Rezaul Karim Environmental Science and Technology Jessore Science and Technology University

Instrumental Technique for Environmental Analysis

Chapter 3 Ultra Violet Spectroscopy

Chapter content and Reference

The UV/Vis spectral region The origin of the absorptions Electronic transitions of organic compounds Chromophore groups Solvent effects: solvatochromism FieserWoodward rules Instrumentation in the UV/Visible UV/Vis spectrophotometers Quantitative analysis: laws of molecular absorption UV Spectra and the Structure of Organic Molecules e.g. Conjugated Diene Systems; Conjugated Ketone Systems; Substitution of Benzene Rings Analytical Applications: qualitative and quatitative

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Reference
1.

2.

Rouessac and Annick, 2007, Chemical Analysis-Modern Instrumentation Methods and Techniques, 2nd Edition, John Wiley & Sons Ltd, The Atrium, Southern Gate, England Robinson, 1995. Undergraduate instrumental analysis, Marcel Dekker, Inc. NY, USA.

The UV/Vis spectral region

This region of the spectrum is conventionally divided into three subdomains termed
near UV (185400 nm), visible (400700 nm) and very near infrared (700 1100 nm).

Most commercial spectrophotometers cover the spectral range of 185 to 900 nm.

The lower limit of the instrument depends upon the nature of the optical components used and of the presence, or not, along the optical pathway of air. Some instruments, on condition that they are operating in a vacuum, can attain 150 nm with samples in the gaseous state. This is the domain of vacuum or far ultraviolet. The long-wavelength limit is usually determined by the wavelength response of the detector in the spectrometer.

The origin of the absorptions

The origin of absorption in this domain is the interaction of photons with ions or molecules of the sample. When a molecule absorbs a photon from the UV/Vis region, the corresponding energy is captured by one (or several) of its outermost electrons. As a consequence there occurs a modification of its electronic energy Eelec, a component of the total mechanical energy of the molecule along with its energy of rotation Erot and its energy of vibration Evib.
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A modification of Eelec will result in alterations for both Erot and Evib resulting in a vast collection of possible transitions obtained in all three cases,. since the polarities of the bonds within the molecules will be disturbed their spectra are given the generic name of charge transfer spectra.

Etot =Erot + Evib +Eelec with Eelec >Evib>Erot

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The UV/Vis spectrum

The interaction of UV and visible radiation with matter can provide qualitative identification of molecules and polyatomic species, including ions and complexes. Structural information about molecules and polyatomic species, e.g. organic molecules.

A typical UV absorption spectrum

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The UV/Vis spectrum

This qualitative information is usually obtained by observing the UV/VIS spectrum, the absorption of UV and visible radiation as a function of wavelength by molecules. The spectrum may be plotted as wavelength vs. Absorbance/ transmittance, / molar absorptivity.

A typical UV absorption spectrum

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Three different aspects of UV/Vis spectra. Spectra of benzene (a) in solution (a band spectrum); (b) in the vapour state (a fine structure); (c) an expansion of a section from the high resolution (0.14nm total interval) line spectrum of iodine vapour.

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The spectrum of benzene vapour, obtained from a drop of this compound, deposited in a silica glass cuvette of 1 cm optical path, provides an excellent test to evaluate the resolution of an UVspectrophotometer. The recorded spectra of compounds in the condensed phase, whether pure or in solution, generally present absorption bands that are both few and broad, while those spectra obtained from samples in the gas state and maintained under a weak pressure yield spectra of detailed fine structure. 11

Electronic transitions of organic compounds

Molecules are composed of atoms that are held together by sharing electrons to form chemical bonds. Electrons in molecules move in molecular orbitals at discrete energy levels as defined by quantum theory. When the energy of the electrons is at a minimum, the molecules are in the lowest energy state, or ground state. The molecules can absorb radiation and move to a higher energy state, or excited state. The process of moving electrons to higher energy states is called electronic excitation.
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Three distinct types of electrons

Sigma () bonds : single bonds, such as those between carbon and hydrogen in alkanes.

The amount of energy required to excite electrons in s bonds is usually more than UV photons of wavelengths 200 nm possess. For this reason, alkanes and other saturated compounds (compounds with only single bonds) do not absorb UV radiation and are therefore frequently very useful as transparent solvents for the study of other molecules. An example of such a nonabsorbing compound is hexane, C2H14 .
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Pi () bond : double and triple (unsaturated) bonds.

