SPECTROSCOPY

Course: B.Sc BiotechSubject: Bio Analytical Technique

Unit: I

1.

Spectroscopy

Spectroscopy is a general term referring to the interactions of various types of electromagnetic radiation with matter.

Exactly how the radiation interacts with matter is directly dependent on the energy of the radiation.

Spectroscopy: Is an analytical method technique wherein the absorbance of specific wavelength of light by the molecules of sample under test is determined. The more the number of molecules in sample, the greater is the absorbance and vice-verse.

Spectroscopy

The higher energy ultraviolet and visible wavelengths affect the energy levels of the outer electrons.

Radio waves are used in nuclear magnetic Resonance and affect the spin of nuclei in a magnetic field.

Infrared radiation is absorbed by matter resulting in rotation and/or vibration of molecules.

INTRODUCTION

Spectroscopic techniques employ light to interact with matter and thus probe certain features of a sample to learn about its consistency or structure.

Light is electromagnetic radiation, a phenomenon exhibiting different energies, and dependent on that energy, different molecular features can be probed.

The basic principles of interaction of electromagnetic radiation with matter are treated in this chapter.

There is no obvious logical dividing point to split the applications of electromagnetic radiation into parts treated separately.

The applications like visible or UV light to probe consistency and conformational structure of biological molecules.

Usually, these methods are the first analytical procedures used by a biochemical scientist.

PRINCIPLE

molecules consisting of some functional groups by which they may incur color or some nature to absorb light of specific wavelength.

This wavelength at which sample absorbs to a greater extent is called as λ max.

When the light beam is passed on to the sample, the electrons in the molecules absorb energy in the light, and go for exited state. During this transition some of the light energy is absorbed while the remaining light falls on the photo-electric detector.

Spectroscopy is suitable for both qualitative analysis and quantitative analysis.

The electric (E) and magnetic (M) field vectors are directed perpendicular to each other.

2.

PROPERTIES OF ELECTROMAGNETIC RADIATION The interaction of electromagnetic radiation with matter is a

quantum phenomenon and dependent upon both the properties of the radiation and the appropriate structural parts of the samples involved.

This is not surprising, since the origin of electromagnetic radiation is due to energy changes within matter itself.

The transitions which occur within matter are quantum phenomena and the spectra which arise from such transitions are principally predictable.

Electromagnetic radiation is composed of an electric and a perpendicular magnetic vector, each one oscillating in plane at right angles to the direction of propagation.

The wavelength ƛ is the spatial distance between two consecutive peaks (one cycle) in the sinusoidal waveform and is measured in submultiples of metre, usually in nanometres (nm).

The maximum length of the vector is called the amplitude.

The frequency ν of the electromagnetic radiation is the number of oscillations made by the wave within the timeframe of 1 s.

The frequency is related to the wavelength via the speed of light c

describes the number of completed wave cycles per distance and is typically measured in 1 cm-1.

QUALITATIVE SPECTROSCOPY This is the technique to know the type of sample molecule there by

one can tell what the sample is and its chemical nature after comparing the obtained analysis curve peaks with that of standard sample from official books like Pharmacopeias or books on chemical standards etc..

A sample is subjected to scanning over entire range of UV or visible radiation.

The point or wavelenght where the sample shows maximum absorbance is noted as it’s λ max.

This λ max is fixed for every sample and and there by any unknown sample can be identified by knowing its λ max after comparing with standard.

Qualitative spectroscopy

Quantitative spectroscopy

3.

QUANTITATIVE SPECTROSCOPY

This is a method to determine the exact concentration of a substance in a given sample.

At a specified wave length (λ max) when a given sample is analyzed by spectroscopy, the concentration in the sample can be known by plotting it against a standard substance graph as shown in the pic.

For this a series of dilution of standard sample and test sample are taken and absorbance is measure by spectroscopy.

The absorbance for difference concentrations of standard and test are plotted on a graph.

From the absorbance of test, the concentration of it can be known by extrapolating it on the graph as shown below in the fig.

PARTICLE NATURE OF RADIATION

Electromagnetic radiation is also described as having the properties of particles.

