The following points highlight the top eight spectroscopy techniques. The techniques are: 1. Infrared (IR) Spectrophotometry 2. Circular Dichromism (CD) Spectroscopy 3. Spectrofluorimetry 4. Luminometry 5. Atomic/Flame Spectrophotometry 6. Electron Spin Resonance (ESR) Spectrometry 7. Nuclear Magnetic Resonance (NMR) Spectrometry 8. Mass Spectrometry.
Technique # 1. Infrared (IR) Spectrophotometry:
IR-light was used in this spectrophotometric analysis, Infra-red spectrophotometry with Gas-Liquid Chromatography or gas analysis techniques often used as a powerful technique for drug metabolism study and also provides a convenient and sensitive means of detecting and measuring differences in the concentration of gases such as carbon monoxide, carbon dioxide and acetylene in biological samples.
The light sources in different spectrophotometric techniques are:
Technique # 2. Circular Dichromism (CD) Spectroscopy:
Information on the three-dimensional structure (conformation) of macromolecules in solution can be obtained by studying their absorption of polarized light, using Circular dichromism (CD) spectroscopy. CD spectroscopy measures this differential absorption of right (R) and left (L) circularly polarized light as a function of wavelength.
The main components of a CD spectrometer are illustrated in Fig. 1.2. Usually L and R circularly polarized radiation is produced from a single monochromator by passing plane-polarized light through an electro-optic modulator.
This is a crystal subject to alternating currents that transits either the R or L component of light dependent on the polarity of the electric field to which it is exposed.
The photomultiplier detector produced a voltage in proportion to the ellipticity of polarization of the combined beam falling on the photomultiplier. CD-spectroscopy is often done to get information about the protein conformation (L, B and random coil) and also to study the conformation of nucleic acids.
Technique # 3. Spectrofluorimetry:
Spectrofluorimetry is a specialized spectroscopic method where two monochromators may be used, one selecting the activating wavelength and the other the phorescent wavelength. No reference cuvette is required, but a calibration curve must be constructed. This method is most accurate at very low concentrations when absorption spectrophotometry is least accurate.
The sensitivity of the instrument is usually easily adjusted over a large range by amplification of the current produced in the photocell circuit. The basic components of a complete spectra-fluorimeter are mentioned in Fig. 1.3.
This includes a continuous spectrum source (mercury lamp or Xenon arc) a monochromator (M) for irradiating the specimen with any chosen wavelength, a second monochromator (M2) which, under conditions of constant irradiating wavelengths, enables the determination of I .v fluorescent spectrum of the specimen; and a detector which is usually a sensitive photocell.
This technique is often used for the quantitative study of Vitamin B,, NADH Cork set and other. Group-specific hydrolases may be readily assayed by measuring the rate of appearance of fluorescence of 450 nm of the amino of 4-methylumbellifore one when the enzyme acts upon an ether of ester derivative of 4-methyl umbelliferone.
Technique # 4. Luminometry:
This is a photometric technique in which light — enlisted as a result of a chemical reaction (luminescence) in contrast to the result of a physical reaction (fluorescence or atomic emission) — is measured in a luminometer.
Luminometers are relatively simple photometers, complicated only slightly by the need to amplify and record the signal from the photocell.
It has a photomultiplier tube with a well-stabilized high voltage power supply to ensure sensitive, reproducible measurement of light emission, a direct current amplifier with a wide range of sensitivity and linear response and a reaction chamber which allows temperature control, adequate mixing of reactants and protection from extraneous light (Fig. 1.4).
This technique is often used for determining ATP concentration, study of bacterial luciferance system, and chemo luminescence.
Technique # 5. Atomic/Flame Spectrophotometry:
Volatilisation of atoms — either in a flame or electro thermally — causes them to emit and absorb light of specific wavelengths. Atomic/flame spectrophotometry takes advantage of the specificity of line spectra to determine the amounts of a specific elements present. Emission flame spectrophotometry measures the emission of light of a specific wavelength by atoms in a flame.
Atomic absorption spectrophotometry measures the absorption of a beam of monochromatic light by atoms in a flame or alternatively by atoms heated electro thermally in a graphite furnace. The energy absorbed is proportional to the number of atoms present in the official path.
The amount of radiation emitted is proportional to the number of excited atoms present, which depends on the temperature and compositions of the flame. Standard additions must always be used to calibrate the system.
The basic components of an atomic (flame) emission spectrophotometer are: nebulizer, burner, monochromator, detector and read-out unit. Nebulizers are usually of the scent spray type, in which a forced stream of air passes over a capillary tube dipping into the test solution.
Large and small drops of sample solution then passes along with air-stream into the burner. Then light thus emitted is passed through the monochromator or filter. Finally, emitted light was detected and read out.
