18 Fourier Transform Infrared Spectroscopy – Useful Analytical Tool for Non-Destructive Analysis

It is highly recognized that the use of Fourier Transform Infrared Spectrometry (FTIR) for chemical substances identification it is not a trivial task to be fulfilled by analytical chemists. The complexity of FTIR characterization comes mainly from the high degree of infrared absorption bands overlapping, that are difficult to be accurately ascribed, despite of the fact that up to date computer-searchable databases of spectra are currently available.

on a list of so-called potential trace evidence, different items which require certain identification; the mentioned list may be formed by objects from various materials: paint, pottery, glass, bricks, liquid residues (oils, wines, and perfumes), fabrics, soil, metals etc.In order to rationalize on the evidences origin, source, transformation and historical age, the archaeologists need a deep analytical study, study that often has to be performed on irreplaceable pieces.Consequently, to accomplish these goals multiple analytical techniques, able to work as non-destructive techniques are used, such as: Fourier Transform Infrared Spectrometry (FTIR), scanning electron microscopy (SEM), polarized light microscopy (PLM) or X-photon spectrometry (XPS).
In our studies, in the amber analysis the main goal was to establish several criteria able to be useful in discrimination between different types of amber, the obtained FTIR information being used to ascribe the origin of the materials found in archaeological sites on the Romanian territory.
Amber is a fossil resin, extremely appreciated for its colour and beauty, and used as a gemstone from very early times.Natural amber deposits are found all around the world, the most famous being the ones from the Baltic sea, the Dominican republic, Sicily, Borneo, Spain, etc. Amber resources are documented in Romania since the late sixteenth century, around the village of Colţi (Buzău County), but they have certainly been exploited only starting with the modern age, after 1828, in the same area (Ghiurcă, 1999;Wollmann, 1996).
The existence of a local amber exploitation on Romanian territory aroused the question of geological origin of amber artefacts found by Romanian archaeologists over time.
There are a few comparative analytical studies of Romanian geological amber, referred to as Rumanite or Romanite, which conclude that Romanian amber is geologically younger, but chemically almost identical, to Baltic amber (Stout et al., 2000).
Regarding archaeological amber from Romanian territory, it was basically unstudied since we started our research project in 2007.Some studies by Nikolaus Boroffka on prehistoric amber (Boroffka, 2006) refer to chemical analyses of amber with an assumed Romanian origin; in another paper, Boroffka (2001) discusses the results published by Banerjee (Banerjee et al. 1999) for two prehistoric beads from Romania, both considered of Baltic origin.
As a hint for the archaeological output, in order to rationalize on the importance of nondestructive analysis, it should be mentioned that the quantity and quality of amber artefacts from Romania is lower than those found in territories placed on the track of the so-called Amber Route (Teodor ES et al, 2010).Nevertheless, from our studies, the Baltic amber is encountered very early, in Neolithic, i.e.only three isolated beads (not published), and only in southern Romania, as a clue that Danube may be considered as a secondary Amber Trail.
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Local amber, Romanite, was also early used, as proved on some beads from the Middle Bronze Age from Pietroasa Mică, in a necropolis placed at the fringes of the Colţi area.In our studies two types of amber have been investigated: artefacts from the largest amber deposit discovered in Romania, in Cioclovina cave (over 3000 beads, most of them in amber) (Teodor ES et al., 2010) and artefacts from Roman Age; discovered in cemeteries dug systematically, for instance in Tomis, Callatis, Beroe, or Carsium cemeteries (Vîrgolici et al. 2010b).

Assessment of amber origin based on FTIR analysis
Taking into account the lack of referential with respect to ascription of amber origin, and considering as critical issue the transformation occurring on amber samples during historical ages strongly influenced by the storing conditions in the deposits where were discovered, in the attempt of establishing few definite FTIR criteria to be useful for amber origin proper assignment we started the analysis not directly with archaeological samples but first, with geological amber, of controlled and certified origin, both from Baltic sources and from Romanian sources.Further obtained results are detailed.

FTIR method
FTIR assays of amber were performed using the FTIR-VAR technique with a beam incidence angle of 45 o , on a Bruker TENSOR 27 instrument, using the OPUS software version 6.0.The samples were used without any pre-treatment, as whole pieces fixed on a gold mirror, and all the spectra were registered versus a background of clean gold foil between 4000 and 600 cm -1 .The spectral resolution was 4 cm -1 , and the co-added scans 96, with an aperture of 4 nm.The FTIR-VAR technique is able to provide the same information as FTIR in terms of transmittance, with a slight loss in signal intensity, but with the advantage of preserving samples integrity.In addition, the bounds between atom plans are not destroyed, and, as consequence, the amount of provided information increases.