Typical examples of compounds with p bonds are alkenes, alkynes, conjugated olefins, and aromatic compounds.

Nonbonding (n) electrons : Electrons that are not involved in bonding between atoms
In saturated hydrocarbons the outer shell electrons of carbon and hydrogen are all involved in bonding; hence these compounds do not have any n electrons. Organic compounds containing nitrogen, O2, N2, S & X2 frequently contain electrons that are nonbonding.
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Various kinds of electronic excitation

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 * transition
This transition appears in the far UV since the promotion of an electron from a bonding MO to a * anti-bonding MO requires a significant energy. This is the reason for saturated hydrocarbons that only contain this type of bonding being transparent in the near UV. Example: hexane (gas state): l max =135nm ( =10 000. Cyclohexane and heptane are used as solvents in the near UV. At 200nm the absorbance of a 1 cm thickness of heptane is equal to 1.

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n * transition
The promotion of an n electron from an atom of O, N, S, Cl to an MO * leads to a transition of moderate intensity located around 180nm for alcohols, near 190nm for ethers or halogen derivatives and in the region of 220nm for amines Examples: methanol: lmax = 183nm ( = 50); ether: lmax = 190nm ( = 2000); ethylamine: lmax=210nm=800; 1chlorobutane: lmax =179nm.

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Transition n * of aniline (a primary amine). This transition corresponds to an increase in the weight of the mesomeric form.

n* Transition

This transition of low intensity results from the passage of an n electron (engaged in a nonbonding MO) to an anti-bonding * orbital. This transition is usually observed in molecules containing a hetero atom carrying lone electron pairs as part of an unsaturated system. The best known is that orresponding to the carbonyl band, easily observed at around 270 to 295 nm. The molar absorption coefficient for this band is weak. Example: ethanal: l =293nm ( =12, with ethanol as solvent).
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* Transition
Compounds possessing an isolated ethylenic double bond reveal a strong absorption band around 170 nm. The precise position depends upon the presence of heteroatom substituents. Example: ethylene: lmax =165nm ( =16 000). A compound that is transparent in a given spectral range of the near UV when it is isolated, can become absorbing if it interact with a species through a mechanism of type donoracceptor (D-A). This phenomenon is linked to the passage of an electron from a bonding orbital of the donor (which becomes a radical cation) towards a vacant orbital of the acceptor (which becomes a radical anion) of an attainable energy level (Figure 9.5).

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Figure: Energy diagram for a donor/acceptor interaction. The excited state is supposed to be essentially in the ionic form The position of the absorption band in the visible part of the spectrum is a function of the ionization potential of the donor and of the electron affinity of the acceptor; the value of is, in general, very large.

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dd transition
Numerous inorganic salts containing electrons engaged in d orbitals are responsible for transitions of weak absorption located in the visible region. These transitions are generally responsible for their colours. That is why the solutions of metallic salts of titanium [Ti(H2O)6 ]3+ or of copper [Cu(H2O)6 ]2+are blue, while potassium permanganate yields violet solutions, and so on.

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Chromophore groups
The functional groups of organic compounds (ketones, amines, nitrogen derivatives, etc.), responsible for absorption in UV/Vis are called chromophores. A species formed from a carbon skeleton transparent in the near UV on which are attached one or several chromophores constitutes a chromogene.

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Characteristic chromophores of some nitrogen-containing groups

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Isolated chromophores:
For a series of molecules having the same chromophore, the position and the intensity of the absorption bands remain constant. When a molecule possesses several isolated chromophores, that is, which do not interact with each other because they are separated by at least two single bonds in the skeleton, then the overlapping of the effects of each individual chromophore is observed.

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Conjugated chromophore systems:

When the chromophores interact with each other the absorption spectrum is displaced towards longer wavelengths (bathochromic effect) with an increase in the absorption intensity (hyperchromic effect). A particular case is that of molecules with conjugated systems, that is, organic structures containing several adjacent to one another unsaturated chromophores (i.e. separated by a single bond). The spectrum is then strongly disrupted with respect to the overlapping effects of isolated chromophores. The greater the number of carbon atoms upon which the conjugated system is extended, the greater the reduction in the difference between energy levels. This results in a very large bathochromic effect.
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FieserWoodwardScott rules Correlation table

the calculation of the position of the absorption maxima (margin 3 nm)

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A chromophore is a group of atoms (part of a molecule) that gives rise to an electronic absorption. An auxochrome is a substituent that contains unshared (nonbonding) electron pairs, such as OH, NH, and halogens. Change wavelength

A shift to longer wavelengths - a bathochromic shift or red shift. A shift to shorter wavelengths - a hypsochromic shift or blue shift.

