An Introduction to Ultraviolet/Visible Molecular Absorption ...

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Chapter 13- An Introduction to Ultraviolet/Visible Molecular Absorption Spectrometry ... Unfortunately, the terms used in spectroscopy and spectrophotometry are ...
Chapter 13- An Introduction to Ultraviolet/Visible Molecular Absorption Spectrometry

A: Measurement of Transmittance and Absorbance

An introduction into any field requires that one learn the terms and symbols associated with work in that field. Unfortunately, the terms used in spectroscopy and spectrophotometry are somewhat confusing. The common terms, symbols, and definitions employed in the measurement of absorption are listed in the table below. The recommended terms and symbols are listed under the column labeled Term and Symbol. (Principles of Instrumental Analysis)

The letters P and Po of the above table represent the power of a beam of light before and after passage through an absorbing species respectively. Absorbance, like the table shows, can be defined as the base-ten logarithm of the reciprocal of the transmittance: A = log 1/T = -log I/Io = -log P/Po It is important to note that the absorbance of a solution increases as the attenuation of the beam becomes greater. Reflection and scattering losses are significant and to compensate for these effects, the power of the beam transmitted by the analyte solution is ordinarily compared with the power of the beam transmitted by an identical cell containing only the solvent. An experimental absorbance that closely approximates the true absorbance is then obtained with the equation A = log Psolvent/Psolution ~ log Po/P

The 0%T adjustment is performed with the detector screened from the source by a mechanical shutter. In this adjustment, the instrument is thus counter signaled so as to read zero in the absence of any radiation from the source and the dark current that many detectors exhibit in the absence of radiation is eliminated. The 100% adjustment is made with the shutter open and the solvent in its light path. Normally the solvent is contained in a cell that is as nearly as possible identical to the one containing the samples. The 100% adjustment may involve increasing or decreasing the radiation output of the source electrically; alternatively, the power of the beam may be varied with an adjustable diaphragm or by appropriate positioning of a comb or optical wedge, which attenuates the beam to a varying degree depending upon its position with respect to the beam.

B: Beer’s Law

Bouguer, and later Lambert, observed that the fraction of the energy, or the intensity, of radiation absorbed in a thin layer of material depends on the absorbing substance and on the frequency of the incident radiation, and is proportional to the thickness of the layer. At a given concentration of the absorbing species, summation over a series of thin layers, or integration over a finite thickness, leads to an exponential relationship between transmitted intensity and thickness. This is generally called Lambert’s law. Beer showed that, at a given thickness, the absorption coefficient introduced by Lambert’s law was directly proportional to the concentration of the absorbing substance in a solution. Combination of these two results gives the relationship now commonly known as Beer’s law. This law states that the amount of radiation absorbed or transmitted by a solution or medium is an exponential function of the concentration of the absorbing substance present and of the length of the path of the radiation through the sample. Beer’s law can be derived as follows. Consider the block of absorbing matter (solid, liquid, or gas) shown in the figure below. A parallel beam of monochromatic radiation with power Po strikes the block perpendicular to a surface; after passing through a length b of the material, which contains n absorbing particles, the beam’s power is decreased to P as a result of absorption. Consider now a cross-section of the block having an area S and an infinitesimal thickness dx. Within this section there are dn absorbing particles; associated with each particle we can imagine a surface at which photon capture will occur. That is, if a photon reaches one of these areas by chance it will be absorbed. The total projected area of these capture surfaces within the section is designated as dS; the ratio of the capture area to the total area, then, is dS/S. On a statistical average this ratio represents the probability for the capture of photons within this section.







