Spectroscopy - Analysis of biomolecules

Spectroscopy

Atoms and molecules interact with electromagnetic radiation and may absorb and/or emit Electromagnetic radiation. The patterns of absorption and/or emissions are called ‘spectra’. Spectroscopy is concerned with the interpretation of these spectra. In other words it is a study of interaction between electromagnetic radiation and matter. Different regions of the electromagnetic spectrum provide different kinds of information as a result of such interactions. A spectrophotometer is an instrument that measures the amount of light or electromagnetic radiation that is absorbed or emitted by a sample. Arnold J. Beckman at the National Technologies Laboratory (NTL) invented the Beckman DU spectrophotometer in 1940. Some of the electromagnetic wave parameters is useful in understanding the spectroscopy.Wavelength (λ ): Wavelength is the distance between the consecutive peaks or crests and is expressed in nanometers 1nm=10^-8 meters.Frequency (ν): Frequency is the number of waves passing through any point per second and is usually expressed as Hertz(Hz).Wave number (ν ): Wave number is the number of waves per cm.Wavelength, Wave number and Frequency are interrelated as,

1/l=ṽ=v/c

Where, λ is wave length, ṽ is wave number, ν is frequency, c is velocity of light in vacuum. i.e., 3 x 10^-8 m/s

The principle of the spectroscopy is based on Beer–Lambert law / Beer –Lambert – Bouguer law which states that the amount of light absorbed is proportional to the concentration of the absorbing substances and to the thickness of the absorbing material (path length).

Log(I/I)=ε cl=A

 Where, I₀- the intensity of incident light I- the intensity of transmitted light, ε - molar absorptivity / molar extinction coefficient in cm 2 mol^-1 or L mol^-1 cm^-1. c - concentration in mol L^-1, l - path length in cm, A- absorbance (unitless).

This law can be used to find the concentration of solutions absorbing in UV or visible region. However there are deviations from this law or the limitations of the Beer–Lambert law is that Beer’s Law successfully describes the behaviour of dilute solutions only. At high concentrations (i,e. greater than 10² M) there is interaction between absorbing particles such that the absorption characteristics of the analyte are affected. The voltage fluctuation, sensitivity changes in the detector and solution containing more than one complex which absorb different wavelength are the other deviating factors of this law. Spectroscopy can be used for qualitative analysis and quantitative analysis. In qualitative analysis the characteristic wavelength are used for a given analyte and through which we can identify the sample and such type of spectrometers are called as photometers (e.g., HPLC detectors). Whereas in quantitative analysis the intensity of absorption or emission from the analyte is used to find out the concentration of the analyte in a given sample and quantitative measurements can be made at any desired wavelength, such type of spectrometers are called as spectrophotometer (e.g.,UV-Visible spectrometer). There are three types of spectrophotometer viz; single, double and split beam spectrophotometer. In single beam all the light passes through the sample and to measure the intensity of the incident light samplemust be removed so that all the light can pass through. It is cheap and less complicated. In double beam spectrophotometer it compares the light intensity between two light paths, one path containing a reference sample and the other the test sample. The readings are stable. The disadvantagesare higher cost, lower sensitivity because of the more complex optics. Split beam is similar to the double beam spectrophotometer but it uses a beam splitter instead of a chopper to send light along the blank. The essential components of a typical spectrometer are,

  Some of the common types of analytical spectroscopes are; Absorption, Fluoresence and Phosphorescence, Emission (atomic with flames, arcs, sparks, and plasmas), Chemilumenesence and Bioluminescence and Reflection spectroscopy. Few important spectroscopy methods are described here.