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Analytical and Bioanalytical Chemistry (v.395, #7)

Victor A. Gault, Neville H. McClenaghan: Understanding bioanalytical chemistry. Principles and applications by Maria Montes-Bayón (pp. 1941-1942).

Nobuhiro Fusetani and William Kem (Eds.): Marine toxins as research tools by Luis M. Botana (pp. 1943-1944).

Robert de Levie: Advanced Excel for scientific data analysis, 2nd ed. by Simon Prikler (pp. 1945-1946).

Non-destructive and microanalytical techniques in art and cultural heritage (Technart 2009) by Demetrios Anglos; Andreas G. Karydas (pp. 1947-1948).

has been a Principal Researcher at the Institute of Electronic Structure and Laser–Foundation for Research and Technology–Hellas (IESL–FORTH), and since September 2009 an Associate Professor of Physical Chemistry at the Department of Chemistry, University of Crete, Greece. His research activities include the study of laser-induced photochemical and photophysical processes in the condensed phase, and the applications of laser spectroscopic techniques (e.g. LIF, LIBS, Raman spectroscopy) in the study and analysis of materials, with emphasis on the development of novel methodology and instrumentation for characterization of works of art and archaeological objects. is a Principal Researcher at the Institute of Nuclear Physics of the National Centre for Scientific Research “Demokritos” in Athens, Greece, and is currently serving as the leader of the XRF group at the International Atomic Energy Agency (IAEA), PCI Laboratories, Vienna, Austria. His main research activities include the study of first and second-order interactions of X-rays with matter, the development of ion beam and X-ray spectrometry techniques (e.g. confocal X-ray microscopy analysis, 3D Micro-XRF, 3D Micro-PIXE, and integrated PIXE–XRF) and their application in interdisciplinary fields. In the field of archaeology, art, and conservation science his group has contributed to advances in novel portable XRF instrumentation and analytical methodologies appropriate for on site, non-invasive studies of cultural heritage objects.

Non-destructive and microanalytical techniques in art and cultural heritage (Technart 2009) by Demetrios Anglos; Andreas G. Karydas (pp. 1947-1948).

The study of bronze statuettes with the help of neutron-imaging techniques by R. Van Langh; E. Lehmann; S. Hartmann; A. Kaestner; F. Scholten (pp. 1949-1959).

Until recently fabrication techniques of Renaissance bronzes have been studied only with the naked eye, microscopically, videoscopically and with X-radiography. These techniques provide information on production techniques, yet much important detail remains unclear. As part of an interdisciplinary study of Renaissance bronzes undertaken by the Rijksmuseum Amsterdam, neutron-imaging techniques have been applied with the aim of obtaining a better understanding of bronze workmanship during the Renaissance period. Therefore, an explanation of the fabrication techniques is given to better understand the data collected by these neutron-imaging techniques. The data was used for tomography studies, which reveal hidden aspects that could not at all or scarcely be seen using X-radiography. For this specific study, the representative bronze ‘Hercules Pomarius’ of Willem van Tetrode (ca 1520–1588) has been examined, along with 20 other Renaissance bronzes from the Rijksmuseum collection.

Keywords: Archaeometry; Neutron tomography; Radiography; Non-destructive testing; Fabrication process; Renaissance bronze

The study of bronze statuettes with the help of neutron-imaging techniques by R. Van Langh; E. Lehmann; S. Hartmann; A. Kaestner; F. Scholten (pp. 1949-1959).

Keywords: Archaeometry; Neutron tomography; Radiography; Non-destructive testing; Fabrication process; Renaissance bronze

Neutron diffraction characterization of Japanese artworks of Tokugawa age by F. Grazzi; L. Bartoli; F. Civita; M. Zoppi (pp. 1961-1968).

Neutron time-of-flight diffraction technique has been used to characterize some Japanese historical artifacts. With this method, metal samples can be analyzed in their bulk properties without need of sampling. Results shown here were obtained at the Italian Neutron Experimental Station (INES@ISIS) located at the pulsed neutron source ISIS (UK). The parallel use of a scanning electron microscope equipped with an energy-dispersive X-ray fluorescence device (SEM-EDX) permitted a full quantitative characterization of the investigated samples, namely four hand-guards (Tsubas) of Japanese swords attributed to the Tokugawa age. In particular, we were able to obtain, in a totally non-invasive non-destructive way, a full quantitative phase characterization of the samples, a detailed Bragg peak broadening analysis, and a quantitative texture determination. These results, complemented with those obtained via the traditional analysis method of SEM-EDX, allowed a full characterization of both the bulk and the surface of the artifacts.

Keywords: Neutron diffraction; Phase analysis; Texture analysis; Non-destructive methods; Metal characterization; Japanese artworks

Neutron diffraction characterization of Japanese artworks of Tokugawa age by F. Grazzi; L. Bartoli; F. Civita; M. Zoppi (pp. 1961-1968).

Keywords: Neutron diffraction; Phase analysis; Texture analysis; Non-destructive methods; Metal characterization; Japanese artworks

Study of metallic components of historical organ pipes using synchrotron radiation X-ray microfluorescence imaging and grazing incidence X-ray diffraction by L. K. Herrera; A. Justo; A. Muñoz-Páez; J. A. Sans; G. Martínez-Criado (pp. 1969-1975).

A comparative study of the composition and microstructure of two different brass alloys from reed pipes, one from a Spanish baroque organ and the other from a modern one, was carried out. This study allowed us to determine the procedure followed to produce the brass used to make ancient reed pipes. Moreover the distribution and correlation of lead and other trace elements present into the main component of the brass, the copper and zinc phases, of the historical tongues and shallots were established. This chemical composition was compared with that of a tongue from a twentieth-century organ. The whole study was accomplished using a combination of laboratory and synchrotron radiation techniques. X-ray fluorescence was the technique used to obtain elemental and chemical imaging of the main phases and the trace elements at a sub-micrometer scale.

