Jan S. Jaworski
University of Warsaw, Poland

The first step in the chemical analysis was to look for possible paints, dyes and pigments that could form the image on the Shroud. It was assumed that the blood ‘traces’ and the image of the body were created in the same way and had the same chemical composition. Already the first research by an American team from the Shroud of Turin Research Project (→STURP) in 1978 showed the falsity of this assumption. Electron spectra (Gilbert and Gilbert 1980) and X-ray analysis (Morris, London and Mottern 1981) showed that blood traces have different physical and chemical characteristics to the body image and therefore different chemical compounds should be sought for them. This was addressed by John H. Heller and Alan D. Adler (see →Blood on the Shroud). In the area occupied by the body image, the presence of metals making up coloured compounds was looked for first and foremost, and then also the presence of such compounds themselves, both inorganic and organic, and compared with the results obtained for samples of pure linen, i.e. pieces of linen on which there is no image. The results of the study will be presented in this order.

The presence of various elements (with an atomic number above 16) in selected areas of the Shroud of Turin’s linen was investigated directly in Turin using X-ray fluorescence by the STURP team (Morris, London and Mottern 1981). Only iron, calcium and strontium were shown to be present. When the position of the sample on the cloth was changed, their contents were not mutually proportional, from which it followed that the sources of these elements must have been, at least in part, different. The apparatus used did not allow the determination of silver, cadmium and tin, and the results for lead were uncertain; it was only found to be less than 15 µg cm^-2. Calcium and strontium were evenly distributed on the surface of the linen: the former averaged 200 ± 50 µg cm^-2, and the latter 2,2 ± 1,0 µg cm^-2. This was confirmed by Heller and Adler 1981; Schwalbe and Rogers 1982; Jumper et al. 1984, members of the STURP team. Roger A. Morris and co-workers suggested that both of these elements may—but only may—originate from the calcareous rock dust present in the air. The total amount of calcium on the Shroud corresponded to about 1% by weight. If this element were distributed throughout the entire volume of the cloth and not just on its surface, the amount would double. A similar content of calcium, strontium and iron using the same method was later confirmed in the USA for three strands from the Shroud taken in 1973. Traces of potassium, chlorine and possibly lead were also detected in them (Schwalbe and Rogers 1982).

The results published by R.A. Morris, J. Ronald London and Robert W. Mottern indicate that the lowest contents (116 µg Ca/cm² and 0.6 µg Sr/cm²) were obtained for measurements from the ‘clean’ cloth area, at a location approximately 4 cm from the blood trail on the forehead hair. The researchers considered this very spot to be characteristic of the area of the original linen, without the image. For the measurement taken 8 cm from the trace of blood flowing from the rear image of the punctured foot and lying twice as far away from the image of this foot, and thus in fact also for the “pure” linen, the calcium and strontium content was higher (191 µg Ca/cm² and 2.9 µg Sr/cm²). One would have to assume that the calcium dust content is higher either in the foot area or in the entire underside of the Shroud (this interpretation was confirmed by Joseph A. Kohlbeck’s later microscopic analysis of the adhesive tape crystals). The same results were interpreted differently by M. Sue Benford and Joseph G. Marino interpreted them differently. They assumed that they indicate an uneven distribution of calcium on the cloth: larger amounts are observed in body image areas than outside the image (Benford and Marino 2008). The scientists hypothesised an additional role for calcium carbonate in the formation of the image according to the Maillard reaction mechanism (→Hypotheses of the Origin of the Image on the Shroud).

For the qualitative determination of heavy metal ions on the linen fibres (taken from the Shroud by Raymond Rogers), J.H. Heller and A.D. Adler used droplet microanalysis, based on colour reactions with added reagents. They found only the presence of iron and calcium (the presence of strontium was not investigated) in both samples from the area with and without the image and in the samples from the water stains; there were no metals such as copper, manganese, nickel, cobalt, aluminium, zinc, lead, arsenic, antimony, tin or chromium. The iron and calcium content of the sample from the centre of the water stain was slightly lower (traces of the water stain were not examined by X-ray fluorescence).

