An introduction to Raman Spectroscopy

Dr.Domenica Masci

UTAGRI, ENEA

prof.Gianfranco Greppi

NRD, Università degli studi di Sassari

WINTER SCHOOL

“Le tecniche spettroscopiche: strumenti innovativi applicati all’analisi dei settori ambientale ed agro-

alimentare- nuove sfide per il futuro”

26-30 gennaio 2015, Milano, CRA-IAA, Via Venezian 26

Raman spectroscopy 1

Raman spectroscopy can provide information about themolecular composition , the bonds , the chemicalenvironment , the phase and the crystal structure of thesamples in question , and is therefore suitable for theanalysis of materials in multiple forms : gas , liquid andsolid amorphous or crystalline

The technique takes advantage of a physical phenomenonThe technique takes advantage of a physical phenomenondiscovered in 1928 by the Indian physicist CV Raman ,which earned him the Nobel Prize in 1931. He discoveredthat a small fraction of the scattered radiation from certainmolecules had power ot energy than that of the incidentradiation , and that the energy difference was related to thechemical structure of the molecules responsible for thespread : the Raman effect.

Raman spectroscopy 2

When a monochromatic radiation strikes the surfaceof an object , the radiation can be :

� absorbed if it has energy equal to a possibletransition to a higher energy level ( eg . UV- Vis,IR) ;IR) ;

� reflected if it does not interact with matter ;

�scattered if it interacts without causing energytransitions ( Raman)

Raman spectroscopy 3

Raman e IR

�With both these techniques we can measure thefrequencies of vibration of the molecule.

�Since these frequencies are characteristic of the molecule in the study and no other , it follows that the spectroscopic properties can serve as a criterion for molecular recognition.

What is the light scattering ?

The scattering of light is one of the most obvious optical effectsand occurs because the molecules have very small comparedto the wavelength of visible radiation ( 400-800 nm )

Being light an oscillating electromagnetic field , it polarizes themolecules that are found in the atmosphere , inducing adipole moment of the oscillating.dipole moment of the oscillating.

The dipole oscillating at the frequency of the incident radiationreemits it with the same frequency in all directions .

The scattering of light consists:

� 1) in the passage of radiant energy from the electromagnetic field to the molecules due to the formation of an induced dipoleformation of an induced dipole

� 2) in the subsequent reemission of light in all directions in space by the induced dipole

the intensity of the scattered light is proportional to the fourth power of the frequency

Raman technique

Raman spectroscopy is a non-invasive and non-destructivetechnique , which is based on the interaction of radiationwith matter .

In particular the radiation emitted by a laser beam interactswith the roto - vibrational motion of the molecules withthe subsequent reemission of light at wavelengths differentfrom that of the incident ( so it is a technique of emission) .from that of the incident ( so it is a technique of emission) .

The spectrum that is obtained , said Raman spectrum , thusprovides a fingerprint of the molecule in question ,allowing the identification

The technique is based on the so-called " Raman effect "

Raman technique and Rayleigh scattering

When monochromatic radiation affects a substance cancause the following effects :

�most of the radiation passes through the sample

�a small part of the radiation elastically spreads in alldirections with no energy loss , that is, at the samedirections with no energy loss , that is, at the samefrequency of the incident radiation ( elastic scatteringor Rayleigh ) ; such diffusion is believed to be causedby elastic collisions between molecules and photons

Scattering Raman Stokes and anti-Stokes

A small part of the radiation spreads inelastically giving(Raman Stokes) or acquiring (Raman anti-Stokes) energy inthe interaction with the molecules, in this way vibrating atfrequencies that differ in the vibrational energy states

The intensity of the scattered radiation ( less than the intensity of the incident radiation ) thus depends on the contribution of the incident radiation ) thus depends on the contribution made by the elastic scattering and by inelastic scattering , and these , in turn, depend on the chemical structure of the molecules responsible for the spread .

Scattering Raman

The Rayleigh scattering has a higher probability of that Raman

( peak are more highest ) because the event linked to it is morelikely; for this reason the line connected to the radiationRayleigh is known as excitation frequency ν0 . In addition theStokes emission is also favored than anti - Stokes ones.

