Original Research Papers

Original Research Papers Application of Thin-Layer Chromatography, X-Ray Fluorescence Spectrometry, and Fourier Transformed Infrared Spectroscopy in the Analysis of Binding Media Present on Mummies of St. Giovanni Olini (1200 AD) and St. Nicolosa Bursa (1500 AD) Iva Rezic´*, Domagoj Mudronja, Marko Obranovic´, Toncˇi Rezic´, and Ksenija �karic´

Key Words Thin-layer chromatography X-ray fluorescence spectrometry Fourier transformed infrared spectroscopy Corpi Santi Mummies Binding media

Summary The main goal of this work was to apply thin-layer chromatography (TLC) in the investigation of different binding media (proteins, sugars, waxes, resins, and oils) found on samples of two mummified bodies of saints originating from 1200 AD and 1500 AD. The historical samples were compared by testing them for the presence of different inorganic and organic compounds. The chemical methods used were TLC, microscopy, X-ray fluorescence spectrometry, and Fourier transformed infrared spectroscopy. The detected similarities in the composition of the binding media coatings on two mummies indicated that those were not applied immediately after death but much later showing resemblance in their preservation treatments. Moreover, according to the composition of the materials detected, the coatings did not seem to have had considerable impact on the mummification of the bodies. The combination of TLC and other chemical methods proved to be an effective and low-cost tool for obtaining valuable information during the archeological investigations.

1 Introduction Mummies are human or animal remains with no preservation of any bony tissue, present in different cultures throughout history [1]. There are two kinds of mummies, natural and artificial, depending on the mummification process [2]. Natural mummification is achieved by environmental factors such as climate, while artificial mummification is a set of intentional procedures performed as a part of mortuary practices. According to Klys et al., the first nation that mummified bodies were not Egyptians but an ethnical group of Chincohoro (northern Chile) who mummified bodies more than 4000 years ago [3]. Although the Egyptian mummies are the most famous, such objects can be found all over the world: in Korea [4], Columbia [5], Peru [6], and other countries [7]. Many mummies around the world are of I. Rezić, Faculty of Textile Technology, University of Zagreb, Croatia; D. Mudronja and K. Škarić, Croatian Conservation Institute, Zagreb; and M. Obranović and T. Rezić, Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia. E-mail: [email protected]; [email protected] Journal of Planar Chromatography 28 (2015) 3, 205–212 Journal of Planar Chromatography 28 (2015) 3 0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest

different origins: some of them can also be the result of natural preservation, such as the mummies in the Capuchin Catacombs in Palermo, Sicily. A distinct category of mummies are the bodies of saints that are in the Roman Catholic tradition considered to be mysteriously preserved due to their holy life. These are called the Holy Incorruptibles (Corpi Santi) and are venerated as relics. The majority of these mummies are placed in Italy, but there are also many of them preserved in other countries (Croatia, France, Poland, etc.) [8]. The Holy Incorruptibles (Corpi Santi) of St. Giovanni Olini and St. Nicolosa Bursa are part of a rich collection of several hundred relics treasured by the parish church of St. Blaise in the small town Vodnjan in Istria (Figure 1). The collection was brought to Vodnjan from Venice by painter and art collector Gaetano Grezler. Grezler donated the collection to the town and the parish of Vodnjan before he left the town in 1818 [9]. According to the legend, St. Giovanni Olini was a priest who lived a life of holiness and helped the sick during the plague. He died in Venice in 1300. St. Nicolosa Bursa was a Benedictine abbess who predicted the very day of her death: Friday, 23rd of April 1510. Multi-slice computed tomography (MSCT) showed that these two relics, unlike the others found in Vodn-