The power of the beam entering the section, Px, is proportional to the number of photons per square centimeter per second, and dPx represents the quantity removed per second within the section; the fraction absorbed is then - dPx/Px, and this ratio also equals the average probability for capture. The term is given a minus sign to indicate that P undergoes a decrease. Thus, - dPx/Px = dS/S

Since dS is the sum of the capture areas present in section, dS must be proportional to the number of particles or dS = αdn where dn is the number of particles and α is the proportionality constant. Substitution into the previous equation and integrating from 0 to n yields - In P/Po = αn/S Upon converting to base ten logarithms and inverting to change the sign, we obtain log Po/P = αn/2.303S Since S (i.e. area under consideration) can be written as V/b where V is the volume of the block then the last equation can be written as log Po/P = αnb/2.303V Noting that n/V has the units of concentration i.e. number of particles per cubic centimeter, conversion to moles per liter yields the following equation log Po/P = NA*αbc/2.303 ×1000 where NA is Avogradro’s number. Finally, collecting the constants into a single term ε gives log Po/P = εbc = A The derivation of this law assumes (a) that the incident radiation is monochromatic, (b) the absorption occurs in a volume of uniform cross- section, and (c) the absorbing substances behave independently of each other in the absorbing process. Thus, when Beer’s law applies to a multi component system in which there is no interaction among the various species, the total absorbance may be expressed as Atotal = ε1bc1 + ε2bc2 + .........+ εnbcn

Applications of Beer’s Law to Mixtures

Beer’s law also applies to a medium containing more than one kind of absorbing substance. Provided that there is no interaction among the various species, the total absorbance for a multicomponent system is given by Atot = A1 + A2 + … + An Where the subscripts refer to absorbing components 1, 2, …, n.

Limitations to Beer’s Law

Few exceptions are found to the generalization that absorbance is linearly related to path length. On the other hand, deviations from the direct proportionally between the measured absorbance and concentration when b is constant are frequently encountered. • Instrumental deviations • Chemical deviations

Real Limitations to Beer’s Law

Beer’s law is successful in describing the absorption behavior of dilute solutions only; in this sense it is a limiting law. At high concentrations (> 0.01M), the average distance between the species responsible for absorption is diminished to the point where each affects the charge distribution of its neighbors. This interaction, in turn, can alter the species’ ability to absorb at a given wavelength of radiation thus leading to a deviation from Beer’s law. Deviations also arise because ε is dependent upon the refractive index of the solution. Thus, if concentration changes cause significant alterations in the refractive index η of a solution, departures from Beer’s law are observed. It is not ε which is constant and independent of concentration, but the expression ε = εtrue* η/( η² + 2)² where η is the refractive index of the solution. At concentrations of 0.01 or less, the refractive index is essentially constant, but at high concentrations the refractive index may vary considerably and so will ε. This does not rule out quantitative analyses at high concentrations, since bracketing standard solutions and a calibration curve can provide sufficient accuracy.

Apparent Chemical Deviations

Chemical deviations from Beer’s law are caused by shifts in the position of a chemical or physical equilibrium involving the absorbing species. A common example of this behavior is found with acid/base indicators. Deviations arising from chemical factors can only be observed when concentrations are changed.

Apparent Instrumental Deviations with Polychromatic Radiation

Unsatisfactory performance of an instrument may be caused by fluctuations in the power-supply voltage, an unstable light source, or a non-linear response of the detector-amplifier system. In addition the following instrumental sources of possible deviations should be understood: Polychromatic radiation. Strict adherence to Beer’s law is observed only with truly monochromatic radiation. This sort of radiation is only approached in specialized line emission sources. All monochromators, regardless of quality and size, have a finite resolving power and therefore minimum instrumental bandwidth. A good picture of the effect of polychromatic radiation can be presented as follows. When radiation consists of two wavelengths, λ and λ1, and assuming that Beer’s law applies at each of these individually the absorbance at λ is given by log ( Po/P ) = A = εbc ⇒ Po/P = 10εbc Similarly, at λ1, P1o/P1 = 10ε1bc The radiant power of two wavelengths passing through the solvent is given by Po + P1o, and that passing through the solution containing absorbing species by P + P1. The combined absorbance is Ac = log ( Po + P1o)/P + P1 Substituting for P and P1, we obtain Ac = log (Po + P1o)/(Po10-εbc + P1o10-ε1bc) In the very special case where ε1 = ε, the above equation becomes Beer’s law. The relationship between Ac and concentration is no longer linear when the molar absorptivities differ; moreover, greater departures from linearity can be expected with increasing differences between ε1 and ε. It is also found that deviations from Beer’s law resulting from the use of a polychromatic beam are not appreciable, provided the radiation used does not encompass a spectral region in which the absorber does not exhibit large changes in absorption as a function of wavelength.