Geological amber FTIR assessment
A large lot of samples from different controlled origins were analysed by FTIR-VAR, from Romania (Colti, Buzău County), from the Baltic region (Palanga and Kaliningrad), from Germany and Poland in order to settle a certain pattern to differentiate Romanian amber from other types of amber, especially the Baltic variety (the material was provided, mainly, from the National Geological Museum from Bucharest).In the case of unsure observations, to clarify some spectral zone from the fingerprint region (1300-900 cm -1 ), or the region 1800-1450 cm -1 , the transmittance spectra of the samples were used for a better evaluation.Spectra were analysed and assigned on the three wave-numbers domains of significance for amber, namely those between 3600-2000 cm -1 , 1820-1350 cm -1 and the 1250-1045 cm -1 regions, which correlate with hydroxyl groups, carboxyl groups, carbonyl groups and with C=C instauration.

Subsequent data acquisition the following observations were drawn:
a.There are no notable differences between Romanite spectra and Baltic spectra in the 3600-3000 cm -1 region mainly in case of geological samples.The noteworthy differences appear (as in any IR technique) in the 'Baltic shoulder' region, 1250-1060 cm -1 , and in the 1161-1155 cm -1 region.For the Baltic amber, the shoulder appears in the region 1275-1155 cm -1 , while that of Romanite has a different shape and is shifted to about 1045-1020 cm -1 , as may be noticed from Fig. 1. b.An important difference in Variable Angle Reflectance spectra is registered for absorption bands from 900-600 cm -1 region (Fig. 1); in this region two species can be differentiated, depending on the age of the polymer.The absence of some characteristic bands indicates that the contraction-reticulation of the polymeric chain is finished (connected with the older age of fossil resin); the absence of a double peak at 667 cm -1 in the Baltic amber spectra and the appearance of the shifted shoulder toward 1045 cm -1 , in the case of Romanite when compared to the Baltic shoulder, that occur at higher wavenumbers (demonstrated also by Fourier spectra de-convolution) provided evidence that Romanite was formed after Baltic amber (i.e., it is younger), as it may be observed in Fig. 1. c.There are vibration frequency shifts determined by the degree of ethers and esters formation, correlated to the number of functional groups from the chain and showing evidence on the intramolecular bounds, which appear frequently in amber type resins with younger ages (for example the OH group, specific to Romanite, is slightly shifted Fourier Transform Infrared Spectroscopy -Useful Analytical Tool for Non-Destructive Analysis 357 due to intramolecular hydrogen bonds toward 1595 cm -1 , if compared to the Baltic amber where it occurs about 1640 cm -1 ).d.The amber has an amorphous structure, and as consequence the reproducibility of determinations is affected by the heterogeneity of the sample, the obtained spectra depending on the analysed part of the sample.As a result of these observations, for each sample were registered two spectra, for different zones of sample, in order to observe if significant differences appear between the two, due to appearance/disappearance of certain absorption bands (specific vibration frequencies of interest).It was observed that difference between two spectra of the same sample appear only in the spectral region related to methyl, methylene, etc. (-CH3, -(CH2)n-, -CH, etc.) chains, namely 2962-2850 cm -1 , and for the specific tensile bands of CH3 from 1375 cm -1 .This led to the conclusion that the presence of functional groups (which are important because they correlate with the origin and the age of the sample, and giving the sample specificity) are equally distributed.Therefore it could be considered that the analytic information supplied by FTIR is acceptable, since the wavenumbers corresponding to carboxylic chains and hydroxy-carboxylic acids at 1547-1423 cm -1 for Romanite and, respectively, 1547-1300 cm -1 for Baltic amber are preserving their region for each duplicate recorded spectrum.
The differences appearing in the 1684-1642 cm -1 band of Baltic amber with respect to the 1547 cm -1 band of Romanite are determined by the shift of asymmetric vibration frequencies for the carboxylic type groups, as a function of the length of hydrocarbon chains.A strengthening-ageing of polymeric chains leads to shifts toward smaller wavenumbers.This can be observed also in 1046-900 cm -1 region, where the shifts toward the smaller wavenumbers are related to the better confirmation of the Baltic origin.The significant regions for each type of amber are summarised in Table 1, corresponding comment with respect to archaeological amber spectra being introduced at this point for a better understanding of the noticed differences.

