaturated (sp2)
  CH stretch at about 3010 cm-1 and a saturated
  (sp3) CH stretch at about 2900 cm-1 these bands
  are typical for alcohols and for aromatic
  compounds containing some saturated carbon.
• Acetylene (ethyne) displays a typical terminal
  alkyne C-H stretch.

46
Examples of infrared spectra
47
Examples of infrared spectra

• Saturated and unsaturated CH bands are shown
  clearly in the spectrum of vinyl acetate (ethenyl
  ethanoate). This compound also shows a typical
  ester carbonyl at 1700 cm-1 and an example of a
  carbon-carbon double bond stretch at about 1500
  cm-1. Both of these bands are shifted to slightly
  lower wave numbers than are typically observed
  (by about 50 cm-1) by conjugation involving the
  vinyl ester group.

48
Example spectra (n-butanol)
49
Example spectra

• n-Butanol CH3CH2CH2CH2OH
• O-H Stretch 3330 cm-1
• C-O Stretch 1070 cm-1
• Hydrogen Bonded

50
Example spectra (Ethanoic acid)
51
Example spectra

• Ethanoic Acid CH3COOH
• O-H Stretch 3050 cm-1
• CO stretch 1715 cm-1
• C-O Stretch 1295 cm-1
• Hydrogen Bonded

52
Example spectra (2-butanone)
53
Example spectra

• 2-Butanone CH3COCH2CH3
• CO Stretch 1715 cm-1

54
Example spectra (ethyl ethanoate)
55
Example spectra

• Ethyl Ethanoate CH3COOC2H5
• CO Stretch 1710 cm-1
• C-O Stretch 1240 cm-1
• C-O Stretch 1050 cm-1

56
Infrared interpretation

• Step 1
• Look first for the carbonyl CO band.
• Look for a strong band at 1820-1660 cm-1. This
  band is usually the most intense absorption band
  in a spectrum. It will have a medium width. If
  you see the carbonyl band, look for other bands
  associated with functional groups that contain
  the carbonyl by going to step 2.
• If no CO band is present, check for alcohols and
  go to step 3.

57
Infrared interpretation

• Step 2
• If a CO is present you want to determine if it
  is part of an acid, an ester, or an aldehyde or
  ketone. At this time you may not be able to
  distinguish aldehyde from ketone.

58
Infrared interpretation

• ACID
• Look for indications that an O-H is also present.
  It has a broad absorption near 3300-2500 cm-1.
  This actually will overlap the C-H stretch. There
  will also be a C-O single bond band near
  1100-1300 cm-1. Look for the carbonyl band near
  1725-1700 cm-1.
• ESTER
• Look for C-O absorption of medium intensity near
  1300-1000 cm-1. There will be no O-H band.

59
Infrared interpretation

• ALDEHYDE
• Look for aldehyde type C-H absorption bands.
  These are two weak absorptions to the right of
  the C-H stretch near 2850 cm-1 and 2750 cm-1 and
  are caused by the C-H bond that is part of the
  CHO aldehyde functional group. Look for the
  carbonyl band around 1740-1720 cm-1.
• KETONE
• The weak aldehyde CH absorption bands will be
  absent. Look for the carbonyl CO band around
  1725-1705 cm-1.

60
Infrared interpretation

• Step 3
• If no carbonyl band appears in the spectrum, look
  for an alcohol O-H band.
• ALCOHOL
• Look for the broad OH band near 3600-3300 cm-1
  and a C-O absorption band near 1300-1000 cm-1.

61
Infrared interpretation

• Step 4
• If no carbonyl bands and no O-H bands are in the
  spectrum, check for double bonds, CC, from an
  aromatic or an alkene.
• ALKENE
• Look for weak absorption near 1650 cm-1 for a
  double bond. There will be a CH stretch band near
  3000 cm-1.  
• AROMATIC
• Look for the benzene, CC, double bonds which
  appear as medium to strong absorptions in the
  region 1650-1450 cm-1. The CH stretch band is
  much weaker than in alkenes.

62
Infrared interpretation

• Step 5
• If none of the previous groups can be identified,
  you may have an alkane.
• ALKANE
• The main absorption will be the C-H stretch near
  3000 cm-1. The spectrum will be simple with
  another band near 1450 cm-1.

63
Infrared interpretation

• Step 6
• If the spectrum still cannot be assigned you may
  have an alkyl halide.
• ALKYL BROMIDE
• Look for the C-H stretch and a relatively simple
  spectrum with an absorption to the right of 667
  cm-1.