Figure 1 Location of Vodnjan, Istra, Croatia. DOI: 10.1556/1006.2015.28.3.2

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TLC, XRF Spectrometry and FTIR Spectroscopy in Analysis of Binding Media

jan, are exceptionally well-preserved mummified bodies with preserved skeletons, inner organs, muscles, and tendons [10]. The knowledge about the possible treatments of the mummified remains in the course of history can be widened by analyzing the materials present on the preserved bodies. This goal can be achieved by proper chemical and physicochemical analyses. The identification of the materials and processes can provide a clue to the question if mummification had occurred naturally or by human intervention [9, 11]. Whenever it is possible, nondestructive methods of analysis should be used. In other cases, it is recommended to apply methods that are able to obtain quality information on small samples. Analytical methods that are generally used for identifying organic compounds are different spectroscopic techniques like ultraviolet–visible (UV–vis) spectroscopy, Fourier transformed infrared (FTIR) spectroscopy, Raman spectroscopy, and mass spectroscopy (MS) coupled with chromatographic techniques (thin-layer chromatography [TLC], high-performance liquid chromatography [HPLC], or gas chromatography [GC]) [12, 13]. Among those, FTIR spectroscopy is probably the most widely used technique for distinguishing different chemical species present on historical material [12]. FTIR spectroscopy also enables detection of deterioration in materials [14]. The stability of organic compounds present on analyzed items depends on the chemical composition. The agents that influence deterioration are photochemical sources, metal oxides, thermal and other environmental conditions [12]. For monitoring the degradation processes, GC coupled with MS is extremely useful [15–18]. Another useful method for analyzing the proteinous binders and for determining the structure of lacquer films is pyrolysis– GC [19, 20]. The structure of fibers present on the mummies (for example, the microstructure of keratin from wool fibers) is usually determined by FTIR spectroscopy [21]. This method can also be combined with infrared microscopy for the direct study of the mummy skin, as was done by Cotte et al. [22]. In addition, a method useful for the investigation of the chemical composition of human skin surface lipids is quantitative TLC [23], while volatile constituents originating from balsam and oils applied on the skin of the mummies (like essential oils from Helichrysum species) are usually analyzed by GC–MS [24–27] or infrared spectroscopy [28]. Nevertheless, the sophisticated methods are rather expensive and often not easily available to many of restoration and conservation laboratories. In contrast to this, TLC is a fast, low-cost, and very efficient methodology for the analysis of many different binding media in archeological samples [29–33]. Therefore, the goal of this work was to show that inexpensive TLC, in combination with other routine methods (like FTIR spectro scopy and X-ray fluorescence [XRF] spectrometry) can provide sufficient information during archeological investigations.

A

B

Figure 2 Fragments sampled from the Croatian mummies of St. Giovanni Olini (A, 1200 AD) and St. Nicolosa Bursa (B, 1500 AD) originating from Venice, Italy.

the course of conservation of the mummies led by the Croatian Conservation Institute between 2009 and 2011. For the purpose of conservation, the mummies were sterilized, disinfected by methyl bromide, cleaned, and placed in the micro A

B

2 Experimental 2.1 Samples

Samples used for the analysis were fragments of the surface-coating materials found on the mummified bodies of St. Giovanni Olini and St. Nicolosa Bursa. The points of sampling are marked in Figure 2A (St. Giovanni Olini, 1200 AD) and 2B (St. Nicolosa Bursa, 1500 AD). The samples were taken in

206

Figure 3 Microphotograph of the sample fragments left after cleaning of the bodies of St. Giovanni Olini (A) and St. Nicolosa Bursa (B).

Journal of Planar Chromatography 28 (2015) 3

TLC, XRF Spectrometry and FTIR Spectroscopy in Analysis of Binding Media B

A

Figure 4 XRF spectra obtained from the bodies of St. Giovanni Olini (A) and St. Nicolosa Bursa (B) obtained by spectrometer Artax (Bruker, AXS).

climate. Samples taken before cleaning, but after disinfection by means of methyl bromide (Figure 3) were used for the chemical analysis. 2.2 Reagents

All reagents (amino acids, sugars, water, chloroform, petrol ether, n-butanol, acetic acid, benzene, methanol, diethyl ether, hydrochloric acid) were of p.a. purity. Standards of the binding media (protein binders, waxes, sugars, and resins) were a part of the collection of the Croatian Restoration Institute. The sample extracts and the standard solutions containing the desired components were stored at 4°C prior to analysis. Ninhydrin reagent was prepared by dissolving the p.a. ninhydrin solid reagent (Merck, Darmstadt, Germany) in p.a. ethanol to obtain 95% solution.

acid (for 24 h on 110°C) for the testing of proteins. In this part of the TLC experiment, the stationary phases were precoated cellulose plates, 10 × 10 cm (CAMAG). The indicators used for the visualization were UV light on 254 nm, ninhydrin (used for amino acids), and iodine (for resins, oils, sugars, and waxes). The mobile phases were as follows: (1) n-butanol–acetic acid– water for proteins (80:20:20), (2) benzene–methanol (95:5) for resins, (3) petroleum ether–diethyl ether–acetic acid (90:10:1) for waxes, and (4) acetonitrile–water (85:15) for sugars. The chromatographic process was performed in a standard manner: after the saturation of the chromatographic chamber with the Table 1 Solid and mobile phases used in TLC investigation of the historical samples.

