Analytical Instrumentation: Ultaviolet (UV) and Visible Absorption Methods

Nikhilbinoy.CAssistant ProfessorICE DepartmentAnalytical Instrumentation:Ultaviolet (UV) and Visible Absorption Methods

BasicsThe most important of all the instrumental methods of analysis are the methods based on the absorption of electromagnetic radiation in the visible, ultraviolet and infrared ranges.

Electromagnetic Radiation

Electromagnetic radiation is a type of energy that is transmitted through space at a speed of Considered as discrete packets of energy called photons.

where E = energy in ergsf = frequency in cycles, andh = Planks constant

Electromagnetic Radiation

For some purpose, electromagnetic radiation can be more conveniently considered as a continuous wave motion in the form of alternating electric field in space.

X-RayUVVisibleIRMicrowave200nm400nm800nmWAVELENGTH(nm)

Interaction of Radiation with MatterWhen a beam of radiation strikes a substanceThe radiation may be transmitted with little absorption taking place, and therefore, without much energy loss.The direction of propagation of the beam may be altered by reflection, refraction, diffraction or scattering.The radiant energy may be absorbed in part or entirely by the substance.

Incident RadiationReflectionAbsorbed RadiationMatterTransmitted Radiation

Scatter/Fluorescence

Absorption SpectroscopyUsually concerned with absorption and transmittance of a beam of radiation through matter.Reflection and scattering must be maintained minimum.Principle is, the amount of absorption that occurs is dependent on the number of molecules present in the absorbing materials.The intensity of the radiation leaving the substance may be used as an indication of the concentration of the material.

Absorption SpectroscopySuppose P0 is the incident radiant energy and P is the energy which is transmitted.Transmittance or % Transmittance

Absorbance

Optical density

Quantum TheoryThe energy states of an atom or molecule are defined.Any change from one energy state to another would require a definite amount of energy.If the exact quantity of energy to bring about a change from given state to another will be provided by photons of one particular frequency supplied by an external source, which may thus be selectively absorbed.The study of frequencies of the photons which are absorbed would thus indicate a lot about the nature of the material.The number of photons absorbed may provide information about the number of atoms or molecules of the materials present in a particular state.Qualitative analysisQuantitative analysis

DE = hn

Quantum TheoryMolecules possess three types of internal energy.ElectronicVibrationalRotationalThe various molecular energy states are quantized and the amount of energy necessary to cause any change in any one of the above energy states would generally correspond to specific regions of the electromagnetic spectrum.The method based on the absorption of radiation of a substance is known as Absorption Spectroscopy.

UV and visible

Near IR

Far IR

Fundamental Laws of PhotometryAbsorption occurs when a photon collides with a molecule and raises that molecule to some excited state.Each molecule can be thought of as having a cross-sectional area for photon capture, and photons must pass within this area to interact with the molecule.The cross-sectional area varies with wavelength.The rate of absorption as a beam of photons passes through a medium depends on the number of photon collision with absorbing atoms or molecules per unit time.The rate of absorption of photons doubles, ifthe number of absorbing molecules is doubled, by doubling either the length of the path of radiation through the medium or the concentration of absorbing species. (Here the factor absorbance is a variable)the beam power doubles, which doubles the number of collision. (Here the factor absorbance is constant)

Fundamental Laws of PhotometryBouguers law.Lamberts law.Beers law.

Lamberts Law

PIf a parallel beam of monochromatic radiation radiant power, P0, traverses an infinitesimally small distance of an absorber, the factor of decrease in power is proportional to distance.

Lamberts Law

PNow consider the entire absorber, integrate this equation0b

This equation is known as Lamberts lawAlso known as Bauguers law, since Bauguer really established this relationship several years before Lambert, but Bauguers publication was not generally known.Lamberts law simply states that, for parallel monochromatic radiation that passes through an absorber of constant concentration, the radiant power decreases logarithmically as the path length increases.

Lamberts LawThe dependence of radiant power on the concentration of absorbing species can be developed in a parallel manner if the wavelength and the distance traversed by the beam in the sample remains constant.The number of absorbing molecules that collide with photons is proportional to the concentration C (unit is g/litre).

followed by integration from dC=0 to C

Lamberts LawsIf both concentration and thickness are variable, then

Convert ln to log

is absorptivity a: Unit is litre g-1cm-1

Beers LawConsider a block of absorbing matter (solid, liquid, or gas) which contains n absorbing atoms/ions/molecules.Consider a small section with cross-section area S and infinitesimal small thickness dx.Image a surface dS at which photon capture will occur.Within this section there are dn absorbing particles.P0Px

Sdxb

Beers LawThe fraction of capture is proportional to dS/S.

