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Sharath .H .N M pharmacy

• INDEX

• introduction

•Raman spectroscopy

• Fluorimetry

• Flame photometry

SPECTROPHOTOMETRY, LIGHT EMISSION AND SCATTERING

ANALYTICALSPECTROPHOTOMETRY,

INTRODUCTION

Measuring light emission, transmittance and scattering are few of the most important techniques used in modern biochemistry laboratory.

• They are used in spectrophotometer, colorimetric, chemiluminescence, ELISA etc. The ease and accuracy of measurement of light emission or transmittance makes it a method of choice in a large number of analytical techniques.

•Some of theLight emission methods used in quantitative analysis are:

•Colourimetry

•Flourimetry

•Flame photometry

•Chemiluminescence

•ELISA

• Atomic emission spectrometry.

Raman Spectroscopy

When radiation passes through a transparent medium, the species present scatter a fraction of the beam in all directions.

In 1928, the Indian physicist C. V. Raman discovered that the visible wavelength of a small fraction of the radiation scattered by certain molecules differs from that of the incident beam and furthermore that the shifts in wavelength depend upon the chemical structure of the molecules responsible for the scattering.

• An important advantage of Raman spectra over infrared lies in the fact that water does not cause interference; indeed, Raman spectra can be obtained from aqueous solutions.

• In addition, glass or quartz cells can be employed, thus avoiding the inconvenience of working with sodium chloride or other atmospherically unstable window materials.

THEORY OF RAMAN SPECTROSCOPY

Raman spectra are acquired by irradiating a sample with a powerful laser source of visible or near-infrared monochromatic radiation. During irradiation, the spectrum of the scattered radiation is measured at some angle (often 90 deg) with a suitable spectrometer. At the very most, the intensities of Raman lines are 0.001 % of the intensity of the source; as a consequence, their detection and measurement are somewhat more difficult than are infrared spectra.

Excitation of Raman Spectra

A Raman spectrum can be obtained by irradiating a sample of carbon tetrachloride with an intense beam of an argon ion laser having a wavelength of 488.0 nm (20492 cm-1). The emitted radiation is of three types:

1. Stokes scattering

2. Anti-stokes scattering

3. Rayleigh scattering

Instrumentation

A Raman system typically consists of four major components:

1. Excitation source (Laser).

2. Sample illumination system and light collection optics.

3. Wavelength selector (Filter or Spectrophotometer).

4. Detector (Photodiode array, CCD or PMT).

Instrumentation

INSTRUMENTATION • Instrumentation for modern Raman spectroscopy consists

of three components:

A laser source, a sample illumination system and a suitable spectrometer.

Source

•

• The sources used in modern Raman spectrometry are nearly always lasers because their high intensity is necessary to produce Raman scattering of sufficient intensity to be measured with a reasonable signal-to-noise ratio.

• Because the intensity of Raman scattering varies as the fourth power of the frequency, argon and krypton ion sources that emit in the blue and green region of the spectrum have and advantage over the other sources.

Sample Illumination System

•

• Sample handling for Raman spectroscopic measurements is simpler than for infrared spectroscopy because glass can be used for windows, lenses, and other optical components instead of the more fragile and atmospherically less stable crystalline halides.

• In addition, the laser source is easily focused on a small sample area and the emitted radiation efficiently focused on a slit.

• Consequently, very small samples can be investigated. A common sample holder for non absorbing liquid samples is an ordinary glass melting-point capillary.

• Liquid Samples:

• A major advantage of sample handling in Raman spectroscopy compared with infrared arises because water is a weak Raman scatterer but a strong absorber of infrared radiation. Thus, aqueous solutions can be studied by Raman spectroscopy but not by infrared. This advantage is particularly important for biological and inorganic systems and in studies dealing with water pollution problems.

•

• Solid Samples:

• Raman spectra of solid samples are often acquired by filling a small cavity with the sample after it has been ground to a fine powder. Polymers can usually be examined directly with no sample pre-treatment.