Keywords: Reed pipes; Brass alloys; Grazing incidence X-ray diffraction (GIXRD); μ-Raman spectroscopy (RS); µX-ray fluorescence (µXRF); Synchrotron radiation

Study on the impregnation of archaeological waterlogged wood with consolidation treatments using synchrotron radiation microtomography by S. Bugani; F. Modugno; J. J. Łucejko; G. Giachi; S. Cagno; P. Cloetens; K. Janssens; L. Morselli (pp. 1977-1985).

In favourable conditions of low temperature and low oxygen concentration, archaeological waterlogged wooden artefacts, such as shipwrecks, can survive with a good state of preservation. Nevertheless, anaerobic bacteria can considerably degrade waterlogged wooden objects with a significant loss in polysaccharidic components. Due to these decay processes, wood porosity and water content increase under ageing. In such conditions, the conservation treatments of archaeological wooden artefacts often involve the replacement of water with substances which fill the cavities and help to prevent collapse and stress during drying. The treatments are very often expensive and technically difficult, and their effectiveness very much depends on the chemical and physical characteristics of the substances used for impregnation. Also important are the degree of cavity-filling, penetration depth and distribution in the structure of the wood. In this study, the distribution in wood cavities of some mixtures based on polyethylene glycols and colophony, used for the conservation of waterlogged archaeological wood, was investigated using synchrotron radiation X-ray computed microtomography (SR-µCT). This non-destructive imaging technique was useful for the study of the degraded waterlogged wood and enabled us to visualise the morphology of the wood and the distribution of the materials used in the wood treatments. The study has shown how deposition is strictly related to the dimension of the wooden cavities. The work is currently proceeding with the comparison of synchrotron observations with the data of the solutions viscosity and with those of the properties imparted to the wood by the treatments.

Keywords: Synchrotron radiation; X-ray microtomography; Waterlogged archaeological wood; Wood conservation; Impregnation

On-site analysis of archaeological artifacts excavated from the site on the outcrop at Northwest Saqqara, Egypt, by using a newly developed portable fluorescence spectrometer and diffractometer by Yoshinari Abe; Izumi Nakai; Kazumitsu Takahashi; Nozomu Kawai; Sakuji Yoshimura (pp. 1987-1996).

Blue-painted pottery was produced in the New Kingdom, Egypt, and decorated with blue, red, and black pigment. In this study, two newly developed portable instruments, a portable X-ray fluorescence spectrometer and a portable X-ray powder diffractometer, were brought to the site on the outcrop at Northwest Saqqara, an archaeological site in Egypt, to verify their performance in on-site analysis of excavated artifacts at the site. Pigments used for the blue-painted pottery and plasters in the New Kingdom were analyzed by these instruments on the basis of both their chemical compositions and crystal-structural information. The blue pigments were identified as two different pigments, Egyptian blue and cobalt blue. The diffraction pattern of the blue pigment of the painted pottery exhibited that of spinel structure. The XRF spectrum of the blue pigment obtained by the same instrument from the same position indicates the presence of Mn, Co, Fe, Ni, and Zn. The possibility of compositional transitions of the cobalt blue pigment with time was revealed on by detailed analysis of the XRF data. The reason for the transitions is considered together with the archaeological background of the New Kingdom, Egypt. Figure On site XRF analysis at an archaeological excavation site has revealed that the typological transition of blue-painted pottery excavated from the site on the outcrop at Northwest Saqqara, Egypt was followed by a chemical compositional transion of the pigment.

Keywords: Archaeometry; Portable XRF; Portable XRD; Cobalt blue; On-site analysis

Characterization of illuminated manuscripts by laboratory-made portable XRD and micro-XRD systems by A. Duran; J. L. Perez-Rodriguez; T. Espejo; M. L. Franquelo; J. Castaing; P. Walter (pp. 1997-2004).

Illuminated Arabic manuscripts have been studied, employing two laboratory-made X-ray diffraction (XRD) systems developed recently in the C2RMF laboratory. The validity of the µ-XRD and XRD portable systems for the study of this type of artworks has been demonstrated. A common observation in all the analyses is the presence of calcite and rutile; also, hematite, goethite, cinnabar, brass, anatase and barite were detected in the various colours. Differences between the results obtained by both techniques due to acquisition mode are discussed. In addition, other techniques such as X-ray fluorescence (XRF) and micro-Raman were used for the complete characterization of the manuscripts.

Keywords: Arabic manuscripts; XRD portable system; Laboratory-made µ-XRD system; Micro-Raman; Archeometry

Combined use of FORS, XRF and Raman spectroscopy in the study of mural paintings in the Aosta Valley (Italy) by Lorenzo Appolonia; Davide Vaudan; Valentina Chatel; Maurizio Aceto; Piero Mirti (pp. 2005-2013).

Mural paintings which decorate the external façade and the internal apsidal wall of a chapel dedicated to St. Maxime and located at Challand St. Victor in the Aosta Valley (Italy) have been analysed with a combined approach involving high-resolution fibre-optic reflectance spectroscopy (FORS), X-ray fluorescence (XRF) spectrometry and Raman spectroscopy. The paintings are attributed to Giacomino from Ivrea, a painter active around the mid-fifteenth century. In order to characterise the palette used by the painter and to yield information useful to restorers, the cited techniques were used either in situ with portable instruments and in laboratory, working on micro samples withdrawn from paintings. The global analytical approach, though not entirely non-invasive, can indeed be considered non-destructive as multiple analyses, including SEM-EDX, could be carried out on the micro samples, exploiting the features of each technique. On the basis of the information obtained, the palette was found to be composed of typical fresco pigments such as calcite, azurite, malachite, vermilion, red and yellow ochres. A particular situation was noted for black pigments since the presence of graphite, rather than wood or lamp carbon, was found, possibly related to the presence of graphite deposits in the Aosta Valley. Furthermore, the presence of smalt superimposed to azurite in areas showing evidence of repainting was detected, suggesting that paintings were subjected to retouching at a relatively early stage after the original execution. Finally, the presence of tin foils, used to decorate haloes of Evangelists, was ascertained.