A typical spectrum of the scattered light is generally given byintensity against frequency shift of the radiation excitingintensity against frequency shift of the radiation exciting

(Δ ν = defined as the difference between the scattered radiationemitted by the sample and that emitted by the source , aparameter therefore independent of the wavelength of thelaser ) .

Stokes and anti - Stokes lines are arranged symmetrically with respect to the Rayleigh line , and the difference in energy than the latter corresponds to the energy purchased or sold by the molecule in varying the initial vibrationallevel .

In the case of fluorescent compounds can have high

Scattering Raman

In the case of fluorescent compounds can have high interference in ' observation Stokes shifts , then you use the anti- Stokes lines , although less intense .

The Raman spectrum

For Raman spectrum is traditionally defined as part of thespectrum containing the Stokes lines .

They correspond to frequencies that differ in vibrationalenergy states ( inelastic scattering , or Raman effect ) .

These differences correspond to the separation of twovibrational levels and frequencies are the emissioncharacteristics of the molecules impacted by the incidentcharacteristics of the molecules impacted by the incidentlight : the set of these differences generates the Ramanspectrum of the compound .

Raman scattering or Raman effect takes on a very limitednumber of events , about 1 in 10 6

Summarizing. In a typical Raman experiment , a beam of monochromatic light is made to affect the sample

and then detect the radiation diffused at an angle perpendicular to the direction of the

incident beam .

When the incident photons collide the molecule , they may lose or

gain energy :

� if the molecule absorbs part of the energy of the photon to switch to an excited state , the photon will reemerge with an energy (frequency ) less than that of incidence going to form the so-called Stokes lines of the Raman spectrum

� but if the molecule is already in an excited state ( the thermal energy at room temperature excites some� but if the molecule is already in an excited state ( the thermal energy at room temperature excites somestates rotational ) can transfer energy to the incident photon that will emerge by the impact with moreenergy going to form the anti - Stokes lines .

The component of the scattered radiation in the same direction of origin without change of frequency is calledRayleigh radiation .

The Raman lines are the result of the inelastic scattering of incident radiation by the sample : the rows shiftedto lower frequencies are produced by molecules that lose energy to photons passing from the ground stateto the first excited ( Stokes lines ) , those at higher frequencies by molecules in an excited vibrational statethat absorb energy from photons ( anti - Stokes lines ) .

The Stokes lines are more intense because of the anti - Stokes to room temperature , the fundamentalvibrational level is more populated and are those used for analytical purposes . The row with the samefrequency of the source , resulting from the Rayleigh scattering of the incident radiation , is by far the mostintense component of Raman spectrum and must be removed .

Interpretation of Raman spectra Raman

The information that the Raman spectrum of a molecule can give derivealmost exclusively from the Stokes lines .

. The radiation Rayleigh provides no information as it has the sameenergy in each sample ; anti - Stokes lines are generally of too lowintensity to be detected and can be exploited only to indicate thetemperature of the sample according to the relationship with theintensity of the Stokes lines

The Stokes lines , however, are related to the functional groups of themolecules of the sample and their modes of vibration , in a mannersimilar to the infrared spectroscopy ( although with differentmechanisms ) , and are therefore exploited diagnostically toqualitatively identify the compounds present in the sample . Also inRaman spectroscopy the quantitative aspect is poorly taken intoaccount because of the diversity of the analyzed surface can affect thereproducibility of a measure .

Examples of Raman spectra

In the Raman spectrum in the ordinate occurs the intensity of emitted light and the abscissa represents the absolute frequency in cm -1 , or , more commonly , the displacement Raman or Raman shift , i.e. the difference in wave numbers between the observed radiation and the incident radiation :

Ds = (s oss - s inc ) cm -1 N.B. the energy associated with the vibration is only the DS, not the know

the absolute λ

Normally, the part of the spectrum with signals Stokes , the most informative , is shown for simplicity in cm -1 positives, although it is actually a negative difference

1): excitation beam

λ = 488 nm

(equivalent to s = 20492 cm -1 ),

Raman signal : 60 cm -1

The Raman Spectrometer

� A laser beam

� a SAMPLING SYSTEM to send the laser beamon the sample and collect the Raman signal ,

� a SYSTEM TO SEPARATE THE RAMANSIGNAL BY COMPONENT OF LIGHTSCATTERED almost elastic ( the so-calledSCATTERED almost elastic ( the so-calledRayleigh scattering )