Thin-layer chromatography of protein binders on historical samples 2.3 Methodology

Solid phase

Precoated cellulose plates, 10 × 10 cm

2.3.1 Microscopy

Mobile phase

n-Butanol–acetic acid–water (80:20:20)

For microscopical analysis, Olympus BX 51 was used, and the photographs of the microscopical samples were taken by Olympus DP 71 camera. This equipment was also used for the identification of natural fibers that are present in textile fragments (Figure 4).

Visualization

Ninhydrine

Solid phase

Silica gel plates 60 F254s, 10 × 10 cm

Mobile phase

Acetonitrile–water (85:15)

2.3.2 Spectrometry

Visualization

Iodine, UV-light

XRF analysis was performed by using XRF spectrometer Artax (Bruker, AXS). FTIR spectroscopy was performed by Shimadzu IR-Affinity-1 spectrometer with the DLATGS detector using KBr pellets.

Thin-layer chromatography of waxes in binders on historical samples

2.3.3 Chromatography

TLC was performed firstly on the chloroform extracts from sample fragments and investigated for the presence of sugars, waxes, oils, and resins. Stationary phases were silica gel plates 60 F254s, 10 × 10 cm (CAMAG, Muttenz, Switzerland). The second part of the sample was hydrolyzed in 6 m hydrochloric Journal of Planar Chromatography 28 (2015) 3

Thin-layer chromatography of sugars in binders on historical samples

Solid phase


Mobile phase

Petroleum ether–diethyl ether–acetic acid (90:10:1)

Visualization

Iodine, UV-light

Thin-layer chromatography of resins in binders on historical samples Solid phase


Mobile phase

Benzene–methanol (95:5)

Visualization

UV light on 254 nm, iodine

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mobile phase (after 15–30 min), the plates with spotted reference materials or samples were placed within the chromatographic chamber and were developed until reaching the length of 8 cm from the starting line. Unknown samples from the fragments of the mummies were analyzed by comparison with the reference samples of proteins and some characteristic amino acids (alanine, arginine, asparagines, asparagines acid, cysteine, glycine, glutamic acid, hydroxyproline, isoleucien, leucine, lysine, phenylalanine, praline, serine, threonine, tyrosine, egg, egg white, egg yolk, casein, animal skin glue, fish glue), resins (amber, benzoine, colophony, copal, dammer, dragon’s blood, elemi, gamboges, Manila copal, mastic, myrrh, shellac and sandarac, resin from marasca tree, gummy mastic, resin from plum tree, resin from cherry tree), sugars (rhamnose, mannose, ribose, fucose, arabinose, xylose, glucose, galactose, gum Arabic, gum tragacanth, cherry gum, dextrin, starch, and guar gum), and waxes (candelilla, carnauba, ceresine, earth wax, spermaceti wax, beeswax, paraffin). Table 1 summarizes solid and mobile phases used during the analysis.

3 Results and Discussion 3.1 Preliminary Investigation by XRF Spectrometry

The result of XRF analysis is presented in Figures 4A and B. As can be seen from this figure, the main chemical elements present in the fragments of the mummies were zinc (Zn), bromide (Br), iron (Fe), and potassium (K), while trace chemical elements were sulfur (S), copper (Cu), and calcium (Ca). All of the present elements are part of human skin and were to be expected. Larger quantities of bromide in the sample originate from the process of disinfection in which the mummies were disinfected by methyl bromide. 3.2 Preliminary Investigation by FTIR Spectroscopy

tion vibrations in the 1200 cm−1 region could be attributed to a high degree of oxidation processes that occur during aging [12]. This is a preliminary proof of the presence of resins (dammar, shellac, mastic, or copal) and proteins (originating from the skin and/or wool and silk fibers). Moreover, FTIR analysis also determined the presence of the inorganic compound ammonium magnesium phosphate hexahydrate (NH4) MgPO4 × 6H 2O, struvite [34, 35]. The results of the FTIR analysis were further compared with the results of chromatographic investigation. 3.3 Thin-Layer Chromatography