The intensity of an absorption bans

An increase in the intensity - hyperchromism; a decrease in intensity - hypochromism.

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Solvents for UV/VIS Spectroscopy

Many spectra are collected with the absorbing molecule dissolved in a solvent. The solute must be soluble in the solvent and the solvent must be transparent over the wavelength range of interest. A molecule will dissolve in a solvent if the formation of a solution leads to a lower energy system. Polar substances dissolve more readily in polar solvents than in nonpolar solvents. It is important for the solute to be dissolved completely; undissolved particles can scatter light from the light source. This can result in serious errors in qualitative and quantitative analyses.
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The solvent may affect the appearance of the spectrum, sometimes dramatically. Polar solvents generally wipe out the vibrational fine structure in a spectrum. Solvents may also shift the position of the absorption band. For the visible region of the spectrum any colorless solvent can be used in which the sample is soluble. At wavelengths shorter than the cutoff wavelength, the solvent absorbs too strongly to be used in a standard 1 cm sample cell. The cutoff is affected by the purity of the solvent. For spectroscopy, the solvents should be of spectral or spectrochemical grade, conforming to purity requirements set by the American Chemical Society.
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Common UV Solvents and their Lower Wavelength Cutoffs

Solvent

Lower wavelength cutoff (nm

330 306 285 265 245 215 205 205 204 195 195 195 190

Acetone Pyridine Toluene Carbon tetrachloride Chloroform Diethyl ether Methanol Cyclohexane 95% Ethanol Hexane Isooctane Water Acetonitrile

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Each solvent has its own characteristic polarity. Since it is known that all electronic transitions modify the charge distribution of the compound in solution, it is obvious that the position and intensity of the absorption bands will vary a little with the nature of the solvent used. The nature of the solvent/solute interactions are a greater indication of the type of transition. Two contrasting effects can be distinguished.

Solvent effects: solvatochromism

Hypsochromic effect (the blue shift) Bathochromic effect (the red shift) Effect of pH

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If the chromophore responsible for the observed transition is more polar in its fundamental state than when it is excited, then a polar solvent will stabilize, by solvation, the form prior to absorption of the photon. Molecules of solvent will be clustered around the solute because of electrostatic effects. More energy would be required to excite the electronic transition concerned, causing a displacement of the absorption maxima to shorter wavelengths than would occur in a nonpolar solvent. This is the hypsochromic effect.

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Hypsochromic effect (the blue shift)

This is rather like the n transition of the ketone carbonyl in solution. The C+O form (characterized by its dipolar moment ) will be more and more stabilized as the solvent becomes more polar. The excited state being rapidly attained, the solvent shell surrounding the carbonyl has no time to reorient itself and bring about stabilization following absorption of the photon. This same effect is observed for the n transition and is accompanied by a variation of the coefficient, .

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Spectra of benzophenone in cyclohexane and in ethanol . Observed here with two different solvents and upon two types of transitions are the bathochromic and hypsochromic effects.
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Bathochromic effect (the red shift)

For less polar compounds the solvent effect is weak. If the dipole moment of the chromophore increases during the transition, the final state will be more solvated. A polar solvent can thus stabilise the excited form, which favours the transition: a displacement is observed towards longer wavelengths when compared with the spectra obtained with a non-polar solvent. This is the bathchromic effect. For example, this is the case for the * transition of ethylenic hydrocarbons of which the double bond is only weakly polar.
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Effect of pH
The pH of the solvent in which the solute is dissolved can have an important effect on the spectrum. Amongst the compounds that present this effect in a spectacular fashion are chemical indicator strips, whose change in colour is used during acidimetric measurements. For example:

The effect of pH upon a solution of phenolphthalein. This compound is not coloured at pH values less than 8 though it is bright pink for those greater than 9.5. In contrast, absorptions towards 500nm appear when the pH becomes alkaline, which are responsible for the well-known colour of the compound. Notable in this example is the modification in the chemical bonding, which is pH dependent.
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The effect of pH upon a solution of phenolphthalein

The graph presented here in 3D perspective reveals that for acid pH there is no absorption in the visible region of the spectrum.

