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IR absorption, Raman, fluorescence and mass spectroscopy

G. Galbács

The infrared range falls between 0.7and 3000 micrometers or ca 3 and

IR absorption spectroscopyVibrational and rotational transitions

and 3000 micrometers, or ca. 3 and14000 cm-1.

EM radiation in the IR (and partiallyin MW) range does not carry enoughenergy to generate electronicexcitation (between two orbitals).Instead it induces transitionsInstead, it induces transitionsbetween rotational and vibrationenergy levels (see the „springmodel” on the right) in covalentbonds of molecules.

mechanical „spring model” of acetaldehyde molecule

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As example, the animated drawings below illustrate the six„vibrational modes” possible for CH2- (methylene) groups.

IR absorption spectroscopyVibrational transitions

„ p 2 ( y ) g p

Symmetrical streching Asymmetrical stretching Scissoring

Rocking Wagging Twisting

The drawings on the

IR absorption spectroscopyVibrational transitions

The drawings on theright illustrate potentialvibrational transitions inthe formaldehydemolecule.

For example, a 1251cm-1 IR radiation willcm IR radiation willexcite the asymmetricbending vibration,thereby increasing theamplitude of thismovement.

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It can be shown that for a non-linear molecule containing natoms (n> 3) 3n-6 vibrational levels (energy storing states) and

IR absorption spectroscopyNormal vibrational modes

atoms (n> 3), 3n 6 vibrational levels (energy storing states) and3 rotational levels are possible. For a linear molecule, 3n-5vibrational and 2 rotational states are available.

These vibrations are called normal modes of vibrations. Thenormal vibrations can be divided into two categories: valencevibrations („bonds are periodically elongating/contracting”) anddeformational vibrations ( periodic change of bond angles)deformational vibrations („periodic change of bond angles).

Vibrational IR selection rule: only such rotational/vibrationaltransitions will appear in the IR spectrum, which areaccompanied by a change in the dipole moment of the molecule.Remember though that a permanent dipole moment is not aprerequisite.

Vibrational IR selection rule: only such vibrational transitionswill appear in the IR spectrum which are accompanied by a

IR absorption spectroscopySelection rule

will appear in the IR spectrum, which are accompanied by achange in the dipole moment of the molecule. Remember thoughthat a permanent dipole moment is not a prerequisite.

Follows from this rule that homonuclear diatomic molecules(such as H2, O2, N2, Cl2, etc.) are infrared inactive.Heteronuclear diatomic molecules, at the same time, can be IRactive as bond stretching changes the dipole momentactive, as bond stretching changes the dipole moment.

Another example is CO2, for which the symmetric stretchingvibration is IR inactive, whereas the asymmetric one is active.

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As there are no identical molecules (in the spring model, the springconstants and masses are different) thus the wavenumbers at

IR absorption spectroscopyCharacteristic bands

constants and masses are different), thus the wavenumbers atwhich the molecule absorbs IR radiation is charasteristic of themolecule, thereby provides qualitative analytical information.

In general however, organic molecules are quite similar to eachother (in terms of the most frequent bond types: C-C and C-H,etc.), thus the most characteristic absorption features will originatef th t f th l l th t t i d bl /t i l b dfrom the parts of the molecule that contain double/triple bonds,hetero atoms („stronger-than-usual spring constant”; „higher thanusual atomic mass”) or functional groups. These absorptionfeatures are therefore called characteristic bands or groupfrequencies. Most of these lie in the 1300 to 4000 cm-1 range.

IR absorption spectroscopyCharacteristic bands

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Between 650 and 1300 cm-1, the infrared spectrum becomesexceedingly complex Bands appearing often can not be directly

IR absorption spectroscopyFingerprint region

exceedingly complex. Bands appearing often can not be directlyassigned, thus it is used as a „fingerprint”. If the IR spectrum fortwo samples in this range fully matches, than the two samples canbe safely considered as identical…

IR absorption spectroscopySample preparation

Sample preparation is veryi i IR b iimportant in IR absorptionspectroscopy.

A large complication iscaused by the fact thatsample cuvettes commonlyused in the UV/Vis rangeabsorb in the IR range.

Similarly, solutionpreparation is alsocomplicated as organicsolvents also have strongIR absorption themselves(see table).