Keywords: Non-destructive; FORS; XRF; Raman; Mural paintings; Aosta

Multilayers quantitative X-ray fluorescence analysis applied to easel paintings by Laurence de Viguerie; V. Armando Sole; Philippe Walter (pp. 2015-2020).

X-ray fluorescence spectrometry (XRF) allows a rapid and simple determination of the elemental composition of a material. As a non-destructive tool, it has been extensively used for analysis in art and archaeology since the early 1970s. Whereas it is commonly used for qualitative analysis, recent efforts have been made to develop quantitative treatment even with portable systems. However, the interpretation of the results obtained with this technique can turn out to be problematic in the case of layered structures such as easel paintings. The use of differential X-ray attenuation enables modelling of the various layers: indeed, the absorption of X-rays through different layers will result in modification of intensity ratio between the different characteristic lines. This work focuses on the possibility to use XRF with the fundamental parameters method to reconstruct the composition and thickness of the layers. This method was tested on several multilayers standards and gives a maximum error of 15% for thicknesses and errors of 10% for concentrations. On a painting test sample that was rather inhomogeneous, the XRF analysis provides an average value. This method was applied in situ to estimate the thickness of the layers a painting from Marco d’Oggiono, pupil of Leonardo da Vinci.

Keywords: X-Ray fluorescence; Quantitative analysis; Multilayers; Varnish; Thickness estimation

Multilayers quantitative X-ray fluorescence analysis applied to easel paintings by Laurence de Viguerie; V. Armando Sole; Philippe Walter (pp. 2015-2020).

Keywords: X-Ray fluorescence; Quantitative analysis; Multilayers; Varnish; Thickness estimation

EDXRF quantitative analysis of chromophore chemical elements in corundum samples by L. Bonizzoni; A. Galli; G. Spinolo; V. Palanza (pp. 2021-2027).

Corundum is a crystalline form of aluminum oxide (Al₂O₃) and is one of the rock-forming minerals. When aluminum oxide is pure, the mineral is colorless, but the presence of trace amounts of other elements such as iron, titanium, and chromium in the crystal lattice gives the typical colors (including blue, red, violet, pink, green, yellow, orange, gray, white, colorless, and black) of gemstone varieties. The starting point for our work is the quantitative evaluation of the concentration of chromophore chemical elements with a precision as good as possible to match the data obtained by different techniques as such as optical absorption photoluminescence. The aim is to give an interpretation of the absorption bands present in the NIR and visible ranges which do not involve intervalence charge transfer transitions (Fe²⁺ → Fe³⁺ and Fe²⁺ → Ti⁴⁺), commonly considered responsible of the important features of the blue sapphire absorption spectra. So, we developed a method to evaluate as accurately as possible the autoabsorption effects and the secondary excitation effects which frequently are sources of relevant errors in the quantitative EDXRF analysis.

Keywords: Corundum; EDXRF; PL; Absorption bands

EDXRF quantitative analysis of chromophore chemical elements in corundum samples by L. Bonizzoni; A. Galli; G. Spinolo; V. Palanza (pp. 2021-2027).

Keywords: Corundum; EDXRF; PL; Absorption bands

Characterisation of foxing stains in eighteenth to nineteenth century drawings using non-destructive techniques by M. Manso; S. Pessanha; F. Figueira; S. Valadas; A. Guilherme; M. Afonso; A. C. Rocha; M. J. Oliveira; I. Ribeiro; M. L. Carvalho (pp. 2029-2036).

The reddish-brown, brown or yellowish stains of circular or irregular shape known as foxing spots have been fully described in conservation literature but still, this phenomenon does not find any scientific agreement since many hypotheses have been raised concerning their origin. In this work a contribution to foxing definition not only focussed on its appearance but also reported on its chemical information. For this purpose foxing stains present in drawings from two Portuguese artists dated from the eighteenth to nineteenth centuries were observed under ultra-violet light and optical microscope and analysed by three non-invasive spectroscopy techniques. The observations carried out on the stains provided information on their surface morphology. The use of energy-dispersive X-ray fluorescence revealed a variation on the elemental content between foxing and paper region. Although the results from X-ray diffraction analysis showed no signs of cellulose degradation in foxing stains, Fourier-transformed infrared analysis revealed the presence of oxide groups. Both the information on the chemical nature and surface morphology of the stains achieved in this study will contribute to increase foxing formation information and develop future protocols for conservation purposes.

Keywords: Foxing; EDXRF; XRD; FTIR; Morphological characterisation

Micro-analytical evidence of origin and degradation of copper pigments found in Bohemian Gothic murals by Silvie Švarcová; David Hradil; Janka Hradilová; Eva Kočí; Petr Bezdička (pp. 2037-2050).

Correct identification of pigments and all accompanying phases found in colour layers of historical paintings are relevant for searching their origin and pigment preparation pathways and for specification of their further degradation processes. We successfully applied the analytical route combining non-destructive in situ X-ray fluorescence analyses with subsequent laboratory investigation of micro-samples by optical microscopy, scanning electron microscopy/energy-dispersive spectroscopy and X-ray powder micro-diffraction (micro-XRD) to obtain efficiently all the data relevant for mineralogical interpretations of the copper pigments origin. Cu salts (carbonates, chlorides, sulphates, etc.) used as pigments exist in a range of polymorphs with similar or identical composition. The efficiency of the micro-XRD for direct identification of such crystal phases present in micro-samples of colour layers was demonstrated in the presented paper. A new, until now unpublished, type of copper pigment—cumengeite, Pb₂₁Cu₂₀Cl₄₂(OH)₄₀—used as a blue pigment on a sacral wall painting in the Czech Republic was found by means of micro-XRD. Furthermore, azurite, malachite, paratacamite, atacamite and posnjakite were identified in fragments of colour layers of selected Gothic wall paintings. We found Cu–Zn arsenates indicating the natural origin of azurite and malachite; artificial malachite was distinguishable according to its typical spherulitic crystals. The corrosion of blue azurite to green basic Cu chloride was clearly evidenced on some places exposed to the action of salts and moisture—in a good agreement with the results of laboratory experiments, which also show that oxalic acid accelerates the corrosion of Cu pigments. Figure In situ XRF measurement of Gothic murals in the Benedictine Monastery in Sazava—here The Joseph’s Doubt scene in the capitular hall

Keywords: X-ray powder micro-diffraction; Copper pigments; Cumengeite; Wall paintings; Corrosion

Chemical and mineralogical characterization on glazes of ceramics from Coimbra (Portugal) from the sixteenth to nineteenth centuries by A. Guilherme; J. Coroado; M. L. Carvalho (pp. 2051-2059).