Instrumental Deviations in the Presence of Stray Radiation

Stray light affects absorption measurements because stray radiation often differs in wavelength from that of the principal radiation and, in addition, may not have passed through the sample. When measurements are made in the presence of stray radiation, the observed absorbance is given by A′ = log( Po + Ps)/(P + Ps) where Ps is the power of nonabsorbed stray radiation. It has been deduced that positive deviations from Beer’s law occurs when stray radiation is absorbed, and negative deviation if it is not.

C: The Effects of Instrumental Noise on Spectrophotometric Analyses

Instrumental Noise as a Function of Transmittance

A spectrophotometric measurement entails three steps: • A 0% T adjustment • A 100% T adjustment • A measurement of % T with the sample in the radiation path The noise associated with each of these steps combines to give a net uncertainty for the final value obtained for T. The relationship between the noise encountered in the measurement of T and the uncertainty in concentration can be derived by writing Beer’s law in the form c = -(1/(b)log T = -(0.434/(b)ln T

Types of Noise

• Shot noise – This noise is generated by current flowing across a P-N junction and is a function of the bias current and the electron charge. The impulse of charge q depicted as a single shot event in the time domain can be Fourier transformed into the frequency domain as a wideband noise. • Thermal noise – In any object with electrical resistance the thermal fluctuations of the electrons in the object will generate noise. • White noise- The spectral density of thermal noise is flat with frequency. • Burst noise – Occurs in semiconductor devices, especially monolithic amplifiers and manifests as a noise crackle. • Avalanche noise – Occurs in Zener diodes are reversed biased P-N junctions at breakdown. This noise is considerably larger than shot noise, so if zeners have to be used as part of a bias circuit then they need to be RF decoupled. • Flicker noise – This noise occurs in almost all electronic devices at low frequencies. Flicker noise is usually defined by the corner frequency FL.

Sources of Instrumental Noise

• Case I: sT = k1 • Case II: sT = k2(T2 + T) ½ • Case III: sT = k3T

Effect of Slit Width on Absorbance Measurements

The ability of a spectrometer to distinguish between two frequencies differing only slightly from each other depends upon the widths of the images produced (relative to the separation of the two images). The width of the image produced is thus an important measure of the quality of the performance of a spectrometer. The figure below shows the loss of detail that accompanies the use of wider slits. It is evident that an increase in slit width brings about a loss of spectral detail.

































Another effect of slit width is the change of absorbance values that accompany a change in the slit width. The figure below illustrates this effect. Note that the peak absorbance values increase significantly (by as much as 70% in one instance) as the slit width decreases.

It is evident from both these illustrations that quantitative measurement of narrow absorption bands demand the use of narrow slits widths. Unfortunately, a decrease in slit width is accompanied by a second-order power reduction in the radiant energy; at very narrow settings spectral detail may be lost owing to an increase in the signal-to-noise ratio. In general, it is good practice to narrow slits no more than is necessary for good resolution for the spectrum at hand.

D: Instrumentation

Instrument Components

Instruments used for measuring the absorption of ultra-violet, visible and infrared radiation are made up of one or more (1) sources, (2) wavelengths selectors (3) sample containers, (4) radiation detectors, and (5) signal processors and readout devices.

Sources

There are several light sources available for use in the ultraviolet- visible region. For the purpose of molecular absorption a continuous source is required whose power does not change sharply over a considerable range of wavelengths.

Deuterium and Hydrogen Lamps

An important feature of deuterium and hydrogen discharge lamps is the shape of aperture between the two electrodes, which constricts the discharge to a narrow path. As a consequence, an intense ball of radiation about 1 to 1.5mm in diameter is produced. Deuterium gives a somewhat larger and brighter ball than hydrogen, which accounts for the widespread use of the former. They both produce a useful continuum spectrum in the region of 160 to 375nm. At longer wavelengths, the lamps produce emission lines, which are superimposed on the sontinuum spectrum. For many applications, these lines represent a nuisance; they can be useful, however, for wavelength calibration of absorption instruments.