• Raman Spectrometers

Raman spectrometers were similar in design and used the same type of components as the classical ultraviolet/visible dispersing instruments.

Most employed double grating systems to minimize the spurious radiation reaching the transducer. Photomultipliers served as transducers. Now Raman spectrometers being marketed are either Fourier transform instruments equipped with cooled germanium transducers or multichannel instruments based upon charge-coupled devices.

• SERS and SERRS•

• Surface-Enhanced Raman Spectroscopy utilizes the following effect. Raman signal from molecules adsorbed on certain metal surfaces can be 5-6 orders of magnitude stronger then the Raman signal from the same molecules in bulk volume.

•

• The exact reason for such dramatic improvement is still under discussion. However, since intensity of Raman signal is proportional to the square of electric dipole moment P = αE, there are two possible reasons - the enhancement of polarizability α, and the enhancement of electrical field E.

•

• The first enhancement of polarizability a may occur because of a charge-transfer effect or chemical bond formation between metal surface and molecules under observation. This is a so-called chemical enhancement.

•

• The second one takes into account interaction of the laser beam with irregularities on the metal surface such as metal micro-particles or roughness profile. It is believed that laser light excites conduction electrons at the metal surface leading to a surface plasma resonance and strong enhancement of electric field E. It is also called electromagnetic enhancement.

APPLICATIONS OF RAMAN SPECTROSCOPY

Raman Spectra of Inorganic Species

•

• The Raman technique is often superior to infrared for spectroscopy investigating inorganic systems because aqueous solutions can be employed.

• In addition, the vibrational energies of metal- ligand bonds are generally in the range of 100 to 700 cm-1, a region of the infrared that is experimentally difficult to study.

• These vibrations are frequently Raman active, however, and peaks with values in this range are readily observed. Raman studies are potentially useful sources of information concerning the composition, structure, and stability of coordination compounds.

• Raman Spectra of Organic Species

Raman spectra are similar to infrared spectra in that they have regions that are useful for functional group detection and fingerprint regions that permit the identification of specific compounds. Raman spectra yield more information about certain types of organic compounds than do their infrared counterparts.

Biological Applications of Raman SpectroscopyRaman spectroscopy has been applied widely for the study of biological systems. The advantages of his technique include the small sample requirement, the minimal sensitivity toward interference by water, the spectral detail, and the conformational and environmental sensitivity.

FLUORIMETRY

INTRODUCTION

• Fluorescence is the emission of visible light by a substance that has absorbed light of a different wavelength. The emitted photon hasa longer wavelength.

• Phosphorescence is related to fluorescence in emitting a photon, however, a phosphorescent material does not immediately re‐emit the radiation it absorbs.

• As the excitation of the molecule is due to the absorption of

a photon (light), these types of luminescence are called photoluminescence.

• At the ground state , the molecular orbital's areoccupied by two electrons. the spins of the two

• electrons in the same orbital must be antiparallel.

This implies that the total spin, S, of the molecule in the ground state is zero [½+( ½)].

•

• This energy state is called “singlet state” and is labelled as S 0.

• The electron spins in the excited state achieved by absorption of radiation may either be parallel or antiparallel.

• Accordingly, this may be a triplet (parallel) ora singlet (antiparallel) state.

• Activation and Deactivation

• The absorption of a photon of suitable energy causes the

molecule to get excited from the ground state to one of the

excited states.

This process is called as excitation or activation

and is governed by Franck‐Condon principle.

• According to this principle, the electronic transition takes

place so fast (~10‐15 s) that the molecule does not get an

opportunity to execute a vibration,

– i.e., when the electrons are excited the internuclear distance does not change.

• The basis for the principle is that the nuclei are very massive as compared to the electrons and therefore move very slowly.

• •The deactivation processes can be broadly

categorised into two groups given below.

• Nonradioactive deactivation

• Radioactive deactivation

FACTORS AFFECTING FLUORESCENCE

AND PHOSPHORESCENCE•

• The common factors affecting the fluorescence are as follows.