Chemical, mineralogical and textural characterizations were performed on glazed pieces prepared in laboratory as well as on faiences fragments collected from the existing remains in “Santa Clara-a-Velha” monastery (Coimbra, Portugal). The chemical investigation was carried out using micro X-ray fluorescence (µ-EDXRF) and wavelength dispersive X-ray fluorescence (WDXRF); the mineralogical results using X-ray diffraction (XRD) and the textural profile was obtained by scanning electron microscopy coupled with an energy dispersive spectroscopy system (SEM-EDS). Attention has been drawn to the glaze mineralogical changes during the firing temperature process, where three different types of glazes were submitted to three different firing temperatures (800 °C, 900 °C and 1,000 °C). Under these conditions, it is possible to relate the mineralogical content of the fragments to their firing temperature. Furthermore, we focused our purposes on identifying the technological aspects of the ceramic production in Coimbra, such as the raw materials, manufacture techniques and firing temperature adopted for the glaze. The latter aspect is highly dependent on the ceramic materials. In the framework of a more general project, this survey has as premise the recognition of a pattern, which is thought to be exclusively typical from the region of Coimbra. The perspective developed in the present work is towards reliable archaeometric criteria, which can be used to characterise scientifically the ceramics from Coimbra.

Keywords: Portuguese faiences; Glaze characterization; Spectrometry techniques; Authenticity; X-ray spectroscopy (XPS | XRF | EDX); Diffraction methods (LEED | X-ray); Archaeometry/fine arts

Chemical and mineralogical characterization on glazes of ceramics from Coimbra (Portugal) from the sixteenth to nineteenth centuries by A. Guilherme; J. Coroado; M. L. Carvalho (pp. 2051-2059).

Keywords: Portuguese faiences; Glaze characterization; Spectrometry techniques; Authenticity; X-ray spectroscopy (XPS | XRF | EDX); Diffraction methods (LEED | X-ray); Archaeometry/fine arts

Byzantine wall paintings from Mani (Greece): microanalytical investigation of pigments and plasters by Anno Hein; Ioannis Karatasios; Dionysis Mourelatos (pp. 2061-2071).

The present case study concerns the technology of Byzantine wall paintings from the Mani Peninsula, Greece. An assemblage of 12 Byzantine churches, constructed in the tenth to fifteenth century, was included in an initial analytical survey. Two random samples of wall paintings were taken in each monument in order to study their micro stratigraphy and the composition of pigment and plaster layers. Polished sections were fabricated for examination with optical microscopy and scanning electron microscopy (SEM). Furthermore, selected samples were powdered and analysed with Fourier-transformed infrared spectroscopy (FTIR) and X-ray diffraction (XRD). The analytical results achieved in this case study provided general conclusions concerning painting techniques for wall paintings in a rather provincial area of the Byzantine Empire. The palette comprised mainly earthen pigments like ochres and carbon black but occasionally also other pigments like cinnabar, minium and ultramarine. In view of future studies, a portable X-ray fluorescence analysis (XRF) set-up was tested. Figure Section of an ultramarine paint layer from the Church Aghios Philippos

Keywords: Wall painting; Pigments; Lime plaster; SEM-EDS; FTIR; XRD

Byzantine wall paintings from Mani (Greece): microanalytical investigation of pigments and plasters by Anno Hein; Ioannis Karatasios; Dionysis Mourelatos (pp. 2061-2071).

Keywords: Wall painting; Pigments; Lime plaster; SEM-EDS; FTIR; XRD

EDXRF imaging of Pb in glazed ceramics using a micropattern gas detector by A. L. M. Silva; C. A. B. Oliveira; A. L. Gouvêa; J. M. F. dos Santos; M. L. Carvalho; J. F. C. A. Veloso (pp. 2073-2080).

A new system for energy-resolved X-ray fluorescence imaging using a microhole and strip plate (MHSP), a new type of micropattern gas detector (MPGD), is proposed. It works as a single photon counting detector with position and energy detection capability. The interaction of X-rays with the gas medium produces electrons via the photoelectric effect, and the number of electrons is proportional to the absorbed X-ray energy. These electrons are further multiplied in the MHSP. Position detection is achieved using the charge division method. The detector has an active area of 28 × 28 mm² and shows good position resolution, about σ = 125 μm, an intrinsic energy resolution of about 14% FWHM for 5.9 keV X-rays, and a counting rate capability of up to 0.5 MHz. The system has shown good properties for energy-dispersive X-ray fluorescence (EDXRF) applications, since it allows efficient energy and position detection of fluorescence X-rays from multielemental samples. In this work, the system was used to study lead depth distributions in eighteenth-century Portuguese faiences from the Santa Clara-a-Velha monastery. The fluorescence images were obtained by irradiating the samples, with a pinhole placed between the sample and the detector to focus the radiation into the detector. The results are presented here, including the elemental map distributions for different samples.

Keywords: X-ray fluorescence imaging; MHSP; Elemental spatial distribution

Analytical characterization of polymers used in conservation and restoration by ATR-FTIR spectroscopy by Ruth Chércoles Asensio; Margarita San Andrés Moya; José Manuel de la Roja; Marisa Gómez (pp. 2081-2096).