John Heller and Alan D. Adler did not observe calcium carbonate crystals on the flax fibres from most samples when they examined them under the microscope. They explained the presence of calcium strongly bound to the cellulose of the flax fibres (as well as most of the iron) by the incorporation of these elements into the cellulose structure during retting (i.e. fermentation), which occurs during flax production and leads to the separation of the flax fibres from the woody parts. Calcium may also have been bound during the growth of the plant.

However, the aforementioned J.A. Kohlbeck, an American specialist from the optical microscopy and crystallography laboratory, recognised distinct crystals of limestone rock on samples taken by R. Rogers in 1978, especially from the hindfoot area. He identified them as aragonite, or more precisely travertine (which is a variety of calcareous necrotic rock, a sedimentary rock composed mainly of calcite and aragonite). As it is usually formed in geological processes in the presence of barium, strontium, iron or lead, it is not surprising to detect strontium and iron on the Shroud by X-ray fluorescence. Research conducted in Jerusalem between 1985 and 1986 under the direction of American archaeologist Eugenia J. Nitowski showed that the rocks of the tomb complex on the site of Jerusalem’s École Biblique, located near the Damascus Gate, had a similar composition. In contrast, they differed from limestones from eight other sites in Israel. This conclusion was supported by secondary ion mass spectrometry performed by Riccardo Levi-Setti at the University of Chicago. Similar secondary ions, both positive and negative, were observed in both samples—from the Shroud and from the tomb—(e.g. positive Ca, CaO and CaOH ions, as well as potassium, sodium, magnesium and aluminium). Some of the ions, e.g. potassium, were attributed to human residues, but it was not ruled out that there may have been a failure to remove flax cellulose residues from the samples. The results suggest the presence of the Shroud in Jerusalem and link it to tombs carved into limestone rocks (Kohlbeck and Nitowski 1986; Sister Damian of the Cross [E.L. Nitowski] 1986).

Iron—the third metal detected on the cloth of the Shroud by X-ray fluorescence—was present in varying amounts in different areas of the cloth. The lowest surface concentration was detected for the ‘clean’ area of the linen (6.8 µg Fe/cm²), at a location approximately 4 cm away from the trace of blood on the hair of the front of the head, the same location that showed the lowest amount of calcium and strontium. The amount of iron at other sites away from the blood trace increased, reaching several micrograms per 1 cm², and was significantly higher for the blood trace: 16.5 µg Fe/cm² for a small drop of blood in the hair on the side of the face, 50.0 µg Fe/cm² for a blood stain leaking from the punctured side and 58 µg Fe/cm² for the end of the leak from the punctured foot. The iron content in subsequent measurements was lower the more the distance from the aforementioned bloodstain on the foot increased, dropping to a value of 24.7 µg Fe/cm² at a distance of 8 cm (Morris, London and Mottern 1981). The researchers found a marked increase in iron content in the areas of the blood traces, about 20 µg Fe/cm² for the blood spill from the foot and about 30–40 µg Fe/cm² for the blood from the wound in the side, lending credence to the hypothesis of the authenticity of the blood on the Shroud. No potassium was detected, but in the control blood samples its peak was an order of magnitude smaller than for iron. No difference in spectral signals was found for the areas covered by the body image and those without, although it was estimated that the sensitivity of the measurements may not have been sufficient to reject the hypothesis that the coloured image is formed by Fe₂O₃ iron oxide (with the eye it is possible to recognise colour already at a concentration of about 2 µg Fe/cm²).