TLC is very suitable for the simultaneous separation and identification of different chemical compounds in historical samples [30–33]. In comparison to HPLC, it offers the advantage of avoiding problems by not demanding purification of samples from impurities prior to introducing samples to the instrumental columns. All plates after development were evaluated by comparing the RF values and colors of spots of unknown samples to the known reference materials. RF values determined for various components that can be present in natural binding media on historical samples are shown in Tables 2–5. Table 2 shows the results obtained after TLC investigation of standards of pure amino acids. Those can be a part of protein binding media such as caseine, animal glue, egg, and others. Table 3 presents the results of the sugar standards, Table 4 the results of wax standards, and Table 5 the results of analysis of standard resin materials [35, 36, 38]. Table 2 R F values of amino acids from protein binders used on historical materials obtained after development on precoated cellulose plates, developed by a mixture of n-butanol–acetic acid–water (80:20:20) as the mobile phase, and recorded after visualization by spraying of the plate with the ninhydrin solution [36*, 38].

Amino acid standard

RF value

Alanine

0.35

Arginine

0.19

Asparagines

0.13

Asparagine acid

0.19

Cysteine

0.05

Glycine

0.23

Glutamic acid

0.31*

Hydroxyproline

0.28*

Isoleucine

0.73

Leucine

0.75

Lysine

0.16

Phenylalanine

0.65

Praline

0.25

Serine

0.21

Figure 5

Threonine

0.30

FTIR spectrum obtained from the fragment left after cleaning of the body of St. Giovanni Olini.

Tyrosine

0.54*

FTIR spectrum is shown in Figure 5. The most prominent lines were 1030, 1235, 1380, 1550, 1655, 2925, and 3450 n, with the pronounced hydroxyl region. The C–O–O deforma-

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TLC, XRF Spectrometry and FTIR Spectroscopy in Analysis of Binding Media Table 3

Table 5

R F values of sugars present in binders on historical materials obtained after development on precoated cellulose plates, developed by a mixture of acetonitrile–water (85:15) as the mobile phase, and recorded after visualization by spraying of the plate with the iodine [36].

R F values of resins used in binders on historical materials obtained after development on silica gel plates 60 F254s, developed by a mixture of benzene–methanol (95:5) as the mobile phase, and recorded after visualization by spraying of the plate with the iodine solution [35, 36].

Sugar standard

RF value(s)

Resin standard

RF value(s)

Arabinose

0.41

Amber

0.0, 0.03, 0.20

Fucose

0.49

Colophony

0.0, 0.03, 0.14, 0.20

Glucose

0.30

Congo copal

0.0, 0.03, 0.14, 0.18, 0.35

Galactose

0.26

Dammar

Galacturonic acid

0.06

0.0, 0.03, 0.07, 0.12, 0.14, 0.18, 0.22, 0.31, 0.41, 0.55, 0.81

Mannose

0.34

Dragons Blood

0.0, 0.03, 0.07, 0.14, 0.18, 0.22, 0.31, 0.41, 0.54, 0.78

Rhamnose

0.63

Elemi

Ribose

0.53

0.0, 0.03, 0.05, 0.09, 0.12, 0.17, 0.25, 0.31, 0.34, 0.41, 0.66, 0.69

Xylose

0.50

Mastic

Cherry gum

0.025, 0.11, 0.30, 0.43, 0.47

0.0, 0.05, 0.10, 0.12, 0.16, 0.18, 0.24, 0.26, 0.33, 0.42

Dextrin

0.019, 0.12, 0.16, 0.33

Myrrh

0.0

Gum arabic

0.025, 0.11, 0.29, 0.43, 0.59

Shellac

0.0, 0.04, 0.10, 0.18, 0.26

Gum tragacanth

0.02, 0.30, 0.43, 0.46

Sandarac

0.0

Starch

0,02, 0.16, 0.33

Sample A

Caseine Animal glue

Sample B

Table 4 R F values of waxes used in binders on historical materials obtained after development on silica gel plates 60 F254s, developed by a mixture of petroleum ether–diethyl ether–acetic acid (90:10:1) as the mobile phase, and recorded after visualization by spraying of the plate with the iodine solution [35, 36].