Molecules exist in a certain number of possible states corresponding to definite amounts of energy.

Molecules can absorb energy and change to a higher energy level called the excited state.

The amount of energy absorbed in this transition is exactly equal to the energy difference between the states.

THE ELECTROMAGNETIC SPECTRUM

Important: As the wavelength gets shorter, the energy of the radiation increases.

4.

ULTRAVIOLET AND VISIBLE LIGHT SPECTROSCOPY

These regions of the electromagnetic spectrum and their associated techniques are probably themostwidely used for analytical work and research into biological problems.

The electronic transitions in molecules can be classified according to the participating molecular orbitals. From the four possible transitions

only two can be elicited with light from the UV/Vis spectrumfor some biological molecules:

transitions are energetically not within the range of UV/Vis spectroscopy and require higher energies.

Molecular (sub-)structures responsible for interaction with electromagnetic radiation are called chromophores.

In proteins, there are three types of chromophores relevant for UV/Vis spectroscopy: peptide bonds (amide bond); certain amino acid side chains (mainly tryptophan and

tyrosine); and certain prosthetic groups and coenzymes (e.g. porphyrine

groups such as in haem).

CHROMOPHORES IN PROTEINS

The electronic transitions of the peptide bond occur in the far UV.

The intense peak at 190 nm, and the weaker one at 210–220 nm is due to the transitions.

A number of amino acids (Asp, Glu, Asn, Gln, Arg and His) have weak electronic transitions at around 210 nm.

Usually, these cannot be observed in proteins because they are masked by the more intense peptide bond absorption.

The most useful range for proteins is above 230 nm, where there are absorptions from aromatic side chains.

While a very weak absorption maximum of phenylalanine occurs at 257 nm, tyrosine and tryptophan dominate the typical protein spectrum with their absorption maxima at 274nm and 280 nm, respectively.

In practise, the presence of these two aromatic side chains gives rise to a band at 278 nm.

Proteins that contain prosthetic groups (e.g. haem, flavin, carotenoid) and some metal–protein complexes, may have strong absorption bands in the UV/Vis range.

These bands are usually sensitive to local environment and can be used for physical studies of enzyme action.

Porphyrins are the prosthetic groups of haemoglobin, myoglobin, catalase and cytochromes.

The spectrum of haemoglobin is very sensitive to changes in the iron-bound ligand.

These changes can be used for structure–function studies of haem proteins.

Molecules such as FAD (flavin adenine dinucleotide), NADH and NAD+ are important coenzymes of proteins involved in electron transfer reactions (RedOx reactions).

They can be conveniently assayed by using their UV/Vis absorption: 438nm (FAD), 340nm (NADH) and 260nm (NAD+).

Chromophores in genetic material The absorption of UV light by nucleic acids arises from transitions of the purine (adenine, guanine) and pyrimidine (cytosine, thymine, uracil) bases that occur between 260nm and 275 nm.

The absorption spectra of the bases in polymers are sensitive to pH and greatly influenced by electronic interactions between bases.

PRINCIPLES

Quantification of light absorption The chance for a photon to be absorbed by matter is given by

an extinction coefficient which itself is dependent on the wavelength (l) of the photon.

If light with the intensity I0 passes through a sample with appropriate transparency and the path length

(thickness) d, the intensity I drops along the pathway in an exponential manner.

Biochemical samples usually comprise aqueous solutions, where the substance of interest is present at a molar concentration c. Algebraic transformation of the exponential correlation into an expression based on the decadic logarithm yields the law of Beer–Lambert.

ABSORPTION OR LIGHT SCATTERING – OPTICAL DENSITY In some applications, for example measurement of turbidity of

cell cultures (determination of biomass concentration), it is not the absorption but the scattering of light that is actually measured with a spectrophotometer.

Extremely turbid samples like bacterial cultures do not absorb the incoming light.

Instead, the light is scattered and thus, the spectrometer will record an apparent absorbance (sometimes also called attenuance).

In this case, the observed parameter is called optical density (OD).

Instruments specifically designed to measure turbid samples are nephelometers, however, most biochemical laboratories use the general UV/Vis spectrometer for determination of optical densities of cell cultures.

