Biological Spectroscopy

Dr. W. Richard ThilagarajSRM UNIVERSITY

Infrared spectroscopyPrinciples and practice

Infrared spectroscopy

Principles and practice

Infrared interpretation Step 1

Look first for the carbonyl C=O 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 C=O band is present, check for alcohols and go to step 3.

Infrared interpretation Step 2

If a C=O 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.

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.

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.

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.

Infrared interpretation Step 4

If no carbonyl bands and no O-H bands are in the spectrum, check for double bonds, C=C, 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, C=C, 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.

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.

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.

Biological applications

Biological Applications of Infrared SpectroscopyBarbara H. Stuart (Editor), David J. Ando (Editor)

ISBN: 0471974145Publisher: John Wiley & SonsPublished: 01 July 1997 Paperback

Infrared spectroscopyUse in determining protein

secondary structure

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.

Protein Structure

Primary

Secondary

Tertiary

Quaternary

Protein secondary structure

Protein secondary structure

The stretching, normal mode of the carbonyl has been shown to have a specific frequency associated with α-helices, β-sheets and other characteristic structures.

Approximate wave numbers corresponding to the three most common features found in proteins are: - α-helix (1650 cm-1) β-sheet (1632 cm-1 and 1685cm-1) Random coil (1658cm-1)

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.

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.

Near infrared spectroscopy

Biological applications

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.

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).

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.

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.

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)

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.

A visible-light image of a human carotid bifurcation exposed at the

beginning of endarterectomy

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

Raman Spectroscopy

Biological applications

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)

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).

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.

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.

Raman spectroscopy

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.

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.

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.

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.

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).

Raman spectra of sucrose

Raman spectrum of amylose

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 (< 1μm in diameter); these spectra allow the identification of species present in that volume.

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.

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.

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.

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 Nd:YAG are especially common). This reduces the risk of damaging the specimen by applying high power.