Saturday, April 12, 2014

Spectroscopy

INFRARED SPECTROSCOPY
INTRODUCTION
The infrared portion of the electromagnetic spectrum is divided into three regions; the near-, mid- and far- infrared, named for their relation to the visible spectrum. The far-infrared, approximately 400-10 cm-1 (1000–30 μm), lying adjacent to the microwave region, has low energy and may be used for rotational spectroscopy. The mid-infrared, approximately 4000-400 cm-1 (30–1.4 μm) may be used to study the fundamental vibrations and associated rotational-vibrational structure. The higher energy near-IR, approximately 14000-4000 cm-1 (1.4–0.8 μm) can excite overtone or harmonic vibrations. Photon energies associated with this part of the infrared (from 1 to 15 Kcal/mole) are not large enough to excite electrons, but may induce vibrational excitation of covalently bonded atoms and groups. 
Figure 1: Electromagnetic spectrum showing the infrared regions
PRINCIPLE
The absorption of electromagnetic radiations in the infrared regions results in vibrational and rotational transitions of the bonding atoms in the molecule leading to asymmetrical charge distribution. Non linear molecules may undergo vibrational motions like stretching and deforming. The energy requirement of stretching vibrations (either symmetrical or asymmetrical) are higher and occur at higher frequencies than deforming or bending (either in plane; scissoring or out of plane; rocking i.e. wagging and twisting) vibrations.
The absorption of infrared radiations doesn't cause excitation of electrons but induces vibrations of bonding atoms. The vibrations which bring about alteration in dipole moment or displacement of charge can be detected. The other types of vibrations are studied by Raman spectroscopy.
Figure 2: Mode of vibrations of molecules in infrared lights


Figure 3: Electromagnetic spectra of IR-Spectroscopy
The fundamental vibrations are those which correspond to V0 – V1 transitions. The intensity of a particular absorption depends on the dipole moment of the molecule in the ground state and vibrational excited state. Higher is the difference in dipole moments, greater will be the intensity of absorption. The IR spectra are observed only in heteronuclear molecules since homonuclear molecules have no dipole moment. For obtaining an infrared spectrum, electromagnetic radiation of increasing wavelength is passed through the sample and percent transmittance or absorbance is measured. The energy of vibrations is quantized. The IR spectrum is functional groups in molecules are highly specific and this characteristic spectrum facilitates identification of compounds according to frequency units rather than wavelength.
INSTRUMENTATION
A beam of infrared light is produced and split into two separate beams. One is passed through the sample, the other passed through a reference which is often the substance the sample is dissolved in. The beams are both reflected back towards a detector, however first they pass through a splitter which quickly alternates which of the two beams enters the detector. The two signals are then compared and a printout is obtained. The IR instruments have following parts;
1. IR source: The source of light is generally an incandescent lamp having output maxima at around 2 cm. However their energy shows a sharp decline (15% of maximum at 15 cm). Neonest-Glober and nichrome alloy wire lamps have been used for this purpose.
Figure 4: Diagrammatic representation of IR-spectroscopy
2. Sample containers: The sample containers used in spectrophotometer such as Glass, quartz, silica cuvettes are impermeable to infrared lights. Non-covalent materials are transparent to infrared light. Thus sodium chloride or common salt plates are used commonly as sample containment. A number of other salts such as potassium bromide or calcium fluoride are also used. The plates are transparent to the infrared light and will not introduce any lines onto the spectra. Some salt plates are highly soluble in water, so the sample and washing reagents must be anhydrous (without water). The window materials for IR-spectroscopy;            NaCl                6000 – 600 cm–1
KBr                 6000 – 450 cm–1
CsI                  6000 – 200 cm–1
Polyethylene   < 600 cm–1
3. Sample Preparation: Gaseous samples require little preparation beyond purification, but a sample cell with a long pathlength (typically 5-10 cm) is normally needed, as gases show relatively weak absorbance.
Liquid samples can be sandwiched between two plates of a high purity salts and measure the absorbance.
Solid samples can be prepared in two major ways. The first is to crush the sample with a mulling agent (usually Nujol; which is hydrocarbon oil, with strong absorption around 2900 and 1400 cm–1) in a marble or agate mortar, with a pestle. A thin film of the mull is applied onto salt plates and measured. The second method is to grind a quantity of the sample with a specially purified salt (usually potassium bromide) finely (to remove scattering effects from large crystals). This powder mixture is then crushed in a mechanical die press to form a translucent pellet through which the beam of the spectrometer can pass.
It is important to note that spectra obtained from different sample preparation methods will look slightly different from each other due to differences in the samples' physical states.
The last technique is the Cast Film technique. This technique is used mainly for polymeric compound. Sample is first dissolved in suitable, non hygroscopic solvent. A drop of this solution is deposited on surface of KBr or NaCl cell. The solution is then evaporated to dryness and the film formed on the cell is analyzed directly. Care is important to ensure that the film is not too thick otherwise light cannot pass through. This technique is suitable for qualitative analysis.
4. Reference: A reference is usually a pure form of the solvent the sample is in. It is used for two reasons:  i. This prevents fluctuations in the output of the source affecting the data.   ii. This allows the effects of the solvent to be cancelled out.  
5. Detector: The detector is a thermopile which comprises of stack of thermocouple junction in an evacuated can having an infrared window.