In the last few decades many new polymers have been synthesized that are now being used in cultural heritage conservation. The physical and chemical properties and the long-term behaviors of these new polymers are determined by the chemical composition of the starting materials used in their synthesis along with the nature of the substances added to facilitate their production. The practical applications of these polymers depend on their composition and form (foam, film, sheets, pressure-sensitive adhesives, heat-seal adhesives, etc.). Some materials are used in restoration works and others for the exhibition, storage and transport of works of art. In all cases, it is absolutely necessary to know their compositions. Furthermore, many different materials that are manufactured for other objectives are also used for conservation and restoration. The technical information about the materials provided by the manufacturer is usually incomplete, so it is necessary to analytically characterize such materials. FTIR spectrometry is widely used for polymer identification, and, more recently, ATR–FTIR has been shown to give excellent results. This paper reports the ATR-FTIR analysis of samples of polymeric materials used in the conservation of artworks. These samples were examined directly in the solid material without sample preparation. Figure

Keywords: Polymers; ATR-FTIR; Synthetic materials; Restoration and conservation of cultural heritage

Non-invasive identification of organic materials in wall paintings by fiber optic reflectance infrared spectroscopy: a statistical multivariate approach by F. Rosi; A. Daveri; C. Miliani; G. Verri; P. Benedetti; F. Piqué; B. G. Brunetti; A. Sgamellotti (pp. 2097-2106).

The aim of this study is to develop a method for the non-invasive and in situ identification of organic binders in wall paintings by fiber optic mid-FTIR reflectance spectroscopy. The non-invasive point analysis methodology was set-up working on a wide set of wall painting replicas of known composition and using statistical multivariate methods, in particular principal component analysis (PCA), for the interpretation, understanding, and management of data acquired with reflectance mid-FTIR spectroscopy. Results show that PCA can be helpful in managing and preliminary sorting of the large amount of spectra typically collected during non-invasive measurement campaigns and highlight further avenues for research. The developed PCA model was finally applied to the case of a Renaissance wall painting by Perugino assessing it predictability as compared to the interpretation of the single spectrum.

Keywords: mid-FTIR; Reflectance; PCA; Binder; Portable equipment

FT-NIR spectroscopy for non-invasive identification of natural polymers and resins in easel paintings by M. Vagnini; C. Miliani; L. Cartechini; P. Rocchi; B. G. Brunetti; A. Sgamellotti (pp. 2107-2118).

In the present study, the analytical strengths and limitations of near-infrared (NIR) spectroscopy to non-invasively characterize organic components in painting materials have been investigated. In spite of the increased amount of information available today from advanced modern analytical instrumentations dedicated to cultural heritage, the non-invasive identification of materials belonging to the wide class of organic compounds historically used in paintings is still a challenging task. Near-infrared spectroscopy offers several attractive features that make this technique particularly suitable to this purpose. In fact, it is non-invasive, allows for non-contact measurements in reflectance mode, gives molecular information on complex macromolecules, and can be performed on-site by means of portable devices. First-derivative transformation of reflectance spectroscopic data has been applied to provide a simple and fast way to deduce more information from NIR spectra. This approach has allowed spectral features to be identified that can be useful to distinguish different compounds belonging to the classes of lipids, proteins, and resins. To this purpose, at first, a spectral database of pure standard has been collected. Our analytical approach was then successfully validated on pictorial models reproducing the typical stratigraphy of an easel painting. As final step, the study of a real painting has been attempted and a drying oil, animal glue, and a terpenic natural resin, as well as an earth pigment were clearly identified, as cross-validated by GC-MS analysis.

Keywords: Near-FTIR; Paintings; Natural organic compounds; Portable equipments

Raman spectroscopy after accelerated ageing tests to assess the origin of some decayed products found in real historical bricks affected by urban polluted atmospheres by M. Maguregui; A. Sarmiento; R. Escribano; I. Martinez-Arkarazo; K. Castro; J. M. Madariaga (pp. 2119-2129).

Bricks, together with stones and mortars, can be considered as one of the most important building materials that constitute our built heritage. Numerous factors which cause several decaying pathologies in bricks can be listed, but it should be emphasised that the most severe and damaging one is the wet and dry deposition of both combustion and greenhouse gases (CO₂, SO x and NO x mainly). For instance, after the impact of CO₂ and SO x, the decayed products promoted in bricks are carbonates and sulphates. Once identified in all these kinds of salts in real samples, it is necessary to make sure that the aggressive atmospheric conditions are sufficient to promote the formation of these salts. Therefore, accelerated exposure test are a good alternative in order to simulate the formation of these decayed compounds and to predict the reactions that promote the decaying mechanism. In this work, brick samples manufactured at different firing temperatures following ancient methods were subjected to humidity/dryness, freeze/thaw, CO₂ and SO₂ (KESTERNICH DIN 50018) accelerated ageing tests followed by a Raman spectroscopy screening in order to verify the formation of sulphate and carbonate salts in bricks on accelerated conditions, simulating the damage caused by a polluted atmosphere throughout many years of exposure. Figure Raman spectra of a white grain after the SO2 ageing experiments. Traces of calcite are observed together with the gypsum signals.

Keywords: Bricks; Accelerated ageing tests; Raman spectroscopy; Micro-ED-X-ray fluorescence; Ion chromatography

Fourier-transform Raman spectroscopic study of a Neolithic waterlogged wood assemblage by M. Petrou; H. G. M. Edwards; R. C. Janaway; G. B. Thompson; A. S. Wilson (pp. 2131-2138).

The use of Fourier-transform Raman spectroscopy for characterising lignocellulosics has increased significantly over the last twenty years. Here, an FT-Raman spectroscopic study of changes in the chemistry of waterlogged archaeological wood of Pinus sp. and Quercus sp. from a prehistoric assemblage recovered from northern Greece is presented. FT-Raman spectral features of biodeteriorated wood were associated with the depletion of lignin and/or carbohydrate polymers at various stages of deterioration. Spectra from the archaeological wood are presented alongside spectra of sound wood of the same taxa. A comparison of the relative changes in intensities of spectral bands associated with lignin and carbohydrates resulting from decay clearly indicated extensive deterioration of both the softwood and hardwood samples and the carbohydrates appear to be more deteriorated than the lignin. The biodeterioration of the archaeological timbers followed a pattern of initial preferential loss of carbohydrates causing significant loss of cellulose and hemicellulose, followed by the degradation of lignin.