Now consider the entire absorber.P0Px

Sdxb

Beers LawCross-sectional area S can be expressed in terms of the volume of the block V and its length b

Concentration C=n/V in moles per liter is

is absorptivity : unit is L mole-1cm-1

Beer-Lambert LawAs radiation passes through a cell, some radiation is reflected at each surface where there is a change in the refractive index.Can be almost entirely compensated by taking P0 as the radiant power transmitted through a cell that contains only pure solvent.The sample to be measured should be placed in a cell as nearly identical as possible.Cells should be free of scratches, dirt, and finger prints, all which scatter radiation.Turbidities in the solvent or the sample also scatter radiation.

Beer-Lambert LawA plot of absorbance versus concentration should be a straight line passing through the origin.

TransmittanceAbsorbanceConcentrationConcentration

Qualitative AnalysisThe sensitivity to concentration is greatest at the peak of an absorption band because the absorptivity is a maximum at the same point.For most quantitative determinations, this peak point (wavelength) is selected and the instrumental controls are not changed during the calibration and measurement.AbsorbanceWavelength

Qualitative Analysis: Simultaneous Spectrophotometric DeterminationWhen there are several components which absorb radiation, different components can be identified by noting the maximas whos wavelength are corresponding to a particular component.Each component have a particular characteristic wavelength.

Quantitative Analysis: Choice of WavelengthThe selection of a suitable wavelength in the spectrum for quantitative analysis of a sample can be made during the course of preparing the calibration curve for the unknown material.The calibration curve is plotted to show the absorbance values of a series of standards of known concentration.Concentration of actual sample can then be read directly using the working curve.AbsorbanceWavelengthAbsorbanceConcentration

Working Curve Method

Quantitative Analysis: Simultaneous Spectrophotometric Determination

Quantitative Analysis: Simultaneous Spectrophotometric DeterminationWhen there are several components which absorb radiation of the same wavelength, their absorbance add together and it would no longer be true that the absorbance of the sample is proportional to the concentration of one component.AbsorbanceWavelengthAbsorbanceConcentration

It is not often true

Quantitative Analysis: Simultaneous Spectrophotometric DeterminationA simplified and more common method is to convert the component under analysis, by adding a chemical reagent which specifically reacts with it to form a highly absorbing compound.Addition of this reagent to the mixture would result in change of the wavelength of the absorption maxima, so that there is no longer interference among the components.AbsorbanceWavelengthAbsorbanceConcentration

How can we identify these components, whose characteristic components (wavelength) are almost equal

Deviation from Beers LawRealInstrumentalChemical

Method of Standard AdditionWhen it is impossible to suppress physical or chemical interferences in the sample matrix, the method of standard addition may be used.The instrument response must be linear function of the analyte concentration over the concentration range and must also have a zero intercept (zero signal for zero concentration).

Method of Standard Addition 1Take the response of the instrument Rx obtained from the solution with unknown concentration x.Rx=KxAdd a small amount of known concentration a to previous sample, and note the instrument response Ra.Ra=K(x+a)

ConcentrationR

Rxx

Raa

Method of Standard Addition 1Unknown concentration of given sample x is

Addition of analyte equal to twice and to half the amount of analyte in the original sample are optimum statistically.All solutions should be diluted to the same final volume so that any interferent in the sample matrix will have an identical effect on each solutionConcentrationR

Rxx

Raa

Method of Standard Addition 2Take the response of the instrument Rx obtained from the solution with unknown concentration x.Add known concentration a of analyte and take the instrument response Ra.Unknown concentration is given by the point at which the extrapolated line intersects the concentration axis.ConcentrationR

Rxx

Method of Standard AdditionWidely used in electro-analytical chemistry to obtain results that are more accurate than those obtained using calibration curves.Atomic absorption and flame emission spectrophotometry use this method with complex sample matrices where viscosity, surface tension, flame effects, and other properties of the sample solution cannot be accurately reproduced in calibration solutions.