� an INTERFEROMETER , a device that exploitsthe interference phenomenon : in fact, whenyou have two or more radiation of the samewavelength , the waves can be added orsubtracted from each other

� a DETECTION SYSTEM now replaced bymodern CCD detectors

The laser source

The necessity of using laser in Raman spectroscopy lies in the following reasons :

o The scattered radiation is very weak compared to the elastic

o The high monochromaticity allows us to observe very small shift and the use ofselective filters , which exclude the diffuse radiation , so allow us to reveal only thecomponents of inelastic scattering

To obtain the Raman spectrum of a sample it can be used a monochromatic source with a wavelength in the near UV , in the visible or near infrared (NIR ) .

Currently between sources LASER used we find :

� UV laser , 244 or 325 nm

� • Argon laser , 488.0 nm or 514.5 nm

� • He-Ne laser , 632.8 nm

� • Ruby laser , 694.3 nm

� • Nd - YAG ( yttrium - aluminum - garnet doped with neodymium ) , 1064 nm

The laser radiation is focused on the sample ; the scattered radiation from the surface is collected , revealed by the detector and shown in the form of the spectrum.

Laser sources

The choice of the excitation source influences the choice of most otherinstrumental characteristics . In most real samples the weak Raman signalsare obscured by the background fluorescence . In general, the fluorescencedecreases if the wavelength changes from the visible region to the near IR (NIR) , i.e if decreases the energy of the excitation source . This means that ,as excitation sources , are selected commonly lasers that emit in the far redand NIR , by reducing the background fluorescence.

Although the fluorescence decreases with increasing wavelength , even the Although the fluorescence decreases with increasing wavelength , even the

intensity of the Raman signal decreases in the same direction.

The ideal situation is to choose the shorter wavelength lasers that prevent the phenomenon of fluorescence without introducing other problems , such as overheating of the sample or the photodegradation .

What ' is the best laser ?

The frequency of the laser used , and then the energy radiated onto thesample , decisively influence the Raman spectrum that you can get .What ' is the best laser ?

You must consider the following aspects :

• the intensity of Raman emission is proportional to the fourth power ofthe frequency of the source , in other words , the emission can beobtained with a UV laser at 244 nm ( 40984 cm -1 ) in theory isenormously more intense than that obtainable with a laser NIR atenormously more intense than that obtainable with a laser NIR at

1064 nm ( 9399 cm -1 )

• the energy involved with laser at higher frequency (UV , visible) is ableto activate electronic transitions in the sample and that can generatephenomena of fluorescence and produce spectra difficult to read ;these phenomena are less obvious with less laser energy as the NIR

• another drawback of the high laser frequency is the damage that cancause to the samples during irradiation , causing photodecomposition ,and then emission of Raman spectra abnormal

What happens whit diferent laser sorces

Importantly, the Raman shift , for a given chemical bond and a givenvibration mode , is independent of λ excitation , that is, the wavelengthof the laser, but depends only on the difference between two vibrationalstates for a given bond .

Raman spectra expressed in units of Raman shifts are in theory identicalwith any laser are produced and are comparable although products withdifferent Raman instruments .

However it is possible that the use of different lasers on a single sampleHowever it is possible that the use of different lasers on a single samplegives Raman signals in the same positions but with different intensities :for example , in the characterization of pigments, occurs experimentallythat the green laser ( 514.5 nm ) is more suitable in obtaining spectra ofpigments blue and green , while the red laser ( 632.8 nm ) is moresuitable for red and yellow pigments .

In some cases this difference is so pronounced that some pigments do notprovide any spectrum if they are not irradiated with the laser moreappropriate

Depth of sampling

From the point of view of the depth of sampling , the analysiscarried out with a Raman spectrometer is surfacedepending : the information comes from a thick layer ofsome uM on the surface

From this it is easy to see that the most useful applications ofRaman spectroscopy are those in which you are interestedto characterize the surface properties of a sampleto characterize the surface properties of a sample

Some instruments have the ability to vary the sampling depthby means of a device known as confocalità , which allows toreceive the information from packets of variable thicknessof the sample, provided that this is transparent to the laserradiation