FACTORS AFFECTING UV/VIS ABSORPTION Biochemical samples are usually buffered aqueous solutions,

which has two major advantages. Firstly, proteins and peptides are comfortable in water as a

solvent, which is also the ‘native’ solvent. Secondly, in the wavelength interval of UV/Vis (700–200 nm)

the water spectrum does not show any absorption bands and thus acts as a silent component of the sample.

The absorption spectrum of a chromophore is only partly determined by its chemical structure.

The environment also affects the observed spectrum, which mainly can be described by three parameters:

• protonation/deprotonation (pH, RedOx);

• solvent polarity (dielectric constant of the solvent); and

• orientation effects.

Vice versa, the immediate environment of chromophores can be probed by assessing their absorption, which makes chromophores ideal reporter molecules for environmental

factors. Four effects, two each for wavelength and absorption

changes, have to be considered:

• a wavelength shift to higher values is called red shift or bathochromic effect;

• similarly, a shift to lower wavelengths is called blue shift or hypsochromic effect;

• an increase in absorption is called hyperchromicity (‘more colour’),

• while a decrease in absorption is called hypochromicity (‘less colour’).

INSTRUMENTATION UV/Vis spectrophotometers are usually dual-beam

spectrometers where the first channel contains the sample and the second channel holds the control (buffer) for correction.

Alternatively, one can record the control spectrum first and use this as internal reference for the sample spectrum.

The latter approach has become very popular as many spectrometers in the laboratories are computer-controlled, and baseline correction can be carried out using the software by simply subtracting the control from the sample spectrum.

The light source is a tungsten filament bulb for the visible part of the spectrum, and

a deuterium bulb for the UV region.

Since the emitted light consists of many different wavelengths, a monochromator, consisting of either a prism or a rotating metal grid of high precision called grating, is placed between the light source and the sample.

Wavelength selection can also be achieved by using coloured filters as monochromators that absorb all but a certain limited range of wavelengths.

This limited range is called the bandwidth of the filter. Filter-based wavelength selection is used in colorimetry, a

method with moderate accuracy, but best suited for specific colorimetric assays where only certain wavelengths are of interest.

If wavelengths are selected by prisms or gratings, the technique is called spectrophotometry.

5.

A prism splits the incoming light into its components by refraction. Refraction occurs because radiation of different wavelengths travels along different paths in medium of higher density.

In order to maintain the principle of velocity conservation, light of shorter wavelength (higher speed) must travel a longer distance.

Diffraction is a reflection phenomenon that occurs at a grid surface, in this case a series of engraved fine lines.

The distance between the lines has to be of the same order of magnitude as the wavelength of the diffracted radiation.

By varying the distance between the lines, different wavelengths are selected.

This is achieved by rotating the grating perpendicular to the optical axis. The resolution achieved by gratings is much higher than the one available

by prisms. Nowadays instruments almost exclusively contain gratings as

monochromators as they can be reproducibly made in high quality by photoreproduction.

The bandwidth of a colorimeter is determined by the filter used as monochromator.

A filter that appears red to the human eye is transmitting red light and absorbs almost any other (visual) wavelength.

This filter would be used to examine blue solutions, as these would absorb red light.

The filter used for a specific colorimetric assay is thusmade of a colour complementary to that of the solution being tested.

Theoretically, a single wavelength is selected by the monochromator in spectrophotometers, and the emergent light is a parallel beam.

Here, the bandwidth is defined as twice the half-intensity bandwidth. The bandwidth is a function of the optical slit width. The narrower the slit width the more reproducible are measured absorbance values. In contrast, the sensitivity becomes less as the slit narrows, because less radiation passes through to the detector.

In a dual-beam instrument, the incoming light beam is split into two parts by a halfmirror.

One beam passes through the sample, the other through a control (blank,reference).

This approach obviates any problems of variation in light intensity, as both reference and sample would be affected equally.

The measured absorbance is the difference between the two transmitted beams of light recorded.

Depending on the instrument, a second detector measures the intensity of the incoming beam, although some instruments use an arrangement where one detector measures the incoming and the transmitted intensity alternately.