Deviation from Beers Law: RealReal deviations arise from changes in the refractive index of the analytical system.Kerturn and Seiler pointed out that Beers law strictly applies only at low concentrations.At low concentrations of 0.001M or less, the refractive index is essentially constant, but at high concentrations, the refractive index varies considerably and does absorptivity.

TransmittanceAbsorbanceConcentrationConcentration

Deviation from Beers Law: InstrumentalBeers law assumed monochromatic radiation, but truly monochromatic radiation is approached in only specialized line emission sources.If a is essentially constant over the instrumental band pass, then Beers law is followed within close limits.

awavelengthNarrow bandWide bandNarrow bandWide band

No apparent change is observed where the curve is linear

Average absorptivity decreases for wide band as compared with narrow band, where the slope of curve changes rapidly.

Deviation from Beers Law: InstrumentalA system appears to follow Beers law if the ratio of change of absorptivity versus wavelength is constant over the wavelength interval passed by the wavelength selector.The wavelength setting must be reproducible.It is the ratio between the spectral slit width and the band-width of the absorption band that is most important.The sensitivity to concentration is greatest at the peak of an absorption band because the absorptivity is a maximum at the same point.For most quantitative determinations, this peak point (wavelength) is selected and the instrumental controls are not changed during the calibration and measurement.

Deviation from Beers Law: Chemical

The dichromate ion absorbs in the visible region at 450nm.Upon diluting a dichromate solution, the equilibrium shifts to left.If an absorbing species is involved in a simple acid-base equilibrium, Beers law fails unless the pH and ionic strength are kept constant.Chemical deviation from Beers law are caused by shifts in the position of a chemical or physical equilibrium involving the absorbing species.

Conventional SpectrophotometerRadiation sources.Filtering arrangement.Monochromator.Detector.Sample holder.

Schematic of a conventional single-beam spectrophotometer

Radiation SourcesFunction of the radiation source is to provide sufficient intensity of light suitable for making a measurement.Tungsten lamp is the most common and convenient source.Consists of a tungsten filament enclosed in a glass envelope.It is cheap, intense and reliable.A major portion of the energy emitted is in the visible region, and only about 15 to 20% is in the IR region.It is desirable to use a heat absorbing filter between the lamp and the sample holder to absorb most of the IR radiation.

X-RayUVVisibleNear IRIR160nm400nm800nm4000nmTungsten

Radiation SourcesHydrogen or Deuterium discharge lamp is used for work in the UV region.The envelope material of the lamp puts a limit on the smallest wavelength, which can be transmitted.Quartz is suitable only upto 200nm.Fused silica is suitable only upto 160nm.There is no emission beyond 400nm.For this reason, both Deuterium and Tungsten lamps are used in UV and Visible.

X-RayUVVisibleNear IRIR160nm400nm800nm4000nmD2, H2Tungsten

Radiation SourcesFor work in the IR region, a tungsten lamp may be used.Due to high absorption of the glass envelope and the presence of unwanted emission in the visible range, tungsten lamps are not preferred.Nernst filament are preferred in IR region.Operated at lower temperature and still radiate sufficient energy.

X-RayUVVisibleNear IRIR160nm400nm800nm4000nmD2, H2TungstenNearnst Filament

Radiation SourcesFor fluorescent work, an intense beam of UV light is required.Xenon arc or mercury vapour lamp is used.Cooling arrangements are very necessary when these types of lamps are used.Mercury lamps are usually run direct from the AC power lines via a series of ballast choke, gives some inherent lamp power stabilization and automatically provides the necessary ionising voltage.Tungsten-Halagen light source has a higher intensity output than the normal tungsten lamp in the changeover region 320-380nm.Has a larger life and doesnot suffering from blackening of the envelope.

X-RayUVVisibleNear IRIR160nm400nm800nm4000nmD2, H2TungstenTungsten-HalagenNearnst Filament

Optical FiltersA filter may be considered as any transparent medium which by its structure, composition, or colour enables isolation of radiation of a particular wavelength.Must have high transmittance at the desired wavelength.Must have low transmittance at other wavelength.In practice, the filter transmit a broad region of the spectrum.

Instrumental Spectral Bandwidth

Natural Spectral Bandwidth

Optical FiltersIn practice, the filter transmit a broad region of the spectrum.Filter is characterized by:The relative light transmission at the maximum of the curve A.The width of the spectral region transmitted NBW (the half width-the range of wavelength between the two points on the transmission curve at which the transmission value equal 0.5A).The residual value of transmission in the remaining part of the spectrum Ares.