Keywords: FT-Raman; Waterlogged wood; Archaeological oak; Archaeological pine; Greece

Fourier-transform Raman spectroscopic study of a Neolithic waterlogged wood assemblage by M. Petrou; H. G. M. Edwards; R. C. Janaway; G. B. Thompson; A. S. Wilson (pp. 2131-2138).

Keywords: FT-Raman; Waterlogged wood; Archaeological oak; Archaeological pine; Greece

Assessment of the ageing of triterpenoid paint varnishes using fluorescence, Raman and FTIR spectroscopy by Austin Nevin; Daniela Comelli; Iacopo Osticioli; Lucia Toniolo; Gianluca Valentini; Rinaldo Cubeddu (pp. 2139-2149).

The assessment of the influence of natural and artificial ageing on the spectrofluorescence of triterpenoid varnishes dammar and mastic is the focus of this work. Both Fourier transform infrared (FTIR) microscopy using attenuated total reflectance and Raman spectroscopy have been employed for complementary molecular analysis of samples. Synchronous fluorescence spectroscopy, excitation emission spectroscopy, and statistical analysis of data have been used to monitor changes in the optical properties of varnish samples. Assessment of naturally and artificially aged samples using excitation emission spectroscopy suggests that extensive exposure to visible light does not lead to easily appreciable differences in the fluorescence of mastic and dammar; cluster analysis has been used to assess changes, which occur with artificial ageing under visible light, indicating that differences in the fluorescence spectra of aged triterpenoids may be insufficient for their discrimination. The results highlight significant differences between the initial fluorescence of films of dammar and mastic and the fluorescence, which develops with ageing and oxidation, and specific markers, which change with ageing in FTIR and Raman spectra, have been identified. Figure Excitation emission spectra of films triterpenoid varnishes mastic and dammar have been recorded as a function of ageing. Using multivariate statistical analysis, dedrograms of contours of fluorescene excitation emission spectra are used to assess the differences in spectra of dammar and mastic

Keywords: Varnish; Fluorescence; Triterpenoid; Mastic; Dammar; Artificial ageing; Light ageing; Raman; FTIR; ATR

Application of synchronous fluorescence to parchment characterization by Bella Dolgin; Valery Bulatov; Israel Schechter (pp. 2151-2159).

A nondestructive method for quantitative parchment characterization and sensitive indication of its deterioration stage was developed. Synchronous fluorescence (SF) measurements were applied for the first time to parchment samples. The method provides detailed spectral features, which are useful for parchment characterization. The discrimination of parchment samples into groups (modern, historical, and artificially aged) was successfully performed. The SF spectra could be resolved into specific fluorophores, which were related to the parchment condition. The spectral data indicate a continuous change in the collagen-to-gelatin ratio during the aging process. Depth-resolved synchronous fluorescence spectra were also measured. The data indicate that parchments possess a layered structure, and the dominant fluorophore in the upper layer is different from those in the lower layers. Layer-resolved profiling allows for quantifying the contribution of each fluorophore in each given layer. This way, significant differences between modern, artificially aged, and historical samples can be observed.

Keywords: Parchment; Archeology; Synchronous fluorescence; Laser ablation; Layer-resolved profile

Application of synchronous fluorescence to parchment characterization by Bella Dolgin; Valery Bulatov; Israel Schechter (pp. 2151-2159).

Keywords: Parchment; Archeology; Synchronous fluorescence; Laser ablation; Layer-resolved profile

Second and third harmonic generation measurements of glues used for lining textile supports of painted artworks by G. Filippidis; K. Melessanaki; C. Fotakis (pp. 2161-2166).

In this study, we present the implementation of non-linear spot measurements for obtaining specific and novel information related to various types of natural and synthetic glues used for lining of painted artworks. Third harmonic generation measurements were employed, in transmission mode, for the accurate and non-destructive thickness detection of lining glues. Furthermore, second harmonic generation signals were collected, in reflection mode, providing complementary information for the discrimination between different types of lining glues.

Keywords: SHG; THG; Lining glues; Painted artworks

Second and third harmonic generation measurements of glues used for lining textile supports of painted artworks by G. Filippidis; K. Melessanaki; C. Fotakis (pp. 2161-2166).

Keywords: SHG; THG; Lining glues; Painted artworks

Applying pyrolysis-gas chromatography/mass spectrometry to the identification of oriental lacquers: study of two lacquered shields by José Carlos Frade; Maria Isabel Ribeiro; José Graça; José Rodrigues (pp. 2167-2174).

Oriental lacquers have been used as coating materials for thousands of years for wooden, ceramics, leather and metal objects. Lacquers are natural polymers obtained from three species growing in different regions of Asia: Rhus vernicifera (China, Japan and Korea); Rhus succedanea (Vietnam and Taiwan); and Melanorrhoea usitate (Myanmar and Thailand). The identification of lacquer films is important for conservation and restoration purposes, as well as for art history studies because it may help in determining the origin of the lacquered objects. In this work, pyrolysis-gas chromatography/mass spectrometry using a filament-type pyrolyser was successfully applied to the characterization of oriental lacquers. A method to identify the three kinds of lacquer was developed and applied to the study of two lacquered shields imported from Asia in the sixteenth century. The materials that constitute the shields were also examined by Fourier-transform infrared microspectroscopy and details of the lacquering technique are reported. Figure Two lacquered shields of unknown origin imported from Asia in the 16th century

Keywords: Oriental lacquer; Rhus vernicifera ; Rhus succedanea ; Melanorrhoea usitate ; Py-GC/MS; FTIR-μs

Comparison of extraction methods for the analysis of natural dyes in historical textiles by high-performance liquid chromatography by Lemonia Valianou; Ioannis Karapanagiotis; Yannis Chryssoulakis (pp. 2175-2189).

