UVVIS and IR Molecular Spectrometry.ppt

,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,*,*,UV-VIS and IR Molecular Spectrometry,Chapter 8,Review,UV-VIS,Utilizes light,Ultraviolet and visible regions of the electromagnetic spectrum,Analyze laboratory samples of molecular compounds and complex ions,Qualitative analysis,Identification of unknowns,Detection of impurities in knowns,Compares absorption or transmission spectra with now spectra,Accomplished with the use of Beers law,Spectra results from electronic transitions in the analyte molecules/complex ions,UV-VIS Instrumentation,Sources-Light,Special light sources used,Provides optimum-quality light beam,For spectrum region utilized,One that emits intense continuous spectrum across entire region of spectrum,Example:visible region,Tungsten filament source,Often referred to as a colorimeter,UV-VIS Instrumentation,Sources-Light,Ultraviolet absorption light source-Deuterium lamp,Called a UV spectrophotometer,Tungsten Filament Lamp,For visible region,Very bright,Emits light over entire visible region,Near infrared region,Intensity varies across range,Deuterium Lamp,Isotope of hydrogen,one neutron(0 neutrons for H,2 neutrons for tritium),Deuterium at low pressure,Constant UV emission,when electricity applied,b/c of presence of deuterium,Emits at 185 to 375nm with varying intensity,Wavelength Selection,To plot absorption spectrum,Must control wavelengths emitted by the source,So you can measure the absorbance at each wavelength,For quantitative analysis with Beers law,Need to carefully elect wavelength of maximum absorption,For plot of absorption at each concentration,Have to filter out unwanted wavelengths,Allow only the wavelength of interest to pass,Wavelength Selection,Reality sezs:,No such thing as a single wavelength,Spectrum of colors continuous,No sharp delineation between green and blue,Where one wavelength ends and the adjacent begins,.:Wavelength selection is a matter of narrowing the selection from a larger band,Called bandwidth,Narrowness of the band is called the resolution,High resolution(narrow bandwidth)ideal,Can use absorption filters=inexpensive way to isolate a wavelength band,Monochromators,Monochromators(“mono”=one;“chromo”=color),More sophisticated than an absorption filter,Isolates the narrow band of wavelengths from visible and UV sources,Three parts,Entrance slit,Dispersing element,Exit slit,Often a network of mirrors,Aligning or collimating beam of light before/after it contacts the dispersing element,Monochromators,Entrance slit,Small circular/rectangular hole in an opaque plate,Size often variable,So intensity of the beam can be adjusted,Wider the opening,the more intense,Where light enters the monochromator from the source,Creates a unidirectional beam of light,Appropriate intensity from the source,Monochromators,Dispersing element,Disperses the light into its component wavelengths,Visible light,Spray of rainbow colors,Violet-blue on one end,Red on the other,Green and yellow in between,ROY G BIV,Monochromators,Exit slit,Selection slit of the particular band of the spectrum,Different narrow wavelength range emerges from the exit slit as it rotates,Can be variable,Makes wider bandwidth,Can be undesirable,Gives the name“monochromator”,Same concept for UV light,Monochromators,Dispersing element,Either a diffraction grating or a prism,Prism,3 dimensional triangularly shaped glass/quartz block,Not used as much as a diffraction grating,Diffraction grating,Highly polished mirror with precisely parallel lines or grooves,Light diffracted,and dispersed into the component wavelengths,Sample Compartment,Follows the wavelength selection,Beam passes into the sample compartment,Holds the sample solution,In the cuvette,Positioned in light path,Has lid,Keeps relatively free of stray light,Detectors,Two types,Photomultiplier,Light sensor with a signal amplifier,Light strikes photosensitive surface,Electrical signal amplified,Dynodes for multiplying the signal,Electrode that emits other secondary electrons,Situated between photocathode and anode,Amplified signal then sent on to the readout,Detectors,Photodiodes,Makes use of semiconductors,Either electron rich(n-type semiconductor),Electron poor(p-type semiconductor),Both amplify signal,Cuvette Selection and Handling,For UV-VIS spectrophotometery,Must be transparent to ALL wavelengths of light,Visible light,Completely clear and colorless,Ordinary plastic and colorless glass are ok,NOT transparent to light in the ultraviolet region,For UV must be made of quartz,Matched curettes,Identical for path length and reflective and refractive properties in pathway,Problems with the cuvette,Avoid,Scratches,fingerprints,water spots,Particulate matter in the sample,Centrifuge sample,Filter sample,Interferences,Deviations,Maintenance,and Troubleshooting,Interferences,Quite common in qualitative and quantitative analysis,A contaminating substance that gives an absorbance signal at the same wavelength range