Natural Spectral Bandwidth

Optical FiltersAbsorption filter.Interference filter.

Natural Spectral Bandwidth

Optical Filters: Absorption FilterUsually consists of coloured media: colour glasses, colour films (gelatin, etc.), and solution of the coloured substances.Has wide spectral band-width.Efficiency of transmission is very poor and is of the order of 5 to 25%.It is possible to obtain more selective light filters from coloured media by increasing their thickness two or more times.Transmission is decreased, but selectivity is increased.Composite filters consisting of sets of unit filters are often used.One set consists of long wavelength sharp cut-off filters, and the other of short wavelength cut-off filters.Combination are available from 360nm to 700nm.

Optical Filters: Absorption FilterTwo types:Glass filter consists of a solid sheet of glass that has been coloured with a pigment, which is either dissolved or dispersed in glass.Gelatin filter consists of a layer of gelatin impregnated with suitable organic dyes and sandwitched between two sheets of glass.Gelatin filters are not suitable for use over long periods.With the absorption of heat, they tend to deteriorate due to changes in Gelatin and bleaching of the dye.

Optical Filters: Interference FilterConsists two semi-transparent layers of silver, deposited on glass by evaporation in vacuum and separated by a layer of dielectric (ZnS or MgF2).Semi transparent layers are held very close.Refractive index of spacer layer is low.Only light with desired wavelength which is reflected twice will be in phase and come out of the filter.

Thickness of transparent spacer film d determines the wavelength transmitted.

Transparent spacer filmSemi transparent silver film

47

Optical Filters: Interference FilterAdvantages:Allow a much narrower band of wavelength to pass and are similar to monochromator in selectivity.Simpler and less expensive.Can be used with high intensity light sources.Continuous selection is possible by using wedge filter.

Transparent spacer filmSemi transparent silver film

48

Optical filters

Absorption filterInterference Filter

MonochromatorsMonochromators are optical systems, which provide better isolation of spectral energy than the optical filters.Efficiency is much better than that of filter.Spectral half bandwidth of 1nm or less are obtained.Prism monochromator.Diffraction gratings.Holographic (Interference) gratings.

Monochromators: Prism

Polychromatic

Ray

Infrared

Red

Orange

Yellow

Green

Blue

Violet

Ultraviolet

monochromatic

Ray

SLIT

PRISM

Polychromatic Ray

Monochromatic Ray

Monochromators: PrismIf a parallel beam of radiation falls on a prism, the radiation of two different wavelengths will be bent through different angles.Refractive index of material is different for radiation of different wavelength.This becomes an important consideration for selection of material for the prism, because only those materials are selected whose refractive index changes sharply with wavelength.

Monochromators: PrismPrism is rotated or exit slit is moved to select wavelength.Scale is non-linear.Same collimating mirror is used for both M1 and M2 to save costs.Glass prisms are used essentially in visible range. Quartz prism can cover the UV spectrum also.Dispersion given by glass is about three times that of quartz.Quartz shows the property of double refraction.Are expensive.

Entrance slitMirrorM1PrismM2ExitslitFocal point of different wavelength will be different

Monochromators: Diffraction GratingsDiffraction grating consists of parallel, equally spaced grooves ruled by a properly shaped diamond tool directly into a highly polished surface.The radiation incident on each groove is diffracted (spread out) over a range of angles.At certain angles reinforcement, or constructive interference occurs, as stated by

In a Littrow mount, the angle of incidence equals the angle of diffraction.

Incident beamReflected beam

Monochromators: Diffraction GratingsThe grating formula shows that the incident energy is diffracted into several orders.The unwanted radiations (higher orders) must be removed with filters, or premonochromators, otherwise it will appear as stray light.By changing the angle at which the radiation strikes the grating, it is possible to alter the wavelength reflected.Compared with prisms, the gratings provide much higher resolving power (mN) and can be used in all spectral regions.Scale is linear.

Monochromators: Holographic (Interference) GratingsConstruction:Coated a glass substrate with a layer of photoresist.Exposed to interference fringes generated by the intersection of two collimated beams of laser light (photo resist is developed, which gives a surface pattern of parallel grooves).Coated with Aluminium to form grating.Compared with ruled grating:The grooves of holographic gratings are more uniformly spaced, smoothly and uniformly shaped.Have much lower stray light levels.Can be produced in much less time.