Different methods for the extraction of Dactylopius coccus Costa, Rubia tinctorum L., Isatis tinctoria L., Reseda luteola L., Curcuma longa L. and Cotinus coggygria Scop. from wool fibres are investigated using high-performance liquid chromatography with diode array detector (HPLC-DAD). The efficiencies of five extraction methods which include the use of HCl (widely used extraction method), citric acid, oxalic acid, TFA and a combination of HCOOH and EDTA are compared on the basis of the (a) number, (b) relative quantities, measured as HPLC peak areas and (c) signal-to-noise ratios (S/N) of the compounds extracted from the wool substrates. Flavonoid glycosides and curcuminoids contained in R. luteola L. and C. longa L., respectively, according to liquid chromatography with mass spectrometry (LC-MS) identifications, are not detected after treating the fibres with HCl. All the other milder methods are successful in extracting these compounds. Experiments are performed using HPLC-DAD to compare the HPLC peak areas and the S/N of the following extracted compounds: indigotin, indirubin, curcumin, demethoxycurcumin, bisdemethoxycurcumin, fisetin, sulfuretin, luteolin, luteolin-7-O-glucoside, apigenin, carminic acid, alizarin, puruprin and rubiadin. It is shown that the TFA method provides overall the best results as it gives elevated extraction yields except for fisetin, luteolin, apigenin and luteolin-7-O-glucoside and highest S/N except for fisetin and luteolin-7-O-glucoside. It is noteworthy that treatment of the fibres with the typical HCl extraction method results overall in very low S/N. The TFA method is selected for further studies, as follows. First, it is applied on silk dyed samples and compared with the HCl method. The same relative differences of the TFA and HCl methods observed for the wool dyed samples are reported for the silk dyed samples too, except for rubiadin, luteolin and apigenin. Thus, in most cases, the nature of the substrate (wool or silk) appears to have negligible effects on the relative difference of the two extraction methods. Second, the selected TFA method is applied to treat wool and silk historical samples extracted from textiles of the Mamluk period, resulting in the identification of several colouring compounds. In all extraction methods mentioned above, DMSO is used to dissolve the dyes, after acid treatment.

Keywords: Archaeometry; Fine arts; HPLC; Natural products; Sampling

Chemical investigation on black pigments in the carved decoration of sixteenth century alabaster tombs from Zaragoza (Spain) by Josefina Pérez-Arantegui; Erika Ribechini; Carlos Pardos; Maria Perla Colombini (pp. 2191-2197).

An analytical protocol based on optical microscopy (OM), scanning electron microscopy (SEM) observation, energy-dispersive X-ray (EDX) analyses, analytical pyrolysis in the presence of hexamethyldisilazane followed by gas chromatographic/mass spectrometric analysis (Py-silylation-GC/MS) and gas chromatography/mass spectrometry (GC/MS) after alkaline hydrolysis, solvent extraction and trimethylsilylation was used to study the origin and nature of black pigments from the carved inscriptions of several panels of two alabaster tombs dated from the mid-sixteenth century. Optical microscopy and SEM observation showed the presence of an amorphous very dark-brown substance, from translucent to opaque. EDX analyses revealed that the samples were mainly made up of C and O, thus highlighting the organic nature of the material used in the inscriptions. Py-silylation-GC/MS and GC/MS analyses provided detailed molecular compositions, highlighting the presence of a wide range of compound classes including diterpenoid acids, tricyclic abietanes, mid- and long-chain monocarboxylic fatty acids, n-alkanols and n-alkanes. The pyrograms, the chromatographic profiles and the presence of characteristic biomarkers indicated that a mixture of pine pitch and beeswax had been used to make the black inscriptions. Chemical characterization of Renaissance black pigments

Keywords: Black pigments; GC/MS; Pyrolysis-GC/MS; Pitch; Beeswax

Combined elemental analysis of ancient glass beads by means of ion beam, portable XRF, and EPMA techniques by D. Sokaras; A. G. Karydas; A. Oikonomou; N. Zacharias; K. Beltsios; V. Kantarelou (pp. 2199-2209).

Ion beam analysis (IBA)- and X-ray fluorescence (XRF)-based techniques have been well adopted in cultural-heritage-related analytical studies covering a wide range of diagnostic role, i.e., from screening purposes up to full quantitative characterization. In this work, a systematic research was carried out towards the identification and evaluation of the advantages and the limitations of laboratory-based (IBA, electron probe microanalyzer) and portable (milli-XRF and micro-XRF) techniques. The study focused on the analysis of an Archaic glass bead collection recently excavated from the city of Thebes (mainland, Greece), in order to suggest an optimized and synergistic analytical methodology for similar studies and to assess the reliability of the quantification procedure of analyses conducted in particular by portable XRF spectrometers. All the employed analytical techniques and methodologies proved efficient to provide in a consistent way characterization of the glass bead composition, with analytical range and sensitivity depending on the particular technique. The obtained compositional data suggest a solid basis for the understanding of the main technological features related to the raw major and minor materials utilized for the manufacture of the Thebian ancient glass bead collection.

Keywords: Ion beam analysis; X-ray fluorescence; Micro-XRF; Portable XRF; Quantification; Ancient glass analysis

Multitechnique characterization of lapis lazuli for provenance study by Alessandro Lo Giudice; Alessandro Re; Silvia Calusi; Lorenzo Giuntini; Mirko Massi; Paolo Olivero; Giovanni Pratesi; Maria Albonico; Elisa Conz (pp. 2211-2217).