64
Useful paper

• Interpretation of Infrared Spectra, A Practical
  Approach
• John Coates
• Encyclopedia of Analytical Chemistry
• R.A. Meyers (Ed.)
• pp. 1081510837
• John Wiley Sons Ltd, Chichester, 2000
• http//infrared.als.lbl.gov/BLManual/IR_Interpreta
  tion.pdf

65
Biological applications
Biological Applications of Infrared
SpectroscopyBarbara H. Stuart (Editor), David J.
Ando (Editor)ISBN 0471974145Publisher John
Wiley SonsPublished 01 July 1997 Paperback
66
Infrared spectroscopy

• Use in determining protein secondary structure

67
Secondary structure of proteins

• Changes in the group frequencies may be used to
  derive information regarding the secondary
  structure of biological molecules.
• The carbonyl group of the amide bond in proteins
  is particularly useful for the determination of
  secondary structure.

68
Protein Structure
Primary
Secondary
Tertiary
Quaternary
69
Protein secondary structure
70
Protein secondary structure

• The stretching, normal mode of the carbonyl has
  been shown to have a specific frequency
  associated with a-helices, ß-sheets and other
  characteristic structures.
• Approximate wave numbers corresponding to the
  three most common features found in proteins are
  -
• a-helix (1650 cm-1)
• ß-sheet (1632 cm-1 and 1685cm-1)
• Random coil (1658cm-1)

71
Protein secondary structure

• Known structures are often used to calibrate the
  vibrational frequency measurements.
• The vibrational spectrum of the amide bond of a
  protein is often complex because of the many
  amide bonds in multiple environments.
• The spectrum can be deconvoluted to provide
  information about the amount and types of
  secondary structures present.

72
Protein secondary structure

• The assumption is usually made that the observed
  vibrational frequency is a linear combination of
  the frequencies associated with the various
  secondary structures that are present.
• Each frequency is weighted by the percent of a
  given structure present.
• Results are compared with known structures and
  with circular dichroism measurements.
• Raman spectroscopy has proved particularly useful
  since water solutions can be used.

73
Near infrared spectroscopy

• Biological applications

74
Infrared region of electromagnetic spectrum
75
Near-Infrared spectroscopy

• Near-IR spectrometry is characterized by low
  molar absorptivities and scattering, which permit
  nearly effortless evaluation of pure materials,
  and broad overlapping bands, which diminish the
  demand for a large number of wavelengths in
  calibration and analysis.
• The near-IR region of the electromagnetic
  spectrum, once regarded as having little
  potential for biological work, has become one of
  the most promising for molecular spectrometry.

76
Near-Infrared spectroscopy

• The advent of inexpensive and powerful computers
  has contributed to the surge of near-IR
  spectrometric applications.
• The near-IR region is usually estimated to
  include wavelengths between 700 nm (near the red
  end of the visible spectrum) and 3000 nm (near
  the beginning of infrared stretches of organic
  compounds).

77
Near-Infrared spectroscopy

• Absorbance peaks in the near-IR region originate
  from overtones and combinations of the
  fundamental (mid-IR) bands and from electronic
  transitions in the heaviest atoms.
• For example, C-H, N-H, and O-H bonds are
  responsible for most major absorbances observed
  in the near-IR, and near-IR spectrometry is used
  chiefly for identifying or quantifying molecules
  including unique hydrogen atoms.

78
Near-Infrared spectroscopy

• Near-IR spectrometry is thus in routine service
  for quantitative analyses of water, alcohols,
  amines, and any compounds comprising C-H, N-H,
  and/or O-H groups. Numerous other elementary bond
  combinations are also likely to generate near-IR
  absorbance peaks.

79
Advantages of near-infrared spectroscopy

• Analysis times under 1 second are possible
• Simultaneous multicomponent analysis is the norm
• No sample preparation is usually required for
  liquids, solids, or gases.
• Non-invasive and non-destructive analysis is
  possible
• Cost per analysis is very low (no reagents are
  used)

80
Advantages of near-infrared spectroscopy

• Physical properties and biological effects can be
  calculated from spectra of samples.
• Automated correction of background and
  interferences is performed in instruments using
  computer algorithms
• Detection limits can be very low
• A wide range of sample sizes can be analyzed.
• Molecular structural information can be derived
  from spectra.

81
Near infrared spectrum of dichloromethane
82
Near infrared spectrum of liquid ethanol
83
A visible-light image of a human carotid
bifurcation exposed at the beginning of
endarterectomy
84
Near-IR images at 6 selected wavelengths (from
top to bottom and left to right, 1678, 1944,
2098, 2180, 2230, and 2312 nm) of the carotid
85
Raman Spectroscopy

• Biological applications

86
Raman Spectroscopy

• This is an alternative approach to studying
  transitions between vibrational energy levels.
• In addition to absorbing light samples also
  scatter light.
• The amount of scattered light is at a maximum at
  90º to the beam.
• Most of the scattered light is at the same
  frequency as that of the incident light (Rayleigh
  scattering)

87
Raman Spectroscopy

• At the molecular level the electric field of the
  light perturbs the electron distribution but no
  transitions between energy levels occurs.
• Scattering is inversely proportional to the forth
  power of the wavelength.
• This is why the sky is blue (Shorter wavelengths
  (blue) are scattered more than longer wavelengths
  (red).

