A Brief Background To Spectrophotometry
UV-VIS SpectrophotometryA Brief Background to SpectrophotometryContentsElectromagnetic SpectrumIntroduction . 1Electromagnetic Spectrum. 1Radiation and the Atom . 2Radiation and the Molecule. 2Electron Transitions . 2Vibration and Rotation . 4Specific Absorption . 4Absorption and Concentration . 5Instrumentation . 6Light Source . 6Monochromator . 7Optical Geometry . 8Sample Handling . 10Detectors . 10Measuring Systems . 11Good Operating Practice . 11Limitations of Beer-Lambert Law . 12Sources of Error . 12Instrument Sources of Error . 12Non-instrument Sources of Error . 14Bibliography . 14Contact Us . 15The electromagnetic spectrum ranges fromGamma radiation, with the smallest wavelength (1pm), to Low Frequency radiation, with the largestwavelength well beyond conventional radio waves(100 Mm or 100000 km). Human beings can onlydirectly detect a very small portion of thisspectrum, with thermal perception of radiant heatbeing a sensitivity to infrared (IR) radiation, andsight is limited to the VIS spectrum. The spectrumis smoothly continuous and the labelling andassignment of separate ranges are appointedlargely as matter of convenience (Figure 1). UVVIS spectrophotometry concerns the UV rangecovering of 200-380 nm and the VIS rangecovering 380-770 nm. Many instruments will offerslightly broader range from 190 nm in the UVregion up to 1100 nm in the near infrared (NIR)region.All electromagnetic radiation travels at the speedof light in a vacuum (𝑐), which equals 3 108 m/s,the distance between two peaks along the line oftravel is the wavelength, (𝜆), and the number ofpeaks passing a point per unit time is thefrequency (𝑣). The mathematical relationshipbetween these three quantities is expressedusing:IntroductionThe spectrophotometer is ubiquitous amongmodern laboratories. Ultraviolet (UV) and Visible(VIS) spectrophotometry has become the methodof choice in most laboratories concerned with theidentification and quantification of organic andinorganic compounds across a wide range ofproducts and processes. Applied across research,quality, and manufacturing, with continuing focuson life science and pharmaceutical environments,they are equally as relevant in agriculture, animalhusbandry and fishery, geological exploration,food safety, environmental monitoring, and manymanufacturing industries to name a few.𝑐 𝜆𝜈Additionally, the laws of quantum mechanicsdefines the energy of a single particle of light, aphoton, as:𝐸 ℎ𝜈Where 𝐸 is the energy of the radiation, and ℎ isPlanck's constant. Combining these twoequations gives:Modern spectrophotometers are quick, accurate,and reliable. They require only small demands onthe time and skills of the operator. However, thenon-specialised end-user who wants to optimisethe functions of their instrument, and be able tomonitor its performance will benefit from theappreciation of the elementary physical lawsgoverning spectrophotometry, as well as the basicelements of spectrophotometer design. This briefbackground to spectrophotometry offers aninsight to support users of Biochrom’s range ofspectrophotometers.𝐸 ℎ𝑐/𝜆Which shows that the energy is inverselyproportional to wavelength. That is to say that theshorter the wavelength the higher the energy.In the visible region it is convenient, and themodern convention, to define the wavelength innm (nanometres), which is 10-9 m. However,1Author: Luke Evans, PhD. Technical Support and Application Specialist at Biochrom Ltd.Issue 1.0
Figure 1: The illustration describes the electromagnetic spectrum with the visible (VIS) range expanded for furthersubdivision.historical literature may display alternative units,such as millimicron (mμ) or Angstrom (Å). Theseare simply converted using:Bohr model to explain the electronic phenomenawhich concerns spectrophotometry.The Rutherford–Bohr model defines an atom ashaving a number of electron shells, n1, n2, n3 andso on, in which the increasing values of nrepresent higher energy levels and greaterdistance from the nucleus. Electrons orbit thenucleus in subshells, designated s, p, d, and f,within each shell. Each n-shell contains aconfiguration of s, p, d, and f subshells and eachsubshell can house a maximum of two electrons(Figure 2). No two electrons can have identicalenergies, but for succinctness they can begrouped related to the n-shell they occupy, 1s, 2s,2p, 3s, 3p, 3d, and so on.1 𝑛𝑚 1 𝑚µ 10 ÅRadiation and the AtomIt is convenient to describe electromagneticradiation as waves. However to clearlydemonstrate the interactions that lead to specificabsorption, it is helpful to consider the radiation asdiscrete packages of energy, or quanta, calledphotons.The phenomena of absorbance depends upon theatomic structure, more specifically the atomicorbitals which each of the electrons of thoseatoms occupy and the associated energy levels ofthose orbitals. Occupied orbitals are finite and welldefined, but an electron can be moved to a moreenergetic orbital, in a process called electronexcitation, providing a quantum of energy equal tothe energy