Absorption Transmission Reflectance Spectroscopy

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Absorption / Transmission / Reflectance (ATR)Spectroscopy

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IntroductionUltraviolet-Visible-Near-Infrared (UV-Vis-NIR) spectroscopy isuseful to characterize the absorption, transmission, andreflectivity of a variety of technologically important materials,such as pigments, coatings, windows, and filters. Thisapplication usually requires recording at least a portion of thespectrum for characterization of the optical or electronicproperties of materials.

A) AbsorptionAbsorbance spectroscopy, commonly referred to asspectrophotometry, is the analytical technique based onmeasuring the amount of light absorbed by a sample at a givenwavelength. Spectrophotometry, particularly in the VIS and UVportions of the electromagnetic spectrum, is one of the mostversatile and widely used techniques in chemistry and the lifesciences.

Molecular absorption spectroscopy in the ultraviolet (UV) andvisible (VIS) is concerned with the measured absorption ofradiation in its passage through a gas, a liquid or a solid. Thewavelength region generally used is from 190nm to about1000nm, and the absorbing medium is at room temperature.However, in some cases (e.g. in enzyme assays)measurements at temperatures above or below roomtemperature may be advantageous or necessary.

A molecule, or part of a molecule, that can be excited byabsorption is called a chromophore. Organic chromophoreswhich absorb strongly in the UV or visible portions of thespectrum nearly always involve multiple bonds such as C=C,C=O or C=N.

Molecular excitation energy is usually dissipated as heat(kinetic energy) by the collision of the excited molecule withanother molecule (e.g. a solvent molecule), as the moleculereturns to the ground state. In other cases, the excitationenergy is dissipated by the emission of light in a process called"fluorescence". In both cases, the intensity of the lighttransmitted by a collection of chromophores is less than theintensity of the incident light.

An excited molecule can possess any one of a set of discreteamounts (quanta) of energy described by the laws of quantummechanics. These amounts are called the "energy levels" of themolecule. In UV/VIS spectrophotometry, the major energy levelsare determined primarily by the possible spatial distributions ofthe electrons and are called electronic energy levels, and to alesser extent by vibrational energy levels, which arise from thevarious modes of vibration of the molecule (e.g. the stretchingand bending of various covalent bonds).

The energy and wavelength of absorbtion is defined by thedifference between energy levels of an electronic transition.This can be expressed by the following (equation 1):

λ = hc/(E2-E1)where:

E1 is the energy level of the molecule before absorption

E2 is an energy level reached by absorption

If all transitions were between only the lowest vibrational levelsof the ground state and the first excited state, then anabsorption spectrum would consist of narrow, discrete peak.However, the transition from one electronic level to the nextlevel can occur between many vibrational levels also.

Since energy differences between vibrational levels within anelectronic level are small compared to the minimum energydifference between electronic levels, the electronic transitionconsists of a cluster of very closely spaced spectral peaks.Each peak has significant width, comparable to the spacingbetween the peaks. This has the effect that the peaks overlapso much that a single broad peak, called an electronicabsorption band, results.

For most molecules, absorption wavelengths corresponding totransitions between the ground state and any vibrational level ofthe first excited state, fall in the range of ultraviolet and visiblelight. Low-energy transitions are also possible betweenvibrational levels within a single electronic level. Thesetransitions produce radiation in the infrared range. Figure 1illustrates the relationship between specific energy transitions inthe energy level diagram (upper graph) and the absorbancespectrum (lower graph).

Absorption / Transmission /Reflectance (ATR) Spectroscopy

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Molecules which absorb photons of energy corresponding towavelengths in the range 190nm to about 1000nm, exhibit UV / VIS absorption spectra. The quantized internal energy(Eint) of a molecule in its electronic ground or excited state can

be approximated, with sufficient accuracy for analyticalpurposes, by the following (equation 2):

Eint = Eel + Evib + Erotwhere:

Eel is the electronic energy

Evib the vibrational energy

Erot the rotational energy

Absorption of a photon results in a change of the electronicenergy, accompanied by changes in the vibrational androtational energies. Each vibronic transition, i.e. a particularelectronic plus vibrational transition, corresponds to anabsorption band consisting of rotational lines. In liquids andsolids, the rotational lines are broad and overlap so that norotational structure is distinguishable.