The latter design is better from an analytical point of view as it eliminates potential variations between the two detectors.

At about 350nm most instruments require a change of the light source from visible to UV light.

This is achieved by mechanically moving mirrors that direct the appropriate beam along the optical axis and divert the other.

When scanning the interval of 500–210 nm, this frequently gives rise to an offset of the spectrum at the switchover point.

Since borosilicate glass and normal plastics absorb UV light, such cuvettes can only be used for applications in the visible range of the spectrum (up to 350 nm).

For UV measurements, quartz cuvettes need to be used. However, disposable plastic cuvettes have been developed that allow for measurements over the entire range of the UV/Vis spectrum.

APPLICATIONS

Qualitative and quantitative analysis Qualitative analysis may be performed in the UV/Vis regions

to identify certain classes of compounds both in the pure state and in biological mixtures (e.g. protein-bound).

Most commonly, this type of spectroscopy is used for quantification of biological samples either directly or via colorimetric assays.

In many cases, proteins can be quantified directly using their intrinsic chromophores, tyrosine and tryptophan.

Difference spectra The main advantage of difference spectroscopy is its capacity

to detect small absorbance changes in systems with high background absorbance. A difference spectrum is obtained by subtracting one absorption spectrum from another.

Common applications for difference UV spectroscopy include the determination of the number of aromatic amino acids exposed to solvent, detection of conformational changes occurring in proteins, detection of aromatic amino acids in active sites of enzymes, and monitoring of reactions involving ‘catalytic’ chromophores (prosthetic groups, coenzymes).

Wavelengths Absorbed by Functional Groups

Again, demonstrates the moieties contributing to absorbance from 200-800 nm, because pi electron functions and atoms having no bonding valence shell electron pairs.

OTHER CONCEPTS IMPORTANT TO UV/VIS SPECTROSCOPY

UV/Vis spectra can be used to some extent for compound identification, however, many compounds have similar spectra.

Solvents can cause a shift in the absorbed wavelengths. Therefore, the same solvent must be used when comparing absorbance spectra for identification purposes.

Many inorganic species also absorb energy in the UV/Vis region of the spectrum.

FLUORESCENCE SPECTROSCOPY

Fluorescence spectroscopy is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light.

Devices that measure fluorescence are called fluorometers.

Principles Fluorescence is an emission phenomenon where an energy

transition from a higher to a lower state is accompanied by radiation. Only molecules in their excited forms are able to emit fluorescence; thus, they have to be brought into a state of higher energy prior to the emission phenomenon.

INSTRUMENTATION

All fluorescence instruments contain three basic items: a source of light, a sample holder and a detector.

In addition, to be of analytical use, the wavelength of incident radiation needs to be selectable and the detector signal capable of precise manipulation and presentation.

In simple filter fluorimeters, the wavelengths of excited and emitted light are selected by filters which allow measurements to be made at any pair of fixed wavelengths.

Simple fluorescence spectrometers have a means of analysing the spectral distribution of the light emitted from the sample, the fluorescence emission spectrum, which may be by means of either a continuously variable interference filter or a monochromator.

In more sophisticated instruments, monochromators are provided for both the selection of exciting light and the analysis of sample emission.

Such instruments are also capable of measuring the variation of emission intensity with exciting wavelength, the fluorescence excitation spectrum.

In principle, the greatest sensitivity can be achieved by the use of filters, which allow the total range of wavelengths emitted by the sample to be collected, together with the highest intensity source possible.

In practice, to realize the full potential of the technique, only a small band of emitted wavelengths is examined and the incident light intensity is not made excessive, to minimize the possible photodecomposition of the sample.

Essential components of a fluorescence spectrometer

6.

Schematics of a spectrofluorimeter

7.

LIGHT SOURCES

Commonly employed sources in fluorescence spectrometry have spectral outputs either as a continuum of energy over a wide range or as a series of discrete lines.

An example of the first type is the tungsten-halogen lamp and of the latter, a mercury lamp.

Mercury lamps are the most commonly employed line sources and have the property that their spectral output depends upon the pressure of the filler gas.