Archaeological amber FTIR assessment
After analysis of geological specimens of amber, the next step was devoted to archaeological samples analysis.The investigated samples were originated from different times (Neolithic, Bronze Age, Roman Age, Byzhantine period, etc) found on different archaeological sites from Romania.
Concerning the archaeological samples, the FTIR-VAR spectra pointed out some differences between the archaeological and geological material.Archaeological samples most generally presented a less intense signal in the 3600-3000 cm -1 region, which correlates with -OH groups and influence the H bonds (Fig. 2); the presence of -OH groups was confirmed by a signal of lower intensity for archaeological samples in the 746-736 cm -1 region, especially for Baltic amber, the relative dehydration of archaeological samples as compared to geological materials being the probable cause (Fig. 2).
The fingerprint zone is different in most of archaeological samples in comparison with geological ones.This region presents adsorption bands of higher intensity for the majority of archaeological samples and some shifts of the specific wavenumbers take place (Figs.2-3).Another difference in VAR spectra of geological origin is registered for absorption bands from 800-600 cm -1 region: 622-621 cm -1 are ascribed to C-O, C=O out of plane bands; 746-736 cm -1 are the confirmation of OH and H bonds; 676-666 cm -1 are the confirmation of CH 2 bonds.The absence of a double peak at 667 cm -1 in the Baltic amber spectra indicates that the contraction-reticulation of the polymeric chain is complete (an aspect related to the older age of the fossil resin, in our case the Baltic species).This region is not so well defined in archaeological samples, especially for Baltic amber (Figs. 2 and 3).Based on the relative comparison with the geological reference material (both Baltic and Romanian amber, see Table 1) and according to the FTIR-VAR bands of archaeological samples from the region 1820-1350 cm -1 , 1275-1020 cm -1 and 900-600 cm -1 , the classification of several archaeological samples is summarised in Table 2. Some spectra obtained for the samples from Nufăru (Roman Age), Cioclovina (Bronze Age) and a Neolithic bead are presented in Figs. 2, 3 and 4.

Sample origin Age FTIR-VAR assignment
Grădistea  These results based on comparison of FTIR-VAR spectra about origin of amber artefacts were clarified and confirmed by other non-destructive analysis (Raman, statistical analysis) and supported by historical context, in some cases.In other cases, the assignment of samples to Baltic amber or local (Romanian) amber contributed to understanding of archaeological (historical) context.
FTIR-VAR is a reliable tool for non-destructive investigating of amber.The spectra are more complex than FTIR-transmittance spectra, the obtained signal are rather highly difficult to ascribe and the variability of results is high, but coupled with statistical analysis and corroborated with other techniques (Raman spectroscopy, X-rays fluorescence) is a consistent method to diagnose the origin of amber in archaeological artifacts.

Applicability of Fourier Transform Infrared technique in the assessment of bio-mimicking systems. Case study-Human low density lipoproteins
In order to sustain the assertion regarding the FTIR versatility as non-destructive analytical technique able to provide valuable information with respect to noteworthy molecular changes, the second exemplification is based on assessment of significant bio-markers used to indicate oxidative modifications occurring on cellular membrane.At the level of living organisms the cellular membranes are the main targets for reactive oxygen species (ROS) and reactive nitrogen species (RNS).Both ROS and RNS induce lipid peroxidation (LPO) of unsaturated fatty acids from membrane phospholipids.At the cellular level, reactive species are responsible for changes in the membrane properties, inducing modification on cell permeability and enzyme activities (Ahsan et al., 2003).
In our experiments we developed a bio-mimetic to be applied in assessment of lipoperoxidation processes using as significant oxidative substrate a component of the cellular membrane, low-density lipoproteins.The system development was based on system deposing a thin lipoproteic layer on the surface of conductive solid support, usually gold sheet, and quantification of the lipoprotein peroxidation degree by electrochemical measurements, the intensity of the registered intensity current at the lipoperoxides specific peak potential being proportional with the amount of formed lipoperoxides.At this point have to be mentioned the fact that we employed the VAR-FTIR in analysis of structural modifications occurring to low-densitylipoprotein as result of the oxidative changes induced by free radicals (ROS) attack.

FTIR method
Starting from the important experimental issue which states that an appropriate development of a bio-mimetic system by immobilization of a compound to a support preserves the structure of the immobilized compound, the first FTIR study was performed on low-density lipoprotein layer deposed on gold support, the results being compared to those obtained for un-bounded lipoprotein.For the deposed layer the reflectance mode was employed, while for free lipoprotein the transmittance mode was used.FTIR spectra were recorded at room temperature using a Bruker Tensor 27 Fourier Transform spectrometer.The spectra were collected and ascribed using Opus software.In transmittance mode the spectral range was 4000 -400 cm -1 , the aperture 4 cm, the spectra resolution 4 cm -1 and 98 scans being acquired for each spectrum, 20 Hz.Samples were pressed into a potassium www.intechopen.com Fourier Transform Infrared Spectroscopy -Useful Analytical Tool for Non-Destructive Analysis 363 bromide (KBr) pellet, the background spectrum being recorded against KBr pellet.In reflectance mode the spectral range was 4000 -600 cm -1 , the background being recorded for an unmodified polycrystalline gold sheet after that being recorded the samples' spectrum.The optimum reflectance angle was 45 degrees, the aperture 6 cm, the spectra resolution 4 cm -1 , 98 scans being acquired for each spectrum, with 2.2 Hz.