difference between the ground andexcited state is delivered. Excited states aregenerally unstable and the electron will rapidlyrevert to the ground state, in a process termedelectron relaxation, losing the acquired energy,described as emission.By considering atoms of sodium vapour, the effectof subjecting an atom to an appropriate radiationcan be demonstrated. Excitation of an electron, inthe outermost subshell of a sodium atom, by aphoton at 589, 330 or 285 nm will promote itstransition to varying excited states; correspondingwith the higher energy (shorter wavelength) of theradiation (Figure 3.).Radiation and the MoleculeElectron TransitionsElectrons in the atom can be considered asoccupying groups of similar energy levels. Themore complicated molecular model showsbonding electrons associated with more than oneWhilst the accepted model of atomic andmolecular structure has arisen from theSchrödinger wave mechanical treatment, it isconvenient to employ the simpler Rutherford–2
SubshellsShellsn1 (2)1 (2)n2 (8)pdf1 (2)3 (6)n3 (18) 1 (2)3 (6)5 (10)n4 (32) 1 (2)3 (6)5 (10) 7 (14)Figure 2: The illustrations above show the spatial geometries of atomicsubshells. Each electron pair can occupy any space of the same colour,allowing for two pairs in each geometry except for the s subshell. They areshown on xyz axes and the specific nomenclature, based on their orientationabout that axis, is shown below each illustration. Within larger atomssubsequent subshells envelope the equivalent subshell of the previous nshell, but maintain the same geometry. The table left shown the subshellcomposition of the first four n-shells. The number in each cell defined thenumber of that given subshell at that n-shell level, and the numbers inbrackets define the maximum number of electrons that can beaccommodated in each shell and subshell.nucleus, and are particularly susceptible tosubshell transitions.transitions from bonding MO’s to their relativeantibonding MO’s, and from non-bonding MO’s toeither antibonding MO’s (Figure 4).The electrons concerned, may be present in oneof two chemical bond types; sigma (σ) bondswhich result from s-subshell overlap, or thegenerally weaker pi (π) bond which results from psubshell overlap.Figure 4: The diagram shows the electron transitionsbetween molecular orbital (MO) types. σ and π-bondswithout an asterisk denote bonding MO’s, while thosemarked with an asterisk denote antibonding MO’s, and ndenotes a non-bonding MO. Due to relatively highstability of σ-bonds, the σ σ* and n σ* transitionsrequire relatively high energy, and are thereforeassociated with shorter wavelength radiation UV.Whereas the relatively low stability of the π-bond, meansthat n π* and π π* are associated with both UV andlarger wavelength VIS radiation.The presence of a carbon-carbon double bond inthe molecule increases the likelihood of π-bonds.Especially if they alternate with single bonds(conjugate double bonds), where one of thebonding MO’s is raised in energy and the otherlowered relative to the energy of an isolateddouble bond. The same applies to the antibondingMO’s. The effect is greater still if the bond containsa highly electronegative atom, such as nitrogen,which attract electrons more strongly. As a result,the transition probability of molecules with πbonds is enhanced, the wavelength of maximumexcitation moves to a longer, less energetic,wavelength and often the likelihood of transitionsto higher excitation states is increased.Figure 3: The diagram illustrates the energy deliveredresulting in alternative subshell transitions.Chemical bonds are formed by overlappingatomic shells that result in one of three types ofMolecular Orbital (MO); bonding (low energy),antibonding (high energy), or non-bonding.Excitation is most typically associated withtransitions induced in electrons involved inbonding MO’s, and the atoms involved are usuallythose containing s and p occupied electrons.Excitation by UV-VIS radiation results in electron3
The probability that transition will occur is closelyrelated to MO structure. If the MO composition isknown, the probability of transition can becalculated with relative certainty and an estimatecan be made of the energy required to induceelectron transition, indicating an approximatevalue for the molar absorptivity of the species.wavelength scale, at which a given substanceshows absorption 'peaks', or maxima, is called anabsorption spectrum (Figure 5).Vibration and RotationThe internal molecular structure may respond toradiant energy in addition to electron transitions.In some molecules the bonding electrons alsohave natural resonant frequencies, giving rise tomolecular vibration, while others exhibit a rotation.The differences in energy levels associated withvibration and rotation are much smaller than thoseinvolved in electron transitions, thereforeexcitation resulting from these phenomena willoccur at comparatively longer wavelengths;vibrational excitation is typically associated withIR radiation, while rotational excitation areassociated with far-IR or even microwaveradiation.Figure 