The Beer-Lambert Law

The Beer-Lambert-Bouguer law, generally called the Beer-Lambert law, may be written for a single absorber, eithergaseous or in solution, as in equation (3):

A = -log10(IT/Io) = -εdcwhere:

IT is the monochromatic radiant power transmitted by theabsorbing medium

Io is the monochromatic radiant power incident on the medium

τi is the internal transmittance (=IT/Io)

ε is the molar (decadic) absorption coefficient

c is the amount concentration

d is the absorption path length

A is the (decadic) absorbance

Internal transmittance τi, i.e. transmittance through the mediumitself, disregarding boundary effects, has to be distinguishedfrom the total transmittance τ. The difference, which is mainlydue to reflection losses associated with cell windows, can becompensated by using a matched cell.

Absorpbance (or absorption factor) α is defined by α = 1 - τwhere reflection is assumed to be negligible.

The Beer-Lambert law holds only if the absorbing speciesbehave independently of each other, and if the absorptionoccurs in a uniform medium.

Figure 1 - Transitions to excited state

Figure 2 - Beer-Lambert Law


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Practical Applications of AbsorptionSpectroscopy

Absorbance measurements allow the following:

• Determination of the concentration of a substance

• Kinetic assay of certain chemical reactions

• The identification of materialsThe most common use of absorbance measurements is todetermine the concentration of a solute. This can be done if theabsorption coefficient (ε) is known and Beer's law is obeyed.

In practice, we do not generally rely on published values of εbecause this quantity may be very sensitive to idiosyncrasies ofreagent preparation and instrument design. Instead, it is normalpractice to derive concentrations of unknown samples from astandard curve. The progress of a chemical reaction can befollowed using a spectrophotometer to measure theconcentration of either a reactant or a product over time. Thisforms the basis for the kinetic assays of many enzymes.

Finally, most substances have characteristic absorbancespectra and can be identified thereby. This can be done bydetermining the complete spectrum and comparing it to spectrapublished in the literature or to the spectra from knownreference libraries.

B) Transmission SpectroscopyTransmission Spectroscopy is highly interrelated to AbsorptionSpectroscopy. This technique can be used for solid, liquid, andgas sampling. Here, light is passed through the sample andcompared to light that has not. The output depends on thepathlength or sample thickness, the absorption coefficient ofthe sample, the reflectivity of the sample, the angle ofincidence, the polarization of the incident radiation, and, forparticulate matter, on particle size and orientation.

In the Beer-Lambert Law (equation 3) the term IT/Io is calledtransmittance. This form of spectroscopy has a setup similar tothe one used for Absorption.

C) Reflection SpectroscopyReflectance Spectroscopy is the study of light that has beenreflected or scattered from a solid, liquid, or gas. As photonsenter a mineral, some are reflected from grain surfaces, somepass through the grain, and some are absorbed. Those photonsthat are reflected from grain surfaces or refracted through aparticle are said to be scattered. Scattered photons mayencounter another grain or be scattered away from the surfaceso they may be detected and measured.

Figure 3 - Illustration of Absorption Spectroscopy set-up


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Basics of Reflection

All materials have a complex index of refraction (equation 4):

m = n – jKwhere:

m is the complex index of refraction

n is the real part of the index

j = (-1)1/2

K is the imaginary part of the index of refraction, sometimescalled extinction coefficient.

When photons enter an absorbing medium, they are absorbedaccording to the Beer-Lambert Law. The absorption coefficientis related to the complex index of refraction by the following(equation 5):

ε = 4K/λ

The reflection of light, R, normally incident onto a plane surfaceis described by the Fresnel equation (equation 6):

R = [(n - 1)2 + K2 ] / [(n + 1)2 + K2]

At angles other than normal, the reflectance is a complextrigonometric function involving the polarization direction of theincident beam.

Scattering can also be thought of as scrambling information.The information is made more complex, and because scatteringis a non-linear process, recovery of quantitative information isdifficult. Consider the simple Beers-Lambert Law in equation 3.In transmission, light passes through a slab of material. Thereis little or no scattering (none in the ideal case; but there arealways internal reflections from the surfaces of the medium).

In reflectance, however, the optical path of photons is a randomwalk. At each grain of photons, a certain percentage areabsorbed. If the grain is bright, like a quartz grain at visiblewavelengths, most photons are scattered and the random walkprocess can go on for hundreds of encounters. If the grains aredark, the majority of photons will be absorbed at eachencounter and essentially all photons will be absorbed in only afew encounters. This process also enhances weak features notnormally seen in transmittance, further increasing reflectancespectroscopy as a diagnostic tool.