Using microscopic analysis and chemical tests, John Heller and Alan D. Adler were able to determine more precisely in which form iron is present in various samples. They distinguished between three forms. In all samples on the flax fibres (even if no iron oxide particles were visible on them under the microscope), at least 90 per cent of the iron was dominated by iron bound to the cellulose, probably forming covalent chelates with the aldehyde and carboxyl groups of the partially oxidised cellulose structure. The scientists explained that this iron comes from the retting of flax fibres, during which the flax structure acts as an ion-exchange resin, selectively binding iron and calcium. Similarly, iron and calcium were detected on control Spanish linen from 300 years ago, as well as flax funerary linen: Coptic from c. 350 BC and Pharaonic from c. 1500 BC. Iron bound in this way to cellulose could be detected without the sample having to be dissolved in acids.

The second form of iron, found only in and near bloodstains, is iron ions strongly bound to haem, forming orange or brown globules insoluble in hydrochloric acid but soluble in royal water or enzyme mixture. These clumps, formed by blood residues, are not birefringent, have a refractive index of less than 1.5 and give reactions characteristic of heme.

The third form of iron is smaller red particles 0.7–1 µm in diameter, soluble cold in concentrated hydrochloric acid, having a refractive index greater than 1.5 and exhibiting birefringence of crystals. They were identified as iron(III) oxide, Fe₂O₃. No manganese, cobalt, nickel or aluminium above 1 per cent were detected in them, as in common iron minerals. The researchers suggested that this iron, too, was originally derived from the retting of flax fibres. In particular, the high accumulation of iron oxide particles on the fibres from the margins of the water stains on the linen of the Shroud suggested to the researchers a resemblance to the production of ‘khaki’, during which the fibres are treated with an iron salt solution, followed by precipitation of the hydroxide in an alkaline environment and dehydration. The observed iron oxide particles could have been formed in a similar way. This supposition was confirmed by the presence of red particles in the core of some flax fibres (Heller and Adler 1981; Jumper et al. 1984). The possibility that the iron oxide was formed during the 1532 fire in Chambéry was also taken into account. Most Shroud researchers considered a hydrological or biological origin of the red iron oxide particles, ruling out a mineral origin, characteristic of inorganic pigments. In recent years, iron oxide particles on Shroud fibres from bloodstain areas have been identified and distinguished from blood residues, which was enabled by much more sensitive analytical methods: energy dispersive X-ray spectroscopy and electron microscopy with a backscattered electron detector (Fanti and Zagotto 2017). Traces of other iron-containing minerals, such as biotite, were also detected on the Shroud sample (Lucotte et al. 2016).

As for other elements, J.H. Heller and A.D. Adler found the sporadic presence of silver (perhaps with trace admixtures of mercury, lead or cadmium), but only in black particles from scorched areas, when dissolved in royal water. They explained the presence of silver by the residue of silver reliquary fragments melted during the 1532 fire at Chambéry.

On a sample from the area of blood flowing from the pierced side, J.H. Heller and A.D. Adler found under the microscope the presence of a bright red cinnabar crystal (mercuric sulphide, HgS), which had previously been described by Walter McCrone. The trace presence of HgS was also confirmed by more recent methods, but only in samples from bloodstain areas, in the 2015–2016 study by G. Fanti and G. Zagotto and G. Lucotte and team (see →Blood on the Shroud).

The presence of other coloured inorganic compounds could not be detected. J.H. Heller and A.D. Adler ruled out the presence of arsenic sulphides (AsS and As₂S₃) and lead oxide (PbO), as the colour of the flax fibres did not dissolve in alkali solutions. Chemical tests confirmed that the body image on the Shroud was not created with inorganic pigments.

The next step in the chemical microanalysis undertaken by J.H. Heller and A.D. Adler was tests to detect specific structures of organic compounds and specific functional groups, which were sought on fibres from areas with the image, areas without the image and burns. Negative results indicated the absence of phenols, riboflavin, steroids, indoles, allylic compounds, starch, porphyrins, pyrroles, creatinine, uric acid and urea derivatives, primary amines and nitro compounds. The presence of soapberry (Saponaria officinalis) extract was also not confirmed. Only aldehyde groups (using Schiff’s reagent) and cellulose carboxyl groups were detected, which induced a colour change in methylene blue and toluidine blue O.