The output from a low-pressure mercury lamp is concentrated in the UV range, whereas the most commonly employed lamps, of medium and high

pressure, have an output covering the whole UV-visible spectrum. Although in many cases the output from a line source will be

adequate, it is rare that an available line will exactly coincide with the optimum excitation wavelength of the sample.

It is therefore advantageous to employ a source whose output is a continuum and the most commonly employed type is the xenon arc.

Xenon arc sources can be operated either on a continuous DC basis or stroboscopically; the latter method offers advantages in the size and cost of lamps.

The output is essentially a continuum on which are superimposed a number of sharp lines, allowing any wavelength throughout the UV-visible region of the spectrum to be selected.

Arc lamps are inherently more unstable than discharge sources and for long term stability a method of compensating for drift is advisable.

The most satisfactory method of doing this is to split the excitation energy so that a small portion is led to a reference detector.

The signal from this reference detector is ratioed with the signal from the detector observing the sample.

Such a ratio-recording system is therefore independent of changes in the source intensity.

All sources of UV radiation will produce ozone from atmospheric oxygen, which should be dispersed, since it is not only toxic, but also absorbs strongly in the region below 300 nm.

For this reason, most lamps will be operated in a current of air and, if the supply fan fails, the lamp should be extinguished immediately.

Lamps must be handled with great care since fingermarks will seriously decrease the UV output.

WAVELENGTH SELECTION

The simplest filter fluorimeters use fixed filters to isolate both the excited and emitted wavelengths.

To isolate one particular wavelength from a source emitting a line spectrum, a pair of cut-off filters are all that is required.

These may be either glass filters or solutions in cuvettes. The emission filter must be chosen so that the Rayleigh-Tyndall

scattered light is obscured and the light emitted by the sample transmitted.

To avoid high blanks it may also be necessary to filter out any Raman scatter.

Recently, interference filters having high transmission (≈40%) of a narrow range (10 – 15 nm) of wavelengths have become available and it is possible to purchase filters with a maximum transmission at any desired wavelength.

UV filters of this type, however, are expensive and of limited range.

A simple filter system is acceptable for much quantitative work, particularly where sufficient chemistry has been carried out to eliminate interfering compounds.

However, it is useful to be able to scan the emission from the sample to check for impurities and optimize conditions.

A convenient method is to make use of a continuous interference filter so that an emission spectrum can be recorded, at least over the visible region of the spectrum.

A further refinement would be to use monochromators to select both the excitation and emission wavelengths.

Most modern instruments of this type employ diffraction grating monochromators for this purpose.

Such a fluorescence spectrometer is capable of recording both excitation and emission spectra and therefore makes full use of the analytical potential of the technique.

If monochromators are employed, it should be possible to change the slit width of both the excitation and emission monochromators independently.

DETECTORS

All commercial fluorescence instruments use photomultiplier tubes as detectors and a wide variety of types are available.

The material from which the photocathode is made determines the spectral range of the photomultiplier and generally two tubes are required to cover the complete UV-visible range.

The S5 type can be used to detect fluorescence out to approximately 650 nm, but if it is necessary to measure emission at longer wavelengths, a special red sensitive, S20, photomultiplier should be employed.

The limit of sensitivity of a photomultiplier is normally governed by the level of dark current.

The dark current is caused by thermal activation and can usually be reduced by cooling the photomultiplier.

Another method of minimizing dark current is to use a stroboscopic source since the ratio of dark current to fluorescence will be very small during each high intensity flash.

During the periods between flashes when the dark current is relatively high, the photomultiplier output can be disconnected.

The overall result is that the dark current no longer becomes the limitation to sensitivity.

The spectral response of all photomultipliers varies with wavelength, but it is sometimes necessary to determine the actual quantum intensity of the incident radiation and a detector insensitive to changes in wavelength is required.

A suitable quantum counter can be made from a concentrated solution of Rhodamine 101 in ethylene glycol which has the property of emitting the same number of quanta of light as it absorbs, but over a very wide wavelength range.

Thus, by measuring the output of the quantum counter at one wavelength, the number of incident quanta over a wide wavelength range can be measured.