Raman Microscopy

In spectrometers Raman equipped with amicroscopy the affected area from theanalysis may be limited to a few units upto a few hundred μm2 , depending on thelaser and lens used . The objectives arenormally used 10x , 20x , 50x , 80x and100x

Among the techniques of molecular analysis, Raman spectroscopy is that with better, Raman spectroscopy is that with betterspatial resolution : can get to distinguishtwo points 1 micron

To exploit this spatial resolution is requiredto know exactly where you are measuringto avoid gross errors ; why Ramanmicroscopes are equipped with a cameracoaxial with the laser , which allows youto view the area where you are aiming

IR and Raman spectroscopy

The Raman spectrum is therefore a vibrational spectrum as the IR spectrum ,but this differs for two basic reasons :

1. the Raman spectrum is generated by the difference of two electronic levels( whose difference is equal to a vibrational level ) ; the source of thespectrometer is so visible , though now are widespread sources in the NIR

2. secondly, the IR and Raman spectra differ for different selection rules of thetransitions . This means that the bands generally have different intensitiesand in some cases there may be bands present in a technique and not inand in some cases there may be bands present in a technique and not inthe other, and vice versa .

3. A IR mode is- active if there is a change of the dipole of the molecule ,while it is Raman - active if there is a change of polarizability .

Raman spectroscopy

�Then the Raman effect is based on the polarity of thechemical bond

�Provides information not only on the identification andquantification of the different chemical species present in asample , but allows you to analyze the vibrational modes ofmolecules : stretching, bending , wagging and othermolecules : stretching, bending , wagging and other

�The bands of Raman spectra are well resolved , allowing toobtain information also on the molecular structure byanalyzing the bands of the s of interest

Interpretation of Raman SpectroscopyThe Raman signals corresponding to the various chemical bonds are obviouslyplaced in the same spectral regions described for infrared spectroscopy , subject tothe differences due to the selection rules . As regards the determination of inorganicsubstances , the Raman signals relating to the metal-ligand bonds are generally inthe range 100-700 cm -1 , a spectral region difficult to use in the IR

Bands of Raman scattering of the main

components of food

� The bands of Raman spectra arewell resolved , allowing to obtaininformation also on the molecularstructure and in particular that ofprotein analyzing the bands of thegroups -CO- NH-

IR and Raman spectra

The differences in energy between the incident radiationand therefore the Rayleigh radiation , and radiationRaman match in terms of wavelengths to the region ofthe mid-infrared or MIR , or in the range of 2.5-50 um( or range of wave number 4000-200 cm -1 ) .

For this reason , in some cases the Raman spectrum andthe IR ones of a compouns can be similar, becauseboth depend from in changes in the vibrational states

Confronto IR e Raman

The two techniques are more complementary thancompetitive. First, the phenomena that are the basis of thetechniques are different :

( selective absorption of radiation that cause transitions inthe IR energy , inelastic scattering of light in the Raman ) .

Also the so-called selection rules , which determine whichvibration modes are active and which are not , are differentvibration modes are active and which are not , are different

�In IR are absorbed energies that cause changes in thedipole moment of a molecule ,

�while in the Raman is required a change of its polariza-bility , properties related to the possibility of distortion ofthe electron cloud .

In consequence of this , some modes of vibration are active inthe IR and not in Raman , and viceversa

IR vs Raman

The two techniques are therefore complementary : in the FT-IR spectrumof the dichloro - acetophenone is evident the stretching of the C = Obond at about 1700 cm -1 , which is almost absent in the FT - Raman .

Advantages of Raman spectroscopy vs IR

The advantages of Raman in front of infrared techniques ofanalysis have long been known , but only recent innovationsinstrumental allowed to exploit them fully .

The main advantage of the technique compared to IR Raman isthat the water is practically transparent and therefore Ramanaqueous samples can be analyzed as such . If you add that theglass has a weak absorption Raman , you can think of analyzingglass has a weak absorption Raman , you can think of analyzingwater samples in simple tubes .

Instead in the analysis of solid samples , the Raman spectrometry, in contrast to the IR techniques , requires no special samplepreparation . For example, the pharmaceutical products can beanalyzed as such , in their final form for consumption. Thisfeature makes the Raman technique potentially useful for theprocess control. The spectrum of the Raman active products isalso , in general , much more intense than that of theexcipients.