In order to produce a beam of radiation with a very narrow band, either a source of white light plus a double monochromator, or a hollow cathode discharge lamp is used. The discharge lamps are specific to the element being assayed.
These techniques are often used to detect the metallic elements present in the inorganic and organic compounds — more than 20 elements in biological samples, particularly Ca8, Mg++, Mn++, Zn++, Ca++, Pb++, Cr++, Ni++ etc. When assaying metal in biological samples it is usual to degrade organic molecules by ashing.
The detection limits for various elements in emission and absorption flame spectrophotometry are depicted below:
Technique # 6. Electron Spin Resonance (ESR) Spectrometry:
This is a technique used for detection of Paramagnetism, i.e. the magnetic movement associated with an unpaired Electron Paramagnetic Resonance (EPR). The technique may be used for detecting transition metal ions and their complexes, free radicals and excited states.
The basic components of an ESR spectrometer are Klystron source, Metal waveguide tube, Field magnet, Sweep coils, Detector, Amplifier, Recorder and Oscillator. Electromagnets generating fields of 50 to 100 millitesla with a uniformity of 1 in 106 are required for accurate work.
Most experiments are conducted at around 330 millitesla, in conjunction with an auxiliary sweep of 10 to 100 millitesla. Klystron oscillator produces the monochromatic miss-wave radiation usually with a wavelength of about 3 × 102 m (9,000 MH2). Samples must be in the solid state, so biological samples are usually frozen in liquid.
Technique # 7. Nuclear Magnetic Resonance (NMR) Spectrometry:
NMR-technique is used for detecting atoms which have nuclei that possess a magnetic movement. These are usually atoms containing an odd number of protons in their nuclei. In the same way that pairs of electrons in the same atomic orbital have opposite spin and no resultant magnetic movement, pairs of protons in a nuclei do not have a magnetic moment.
However, an odd portion in a nudens imports a magnetic moment to the molecule which can interest with an applied magnetic field. This interaction is major concern of nuclear magnetic resonance Spectrometry.
In a magnetic field of several hundred millitesla (several thousand gauss) such nuclei absorb radiation in the resonance spectrum, giving rise to the phenomenon known as nuclear magnetic resonance (NMR). Most studies are conducted using Hydrogen (H).
The basic components of an NMR Spectrometer are similar to those illustrated for an ESR-Spectro- meter. A radio frequency transmitter in place of a Klystron source used to irradiate the sample. For DMR the sample must be dissolved to a relatively high concentration in a solvent which lacks protons, such as D20 or CDCl2.
To minimize variations in the magnetic field, the sample is contained in a tube of high precision diameter and is usually rotated at high speed by an air turbine. The absorption signal, detected by a radio receiver, is amplified and recorded.
The major use of NMR is for the study of the molecular structure of relatively simple organic molecules. This structural information relevant to the biological action of the antibiotics has also been obtained from NMR studies. NMR has also been particularly useful in the study of phosphate compounds viz., AMP, ADP, ATP, and phosphocreatine.
Technique # 8. Mass Spectrometry:
It is based upon the principle that moving ion may be deflected by a magnetic field to an extent that is dependent upon its mass and velocity. Thus ions of a larger momentum are deflected less than ones of lower momentum, whilst a mixture of ions of different mass but constant velocity will be deflected in proportion to their mass.
So, in a Mass Spectrometer, molecules of a compound are ionised, either by ejection of an electron or capture of a proton, to give the parent molecular ion the energy which is such that some fragmentation occurs to give a series of fragment ions. Knowledge of the mass of the molecular ion and its major fragment ions is frequently sufficient to enable the structure of the parent compound to be uniquely deduced.
This method is very sensitive and often used for as little as 10-6 to 10-9 g of material. Mass spectra show a series of peaks or lines corresponding to the m/c values of the positive ions produced from the compound. The height of the peaks corresponds to the relative abundance of the ions. A reference ion of similar m/c value to that of the parent ion is used to calibrate the mass axis (abscissa) of the intensity spectrum.
The parent ion is the peak with the greatest mass, although it is not necessarily the most abundant (base peak). Ion intensities in a mass spectrum are usually recorded as percentage of the intensity of the base peak. The basic components of a Mass Spectrometer are Ionization chamber vacuum pump unit, Electrostatic field, Ion trajectory, Magnetic field, Detector, Amplifier and Recorder.
The earliest biochemical uses of mass spectrometry were in the study of metabolic pathways — particularly the determination of chemical structure and, hence, the identification of compounds. This technique has also been used to determine the amino acid sequence of oligopeptides derived from protein hydrolysate and other sources.