READ-OUT DEVICES

The output from the detector is amplified and displayed on a readout device which may be a meter or digital display.

It should be possible to change the sensitivity of the amplifier in a series of discrete steps so that samples of widely differing concentration can be compared.

A continuous sensitivity adjustment is also useful so that the display can be made to read directly in concentration units.

Digital displays are most legible and free from misinterpretation. Improvement in precision is obtained by the use of integration techniques

where the average value over a period of a few seconds is displayed as an unchanging signal.

Microprocessor electronics provide outputs directly compatible with printer systems and computers, eliminating any possibility of operator error in transferring data.

SAMPLE HOLDERS

The majority of fluorescence assays are carried out in solution, the final measurement being made upon the sample contained in a cuvette or in a flowcell.

Cuvettes may be circular, square or rectangular, and must be constructed of a material that will transmit both the incident and emitted light.

Square cuvettes, or cells will be found to be most precise since the parameters of pathlength and parallelism are easier to maintain during manufacture.

However, round cuvettes are suitable for many more routine applications and have the advantage of being less expensive.

The cuvette is placed normal to the incident beam. The resulting fluorescence is given off equally in all directions, and may be collected from either the front surface of the cell, at right angles to the incident beam, or in-line with the incident beam.

Some instruments will provide the option of choosing which collecting method should be employed, a choice based upon the characteristics of the sample.

A very dilute solution will produce fluorescence equally from any point along the path taken by the incident beam through the sample.

Under these conditions, the right-angled collection method should be used since it has the benefit of minimizing the effect of light scattering by the solution and cell.

This is the usual measuring condition in analytical procedures. Although fluorescence takes place from every point along the light path,

only a small fraction of this emission is actually collected by the instrument and transmitted to the detector.

The result is that much of the solution does not contribute to the fluorescence emission and the same intensity will be observed from a much smaller volume of solution contained in a microcell whose dimensions more closely match the optical considerations of the instrument

Fluorescence emission from a microcell whose dimensions closely match the optical considerations of the instrument

8.

As the absorbance of the solution increases, the fluorescence emission becomes progressively distorted until a point is reached where little actually penetrates the main bulk of the solution and the fluorescence will be confined to the front surface of the cuvette.

Front surface collection will still allow measurements to be made, although the contribution due to light scattered from the cuvette wall will be large.

Front surface collection will at least always show emission from a fluorescent sample, whereas the fluorescence obtained from 90° collection falls rapidly as the absorbance of the solution increases.

It is possible to dismiss a potential sample as non-fluorescent simply because the concentration is too high.

Wherever possible, the absorbance of a completely unknown solution should be measured before attempting a fluorescence check and the concentration adjusted to provide a solution of absorbance <0.1 A.

FACTORS AFFECTING QUANTITATIVE ACCURACYNon-linearity The proportional relationship between light absorption and fluorescence

emission is only valid for cases where the absorption is small. As the concentration of fluorophore increases, deviations occur and the plot

of emission against concentration becomes non-linear. With right-angle viewing, the principal distortion arises from the absorption

of the excited light before it can penetrate to the heart of the cell, where the emission produced is accepted by the detector optics.

Temperature effects Changes in temperature affect the viscosity of the medium and hence the

number of collisions of the molecules of the fluorophore with solvent olecules.

Fluorescence intensity is sensitive to such changes and the fluorescence of many certain fluorophores shows a temperature dependence.

In such cases the use of thermostatted cell holders is to be recommended. Normally it is sufficient to work at room temperature with the proviso that

any sample procedure involving heating or cooling must also allow sufficient time for the final solution to reach ambient before measurement.

pH effects Relatively small changes in pH will sometimes radically affect the intensity

and spectral characteristics of fluorescence. Accurate pH control is essential and, when particular buffer solutions are

recommended in an assay procedure, they should not be changed without investigation.

Most phenols are fluorescent in neutral or acidic media, but the presence of a base leads to the formation of nonfluorescent phenate ions.

5 hydoxyindoles, for example, serotonin, show a shift in fluorescence emission maximum from 330 nm at neutral pH to 550 nm in strong acid without any change in the absorption spectrum.