selected for the analyte,Qualitative analysis,Show up as an incorrect absorption spectrum,Leading to incorrect conclusions if contaminant not known,Quantitative analysis,Higher absorption,Absorptions are additive-subtract absorbance of interference at the wavelength from total to achieve actual absorbance,Interferences,Deviations,Maintenance,and Troubleshooting,Deviations,Beers law deviations,Plot not linear,Observed at higher concentrations of the analyte,Either chemical or instrumental,Instrumental deviations,Not possible for instrument to be accurate at high or low transmittance values-approaching either 0 or 100%,Normal is 15-80%transmittance or 0.1 to 0.82 absorbance,Chemical interferences,High or low concentration of analyte that causes chemical equilibrium shifts in the solution,Interferences,Deviations,Maintenance,and Troubleshooting,Maintenance,Safety and cleanliness,Cleanliness=longer instrument live and lower chance for contaminations,Troubleshooting,Checking electrical components,Mechanics of a procedure,Contamination,Fluorometry,Fluorometry,Utilizes the ability of some substances to exhibit luminescence,The phenomenon where substance appears to glow when light shines on it,.:when a light is absorbed,light of a different wavelength is emitted,Can occur with molecules,complex ions,and atoms,Fluorometry,Fluorometry,Atoms,molecules,and complex ions,Seek lowest possible energy state at all times,When raised to excited energy state through light absorptions,No longer in“ground”state,Will lose energy gained to return to ground state,Called fluorescence,The luminescence that results is called phosphorescence,Also called delayed fluorescence,It is the energy lost from going from the excited state to the ground state,Energy lost in the form of light,Simplified energy level diagram(fig.8.11,pg.216),Fluorometry,Fluorescence,Simplified energy level diagram,Only shows one wavelength,Reality=many wavelengths can be absorbed,Many can also be emitted,Called a fluorescence spectrum,Uses filters to isolate desired wavelength of emission,Called filter fluorometers,Need two filters,One to isolate the wavelength from the source to be absorbed(wavelength of maximum absorbance,One to isolate the wavelength of maximum fluorescence to be detected,Fluorometry,Measures the intensity of the light emitted,Intensity=concentration,Intensity is proportional to concentration,Measurement of a quantitative analysis,Measure series of standard solutions of the fluorescing analyte,Graph intensity vs.concentration,Expected to be linear in the concentration range studied,Fluorometry,Compounds to be measured,Very limited,Benzene ring systems(riboflavin or thiamine)highly fluorescent compounds,Metals-complex ion formation,Fluorometry vs.Absorption Spectrophotometery,Fluorometry vs.Absorption Spectrophotometery,Competing techniques,Molecular species and complex ions,Each offers own advantages and disadvantages,Fluorescence,Limited number of species to test,Those that do are generally very intense,Absorption more universally applicable,Fluorometry suffers less from interferences and more sensitive,If compound does fluorescence,fluorometry likely to be chosen for the analysis,Foods and pharmaceutical preparations for vitamin content,Introduction,Different from UV-VIS,Absorption of IT light results in vibration energy transitions,Can analyze pure liquids AND undissolved solids,Polymer films,gases,etc,Sampling containers are called“liquid sampling cells”,Have very short path lengths.often fractions of millimeters,Use large polished inorganic salt crystals as windows(cell walls)for IR light.,Solvents MUST be organic liquids,Do not dissolve inorganic salt crystals,NO WATER ALOWED!,Glass and plastic have significant disadvantages and are not usually used,Introduction,Different from UV-VIS,IR spectra usually transmission spectra rather than absorption spectra,Also wavenumber,rather than wavelength,is plotted on the x-axis,Characterization,Sharp absorption bands,Each characteristic of a particular covalent bond in the sample,IR spectra are molecular fingerprints,Better for qualitative analysis because of the specificity of the absorption bands,Require an interferometer(instead of light)between the source and the sample,Requires signal processing circuit for mathematical operation,Fourier Transformation to obtain spectra=,FTIR,IT Instrumentation,FTIR,IR light from source enters an interferometer,Device that creates a pattern of light,From the combined constructive and destructive interference of all component wavelengths,Pattern called an interferogram,Absorbing sample,Placed in the path of the light,Some wavelengths absorbed sharply,Creates different interferogram at the detector,Comparing the two interferograms using Fourier transformation signal processing results in the absorption spectrum of the sample,Advantages over dispersive techniques,Faster,Make it possible to be incorporated into chromatography schemes,Energy reaching the