Wax standard

RF value(s)

Candelilla

0.0, 0.11, 0.17, 0.43, 0.70, 0.90, 1.0

Carnauba

0.0, 0.07, 0.10, 0.14, 0.86

Ceresine

0.98, 1.0

Earth wax

0.0, 0.13

Spermaceti wax

0.84

Beeswax

0.0, 0.08, 0.13, 0.31, 0.42, 0.50, 0.72, 0.86, 1.0

Paraffin

1

Based on the comparison of the results of analysis of standard materials and unknown samples, a conclusion on the chemical composition can be drawn. Therefore, other plates were developed for the comparison of preselected components and unknown samples. Final results of those TLC investigations, obtained by comparing the chosen samples to the mostly corresponding reference materials, are presented in Figures 6–8. Figure 6 presents the comparison of the hydrolyzed protein solutions of the unknown samples with the hydrolyzed protein reference materials of casein and animal glue, in which the samples correspond to the animal glue. Figure 7 shows the comparison of the organic extract solutions of the unknown hisJournal of Planar Chromatography 28 (2015) 3

Figure 6 Result of the TLC investigation of proteins in unknown historical sample (solutions of proteins were collected after 24 h of the acidic hydrolysis), performed on precoated cellulose plates, developed by mixture of n-butanol–acetic acid–water (80:20:20) as the mobile phase, and recorded after visualization by spraying of the plate with ninhydrin solution.

torical sample and organic extracted sugar reference materials (in which the sample of St. Olini corresponds to gum Arabic, and the sample of St. Bursa corresponds to starch). Figure 8

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Sample Sample Gum Cherry Starch A B arabic gum

Figure 7 Result of the TLC investigation on sugars in unknown historical sample, performed on silica gel plates 60 F254s, developed by a mixture of petroleum ether–diethyl ether–acetic acid (90:10:1) as the mobile phase, and recorded after visualization by spraying of the plate with the iodine solution. Sample A is the extract of St. Olini sample from 1200 AD, while sample B is the extract from St. Bursa from 1500 AD.

Sample A and B Copal Colophony Matiks Schellac Oil

Figure 8 Result of the TLC investigation on resins in unknown historical samples, performed on silica gel plates 60 F254s, developed by a mixture of benzene–methanol (95:5) as the mobile phase, and recorded after visualization by spraying of the plate with the iodine solution. Sample A is the extract of St. Olini sample from 1200 AD, while sample B is the extract from St. Bursa from 1500 AD.

shows the comparisons of the results of the extracted resin standards solutions to the extracted solution of the unknown sample. In resin investigations, the samples also differed: while the St. Giovanni Olini (1200 AD) sample contained shellac resin and oil component, the sample of St. Nicolosa Bursa (1500 AD)