5: Shows an example VIS range absorptionspectrum.An absorption spectrum of a compound is a usefulphysical characteristic, for both (concentration) analysis. In its simplest form,absorption of wavelengths at the red end of theVIS spectrum, and reflection of unabsorbedwavelengths will result in the compoundappearing green/blue (Figure 6).Despite vibrational and rotational excitation beingprimarily associated with spectral regions otherthan UV-VIS, they do have an effect on electrontransitions within this range. The principal effect isof ‘broadening’, that is the deviation of anobserved absorption region from its predictedregion.For most species, especially in solution, excitationdoes not appear as sharp absorbance points athighly differentiated wavelengths, but rather asbands of absorbance over a range ofwavelengths. A principal reason is thatabsorbance at the electron transition level arefrequently accompanied by smaller structures atthe vibrational level. In the same way eachvibrational structures may have even smallerassociated structures at the rotational level, so anabsorbance spectrum due to electron transitionsmay display far more complex structures thanexpected.AbsorbedWavelength700 nm600 nm550 nm530 nm500 nm450 nm400 d-VioletRedOrangeYellowFigure 6: Shows the colour of absorbed light and itscomplimentary reflected colour from the VIS spectrum.The given wavelengths are for estimating the spectralregion only.The chemical group that most strongly influencesthe absorbance of a compound is referred to asthe chromophore. As discussed earlier,chromophores which can be excited by UV-VISradiation involve a multiple bond (such as C C,C O or C N). They may be conjugated with othergroups to form complex chromophores, andincreasingly complex chromophores move theassociated absorption peak towards longer, lessenergetic, wavelengths and generally increasethe degree of absorbance at the absorptionmaxima (Figure 7).Specific AbsorptionEach electron in a molecule has a unique groundstate energy, and as the distinct levels it may bepromoted to are also unique, there is a finite andpredictable set of transitions available to electronsin any given molecule. Each transition, resultingfrom the absorption of a photon, will have a directand permanent relationship between thewavelength of the photon and the particulartransition that it stimulates, known as specificabsorption. A plot of those points along the4
Figure 7: Shows a composite of UVVIS range absorption spectrabasedonthecomparativecomplexity of benzene and abicinchoninic acid (BCA)-coppercomplex, and the effect it has ontheir respective absorption maxima(λmax).1760). Therefore successive layers of equalthickness will transmit an equal proportion of theincident energy. It is defined by the equation:The correlation between molecule complexity anddetectability using UV-VIS spectrophotometrylends itself to the measurement of organiccompounds. However, a wide range of inorganiccompounds offer themselves to similar methodsof analysis. Species with a non-metal atom doublebonded to oxygen absorb in the UV region, andthere are several inorganic double-bondchromophores that show characteristic absorptionpeaks. In some instances, measurement ofinorganic materials may demand a secondaryprocess, such as complexation with a colourforming reagent or oxidation. For example,manganese (II) oxide (MnO) oxidised tomanganese (VII) oxide (Mn2O7), and measured asthe permanganate ion (MnO4-).𝐼 𝑇𝐼0Where 𝐼 is the intensity of the transmitted light, 𝐼0is the intensity of the incident light, and 𝑇 is thetransmittance. It is typical to expresstransmittance as a percentage of the incidentlight, which is defined as follows:𝐼 100 %𝑇𝐼0The second, Beer’s Law states the absorption oflight is directly proportional to both theconcentration of the absorbing medium and thethickness of that medium (Beer, 1852).Absorption and ConcentrationFor analytical purposes, two main propositionsdefine the laws of light absorption. The first,Lambert's Law states the proportion of incidentlight absorbed by a transparent medium isindependent of the intensity of the light (Lambert,A combination of the two laws, the bance and transmittance.Figure 8: Illustrates the conditionswhen three samples with identicalabsorption are introduced into abeam of monochromatic light, eachtransmitting half of the intensity ofthe incident (50 %T).5