Although all the fibres became stained under the influence of the added indicators, an increasingly strong colouration was observed in the following order: ‘pure’ fibres, i.e. without image (normally faint yellow), fibres forming an image with a yellow colour and fibres from slightly burnt areas, with a darker colour. Thus, in this order, the number of carboxyl groups increased and, in the same order, increasingly strong destruction of the fibre surface was observed under the phase-contrast microscope. In turn, R. Rogers observed that the adhesive tape from the area with the image could be more easily detached from the cloth of the Shroud, and that the fibres on it were shorter and more broken than those from the area without the image. John Heller and Alan D. Adler also carried out attempts to extract the yellow staining forming the image on the surface of the linen fibres. This could not be done (or even weaken the colour) using water, many inorganic bases and acids and 14 organic solvents. It was only possible to bleach the yellow colour of the fibres by using very strong reducing agents (hydrazine bleached the colour slowly, and diimide/diazene—HN=NH, formed from hydrazine and hydrogen peroxide in boiling pyridine—immediately) and a strong oxidant: hydrogen peroxide in an alkaline medium. In contrast, the control Spanish linen fibres could be dyed a yellow colour, similar to the fibres forming the image, by acting on them for half an hour with concentrated sulphuric acid.

These observations suggested that the aldehyde and carboxyl groups detected were formed by oxidation of the cellulose of the linen itself in a similar way to that for known linen ageing processes (Heller and Adler 1981; Schwalbe and Rogers 1982; Jumper et al. 1984). Summarising the chemical studies of body image areas, STURP scientists found that oxidation of cellulose hydroxyl groups and dehydration led to chromophores with conjugated double bonds (>C=O and >C=C<) and different chain lengths. In other words, there was a more advanced decomposition of cellulose in areas of the body image, characteristic of natural ageing, resulting in the formation of conjugated carbonyl systems and a consequent shift in light absorption from the ultraviolet to the visible part. Researchers have proposed more than a dozen general chemical reactions (oxidation, condensation, dehydration) that could take place in cellulose fragments to form such chromophores, but have not discussed them in more detail or supported them with any experiments (Jumper et al. 1984).

The presence of only cellulose and hemicelluloses (and the absence of other compounds) in the image area samples taken from the Shroud in 1978 was indirectly confirmed a few years later at the University of Nebraska at Lincoln. After pyrolysis of the aforementioned samples, signals of glucose, furfural and hydroxymethylfurfural (HMF), i.e. products of cellulose decomposition (occurring via glucose to HMF) and hemicellulose decomposition (occurring via glucose and galactose to HMF or via xylose and arabinose to furfural) were detected by mass spectrometry (Rogers 2005). Chemical tests, carried out after radiocarbon dating of the Shroud and designed to demonstrate the different chemical composition of the samples taken for this purpose from the rest of the linen, confirmed that samples of adhesive tapes from areas outside the body image taken in 1978 do not contain significant amounts of heavier elements. Alan Adler, Russell Selzer and Frank DeBlase, using electron microscopy, confirmed by energy dispersive X-ray spectroscopy (EDS) that the fibres from this area contained 93% by weight carbon and 3.2% oxygen of the cellulose material, 1.7% sodium and 1.5% chlorine and only 0.4% each of calcium and iron and 0.1% each of aluminium, potassium and copper. In the area of water stains, the amount of carbon and oxygen decreased while the amount of iron increased up to 5.5%, consistent with J.H. Heller and A.D. Adler’s hypothesis of iron binding by cellulose. In contrast, matrix strands from a sample taken for radiocarbon dating varied considerably in quantitative elemental content (→radiocarbon dating). On the other hand, FT-IR spectra in the vibrational range of the carboxyl group (1650–1540 cm^-1) and conjugated ketones (1680–1640 cm^-1) showed a gradual increase in the intensity of the bands indicative of increasingly strong oxidation of cellulose in the sequence from areas without image (weak peaks 1593 and 1643 cm^-1), through areas with water traces, then image areas (strong peak 1694 and shoulder 1645 cm^-1) and linen burns, and finally fibres from areas sampled for radiocarbon studies (Adler, Selzer and DeBlase 2002). The gradual oxidation of cellulose was thus fully confirmed.