Monochromator Structure

Stray LightIt refers to the idea that an optical system will contain light not intended in the design.Similar to signal-to-noise ratio and contrast ratio.Stray light sets a limit on how dark the system can be.Optical measuring instruments that work with monochromatic light, such as spectrophotometers define stray light as light in the system at wavelengths (colours) other than the one indented.One method to reduce stray light is the use of double monochromators.The terms used for the indication of stray light is Stray Radiant Power (SRP) and Stray Radiant Power Ratio (SRPR).

Stray LightThe source of stray light are:Ghost orders (in diffraction grating).Scattered light (towards a telescope from particles along the optical path to a star).Reflections (from lens surface).Use of anti-reflective coating is to reduce stray light.

Optical ComponentsMaterials used for the construction of optical components areOrdinary silica glasses (350nm to 3000nm).Special corex glass (300nm to 350nm).Quartz or fused silica (210nm to 300nm for quartz).Reflection from glass surface and scattering are reduced by coating with magnesium fluoride.Is soft and has poor chemical resistance.Silica or synthetic quartz coating is the better solution.To minimize light losses, lenses are sometimes replaced by front surfaced mirrors.Chromatic aberrations and other imperfections of the lenses are minimized.

DetectorsEarlier detectors were the eye or film.For good detectors want:High sensitivity.Good signal to noise ratio.Constant response over range of interest.Fast response.Little or no signal in absence of light (dark current).

Detectors: Photo Voltaic Cell or Barrier CellConsists:Semiconductor substance, selenium deposited on iron metal base.Iron acts as one electrode.Selenium is covered with Silver/Gold as thin layer.Silver/Gold coating is done by (deposition) cathodic deposition in vacuum. This layer acts as collecting electrode.Light of sufficiently high energy passes through the thin transparent silver layer and hits Selenium causing electrons to be released which move across barrier toward silver layer (electro positive) and collected at iron layer to neutralize selenium layer.Current produced is proportional to photons hitting surface.Maximum response at 500nm (350nm to 700nm)

Detectors: Photo Voltaic Cell or Barrier CellAdvantages are:Very robust in construction.Cheap.Rugged.No external power source.Good for portable instruments.Linear relationship with the incident light intensity at constant temperature.Disadvantages are:Not very sensitive (sensitivity is greater within the visible region).Shows fatigue (decrease in response with continued illumination).Difficult to amplify the signal.

Detectors: High Vacuum Photoemissive CellsConsists two electrodes.Cathode having a photo sensitive layer of metallic cesium deposited on a base of silver oxide.Either axially centered or a rectangular wire anode.Electrodes are sealed within an evacuated glass envelope.when a beam of light falls on the surface of the cathode, electrons are released from it, which are drawn towards the anode.Anode is maintained at a certain positive potential.Give rise to photo current.

Detectors: High Vacuum Photoemissive CellsSpectral response depends on the nature of substance coating cathode.Cesium-silver oxide. Sensitive to near IR wavelength.Potassium-silver oxide or Cesium-antimony. Maximum sensitivity in visible and UV region.Large values of photo tube load resistor are employed to increase the sensitivity.Advantages:Sensitive.Signal easily amplified.Disadvantages:Some dark current.

Detectors: Photomultiplier TubeAre used to detect very weak light intensity.The tube consists of:Photoemissive cathode.Anode.Dynodes (cascade stages of electron amplification).The electrons generated at the photocathode are attracted by the first dynode, which gives out secondary electrons.A tube may have 9 to 16 dynodes.The dynode consists of a plate of material coated with a substance having a small force of attraction for the escaping electrons.With the fall of electron, each dynode discharge secondary electrons under the influence of positive potential.

Dynodes all covered with photoemissive material

Detectors: Photomultiplier TubeAdvantages:Very sensitive to low intensity (sensitivity can be varied by regulating the voltage of amplification).Very fast response.The response is linear due to relative small potential difference between the dynodes.Disadvantages:Need high voltage power supply.Intense light damage.Devices are sensitive to electromagnetic interference.Are more costly.May be damaged if excessive current is drawn from the final anode.

Sample CellMust be transparent at the wavelength used.Quartz or fused silica for UV (