• Temperature

• pH

• Dissolved oxygen

• Solvent

The essential components of an instrumentused to measure fluorescence of the sampleare:

• Excitation light sources

• Filters or Monochromators

• Sample holder

• Detector

• Readout device

Flame Photometry

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.

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.

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.

Flame photometry

Principal of Flame Photometry:

• It is the conversion of molecules to their component atoms in gaseous state ; and it is carried out by introduction of the molecules solution in the flame in very fine droplet Atoms in gaseous state in the flame absorb thermal energy from the flame itself .

• some of the atoms get excited & as they return back to the ground state they emit radiation having energy equal to that absorbed.

• The emission is proportional to the number of excited atoms, which is proportional to the total number of atoms in the flame i.e. the sample concentration

• When a metallic salt solution is aspirated into path of flame, following events takes place;

1) Vaporization: The solvent is vaporized leaving particles of solid salt.

2) Atomization : The salt is vaporized and converted into free neutral gaseous atoms or radicals

3) Excitation: Some of these atoms are excited by the thermal energy of the flame to higher energy levels. The various events are as follows,

2) INSTRUMENTATION OF FLAME EMISSION SPECTROSCOPY:• „ 1-Flame atomizer.

„ 2- Monochromator

„ 3- Detector.

„ 4- Readout meter.

• 1-Burner:

• Atomization of the sample is carried out in the path of flame. Thermal energy is used to excite the atoms.

• The atomizer is composed of : Nebulizer and burner. Burners are of two types;

• Total Consumption Burner and Premix or Laminar flow Burner.

• Total Consumption Burner:

• The fuel gas, oxidant gas and liquid samples are drawn to the opening of the burner through separate inlets. The fuel gas burns in presence of oxygen to produce the flame attached to the base of burner. Nebulizer is attached to the base of burner. ( Nebulizer: is a device by which sample solution is divided into very fine droplets which are aspirated into fine spray or aerosol.)

Advantages:

1) Entire sample enter into flame and hence it is more sensitive.

2) The flame can be adjusted to produce high temperature.

• Premix or Laminar Flow Burner:

• The fuel, oxidant gas and liquid sample are thoroughly mixed before entering the flame. In premix burner only small droplets of the sample are reach the flame and large droplets are trapped by baffles and drained off and sample is wasted.

Advantages: 1) The flame produced is stable, no turbulent, and noiseless.

2) Vaporization, atomization and excitation of atoms takes place efficiently.

3) Sensitivity and reproducibility is good.

• 2- Monochromator :

• Either grating or interference filters which allow the resonance wavelength to pass to the detector

• 3) Detector and Read out Device:

• The light selected by the monochromator is directed onto a detector that is typically a photomultiplier tube , whose function is to convert the light signal into an electrical signal proportional to the light intensity. The processing of electrical signal is fulfilled by a signal amplifier . The signal could be displayed for readout , or further fed into a data station for printout by the requested format.

• 3) Application of Flame Emission Spectroscopy

F.E.S. Used for Qualitative analysis and Quantitative analysis of Alkali and alkaline earth metals.

‰ Soil analysis: Na ,K ,Ca ,Mg .

‰ Fertilizer : Na ,K ,Ca ,M g.

‰ Biological Fluids : Na ,K ,Ca , Mg.

‰ Petrol : Lead

‰ Cement: Ca and Mg.

‰ Glass Analysis: Na ,Ca ,Mg , K ,and Li.

‰ Organic Compounds : Boron

‰ Physiology and Clinical Studies : Electrolyte Balance.

• Quantitative Analysis

• Calibration Curve

• A calibration curve is used to determine the unknown concentration of an element in a solution. The instrument is calibrated using several solutions of known concentrations.

• The emission intensity of each known solution is measured and then a calibration curve of emission intensity v/s concentration is plotted.

• References :

• Analytical chemistry by David Harvey

• Instrumental method of analysis by P.D . Sethi

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