210

contained copal and oil. No wax component was determined on any of the investigated samples. As it can be clearly seen from the obtained results, the mummies were treated with resins, waxes, and oils (TLC investigation) and wrapped with textiles made of flax (St. Giovanni Olini 1200 AD, sample A) and silk (St. Nicolosa Bursa 1500 AD, sample B). It is hard to conclude if this treatment occurred immediately after the death of St. Giovanni Olini and St. Nicolosa Bursa, being the cause of perfect preservation of the mummies, or this treatment was applied several decades or hundreds of years afterwards, in a period between 1600 and 1800 in Croatia, during their preservation and conservation in the collection of relics. Nevertheless, due to the similarities in the obtained chemical results, the second explanation could be more probable. Therefore, we can conclude that both mummies were treated with organic and inorganic materials during their conservation treatments many years after the persons’ death and after bringing the mummies to Croatia from Italy. The combination of the proposed chemical methods (XRF spectrometry, FTIR spectroscopy, and microscopy) to TLC proved to be very suitable for the analysis of human mummies, since none of those methods required large sample amounts for the analysis. Nevertheless, they were able to provide very useful data concerning the materials and chemical compounds present on the samples taken from the historical objects. Therefore, they are a very efficient tool for interdisciplinary study which combines forensic investigation, conservation of historical materials, and instrumental chemical analysis. The application of non-destructive methods (portable PIXE-alpha and portable X-ray diffraction [XRD] techniques [37]) allowed performing a systematic investigation in situ, while their combination to different spectroscopic and chromatographic techniques enabled understanding technological conditions implemented for the mummification of the Corpi Santi. This work is not the first attempt to use TLC methodology in the analysis of historical objects [39], but it presents a possibility to apply TLC for the analysis of different binding media in the same sample (proteins, waxes, sugars, and resins) on historical materials. Moreover, historical textiles are not the only target group of TLC analysis, since the analysis of modern textiles can benefit from TLC methodology to a large extent [40, 41]. Although TLC cannot offer such low limits of detection for particular analytes, which are reachable by sophisticated analytical procedures [42–47], the advantage of TLC is the fact that it is approachable under low cost, and real samples can be analyzed without a need of paying special interest to column cleaning and regeneration [5, 48, 49]. The importance of TLC in future investigation of historical materials and mummies is hard to predict. Although some researchers predict that TLC is going to be replaced by modern methods (GC, pyrolysis–GC, HPLC, and capillary electrophoresis) in the analysis of historical materials and mummies [50], in our opinion, the laboratories that are working with low costs will always be in need of TLC methodology. However, it is true that complex proteomics and hyphenated techniques, such as nano-liquid chromatography–nano-electrospray ionization/collision quadrupole time-of-flight tandem MS, which have been recently applied to the identification and determination of proteinaceous binders, may provide significant information on the samples analyzed [50]. Nevertheless, the development of new TLC systems (including new visualization reagents, new staJournal of Planar Chromatography 28 (2015) 3


tionary and mobile phases) may emphasize the effectiveness of TLC in the analysis of different samples [51]. Therefore, it can be expected that TLC will stay as an unavoidable methodology for monitoring different analytes present in various samples, in chemical analysis or chemical and biochemical processing [52–54]. Many relevant studies use TLC in the analysis of archeological samples and mummies. For example, in a recent study, Giuffra et al. emphasized that the knowledge of the embalming methods used on mummies in the Renaissance Italy (and northern Croatia) comes not only from the literary texts of that period, but also from the artificial mummies which have been found [55]. Their study was carried out with a multidisciplinary perspective which allowed them to reconstruct the embalming techniques including the substances used for mummification by applying TLC. In addition, the lipid content of a Ptolemaic Egyptian mummy was easily monitored using TLC by Barraco et al. who determined that thin-layer chromatograms of the lipid material stained positive for cholesterol and sterol esters but negative for phospholipids and ganglioside sialic acid, which helped them in the characterization of the mummies [56]. Therefore, it is to be expected that TLC will stay as a valuable analytical tool for laboratories dealing with historical samples. However, some modifications of the current procedures (like enhancing the quantification methodology [57], coupling TLC with sophisticated methods for the analysis of binding media used during the mummification processes [58], or coupling the existing techniques with some chemometrical tools [59, 60]) might further enhance the usefulness of TLC in the analysis of archeological objects.

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4 Conclusion The TLC analysis of the fragments sampled on the mummies’ bodies from 1200 AD and 1500 AD revealed the presence of proteins (animal glue), sugars (starch), and resins (copal and shellac), which was confirmed by FTIR spectroscopy. In addition, the results of inorganic analysis by XRF have shown that the main chemical elements present in fragments of the mummies were Zn, Br, Fe, and K, while trace chemical elements were S, Cu, and Ca. Since the two bodies dated from different historical periods, the similarities in the composition of the coatings could indicate that the coatings were probably not applied as embalming and mummifying agents but were applied much later. In addition, according to their compositions, the coatings most likely did not have any considerable impact on the preservation of the bodies. Therefore, we conclude that the combination of the used microscopy and spectrometric methods with TLC is an excellent tool for the investigation of mummies and other valuable historical items and can be recommended for laboratories connected to restaurateurs and conservators. References [1] N. Lynnerup, Mummies, Ybk. Phys. Anthropol. 50 (2007) 162– 190. [2] A.C. Aufderheide, The Scientific Study of Mummies, Cambridge University Press, Cambridge, 2003. [3] M. Klys, T. Lech, J. Zieba-Palus, J. Bialka, Forensic Sci. Int. 99 (1999) 217–228.


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