88
Raman Spectroscopy

• Rayleigh scattering is observed at all
  wavelengths.
• The intensity of the scattered light is related
  to the polarisability of the molecules.
• A small number of molecules return to a different
  vibrational energy level after scattering.

89
Raman Spectroscopy

• The vibrational energy level can be either higher
  or lower than the initial state.
• As a result of this change some of the scattered
  light will be at a slightly higher or lower level
  frequency than the incident light.
• This is called Raman scattering.

90
Raman spectroscopy
91
Raman spectroscopy

• The Raman effect occurs when light impinges upon
  a molecule and interacts with the electron cloud
  of the bonds of that molecule.
• A molecular polarizability change, or amount of
  deformation of the electron cloud, with respect
  to the vibrational coordinate is required for the
  molecule to exhibit the Raman effect.
• The amount of the polarizability change will
  determine the intensity, whereas the Raman shift
  is equal to the vibrational level that is
  involved.

92
Raman spectroscopy

• The incident photon (light quantum), excites one
  of the electrons into a virtual state.
• For the spontaneous Raman effect, the molecule
  will be excited from the ground state to a
  virtual energy state, and relax into a
  vibrational excited state, and which generates
  Stokes Raman scattering.
• If the molecule was already in an elevated
  vibrational energy state, the Raman scattering is
  then called anti-Stokes Raman scattering.

93
Raman Spectroscopy

• A very intense light source is required to
  observe Raman scattering because only a very
  small amount of the scattered light displays a
  change in frequency.
• The advent of lasers has permitted this to be
  done routinely.
• Higher concentrations are often required.
• Raman spectroscopy is a scattering phenomenon as
  compared with absorption spectroscopy.

94
Advantages of Raman Spectroscopy

• A permanent dipole moment is not required.
• It is only necessary for the polarisability of
  the molecule to change between different
  vibrational energy levels.
• Visible light may be used rather than infrared
  light so than Raman spectra may be readily
  obtained in water.
• Crystals and films may also be studied.

95
Raman Spectroscopy

• Due to the fact that the intensity of the Raman
  lines is weak, intense light sources and higher
  concentrations than those needed for infrared
  spectroscopy are often needed.
• Raman and infrared are often regarded as
  complimentary techniques.
• Since infrared depends on a permanent dipole
  moment and Raman on the polarisability a
  vibrational transition is usually observed in
  either the infrared or Raman spectrum (but not
  both).

96
Raman spectra of sucrose
97
Raman spectrum of amylose
98
Raman microscopy

• Raman spectroscopy offers several advantages for
  microscopic analysis. Since it is a scattering
  technique, specimens do not need to be fixed or
  sectioned.
• Raman spectra can be collected from a very small
  volume (lt 1µm in diameter) these spectra allow
  the identification of species present in that
  volume.

99
Raman microscopy

• Water does not interfere very strongly. Thus,
  Raman spectroscopy is suitable for the
  microscopic examination of minerals, materials
  such as polymers and ceramics, cells and
  proteins.
• A Raman microscope begins with a standard optical
  microscope, and adds an excitation laser, a
  monochromator, and a sensitive detector (such as
  a charge-coupled device (CCD) or photomultiplier
  tube (PMT)). FT-Raman has also been used with
  microscopes.

100
Raman microscopy

• In direct imaging, the whole field of view is
  examined for scattering over a small range of
  wavenumbers (Raman shifts). For instance, a
  wavenumber characteristic for cholesterol could
  be used to record the distribution of cholesterol
  within a cell culture.

101
Raman microscopy

• Another approach is hyperspectral imaging, in
  which thousands of Raman spectra are acquired
  from all over the field of view. The data can
  then be used to generate images showing the
  location and amount of different components.
  Taking a cell culture example, a hyperspectral
  image could show the distribution of cholesterol,
  as well as proteins, nucleic acids, and fatty
  acids. Sophisticated signal- and image-processing
  techniques can be used to ignore the presence of
  water, culture media, buffers, and other
  interferences.

102
Raman microscopy

• Raman microscopy, and in particular confocal
  microscopy, has very high spatial resolution. For
  example, the lateral and depth resolutions are
  250 nm and 1.7 µm, respectively, using a confocal
  Raman microspectrometer with the 632.8 nm line
  from a He-Ne laser with a pinhole of 100 µm
  diameter.
• Raman microscopy for biological and medical
  specimens generally uses near-infrared (NIR)
  lasers (785 nm diodes and 1064 nm NdYAG are
  especially common). This reduces the risk of
  damaging the specimen by applying high power.

103
Raman Imaging

• Mapping of Wheat Grain Kernels

104
Raman spectrum of kernel
105
Raman mapping of components
Raman analysis was carried out with a LabRAM
Raman microscope, using a 633 nm HeNe laser
(typically 8 mW on sample). Raman images
consisting of 15x11 point analyses were recorded
on 50 µm thick solid sections, obtained with a
cryomicrotome.