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FieserWoodward rules
Structural analysis from electronic spectra is a fairly vague exercise, in the sense that the relative simplicity of the spectrum has for corollary a poor yield of information. The best known of these are the empirical rules due to Fieser, Woodward and Scott, which concern unsaturated carbonyl, dienes or steroids compounds. The table presents in the form of increments to max a variety of factors and structural features affecting the position of the absorption band * for these conjugated systems.

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There is good agreement between the calculated values and those derived from experiment for the four examples displayed:

Comparison between calculated and experimental values for some unsaturated organic compounds.
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Instrumentation: three system

the source, the dispersive system and the detection system

These components are typically integrated in a unique framework A sample compartment either before or after the dispersive system.
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UV/Vis spectrophotometers

UV/Vis spectrometers are classified into three optical designs:

a fixed spectrometer with a single light beam and sample holder; a scanning spectrometer with double light beams and two sample holders for automatic measurement of absorbance; and a non-scanning spectrometer with an array detector for simultaneous measurement of multiple wavelengths.
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Light sources

All spectrometers require a light source.

for the visible region of the spectrum, an incandescent lamp fitted with a tungsten filament housed in a silica glass; for the UV region a deuterium arc lamp working under a slight pressure to maintain an emission continuum (<350nm; for the entire region 200 to 1100 nm, a xenon arc lamp can be used for routine apparatuses.
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The deuterium discharge lamp

The deuterium discharge lamp contains two electrodes bathed in an atmosphere of deuterium. Between the electrodes a metallic screen is placed which is pierced with a circular hole of approximately 1mm diameter. The migration of the electrons toward the anode creates a discharge of current generating an intense arc at this hole, which is in front of the anode. Subjected to this electron bombardment the molecules of deuterium, D2 dissociate, emitting a continuum of photons over the wavelengths range between 160 and 500 nm.

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Lamp, view from above 1 - Silica glass envelope 2 - Filament (cathode) 3 - Metal screen 4 - Anode
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Sequential instruments

Dispersive systems and monochromators

The light emitted by the source is dispersed through either a planar or concave grating which forms part of a monochromator assembly. The wavelength can be varied gradually by pivoting the grating. Optical paths with long focal lengths (0.2 to 0.5 m) yield the best resolution. This category of instrument functions according to the spectrograph principle. The light beam is diffracted after travelling through the measuring cell.
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Simultaneous instruments

Detectors

The detector converts the intensity of the light reaching it to an electrical signal. Two types of detector are used, a photomultiplier tube PMT or a semiconductor (charge transfer devices / silicon photodiodes). For both of which the sensitivity depends upon the wavelength. For simultaneous instruments which do not possess a monochromator. A dispersive system, the light intensity at all wavelengths is measured practically at the same instant by aligning a large number of detectors in the form of a diode array.
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The photomultiplier tube (PMT).

A PMT is a sealed, evacuated transparent envelope (quartz or glass) a photoemissive cathode - coated with elements (e.g., Na/K/Cs/Sb or Ga/As) that emits electrons when struck by photons an anode, and several additional electrodes / dynodes. - multiply the available electrons.

A commercial PMT may have nine or more dynodes. The gain may be as high as 109 electrons per photon.

The efficiency of the PMT depends upon

the yield at the photocathode, which varies with the wavelength (e.g. 01e/photon at 750 nm), and on the signal gain provided by the cascade of dynodes (e.g. a gain of 6x105).
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Schematic of a PMT
Impinging photons pass through the quartz envelope and liberate electrons from the lightsensitive cathode. The electrons are accelerated to the first dynode, where each electron liberates several electrons on impact. The process is repeated at the other dynodes, resulting in a cascade of electrons for every photon hitting the PMT

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How does it work?