More than 30 years after the STURP team’s research on the production and ageing of paper, which, like flax, consists mainly of cellulose, it was still claimed that the yellowing of cellulose was the result of the formation of a mainly dicarbonyl system, made up of two >C=O carbonyl groups lying at adjacent carbon atoms in the glycosidic ring of cellulose. In contrast, single aldehyde and carbonyl groups weakly affect the absorption of light in the visible range. The carboxyl groups themselves do not absorb light in this range, but accelerate the colour changes induced by the carbonyl groups (Mosca Conte et al. 2012; Ahn et al. 2019). Such dicarbonyl chromophores are still considered to be responsible for the image of the body on the Shroud (Di Lazzaro et al. 2012), even though no direct chemical test has actually detected the presence of a carbonyl group on the Shroud’s linen, as the Schiff reagent used only allows confirmation of the existence of an aldehyde group. The answer to the question of how the chromophores forming the image of the body on the Shroud are structured seems to require further experimental research. Recently, a hypothesis has been put forward that it might be quinoid systems, formed in cellulosic materials in much lower concentrations than conjugated carbonyl systems, but much more strongly absorbing light (Jaworski 2020).

The researchers from the STURP team did not answer the question of whether the ageing of the cellulose, which caused the yellow image, was rapid (as in artificially conducted high-temperature processes) or a prolonged process, or what its mechanism was. However, they ruled out the effect of temperatures above 200^oC, as other decomposition products are then produced, and the linen shows an orange-red fluorescence, which was observed on the Shroud only for areas slightly burnt during the Chambéry fire.

The chemical studies carried out after the radiocarbon dating of the Shroud raised the issue of the presence of starch on the surface of the linen. The chemist R.N. Rogers, in his work, put forward a competing →hypothesis of the origin of the image, according to which the image of the body formed only on a thin coating of starch (perhaps with traces of other polysaccharides) covering the linen fibres throughout the linen of the Shroud. Loose fragments detached from this coating, which have been called ‘ghosts’, are in places clearly visible under the microscope. As suggested by specialists at the Egyptian Museum in Turin, Rogers believed that during the fabric production the warp yarn was protected by starch, giving it stiffness. Indeed, the starch layer is difficult to remove later from the fibres by rinsing in water and soapstone solution. On the other hand, starch, a biopolymer composed of amylose (with a structure similar to cellulose) and amylopectin, is less thermally stable than cellulose and is more easily dyed in various reactions. In the book, already published after the death of R.N. Rogers, there is information about the detection by microchemical tests of an admixture of starch on the surface of fibers from the Shroud (Rogers 2008). In it, the researcher referred to work of W. McCrone, presented at a meeting of scientists from the STURP project in 1979, adding without any details that they had confirmed it with an iodine test (when iodine is added, a dark blue starch colour is produced). In a joint article by R. Rogers and Anna Arnoldi, on the other hand, reported that, using a test to check for the presence of sulfoproteins in areas of blood traces (i.e. adding sodium azide with iodine), they obtained a reddish background colour, characteristic of amylose (they may have meant amylopectin) from starch. However, they did not give the location on the Shroud of the samples from which the fibres tested came (Rogers and Arnoldi 2003). Such a test is described in detail by R.N. Rogers in his book (Rogers 2008, p. 73), but it only concerns the formation of a red colour under the influence of iodine for the cotton fibre from G. Raes’ sample, indicating the presence of a starch fraction insoluble in cold water. In his book, he also regrets that in his later work only samples from the Shroud were available to which W. McCrone had glued microscope slides, so it was difficult to study the surface of flax fibres with the original covering destroyed. Thus, it is possible to consider that R. N. Rogers detected traces of starch in the sample of G. Raes, and perhaps also in the samples of blood traces on which he found sulfoproteins, but the generalisation of this conclusion to the area of the entire Shroud is considered by many researchers to be unjustified (Fanti et al. 2010). Indeed, let us recall that J.H. Heller and A.D. Adler, using the iodine test, did not detect starch on many samples, including those from the area of the image on the Shroud. Other researchers considered the ‘ghosts’ to be yellow fragments of the outer covering of the flax fibres, which are probably the primary cell wall. The hypothesis of R.N. Rogers does not explain the uniformity of the starch layer around the entire periphery of the fibre, including at the points of contact with neighbouring fibres (Fanti et. al. 2010). It requires further verification, in particular the detection of starch on a larger number of samples taken from different locations on the Shroud. It should be added that spectroscopic studies (including infrared and X-rays) of three strands from the corner of the Shroud (samples taken by G. Raes in 1973 and 1988 for radioactive carbon dating), during which the presence of various elements and organic fragments was determined, led to the conclusion that the composition of these strands differs markedly from that of the main part of the Shroud (Villarreal et al. 2009) (cf. →Age of the Shroud and →Physical Analyses of the Shroud). Scientists have also attempted to link the yellowing processes of the linen fibres caused by their ageing quantitatively to the depolymerisation of cellulose, the degree of which can be determined by X-ray measurements (De Caro et al. 2020), but this could not be used to study the Shroud.