Probably a deuterium lamp (about 3 inches long). The tube was sealed into a metal tube and clamped to a heatsink. One reason for the sealing is probably to reduce ozone, which can be smelt strongly after the tube is run for just a few seconds.





















Tungsten Filament Lamps

Tungsten-filament ‘incandescent’ lamps are commonly used in the visible and near-infrared region. These are thermal or ‘black body’ sources in which the radiation is the result of high temperature of the solid filament material. These sources provide continuous radiation from about 320 to 3000 nm - most of it, unfortunately in the near infrared. At the usual operating temperature of about 3000 K, only about 155 of the total radiant energy falls in the visible region. The lifetime of a tungsten filament lamp can be greatly increased by the presence of a low pressure of iodine or bromine vapor within the lamp. Most work in the ultraviolet region is done with hydrogen or deuterium electrical-discharge lamps typically operated under low-pressure DC conditions (about 40 V with 5 mm gas pressure). These lamps provide a continuous source of radiation form 375 nm down to about 160 nm hence quartz windows must be used with these types of lamps since glass absorbs strongly at wavelengths less than about 350 nm.

Tungsten Filament Lamps

This lamp produces intense radiation by the passage of current through an atmosphere of xenon. The spectrum is continuous over the range between about 200 to 1000nm, with the peak intensity occurring at about 500nm.

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Sample Containers

In common with other optical elements of an absorption instrument, the cells, or cuvettes that hold the sample must be constructed of a material that passes radiation in the spectral region of interest. The best cells have windows that are perfectly normal to the direction of the beam in order to minimize reflection losses.

Types of Instruments

Four general types of spectroscopic instruments will be discussed: • Single-beam • Double-beam in space • Double-beam in time • Multichannel

Single-Beam Instruments

The figure below contains diagrams showing instrument designs for photometers and spectrophotometers.





The simplest type is the single-beam instrument shown as (a) in the figure below. There is only one light beam or optical path from the source to the detector. The wavelength selector is either a filter or monochromator and the determination of transmittance involves three successive steps that are separated in time; (1) the 0%T setting with a shutter in place; (2) the 100% T adjustment with the solvent in the light path and (3) the measurement of the %T with the sample in place. In single beam instruments there is only one light beam or optical path from the source to the detector.

Double-Beam Instruments

Alternatively, in double beam instruments (i.e. diagram (b)), the reference and the sample may be compared several times a second. The light from the source after passing through the monochromator, is split into two separate beams - one for the sample and the other for reference.

[pic]

Multichannel Instruments

The most recent type of spectrophotometer, which appeared on the market in the early 1980s, is a single beam instrument based upon the diode array transducer described in Chapter 7. The entire cycle of this instrument takes a few milliseconds. The monochromator slit width of a diode array instrument is usually made identical to the width of one of the silicon diodes.

Some Typical Instruments

Under this heading a few of the typical photometers and spectrometers (instruments incorporating a monochromator ) are described.

Photometers

Photometers provide relatively inexpensive tools for performing absorption analyses. Filter photometers are often more convenient, more rugged, and easier to maintain and use than are the more sophisticated spectrometers. The figure below presents the schematics for two photometers. The upper figure illustrates the single-beam, direct reading instrument consisting of a tungsten filament lamp, a lens to provide a parallel beam of light, a filter, and a photovoltaic cell. Diagram (a) is that of a single-beam photometer while diagram (b) is a double-beam photometer.

For the single-beam photometer, the optical path is simply from the light source, through the filter and the sample holder, and to the detector. Light from the tungsten filament lamp in the reflector is defined in area by fixed apertures in the sample holder and restricted to a desired band of wavelengths by an absorption or interference filter. After passing through the sample cuvette, the light strikes the surface of a photovoltaic cell, the output of which is measured by the deflection of a rugged light-spot galvanometer. Yet another type of photometer is the one illustrated below. It is classified as a probe-type photometer and it employs an optical fiber to transmit light from a source to a layer of solution lying between the glass seal at the end of the fiber and a mirror. The reflected radiation from the latter passes to photo diode detector via a second glass fiber. The photometer uses an amplifier with an electric chopper that is synchronized with the light source; as a result, the photometer does not respond to extraneous radiation. Absorbance is measured by dipping the probe into the solvent and then into the solution to be measured.