Advantages of Raman spectroscopy

� It is easy to perform

� The signals are stable

� Glass and water do not interfere in the measurements due to the Raman no-active vibrational modes

� It is a noninvasive spectroscopy

� Possibility of in - situ measurements using portable � Possibility of in - situ measurements using portable systems

� Possibility to perform mapping 2D and 3D coupling the excitation laser and the detector to a confocalmicroscope

� Ability to follow structural changes molecular time-resolved with laser pulsed also the order of picoseconds

Disadvantages of Raman spectroscopy

� In most real samples the weak Raman signals areobscured by background fluorescence , which is thereason to choose commonly as excitation sourceslasers or laser diodes which emit in the far red andNIR , by reducing the background fluorescence.lasers or laser diodes which emit in the far red andNIR , by reducing the background fluorescence.

� Risk of photodegradation of the sample at high power laser excitation

FOR INORGANIC COMPOUNDS, Raman signals related to the metal-ligand bondsare generally in the range 100-700 cm -1 ,( in the IR spectral region difficult to use )

Application

� The Raman is finding an increasing number of applications in thepharmaceutical and materials science . Interesting applications ,although limited , are those of the recognition of natural gemstonesfrom synthetic ones , through the analysis of inclusions, in archeologyand in criminology in recognition of traces of gunpowder.

� The future of the Raman though is in the agricultural sector ,environmental , biological and medical applications , where youenvironmental , biological and medical applications , where youhave to analyze samples with high water content .

� Among the applications in the experimental phase , we can mention ,for example , that the Raman has been used for the recognition ofcancerous cells in human breast tissues .

Raman spectroscopy and natural pigments

The natural pigments can be classified based on their structural and / or functional groups [ 1] . Only the most important group in regard of application in Raman spectroscopy and those more significant for the agroofood will be presented below. Of course over the chlorophyll they are:

I. Carotenoids

II. Flavonoids

III. Anthraquinones

IV. Indigoids

The concentration of natural dyes in plants is generally low and it varies depending on species and pigment. For anthocyanins these amounts are about 0.1–5 g/100g dry matter, whilst for lycopene and ß-carotene the mean levels are between 4.5–6.3 and 0.8–1.6 mg/100 g of fresh matter, respectively

CarotenoidsCarotenoids are composed of eightisoprenoid units and belong to the class ofhydrocarbons (carotenes) and theiroxygenated derivatives (xanthophylls ) .

Their main characteristic is to have a longcentral chain of double bonds coordinatedacting as chromophore and which areindeed responsible for giallor colors ,orange, red of these compounds.

A part of the chlorophyll they are the mainA part of the chlorophyll they are the mainpigments involved in photosynthesis .They have two well known functions : a)they are accessory pigments for thecollection of electromagnetic radiation ; b )are photoprotectors against oxidativedamage .

It is interesting to emphasize that only theplants and bacteria are able to synthesizecarotenoids de novo.

Carotenoids were also isolated in marineorganism

The Raman analysis of carotenoids is well documented [ 2 ]. Carotenoids occur in plants as a secondarymetabolites on the ppm level, and, due to high sensitivity of the Resonance Raman spectroscopy (with thelaser excitation from the visible range), even such small amounts are possible to be detected [ 3 ]

Moreover, it is known that the wavenumber location of the carotenoid bands, especially C = C, is correlatedwith the length of the polyene chain [table ] i.e. the position is red-shifted with the extent of theconjugation length of the polyene chain due to an electron-phonon coupling.

Anyway the band position could be slightly influenced by the carotenoid side group and bonding to otherplant constituents. It is explained by the fact that the C = C stretching wavenumber of carotenoids issensitive to the complexity of the matrix in which this compound is involved .Even though the carotenoids were analyzed not only in solution but also in the algal cell, in human eyes,human blood and skin. Raman spectroscopy can be used for in situ quantitative measurements ofcarotenoids. The quantitative analysis of crocetin in saffron ( Crocus sativus L.) was carried out withapplying of RS and chemometrics [ 4 ].

A rapid method for determining the crocetin esters and colouring strength directly in saffron samples,using NIR Raman spectroscopy (785 nm) together with partial least-square regression (PLS) wasdemonstrated [ 4 ] As a reference methods HPLC and UV -Vis spectroscopy were used.