detector is much greater,Increases the sensitivity,Sampling,Sampling,The method by which a sample is held in the path of the IR light.,3 types,Liquid sampling,Solid sampling,Gas sampling,NOTE:glass and plastic are undesirable materials for the cells-they are molecular(covalent)materials and would absorb IR light and interfere with reading the sample,NOTE:inorganic salts-NaCl or KBr-are inorganic materials and do not absorb IR light because ionic bonds cannot undergo vibrational energy transitions.Covalent bonds can vibrate while ionic bonds cannot.,Sampling,Liquid sampling,Primarily done with the liquid sampling cell,Used with pure liquids,Called neat liquids,Liquid solutions,Sandwiching in a thin layer of liquid,Between two large NaCl or KBr crystals,IR light from the interferometer passes directly through the assembly from one window,through sample,then through the other window to the detector,KBr windows called salt plates,Uses either a sealed cell,a demountable cell,or a combination sealed demountable cell,Liquid sampling,Liquid sampling-,sealed cells,Permanente fixtures for the windows,Never disassembled during lifetime of use,Fixed path length,Useful for quantitative analysis,Path length is reproduced from one standard to another and for the unknown,Defined by the thin polymer film spacer mentioned,Leaves space to be filled by small volume of sample,Fill via luer-lock syringe ports,On the port side of cell,hole drilled in it to move sample from the syringe,through the inlet port to the space,then out the outlet port,Liquid sampling,Liquid sampling-,demountable cells,Cam be disassembled,May or may not use a spacer,w/o spacer,Liquid sample simply applied to one salt plate,Second plate positioned over the first to smear the liquid out between the plates,Nor useful for quantitative analysis,Path length is undefined,Not reproducible,W/spacer,Its positioned over one of the plates,Sample applied to this plate in the cutout space,Second plate positioned over the first,No inlet/outlet ports,Liquid sampling,Liquid sampling-,sealed demountable cells,Features of both,Sealed but meant to be disassembled,Disassembled for changing the spacer,Not for introducing the sample,Use inlet port with the syringe,Useful for both qualitative and quantitative analysis,Path length defined,Reproducible as long as cell not disassembled between standards and unknowns,Liquid sampling,Things in common,Use neoprene gaskets,Cushions fragile salt crystals from metal frame,Have hoes drilled to coincide with inlet and outlet ports for filling,Liquid sampling,Filing the cell,Can be troublesome!,NO AIR!,Push on plunger of the inlet syringe,Pull on the plunger of the empty syringe,Eliminates excessive pressure on the inlet side,Reduces possibility of damaging the cell,Refilling,Excess liquid may be used to rinse the cell,Eliminates residue from the previous one,Or rinse with dry volatile solvent and solvent evaporated before introducing the next sample,Liquid sampling,Salt crystals(KBr),PROTECT FROM WATER!,VERY WATER SOLUABLE,Severe damage caused by water,Liquid sampling,Liquid samples,Pure or mixed with a solvent(solution),Pure,Used when the purpose is either identification or the determination of purity,ID possible b/c spectrum is a fingerprint when no solvent or contaminant present,Solutions,Both analyte and solvent present in the spectrum,Can hinder results,Use solvents with simple spectra,Carbon tetrachloride-only C-Cl bonds,Choloroform-CHCl,3,Methylene chloride,Solid Sampling,Solution Prepared and Placed in a Liquid Sampling Cell,Solid dissolved in a suitable solvent,Measured using liquid sampling cell,Only method of solid sampling suitable for quantitative analysis,Only one that has a defined and reproducible path length,Solid Sampling,Thin Film Formed by Solvent Evaporation,Simple method for solids,Solution of the solid prepared,Several drops of the solution placed on the surface of a single salt plate,Evaporate,Leaves a thin film of solid on the plate,Fixed in the path of light,Measured same as the liquid sampling devices,Uses“disposable”IR cards,Polyethylene windows for applying the solution,Evaporate solvent,Test“solid”,Subtract out the polyethylene from the absorption bands,Solid Sampling,KBr Pellet,KBr as a dry,finely powdered substance,Squeezed under high pressure into a transparent disc,To both IR and visible light,Must be dry,Makes for good pellet,Avoids absorption bands due to water(-OH),0.1-2.0%analyte added to dry power before pressing,Use mortar and pestle to grind and mix KBr and sample,Wafer formed,Placed in light path,IR beam passes from the interferometer,thorough the wafer,to the detector,Solid Sampling,KBr Pellet,Pressing the KBr,Uses special die for this purpose,Bolt placed in the die,Surface covered with the mixture,Second bolt turned down onto the sample,Squeezing into the pellet or wafer,Two bolts,carefully,removed,Pellet should remain in the die,Appear transparent or nearly so,Die placed in the instrument s