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IR absorption spectroscopySample preparation

IR liquid sample holders aretherefore complex constructions,

i i i d d fcontaining windows made ofinorganic salts such as NaCl, LiF orKBr. These windows must beprotected from humidity/water.Filling of the cell can beaccomplished by using a syringe.

Solid samples can also only bep ypresented in a dried form. Powderedsamples are typically pelletizedafter mixing it with a suitable salt(e.g. KBr) or oil (e.g. Nujol).Creating a suspension and handlingit as a liquid is another popularapproach.

IR absorption spectroscopyAnalytical applications - qualitative

Main application area for IRspectroscopy is qualitative analysis,that is identification of the presence offunctional groups, heteroatoms,confirmation of suggested structure, etc.

This is possible after a painstakingexamination of the absorption peakfeatures/patterns in the spectra, orrecently by a database searchrecently by a database search.

This is facilitated by the fact that – incontrast to e.g. UV/Vis spectroscopy –the peaks are not broad and spectralfeatures are influenced to a much smallerextent by the temperature or the solvent.Spectrum of stearic acid

at two largely different temperatures

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IR absorption spectroscopyAnalytical applications - quantitative

Beer-Lambert law is equally valid in the IR region as well, thustit ti li ti i l ibl i ll i th NIRquantitative application is also possible, especially in the NIR

range. However, IR spectroscopy is generally not employed thisway, for several reasons:

• poor reproducibility of absorbances due to sample cell degradation and the small cell thickness)

• spectra can be very complicated with many overlapping peaks, thus baseline correction is difficult

• many IR spectrometers (non FT types) have not enough resolution to fully resolve the peaks

• lack of ideally suitable (non-absorbing) solvents in the mid-IR, which results in the neccessity of using higher sample concentration that is bound to create deviations from the Beer-Lambert law

IR absorption spectroscopyRotational spectra

The rotational energy levels of a covalent molecule is also quantizedaccording to quantum theory, thus transitions between these levels givei t b ti k i IR R t ti t iti d f lrise to absorption peaks in IR. Rotation transitions need far less energy

than vibrations (appear in far IR or MW), and consequently are usuallysuperimposed onto vibrational peaks. Rotational IR selection ruledictates that the molecule has a dipole moment (asymmetry allowsfor EM radiation to exert a „torque” on the molecule). Application ispurely qualitative and is less frequent than that of vibrational IR spectra.

CO

H2O

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Raman (IR) spectroscopy

Raman spectroscopyThe phenomenon

Raman spectroscopy is based on inelastic scatter of light. It can beobserved that EM radiation is scattered by a molecule not onlyy yelastically (at the same wavenumber, Rayleigh scatter) but alsoinelastically (at different wavenumbers, see the drawings below). It isalso interesting that in the latter case, scattered radiation can bedetected both at lower (Stokes peaks) and higher (Anti-Stokes peaks)wavenumbers. The difference in energy (between incoming andscattered wavenumbers) can be assigned to vibrational transitions.

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Raman spectroscopyRelation to IR vibration spectroscopy

The selection rule for Raman spectroscopy is that thep pypolarizability needs to be changing during transition.Homonuclear diatomic molecules are therefore Raman actives,but IR inactive.

These two methods complement each other very well.

According to a practical observation, if there is a symmetrycenter in the structure of the molecule then IR and Ramancenter in the structure of the molecule, then IR and Ramanspectra will be intense in a complementer way.

Raman spectroscopyRelation to IR vibration spectroscopy

on

IR t

ransm

issi

inte

nsi

ty

2,5-dichlor-acetophenon

Raman spectra are plotted as a function of the „Raman shift” (difference of the peak position with reference to the excitation wavenumber)

Ram

an

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Raman spectroscopyPracticalities

Advantages of Raman spectroscopy include that it needs no specialsample holder and that the wavelength of the excitation source cansample holder and that the wavelength of the excitation source canbe freely selected (only the shift is important).

Raman intensities strongly depend on the excitation intensity, hencenowadays only focused laser light sources are used, mainly in the Visor NIR ranges. Laser wavelegth should also be chosen so that it doesnot induce strong fluorescence.

G lid l ti ll b d C h t bGases, solids or solutions can equally be measured. Care has to beexercised to filter off any particles from liquid samples (could induceincreased elastic scatter) and to avoid the local overheating ofsamples. Even sample solutions with mM koncentrations can also bemeasured.