In a mixture of light and dark grains (e.g. quartz and magnetite)the photons have such a high probability of encountering a darkgrain that a few percent of dark grains can drastically reducethe reflectance, much more than their weight fraction. A generalrule with mixtures is that at any given wavelength, the darkercomponent will tend to dominate the reflectance. The amount oflight scattered and absorbed by a grain is dependent on grainsize. A larger grain has a larger internal path where photonsmay be absorbed according to the Beers-Lambert Law. It is thereflection from the surfaces and internal imperfections thatinfluence the scattering.

In a smaller grain there are proportionately more surfacereflections compared to internal photon path length, or in otherwords, the surface-to-volume ratio is a function of grain size.Absorptions in a spectrum have two components: continuumand individual features. The continuum is the "backgroundabsorption" onto which other absorption features aresuperimposed. This may be due to the wing of a largerabsorption feature.

Applications of ReflectionSpectroscopy

(1) External Reflection Spectroscopy: In this technique, alsoknown as specular reflectance, light is reflected from a smooth(mirror-like) sample to record its spectrum. External reflectanceis a non-destructive, non-contact technique. It is particularlyuseful for film thickness and refractive index measurements, aswell as recording spectra of thin films on metal substrates.

External reflection spectra may look different from transmissionspectra in many ways, e.g. bands may be shifted to higherwavenumbers, spectra may follow the dispersion in therefractive index, and spectral contrast may not depend linearlyon sample thickness.

(2) Internal Reflection Spectroscopy: For IRS, light is introducedinto a suitable prism at an angle exceeding the critical angle forinternal reflection. This produces an evanescent wave at thereflecting surface. A sample is brought into contact with or inclose proximity to the reflecting surface. From the interaction ofthe evanescent wave with the sample, a spectrum can berecorded with little or no sample preparation. The IRStechnique is non-destructive and can be used for solids, liquids,and powders.

For thin films, the IRS spectra are the same as transmissionspectra. For thick films, the absorption bands are more intenseat longer wavelengths. As the angle of incidence approachesthe critical angle, the bands tend to broaden on the longwavelength side and the minima are displaced to longerwavelengths (lower wavenumbers). Dispersion type spectra areobserved very close to and below the critical angle.


(3) Diffuse Reflection Spectroscopy: Spectra of powders andrough surfaces can be recorded by illuminating these surfacesand, with the aid of the appropriate optics, collecting sufficientscattered light for spectral analysis. This has been doneroutinely in the UV-VIS with integrating spheres, and in theinfrared with ellipsoids and paraboloids to collect the scatteredradiation. Reaction chambers for diffuse reflectance allow thestudy of catalysis and oxidation reactions in situ.

Since diffuse reflection spectra are the result of the interactionof light with the sample in every conceivable way, the spectramay exhibit features of transmission, external reflection and/orinternal reflection.

Recommended Products forATR SpectroscopyThe ideal detection system for an absorption setup consists ofan ICCD or EMCCD camera hooked to an imagingspectrograph. Andor's range of iStar ICCD cameras andNewtonEM EMCCD cameras provide for the highest possiblesensitivity among scientific grade cameras. The iStar's ability tobe gated down to a few hundered pico-seconds and theNewton's ability to run at 2.5MHz allow the two cameras to berun with high temporal precision and frame rate respectively,providing the user the ability to opt for time-resolved basedexperiments and also test samples that have very lowabsorption co-efficients.

Andor Shamrock

Andor's range of Shamrock spectrographs provide the user theoption for a low resolution 163mm focal length spectrometer, i.e.SR-163 or the higher end 303mm focal length SR-303. Theuniqueness of these systems is that Andor offers various kindsof sample chambers which can be attached in front of theentrance port of the Shamrock providing tremendousconvenience to the user. Different configurations allow forReference and Signal from different cuvettes sitting inside thechamber, without the user having to replace the cuvette eachtime.

This fully motorised 303mm focal length triple-grating imagingspectrograph designed for high throughput and resolution for arange of low light spectroscopy applications. When combinedwith an Andor CCD detector and software, it ships as aseamless solution.

Andor also offers an extensive range of spectroscopyaccessories. Please refer to pages 210 - 231 in the ProductsAccessories section for further details

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Andor's iDus is a family of affordable yet high-end spectroscopyCCD detectors that house a range of sensor formats, bothfront- and back-illuminated, offering vacuum Thermoelectric(TE) cooling to –90°C. USB 2.0 connectivity provides plug-and-play operation.

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Absorption Transmission Reflectance Spectroscopy