Separate studies were carried out on particles taken with a mini vacuum cleaner by Giovanni Riggi di Numan in 1978 and 1988 from the space between the Shroud and the reinforcing Dutch cloth, which were deposited on special filters. Optical microscope and scanning electron microscopy images were used to classify the particles, ranging in size from 3 to 30 µm, from the five filters, and for some particles the elemental content was examined by EDS X-ray spectroscopy (Calliari and Canovaro 2015). The presence of a large amount of calcium was confirmed on the flax fibres. EDS analysis of the composition of the reddish mineral particles indicated the presence of calcium, oxygen, silicon and aluminium, as well as in smaller amounts magnesium and iron, which gave them their colour. Amir Sandler of the Geological Survey of Israel compared the composition of these particles with minerals from soil in Jerusalem, particularly from Mount Zion, finding a high agreement of EDS spectra (Fanti and Malfi 2020). Interesting results were also obtained by Giulio Fanti and Claudio Furlan, analysing by EDS spectroscopy the chemical composition of 17 gold dust crumbs ranging in size from 1 to 10 µm and comparing them with the composition of a medal and 32 Byzantine coins from the reigns of Emperors Justinian II and Alexis III (c. 692–1197). Five crumbs from the Shroud and four coins contained pure 24-carat gold, while nine crumbs from the Shroud were an alloy of gold with silver and a small admixture of copper with a composition like that of the Byzantine alloy called electrum, from which the 19 coins analysed were struck (with six crumbs having a gold content between 70–90% and one 32%, which corresponded exactly to the composition of some of the coins analysed). These results are consistent with the hypothesis that the Shroud as Mandylion of →Edessa was at the imperial court in Constantinople between 944 and 1204, where it came into contact with gold jewellery (Fanti and Furlan 2020).

Summarising the chemical study of the Shroud, it must be said that the results obtained so far exclude the formation of the body image by added elements or coloured compounds (both inorganic and organic) and indicate only a surface modification of the cellulose fibres of the linen fabric itself, causing yellowing analogous to the natural ageing processes of cellulose. It was not possible to explain by what process an image with such properties could have been produced. The natural presence of calcium admixed with strontium and iron throughout the linen was convincingly explained. Chemical studies of the bloodstains on the Shroud have confirmed its authenticity, while the analysis of the crumbs of dirt from the spaces between the clothes is consistent with the hypothesis that the Shroud was located in Jerusalem and Byzantium.

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