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IN 504 Analytical Instruments

Reference Text: R S Khandpur Handbook of Analytical Instrumentation1 Presented by; Anju Sunny CUSAT

IntroductionWhat are Analytical Instruments ?Instruments that are used to analyze materials and to establish the composition.Provide, qualitative informationquantitative data

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Elements of an Analytical Instrument3

Absorption SpectroscopyMost of the instrumental analysis methods are based on the absorption of electromagnetic radiation in the visible, ultraviolet and infrared ranges.

The method based on absorption of radiation of a substance is known as Absorption Spectroscopy or Absorption Spectrophotometer.

Advantages:High speedSensitivity to very small amounts Simple operational method4

Laws of Photometry1) Lamberts LawStates that each layer of equal thickness of an absorbing medium absorbs an equal fraction of the radiant energy that traverses it.Lamberts law is expressed as: Transmittance T= I / I0 Absorbance = Log 10 (1/T) where I0 incident radiant energy I energy which is transmitted

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Laws of Photometry2) Beers LawStates that absorption of light is directly proportional to both concentration of the absorbing medium and the thickness of the medium in the light path.Based on this, for a fixed path length, Absorption spectroscopy can be used to determine the concentration of the absorber in a solution.

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Laws of Photometry3) Beer - Lambert LawDefines relationship between Absorbance (A) and Transmittance (T).States that the concentration of a substance in solution is directly proportional to the Absorbance, A of the solution.Absorbance, A = c bwhereA measuredabsorbance, inAbsorbanceUnits (AU) constant known as themolar absorptivity (function of wavelength) (dm3 mol-1 cm-1) c concentrationof the absorbing species (mol dm-3 ) b path length through the sample (cm)

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Limitations of Beer - Lambert LawOnly applicable to monochromatic radiations.Non-linearity arises at high concentration. Chemical & Instrumental factors which causes non-linearityDeviations in absorpitivity coefficient at high concentrationScattering of light due to particulates in sampleFluorescence or phosphorosence of the sampleChanges in the refractive indexShifts in chemical equilibrium Non-monochromatic radiationStray light

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Types of Absorption SpectrophotometerCommonly used Absorption Spectrophotometer is :UV Vis - NIR Spectrophotometers (Ultra Violet Visible Near Infra Red Spectrophotometer)

This means it uses light in the UV, visible and near-infrared (NIR) ranges.9

UV - VIS SPECTROPHOTOMETERS 10

RADIATION SOURCESProvide sufficient intensity of light for making a measurement.Blackbody Sources :- A hot material like electrically heated filament which emits a continuous spectrum of light.Discharge lamps :- When electric current pass through a rare gas or metal vapour, the electrons collide with gas atoms, exciting them to higher energy levels and then decay to lower levels by emitting light.Lasers :- Laser beam is highly directional, monochromatic and provide high density energy which can be finely focused.

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Blackbody sourcesTungsten filament lamps (350 to 2500 nm) visibleGlowbar lamps (1 to 40 m) infraredNernst glower lamps (400 nm to 20 m) infrared12

Discharge lampsHydrogen or deuterium lamps (160 to 380 nm) ultravioletMercury Lamps ( 253.7 nm) visible and near UVNe, Ar, Kr, Xe discharge lamps (300 13 nm) near UV to near IR13

Wavelength Selectors or Filtering ArrangementFor selection of a narrow band of radiant energy.Requirements of filters:- -- High transmittance at desired wavelength -- Low transmittance at other wavelengthIt can be;Optical FiltersAbsorption filterInterference filterMonochromatorsPrism monochromatorsDiffraction gratingReflection Gratings

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Absorption Filters

Used in the visible range.Have effective bandwidths from 30 to 250 nm.Less expensive than interference filters.

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Three types of absorption filters:

1) Coloured glass :- The filters absorb all wavelengths of light except for particular wavelengths which they pass.2) Dyed gelatin :- do not last long and must be frequently replaced.3) Sharp cutoff (band pass filter) :- consists of two filters put together.