FTIR assessment of native and oxidised low-density lipoprotein
As mentioned in the previous section, the first FTIR studies were performed to evaluate the suitable deposition of the low-density lipoprotein on the gold support in order to preserve the lipoprotein structural characteristics.This is an important experimental issue, because the protein structure and surface charging are the most important structural features necessary to be maintained to ensure feasible "ex-vivo" analytical information with respect to oxidative changes induced on cellular membrane by ROS.
The main regions corresponding to FTIR absorption bands and specific for native LDL are given in table 3. Absorption bands from 2800-3000 cm -1 corresponding to methyl groups are well defined, no matter the type of the sample, either free or deposed LDL.

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As observable from figures 5 and 6, the amide specific band absorptions for proteins, amide I band around 1654 cm -1 and amide II band around 1541 cm -1 (Firth et al. 2008;Banuelos et al. 1995) are not changed when LDL was deposited on the gold support.This observation is important because it proved that the secondary structure of protein is preserved subsequent deposition therefore it can be concluded that the deposition on solid support did not affect theLDL functionality, and, consequently, that deposed LDL is expected to react with free radicals according to the same pathway as free LDL.Moreover, it should be mentioned that this argument is consistent with the data published by Paker (Paker, 1991) where it is mentioned that LDL ex vivo peroxidation pathway is similar as in vivo peroxidation pathway.
Fig. 5. FTIR spectra of LDL free, on transmittance (KBr pelleted) When lipoprotein are subjected to the attack of peroxyl radicals, thermally induced from aqueous solutions of azo-initiator 2,2'-azobis (2methylpropionamidine).dihydrochloride (AAPH), the lipoperoxidation occurs according to a radical pathway (Litescu et al, 2002;Tache et al., 2011) leading to lipo-peroxides formation on the LDL layer.In our experiments the FTIR analysis was performed first on the LDL deposed on the solid support, then the LDL was subjected to free radicals attack for 10 minutes, allowed to dry on inert atmosphere and after that the oxidised LDL layer was once more assessed by FTIR.The performed studies proved that in the presence of free radicals (Fig. 7), the IR absorption bands corresponding to ester groups from lipid residues at 1740 cm -1 were changed in intensity and shifted as wavenumber, thus proving the lipoperoxides formation.
Another proof of lipoperoxides existence was the presence of HO absorption bands from 3600-3700 cm -1 and 917 cm -1 .In detail, the signal ascription are as follows: the band from 3278 cm -1 increasing in intensity and shoulders rise at 3334 cm -1 , 3304 cm -1 , 3220 cm -1 and 3197 cm -1 assigned to O-H and N-H vibrations.The bands from 3072 cm -1 are corresponding to hidroxil and amine groups involved in hydrogen bonds.In the same time, it is important to mention that several changes arose around 1717 cm -1 indicating the formation of new C=O groups.
The changes observed on FTIR spectra of deposed LDL correlated perfectly with electrochemical and MALDI information (Litescu et al, 2011), proving the usefulness of nondestructive FTIR analysis in ascribing the oxidative modification occurring on lipoproteic components of the cellular membrane.

Conclusions
The use of FTIR, especially as variable angle reflectance technique, proved to be of crucial importance in two main types of applications where the critical issues are the importance to conserve the sample integrity and the limited amount of available sample.
Two examples of FTIR non-destructive analysis employment in two very different research domains, archaeology and modified surfaces are supporting the highest utility of the technique when the analysis is dealing with valuable samples, sometimes of patrimony value, which have to remain un-affected subsequent analysis, as it was the amber samples case study, or when the analysis have to provide arguments on sharp and specific structural changes, as it was the case of lipoprotein oxidation study.
It could be concluded that FTIR proved its efficiency in evaluation of the oxidative modifications on the level of one of the main components of the cellular membrane, oxidative process which plays, generally, as key-event in several age -related and degenerative pathologies.

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Fourier Transform Infrared Spectroscopy -Useful Analytical Tool for Non-Destructive Analysis 367 In the same time, it should be emphasized that, in our opinion, FTIR-VAR technique use as non-destructive way to analyse archaeological artefacts is strongly recommended, especially when coupled with statistical analysis.

Acknowledgment
This work was partially supported by EU FP7-SME project Sensbiosyn, contract no 232522/2009.

Table 1 .
The main characteristics of spectral domains in Baltic amber and Romanite and comparison between geological and archaeological samples) www.intechopen.comFourierTransform Infrared Spectroscopy -Useful Analytical Tool for Non-Destructive Analysis

Table 2 .
The main categories of archaeological samples analyzed by FTIR-VAR and the assignment of amber origin in comparison with geological samples