Visible Photometers

Probe-Type Photometers

Filter Selection

Ultraviolet Absorption Photometers

Spectrophotometers

Several spectrometers designed to operate in the wavelength range of about 380 to 800 nm are available from commercial sources. These instruments are frequently simple, single-beam grating instruments that are relatively inexpensive. The figure below shows a simple and inexpensive spectrometer, the Spectronic 20. The original version of this instrument first appeared in the market in the mid-1950s, and the modified version shown in the figure is still being manufactured and widely sold.

[pic]

1) On/Off switch and zero transmission adjustment knob 2) Wavelength selector/Readout 3) Sample chamber 4) Blank adjustment knob 5) Absorbance/Transmittance scale

Instruments for the Visible Region

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Single-Beam Instruments for the Ultraviolet/Visible Region [pic] Fig. 1 Schematic diagram of a single-beam spectrophotometer [pic]

Single-Beam Computerized Spectrophotometers [pic]

Inside of a single-beam spectrophotometer connected to a computer.

Double-Beam Instrumetents Numerous double-beam spectrophotometers for the ultraviolet/visible region of the spectrum are now available. Generally, these instruments are more expensive than their single beam counterparts. A current studies involving UV-Vis spectrometry includes “Variable Path Length Transmittance Cell for Ultraviolet, Visible, and Infrared Spectroscopy and Spectroelectrochemistry” by Paul A. Flowers* and Sherry-Ann Callender, which was found in Analytical Chemistry, 68 (1), 199 -202, 1996. The abstract discusses the design and characteristics of a transmittance cell for ultraviolet, visible, and infrared spectroscopy and spectroelectrochemistry. Through modification of a previously reported design, this cell employs threaded glass connectors as insertion ports for either quartz- or silicon-windowed tubes, thus permitting essentially continuous variation of the optical path length from ~0.050 to 200 mm. Though the initial fabrication requires skillful glassblowing, once constructed, the cell's simple design allows for rapid and reproducible disassembly/reassembly between experiments. The utility of the cell for a diversity of fluid samples is demonstrated through applications to water, aqueous ferricyanide, ferrocene in methylene chloride, and acetone vapor. Ions were quantitated with estimation errors below 10 mM using this procedure. In addition, the species' spectral profiles were recovered, and optimum estimates of the system's equilibrium constants were determined.

Double-Dispersing Instruments

Diode Array Instruments

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Diode array technology is unique in the sense that it doesn't use any moving parts in the optics. This greatly improves the stability of the instrument as there are no parts that can be worn out or misaligned. The result is an instrument which requires much less maintenance than when moving parts are used, as for example is the case with scanning monochromators. The simple picture above shows the optical principle of the DA 7200. 1. A lamp illuminates the sample with white light. Some of the light is absorbed (depending on the composition of the sample) and the rest is reflected. 2. The light which is reflected hits a stationary grating, which separates the light by wavelength. Instead of white light, we now have a "rainbow". 3. Each wavelength is measured by a dedicated detector.

References

http://www.anachem.umu.se/jumpstation.htm http://userwww.service.emory.edu/~kmurray/mslist.html http://www.anachem.umu.se/jumpstation.htm http://userwww.service.emory.edu/~kmurray/mslist.html http://www.anachem.umu.se/jumpstation.htm http://www1.shimadzu.com/products/lab/spectro/uv3150.html http://www.perten.com/product_range/diode_array/da_technology.html http://repairfaq.ece.drexel.edu/sam/CORD/leot/course10_mod03/mod10-03.html http://las.perkinelmer.com/catalog/Product.aspx?ProductID=L950 http://www.olisweb.com/products/upgrades/ir983.php http://imagers.gsfc.nasa.gov/ems/visible.html http://biology.easternct.edu/courses/spectwenty.htm http://www.cairnweb.com/systems/prod_lamp.html http://www.odyseus.nildram.co.uk/RFIC_Theory_Files/Noise_Tutorial.pdf ----------------------- [pic]

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