The photocurrent was limited to the electrons ejected from the cathode The ejected electrons are attracted to a dynode that is maintained at a positive voltage with respect to the cathode. Upon arrival at the dynode, each electron strikes the dynodes surface and causes several more electrons to be emitted from the surface. These emitted electrons are in turn attracted to a second dynode, where similar electron emission and more multiplication occurs. The process is repeated several times until a shower of electrons arrives at the anode, which is the collector. The number of electrons falling on the collector is a measure of the intensity of light falling on the detector. In the process, a single photon may generate many electrons and give a high signal. The dynodes are therefore operated at an optimum voltage that gives a steady signal.
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LambertBeer law:

Quantitative analysis: laws of molecular absorption

This law, which concerns only that fraction of the light absorbed can be verified by application of the following conditions:

the light used must be monochromatic the concentrations must be low the solution must be neither fluorescent or heterogeneous the solute must not undergo to photochemical transformations the solute must not undertake variable associations with the solvent.
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Empirical rules based on thousands of laboratory observations have been developed over the years relating the wavelengths of the UV absorption maxima to the structures of molecules. R.B. Woodward in 1941 and then L. Fieser and M. Fieser developed rules for predicting the absorption maxima of dienes, polyenes, and conjugated ketones by studying terpenes, steroids, and other natural products. There are essentially four organic molecular systems:

1. 2. 3. 4. conjugated dienes, conjugated carbonyl systems Mono-substituted benzene rings, Di-substituted benzene rings

UV spectra and the structure of organic molecules

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Conjugated Diene Systems

The parent diene of conjugated diene systems is C=C-C=C. This system in a hexane solvent absorbs at 217 nm. If the conjugated system is increased, the wavelength of the absorption maximum is increased

30 nm for every double bond extension. 5 nm by an alkyl group 0 nm for by O-alkyl

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Empirical Rules for Calculating the Absorption

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Example

We have a diene (the two double bonds) in a ring with an alkyl group attached to one of the diene carbon atoms. Since we have a diene, the value assigned to the parent diene is 217 nm. Diene system within a ring; therefore we add 36 nm to the absorption maximum. One is obviously the ethyl group,C2H5, but the groups in positions A and B are also alkyl substituents. Each alkyl group adds 5 nm to the absorption maximum, so the peak should appear at 217+36+ 3(5) =268 nm.

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The parent diene is at 217 nm. The diene system is within a ring, so we add 36 nm. There is one double bond extension to the system. As you can see, the conjugated system consists of three double bonds rather than two double bonds. This adds 30 nm to the absorption maximum. There is one exocyclic double bond between carbons C and D, which adds 5 nm to the absorption maximum. The double bond between carbons E and F is also within a ring, but neither carbon touches the other ring, so this double bond is not exocyclic to a ring. The same thing is true for the double bond between carbons A and B;

There are five alkyl substitutes as follows: two on carbon A, one on carbon B, one on carbon D, and one on carbon F. The alkyl substituents on carbons D and F are designated by asterisks.
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Conjugated Ketone Systems

The parent system is C = C-C = C-C=O The absorption maximum assigned to this parent system is 215 nm. the absorption maxima for conjugated ketones are modified by extension of the double bond substitution and position relative to rings and relative to the carbonyl group. The carbons are labeled , , and and substitutions in these positions change the shift of the absorption maximum.

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Rules for ,-Unsaturated Ketone and Aldehyde Absorptions

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Example

It should again be emphasized that the parts of the molecule that do not touch the conjugated or parent system do not shift the absorption maximum. This is true even for complex molecules, as some of these examples demonstrate. This makes prediction of the absorption maximum easier but the absorption maximum gives no information as to the structure of the entire molecule.

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Substitution of Benzene Rings

Benzene is a strong absorber of UV radiation and particularly in the gas phase shows considerable fine structure in its spectrum. Substitution on the benzene ring causes a shift in the absorption wavelengths. It is not uncommon for at least two bands to be observed, and frequently several more. If the benzene ring is di-substituted, then calculations are necessary to predict the absorption maximum, because a list containing all the possible combinations would be very long.

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Absorption Maxima of Mono substituted Benzene Rings Ph-R

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An electron-accepting group, such as NO2, and an electron-donating group, such as OH, situated ortho or para to each other tend to cancel each other out and provide a spectrum not very different from the monosubstituted benzene ring spectrum (Table 5.9). Two electron-accepting / donating groups para to each other produce a spectrum little different from the spectrum of the monosubstituted compound. An electron-accepting group and an electrondonating group para to each other cause a shift to longer wavelengths.

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Some rules of di-substitution of benzene rings

Example

To become an expert in the interpretation of UV spectra requires reading of more detailed texts, as well as practice. It can be seen that the absorption maxima can be calculated with reasonable accuracy if the structure is known, usually within 5 nm or so.
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