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Interference Filters

Use optical interference to provide narrow bandwidths of radiation.Consists of a dielectric insulator like MgF2 or CaF2 which is sandwiched between two semitransparent metallic films. These three layers are then sandwiched between two plates of glass or transparent materials.

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Interference Filters - working

Thickness of the dielectric layer determines the wavelength of the transmitted radiation. When the beam of radiation strikes this filter, some of the radiation passes through the first metallic layer while the rest is reflected. The remaining light then strikes the second metallic layer and some is passed while the rest is reflected.

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Interference Filters - working

If the reflected light from the second layer is of the proper wavelength, it is partially reflected from the inside surface of the first layer in phase with incoming radiation of the same wavelength. The result is that the desired wavelength is reinforced while the others wavelengths, being out of phase, undergo destructive interference.Interference filters are used throughout the ultraviolet and visible regions and about 14 m into the IR region.

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InterferenceInterferenceis a phenomenon in which twowavessuperimpose to form a resultant wave of greater or loweramplitude.

Two types ;Constructive interference :- Constructive interference occurs when thephasedifference between the waves is a multiple of 2. Destructive interference :- Destructive interference occurs when the difference is an odd multipleof .

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Resultant wave

Wave 1

Wave 2 Constructive interference Destructive interference

Monochromators

Principle is based on Refraction.Allows only certain wavelengths to be selected and used.Types; PrismGratingDiffraction GratingReflection Grating

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Prism MonochromatorBasic principle : The isolation of different wavelengths in a prism monochromator is based on refractive index of materials is different for radiation of different wave lengths.Optical elements:Entrance slitCollimating lensPrism or gratingFocussing element Exit slit

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Basic Principle24

Basic Setup Prism Monochromator25

Working - How select one particular wavelength?26

h1sinq1 = h2sinq2Snells Law of Refraction : Also, remember that no refraction occurs if light at normal or 1 = 0 So, light must hit prism at an angle.Most common is a 60o prism of glass or quartz.High resolution prism: mixture of Silicon dioxide, Sodium chloride and Potassium bromide.

Grating MonochromatorTwo types:Diffraction GratingReflection Grating 29

Diffraction /Transmission Grating Basic Principle: DiffractionDiffraction phenomenon is described as the apparent bending of waves around small obstacles and the spreading out of waves past small openings.

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Interference pattern from two-slit diffraction.

Diffraction GratingA diffraction grating consists of a series of parallel grooves or slits on a highly polished reflecting surface.When light is incident on a diffraction grating, diffractive and mutual interference effects occur, and light is reflected or transmitted in discrete directions.The separation of grooves in the direction of radiation is a whole number of wavelength, then the waves would be in-phase and radiation would be reflected undistributed.31

Diffraction Grating32

Relation between , d, and wavelength of radiationd distance between grooves angle at which the radiation is reflectedm order of interferenceN total no. of grooves

33m = 2 d sin Resolving Power= mN

Diffraction Grating in Monochromator34

Reflection GratingOn contrary to the above, if the plate is mirrored, we get reflection grating which have got vast applications in spectrophotometry.Most commonly used grooved surface with reflective coating (Al, Au, Pt) .

35nl = d(sin b + sin )

Effect of Slit Width36

Comparison between Grating & Prism37Type of DispersionSizeStray Lightl range of useGratinguniform dispersion vs. lSmallerHigher stray lightunlimitedPrismshorter l better separatedlargerLess of problemLimited(l 350 nm)

Increase size of either prism or grating will give better dispersion. Stray light can be removed with filters.