Luminescence spectroscopy

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Luminescence (fluorescence) spectroscopyThe phenomenon

During photoluminescence, the electrons of the sample isexcited by irradiation of UV or Vis photons and the absorpedexcited by irradiation of UV or Vis photons and the absorpedenergy is „later” emitted also in the form of raditation, at the sameor at a longer wavelength. Photoluminescence can be observed foratoms, ions and molecules equally, but here we will discuss thecase of molecules only.

In most cases, the emission takes place shortly after theabsorption (ns-µs scale). This is called fluorescence. If the

i i d t ithi h t ti b t i i t i demission does not cease within a short time, but is maintained(with decreasing intensity) for as much as 100 seconds, we arefacing phosphorescence. In chemical analysis, we mostly usefluorescence of the two.

Luminescence (fluorescence) spectroscopyEmitted intensity

Quantum efficiency is the quotient of the number of emitted andabsorbed photons. Clearly, efficient fluorescence needs an as large asabsorbed photons. Clearly, efficient fluorescence needs an as large aspossible portion of the absorbed energy to be released in the form ofphotons (as opposed to collisions or rotations), therefore it isunderstandable that the molecules show strong fluorescence have arigid, planar structure. Conjugated or aromatic double bonds alsopromote fluorescence, as they these electrons will absorb concertedly.Examples include naphtalene, anthracene, fenanthrene, etc.

I l th i t it f th itt d di ti i ti l t thIn general, the intensity of the emitted radiation is proportional to thefollowing parameters:

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Luminescence (fluorescence) spectroscopySelf-absorption (filter effects)

Proportionality with concentrationp yonly holds for dilute solutions, forup to 10-4 mol/dm3. Above thisconcentration the calibration curvetypically shows a floding-backcharacteristic. In most cases this iscaused by self-absorption,especially if resonance fluorescenceis studiedis studied.

Fluorescence intensity is also ofteninfluenced by the pH or quality ofsolvent.

Luminescence (fluorescence) spectroscopyAbsoprtion and fluorescence

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Luminescence (fluorescence) spectroscopyThe instrument and its application

Luminescence atmany wavelengthsFluorescence spectroscopy is a very

selective and sensitive quantitativemethod. This is due to the combinedeffect of:

a) high excitation light intensitya) high excitation light intensity,b) the double monochromator,c) small background signal

It is employed for the analysis of manychemicals, such as pharmaceuticals,pesticides, clinically relevant samples,food components, etc.

Mass spectroscopy

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Mass spectroscopyThe concept

Mass spectroscopy is amethod whose primaryp yapplication is thedetermination of themass of atoms andmolecules present in thesample. It is based on theionization/fragmentationof the sample consituentsand then their

ti / tisorting/countingaccording to their m/zratio.

It also helps structureelucidation by identifyingthe fragments of themolecule.

Mass spectroscopyElectron or chemical impact ionization (EI and CI)

Ionization can be done in one of two ways: by impact of anaccelerated electron beam (EI), or by impact of a beam of chemicallyreactive ions (e g CH + from methane CI)reactive ions (e.g. CH5

+ from methane, CI).

Out of the two methods, EI usually gives rise to a more complexspectrum. In CI spectra, the peak of MH+ ion is usually present (ionof largest mass, helps assignation), whereas it is unseen in EI, asthen it fragments.

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Mass spectroscopyElectrospray sample introduction and ionization

For large molecules, ESI-MS is an advantageous combined sampleiontroduction/soft ionization technique. This device applies highvoltage to break down solution samples. ESI-MS spectra aresimple, as they typically contain only protonated molecules but nofragments.

Mass spectroscopySpectrum evaluation

The interpretation of mass spectra is generally difficult.

One complication is caused by fragmentation, which reasults inthe appearance of several „daughter” ions. The more complex isthe molecule, the more fragments are formed.

Another complication is caused by isotopes. Due to the fact thateach and every atom in the structure of a molecule is presented byone of the natural isotopes of the element (according to the naturalabundances), the mass spectrum becomes even more complex. Allpossible combinations will be present and peak intensities will bepossible combinations will be present and peak intensities will beproportional to the abundances.

Because of the above said things, MS is basically only used for theanalyis of pure samples. The „purification” can also be done in-line, viacoupling to chromatography. MS is primarily a qualitative method.