𝐴 𝑙𝑜𝑔𝐼100 𝑙𝑜𝑔 𝜀𝑐𝑙𝐼0𝑇that can then be applied to subsequent sample ofunknown concentrations of that same molecule.This avoids the relatively time consuming processof a plotting calibration curve.Where 𝐴 is the absorbance, which has no units,although it is often referred to in absorbance units(AU). 𝜀 is the molar attenuation coefficient of themedium (M-1cm-1), 𝑐 is the molar concentration(M), and l is the pathlength (cm). It is important tonote that the molar attenuation coefficient is afunction of wavelength, therefore Beer-Lambertlaw is only true at a single wavelength, also knownas monochromatic light.In a scenario where three identical samples of 50%T are placed sequentially in a beam of incidentradiation (100 %T), then the intensity after eachsample will be halved (Figure 8). The threesamples may be considered as knownconcentrations of an absorbing medium and it istherefore possible to plot transmission againstconcentration (Figure 9). This graph will follow anexponential curve, and so is of limited value.Figure 10: Shows the linear relationship associated withabsorbance plotted against concentration.To calculate the factor (𝑘), the absorbance of aknown concentration of the molecule of interestneeds to be determined. These two parameterscan then be used as detailed in the equation.𝑘 ��𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒This factor can then be used in the followingequation to calculate the concentration (𝑐) fromthe absorbance (𝐴) of a sample of unknownconcentration.𝑐 𝑘 𝐴Figure 9: Shows the exponential curve associated withtransmission plotted against concentration.InstrumentationHowever, providing the light is monochromaticand the Beer-Lambert law is obeyed, it becomespossible to define the process in terms ofabsorbance units (AU).In their simplest form, a spectrophotometer has alight source, a monochromator, a samplecompartment, and a detector coupled with ameasurement system and result readout.For the same example, converting %T to AU thenplotting absorbance against concentration showsa linear relationship (Figure 10). Making theresults more convenient to be expressed inabsorbance, rather than transmission, whenmeasuring samples of unknown concentration,given that linear calibration curves are available.Light SourceAn alternative use of the linear relationshipbetween absorbance and concentration, is tocalculate a factor for a specific molecule of interestVIS light is normally supplied by a tungsten basedlamp, with tungsten-halogen lamps havingUV light is generally derived from a deuterium arclamp that provides emission of high intensity andsuitable continuity in the 190-380 nm range. Aquartz envelope is necessary to transmit theshorter wavelengths of UV radiation.6
KeyTungsten-halogenDeuteriumXenonRed LEDBlue LEDGreen LEDFigure 11: Shows approximateemission spectra of tungstenhalogen, deuterium, xenon, andLED light sources as per the keyabove. In a system that combinesdeuterium and tungsten lamps, theUV-VIS crossover is usually set toapproximately 360 nm.relatively higher output in the UV-VIS crossoverregion (Figure 11). The long wavelength limit isusually the cut-off of the glass or quartz envelopeof the lamp, but is normally beyond the usefulvisible limit at 900 nm.MonochromatorThe monochromator is responsible for producinga selectable beam of monochromatic (singlewavelength) radiation from the wide range ofwavelengths provided by the light source. Theycomprise of any number or combination ofLenses, filters, gratings, mirrors, and slits (Figure12).Xenon flash lamp light sources are an alternativeto combined deuterium-tungsten systems. Xenonflash lamps cover the UV and VIS range, and havea very long lifetime. However, additionalprocesses to account for higher levels of straylight, as well as less energy in the VIS region,means that the instruments are limited toapplicationswherehigherinstrumentspecifications are not required.Two basic methods of wavelength selection exist;filters, and dispersing elements such as diffractiongratings.Filters of coloured glass or gelatine are thesimplest form of selection, but they are limited inusefulness due to cost-effective filters beingrestricted to the VIS region, as well as having poorwavelength resolution: Typically they cannotisolate wavelength ranges smaller than 30-40 nm.More sophisticated interference filters canachieve wavelength resolutions of 10 nm or less,Light Emittin
appreciation of the elementary physical laws governing spectrophotometry, as well as the basic elements of spectrophotometer design. This brief background to spectrophotometry offers an insight to support users of Biochrom’s range of spectrophotometers. Electrom
Book · September 2015 CITATIONS 0 READS 68,531 . All content following this page was uploaded b y Cosimo A. De Car o on 12 No vember 2017. The user has r equested enhancement of the do wnloaded file. UV/VIS Spectrophotometry UV/VIS Spectrophotometry . whereas infrared light has less energy than visible light due to its longer wavelength.
applications using molecular absorption, fluorimetry and resonance light scattering spectrophotometry are presented. Based on the literature data and the experience in the field, challenges and perspectives in the ion-pair spectrophotometry are also considered. KEYWORDS: Ion-pair spectrophotometry, pharmaceuticals, UV-VIS absorption,
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Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of à -Lactam Antibiotics 113 2.2 Direct spectrophotometry enrich ed by chemometric procedures The other way of improving the selectivity of direct spectrophotometry for the determination of Ã-lactam antibiotics is the enrichment of
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