By using the Ft-Ramanmicroi-maging, the astaxanthin(classified as xanthophyll) wasin situ and in vivo detected inHaematococcus pluvialis [5].Raman maps of carotenoiddistribution in the algalspecies Neochlorisoleobundans was reported inref. [6] and the carotenoid

Fig. Raman spectra of Calendula officinalis L.measured in three different points showing the presence of 7-, 8-, and 9-conjugated carotenoids (a). Picture of Calendula officinalis L. flower (b) and corresponding Raman maps coloured according to the band intensity at 1,536 (c),1,530 (d), and 1,524 cm (e) related to the content of 7-, 8-, and 9-conjugated double bond carotenoids, respectively.

ref. [6] and the carotenoidcompound was detectedsimultaneously with lipids(triglyceride).

Raman measurements to estimate the ripenessof pigmented fruitsIt is well known that during ripening the composition of the carotenoid fractionsometimes changes dramatically. Also in this context Raman spectroscopy was successfullyapplied e.g. to characterize the content of lutein, ß-carotene and capsanthin in the individual ripening stages of bell pepper (Capsicum annum) [7]. Quite recently, confocalRaman has been used to identify in parallel lycopene, ß-carotene,and lutein in tomatofruits [8] The authors mention that this technique provides some advantages for the foodindustry in order to get a rapid information of fruit quality during processing. this findingwas possible due to the ability to separate different Raman bands to ß-carotene (1,520 cm ) ,a-carotene and lutein (1,527 cm),a-carotene and lutein (1,527 cm)

Fig. FT-Raman spectra of pure carotenoidsstandards:-carotene (A), -carotene (B), and lutein (C).

Raman identification of carotenoids in some varieties of grape at different ripening

The carotenoids are available in the grapes in great quantity before the grapes colouring and their level decreases during the ripeness. The concentrations of carotenoids in the grapes however are influenced also by the type of cultivar, by the pedo-climatic conditions, by the degree of exposure to the light .

Raman spectroscopic studies of carotenes and carotenoids in grapes berries have been undertaken and the characteristic bands of C=C, C–C stretching and C–CH3 bending have been recorded under different excitation sources (785 nm, 1064 nm) with a portable Raman analyzer. Raman spectra can be obtained directly from the single plant cells As shown in fig. especially with the excitation laser with emission at 1064 nm the fluorescence fig. especially with the excitation laser with emission at 1064 nm the fluorescence contribution is minimized

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As shown in figure first results taken at differentripening degree seem to confirm what it wasdemonstarted also with HPLC mesurament , in particularly that meaningful differences in the carotenoids quantities are visible during the grape ripeness. Such different degradation could provide useful indications in the control of the potential aroma of the grapes assigned to the vinification.

Bibliography� 1) Wissgott U, Bortlik K (1996) Prospects for new natural food colorants. Trends

Food Sci technol 7: 298–302

� 2) Schulz H, Baranska M, Baranski R (2005) Potential of NIR-Ft-Raman spectroscopy in natural carotenoid analysis. Biopolymers 77: 212–221

� 3) Bode S, Quentmeier CC, Liao P-N, Hafi N, Barrost, Wilk L, Bittner F, Walla PJ (2009) PNAS 106:12311(2009) PNAS 106:12311

� 4) Anastasaki Eg, Kanakis Hd, Pappas C, maggi L, Zalacain A, Carmona M, Alonso GL, Polissiou Mg (2010) Quantification of crocetin esters in saffron (Crocus sativus L.) using Raman spectroscopy and chemometrics. J Agric Food Chem58: 6011–6017

� 5) Kaczor A, Turnau K, Baranska m (2011) In situ Raman imaging of astaxanthin in a single microalgal cell. Analyst 136: 1109–1112

� 6) Huang YY, Beal Cm, Cai WW, Ruoff RS, Terentjev Em (2010) micro-Raman spectroscopyof algae: composition analysis and fluorescence background behavior. Biotechnol Bioeng 105: 889–898

� 7) Baranski R, Baranska M, Schulz H (2005) Planta 222:448

� 8) Pudney PdA, Gambelli L, Gidley M (2011) Appl Spectr 65:127

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