Stray lightStray light is light of wavelengths different from those wanted.Sources of stray light:incompletely removed higher order wavelengths in grating instrumentscattering of light by dustreflection of light by lens and grating mountingsPrevention of stray light:use a double monochromatorfirst monochromator selects wavelengths and passes them to a second which refines the wavelengthspaint all components except for reflecting surfaces which are desired blackenclose system to keep out dust

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Bandwidth is defined as the frequency band around the carrier frequency containing 99 percent of the signal power.The bandwidth for an individual AM station is about 10,000 Hz.The bandwidth for an individual FM station is about 200,000 Hz.Signal bandwidth

frequency

bandwidthfc =carrier frequencyfc+fm=upper sidebandfc+fm=lower sideband

ResolutionIt is the smallest amount of input signal change that the instrument can detect reliably.40

Photosensitive DetectorsPurpose is quantitative measure of radiation intensities.In photosensitive detector, the light energy is converted into electrical energy.Electric current produced by this can be measured with a sensitive galvanometer. Requirements of a good detectorHigh sensitivityLinear response over the wavelength range of interestFast responseLittle or no signal in absence of light (dark current)

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Photosensitive Detectors

Different types:Photovoltaic CellPhoto-emissive CellSilicon Diode DetectorsPhoto Diode Array (PDA)

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Photovoltaic or Barrier Layer Cell43

Photovoltaic CellAdvantages: Robust in constructionNeed no external power sourceGood for portable instrumentSensitive to almost the range of wavelength of the spectrum.

Disadvantage: Shows fatigue (decrease in response with continued illumination), Difficult to amplify signal, since small internal resistance of selenium (Ohms law: I=(V/R)).

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Photo-emissive CellRequires an external power supply to facilitate flow of electrons.Amplifier circuits are employed for the amplification of the current.Three types;High vacuum Photo-emissive cellGas-filled Photo cellPhotomultiplier Tube (PMT)

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High vacuum Photo-emissive cell

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High vacuum Photo-emissive cellThe spectral response depends upon the nature of the substance coating at the cathode.Cesium-Silver oxide cell sensitive to Near InfraRed wavelength.Potassium-Silver oxide/ Cesium-Antimony sensitive to visible & UV wavelength.Current number of photons.Smaller current than photovoltaic cell, but can be amplified.

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Gas-filled Photo cellSometimes inert gas like Ar, is present in the tube. As e- collide with gas, more e- and ions produce results in an increase in current.Presence of the small quantities of the gas prevent the phenomenon of saturation current, when higher potential difference are applied between the cathode & anode.

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Photomultiplier Tube (PMT)49

photochathodeanodehigh voltagevoltage divider networkdynodes

lightelectronse-

Photomultiplier Tube - WorkingIt is a very sensitive device in which electrons emitted from the photosensitive cathode strike a second surface called dynode which is positive with respect to the original cathode.Additional electrons are generated at each dynode.If the above process is repeated several times, so more than 105 to 107 electrons are finally collected for each photon striking the first cathode.The amplification depends upon the number of dynodes and accelerating voltage.

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Photomultiplier Tube Advantages: very sensitive to low intensity.very fast response.Disadvantages: need a stabilized high voltage power supply. intense light causes damages. Large & expensive

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Silicon Diode Detectors52Silicon diode/Photo diode can be powered from a low voltage source. And signal can be amplified by a low noise op-amp.This type is not as sensitive as PMTs, but are small and robust.Commonly used semiconducting materials;SiGeInAs Indium ArsenideInSb Indium Antimonide

Silicon Diode Detectors53Diagram (Refer Note)

Photo Diode Array (PDA)54Diagram (Refer Note)

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Scanning Double Beam Spectrophotometer

56Scanning Double Beam Spectrophotometer In double beam arrangement, the light alternately passes through the sample and reference (blank), directed by rotating half-sector mirror (chopper) into and out of the light path.

When light passes through the sample, the detector measures the P. When the chopper diverts the beam through the blank solution, the detector measures P0.

The electronic circuit at the detector automatically compares P and P0 to calculate absorbance

57Advantages of double beam instruments

Automatic correction for changes of the source intensity and changes in the detector response with time or wavelength because the two beams are compared and measured at the same time.

Automatic scanning and continuous recording of spectrum (absorbance versus wavelength).

58 Applications of Ultraviolet/Visible Spectrophotometry Molecular spectroscopy based upon UV-Vis radiation is used for identification and estimation of inorganic, organic and biomedical species. Molecular UV-Vis absorption spectrophotometry is employed primarily for quantitative analysis. UV/Vis spectrophotometry is probably more widely used in chemical and clinical laboratories throughout the world than any other single method.