USES AND APPLICATIONS

1.      Infrared spectroscopy is widely used in both research and industry as a simple and reliable technique for measurement, quality control and dynamic measurement. The instruments are now small, and can be transported, even for use in field trials. With increasing technology in computer filtering and manipulation of the results, samples in solution can now be measured accurately (water produces a broad absorbance across the range of interest, and thus renders the spectra unreadable without this computer treatment.
2.      The main application of infrared spectroscopy in biochemical investigations is in elucidation of structure of purified biological molecules of intermediates size such as small peptides, metabolic intermediates, drugs etc.

3.      It is employed for examining the secondary structure of proteins. Polypeptides give intensity bands at 3290, 1650 and 1535 cm–1 providing clues about the secondary structure. E. g. In alpha helix, amide I bands splits into two absorbance maxima of 1650 cm–1(strong one) and 1652 cm–1(medium one) intensity. While amide II give weak intensities at 1516 and 1546 cm–1 only.
4.      By measuring at a specific frequency over time, changes in the character or quantity of a particular bond can be measured. This is especially useful in measuring the degree of polymerization in polymer manufacture. Modern research machines can take infrared measurements across the whole range of interest as frequently as 32 times a second. This can be done whilst simultaneous measurements are made using other techniques. This makes the observations of chemical reactions and processes quicker and more accurate.
5.      Infrared spectroscopy has been highly successful for applications in both organic and inorganic chemistry. Infrared spectroscopy has also been successfully utilized in the field of semiconductor microelectronics: for example, infrared spectroscopy can be applied to semiconductors like silicon, gallium arsenide, gallium nitride, zinc selenide, amorphous silicon, silicon nitride, etc.

6.      Isotope effects: The different isotopes in a particular species may give fine detail in infrared spectroscopy. For example, the O-O stretching frequency of oxyhemocyanin is experimentally determined to be 832 and 788 cm-1 for ν (16O-16O) and ν (18O-18O) respectively.


LIMITATIONS OF IR-SPECTROSCOPY

  •  It will measure only sample molecules with a dipole moment.
  • It is very difficult to reproduce exact results in IR-spectroscopy because of changes in pathlength & changes in concentration.
  • If absorption is not in the 4000-400 cm-1 range it will not show on spectrum graph in IR-spectroscopy.
  • Absorption is too weak to be observed.
  • Absorptions can be too close to each other.
  • Additional weak bands of vibrations are observed.



Bacteria in Photos

Bacteria in Photos