Inner-filter effects

Fluorescence intensity will be reduced by the presence of any compound which is capable of absorbing a portion of either the excitation or emission energy.

At high concentrations this can be caused by absorption due to the fluorophore itself.

More commonly, particularly when working with tissue or urine extracts, it is the presence of relatively large quantities of other absorbing species that is troublesome.

The purpose of extraction procedures is usually to eliminate such species so that the final measurement is made upon a solution essentially similar to the standard.

QUENCHING

Decrease of fluorescence intensity by interaction of the excited state of the fluorophore with its surroundings is known as quenching and is fortunately relatively rare.

Quenching is not random. Each example is indicative of a specific chemical interaction, and the common instances are well known.

Quinine fluorescence is quenched by the presence of halide ion despite the fact that the absorption spectrum and extinction coefficient of quinine is identical in 0.5M H2SO4 and 0.5M HCl.

APPLICATIONS

Fluorescence spectroscopy is used in, among others, biochemical, medical, and chemical research fields for analyzing organic compounds. There has also been a report of its use in differentiating malignant, bashful skin tumors from benign.

Atomic Fluorescence Spectroscopy (AFS) techniques are useful in other kinds of analysis/measurement of a compound present in air or water, or other media, such as CVAFS which is used for heavy metals detection, such as mercury.

Fluorescence can also be used to redirect photons, Additionally, Fluorescence spectroscopy can be adapted to the

microscopic level using microfluorimetry. In analytical chemistry, fluorescence detectors are used with HPLC.

SPECTROSCOPY APPLICATIONS:

1. Spectroscopy is the important detector system in advanced methods like HPLC, HPTLC etc.

2. It is also important and a main detector system in multi sample analyzer instruments like Elisa plate reader, micro-plate reader, auto-analyzers etc.

3. It is also a part of continuous culture broths like in fermentation tanks to keep the concentration of microbes or any chemical substance at a constant and helps regulate the rate of addition or deletion into the tank.

4. It is useful to determine bio molecules like corticosteroids, testosterone, aldosterone etc.

5. It is also useful in analysis of phyto-chemicals like glycosides, tannins, alkaloids etc.

6. It is also useful in determination of inorganic substance like Fe, Mg, Ca, Cu and other salts and their derivatives. Further chemicals like potassium permanganate, Ferrous sulphate etc.

SPECTROSCOPY APPLICATIONS:

1. Spectroscopy is the important detector system in advanced methods like HPLC, HPTLC etc.

2. It is also important and a main detector system in multi sample analyzer instruments like Elisa plate reader, micro-plate reader, auto-analyzers etc.

3. It is also a part of continuous culture broths like in fermentation tanks to keep the concentration of microbes or any chemical substance at a constant and helps regulate the rate of addition or deletion into the tank.

4. It is useful to determine bio molecules like corticosteroids, testosterone, aldosterone etc.

5. It is also useful in analysis of phyto-chemicals like glycosides, tannins, alkaloids etc.

6. It is also useful in determination of inorganic substance like Fe, Mg, Ca, Cu and other salts and their derivatives. Further chemicals like potassium permanganate, Ferrous sulphate etc.

BOOK AND WEB REFERENCES http://mtweb.mtsu.edu/nchong/Spectroscopy-CHEM6230.pdf http://www.chm.davidson.edu/vce/spectrophotometry/

Spectrophotometry.html http://www.rajaha.com/spectroscopy-principle-applications/ Book name: Wilson and Walker http://www.oneonta.edu/faculty/kotzjc/LAB/Spec_intro.pdf http://www2.bren.ucsb.edu/~keller/courses/esm223/

Spectrometer_analysis.pdf http://www.wikihow.com/Do-Spectrophotometric-Analysis

IMAGE REFERENCES

1.http://jtgnew.sjrdesign.net/images/spectroscopy_pinkfloyd_albumcover.jpg

2.,3.,5.,6.,7.,8. Book Name: Principles and Techniques of Biochemistry and Molecular Biology, by Wilson n Walker

4.http://chemwiki.ucdavis.edu/@api/deki/files/8475/spectrophotometer_structure.png

1.