Lapis lazuli is one of the oldest precious stone, being used for glyptic as early as 7,000 years ago: jewels, amulets, seals, and inlays are examples of objects produced using this material. Only a few sources of lapis lazuli exist in the world due to the low probability of geological conditions in which it can form, so that the possibility to associate the raw material to man-made objects helps to reconstruct trade routes. Since art objects produced using lapis lazuli are valuable, only nondestructive investigations can be carried out to identify the provenance of the raw materials. Ionoluminescence (IL) is a good candidate for this task. Similar to cathodoluminescence (CL), IL consists in the collection of luminescence spectra induced by megaelectronvolt ion (usually protons) irradiation. The main advantage of IL consists in the possibility of working in air while measuring simultaneously the composition of major and trace elements by means of complementary ion beam analysis techniques like particle-induced X-ray emission (PIXE) or particle-induced gamma-ray emission (PIGE). In the present work, a systematic study of the luminescence properties of lapis lazuli under charged particle irradiation is reported. In the first phase, a multitechnique approach was adopted (CL, scanning electron microscopy with microanalysis, micro-Raman) to characterize luminescent minerals. This characterization was propaedeutic for IL/PIXE/PIGE measurements carried out on significant areas selected on the basis of results obtained previously. Criteria to identify provenance of lapis lazuli from four of the main sources (Afghanistan, Pamir Mountains in Tajikistan, Chile, and Siberia) were proposed.

Keywords: Lapis lazuli; Provenance; Ionoluminescence; Cathodoluminescence; Archaeometry; Ion beam analysis

Quantitative X-ray fluorescence analysis of an Egyptian faience pendant and comparison with PIXE by L. de Viguerie; A. Duran; A. Bouquillon; V. A. Solé; J. Castaing; P. Walter (pp. 2219-2225).

The X-ray fluorescence (XRF) technique is a common choice in the archaeometric field for in situ investigations with portable instruments. This work shows that XRF portable systems can be used for quantitative analyses using appropriate software, obtaining a similar accuracy to that provided with other techniques such as particle-induced X-ray emission (PIXE), as shown for an Egyptian faience pendant and for two glass standards.

Keywords: X-ray fluorescence; Quantitative analyses; PIXE; Egyptian faience

Quantitative X-ray fluorescence analysis of an Egyptian faience pendant and comparison with PIXE by L. de Viguerie; A. Duran; A. Bouquillon; V. A. Solé; J. Castaing; P. Walter (pp. 2219-2225).

Keywords: X-ray fluorescence; Quantitative analyses; PIXE; Egyptian faience

Isotopic analysis for degradation diagnosis of calcite matrix in mortar by E. Dotsika; D. Psomiadis; D. Poutoukis; B. Raco; P. Gamaletsos (pp. 2227-2234).

Mortar that was used in building as well as in conservation and restoration works of wall paintings have been analysed isotopically (δ¹³C and δ¹⁸O) in order to evaluate the setting environments and secondary processes, to distinguish the structural components used and to determine the exact causes that incurred the degradation phenomena. The material undergoes weathering and decay on a large proportion of its surface and in depth, due to the infiltration of water through the structural blocks. Mineralogical analysis indicated signs of sulphation and dissolution/recrystallisation processes taking place on the material, whereas stable isotopes provided information relative to the origin of the CO₂ and water during calcite formation and degradation processes. Isotopic change of the initial δ¹³C and δ¹⁸O in carbonate matrix was caused by alteration of the primary source of CO₂ and H₂O in mortar over time, particularly by recrystallisation of calcite with porewater, evaporated or re-condensed water, and CO₂ from various sources of atmospheric and biogenic origin. Human influence (surface treatment) and biological growth (e.g. fungus) are major exogenic processes which may alter δ¹⁸O and δ¹³C in lime mortar.

Keywords: Stable isotope; Mortar; Degradation; Greece

Isotopic analysis for degradation diagnosis of calcite matrix in mortar by E. Dotsika; D. Psomiadis; D. Poutoukis; B. Raco; P. Gamaletsos (pp. 2227-2234).

Keywords: Stable isotope; Mortar; Degradation; Greece

Plasma reduction of bronze corrosion developed under long-term artificial ageing by Jelica Novakovic; Olga Papadopoulou; Panayota Vassiliou; Eleni Filippaki; Yannis Bassiakos (pp. 2235-2244).

Reference Cu-based alloy with chemical composition and micro-chemical structure similar to that of ancient alloys has been used for carrying out the artificial long-term degradation test based on chloride enriched soil (chemical + soil) degradation. The results show that such degradation procedure produces natural like “patinas” as the ones grown on archaeological artefacts, from a chemical, structural and micro-morphological point of view. Glow discharge plasma technique has been employed for the treatment of the as-corroded bronze coupons. The gradual elimination of chloride-containing corrosion products in favour of the formation of more stable species and even the complete reduction back to copper has been observed. The chemical and metallurgical features have been determined by combined use of different analytical techniques such as scanning electron microscopy with energy dispersive X-ray micro-analysis (SEM-EDS), X-ray diffraction (XRD) and optical microscopy.

Keywords: Cu–Sn alloys; Artificial corrosion; Plasma reduction

Plasma reduction of bronze corrosion developed under long-term artificial ageing by Jelica Novakovic; Olga Papadopoulou; Panayota Vassiliou; Eleni Filippaki; Yannis Bassiakos (pp. 2235-2244).

Keywords: Cu–Sn alloys; Artificial corrosion; Plasma reduction

An integrated study for mapping the moisture distribution in an ancient damaged wall painting by Donatella Capitani; Noemi Proietti; Marco Gobbino; Luigi Soroldoni; Umberto Casellato; Massimo Valentini; Elisabetta Rosina (pp. 2245-2253).

An integrated study of microclimate monitoring, IR thermography (IRT), gravimetric tests and portable unilateral nuclear magnetic resonance (NMR) was applied in the framework of planning emergency intervention on a very deteriorated wall painting in San Rocco church, Cornaredo (Milan, Italy). The IRT investigation supported by gravimetric tests showed that the worst damage, due to water infiltration, was localized on the wall painting of the northern wall. Unilateral NMR, a new non-destructive technique which measures the hydrogen signal of the moisture and that was applied directly to the wall, allowed a detailed map of the distribution of the moisture in the plaster underlying the wall panting to be obtained. With a proper calibration of the integral of the recorded signal with suitable specimens, each area of the map corresponded to an accurate amount of moisture. IRT, gravimetric tests and unilateral NMR applied to investigate the northern wall painting showed the presence of two wet areas separated by a dry area. The moisture found in the lower area was ascribed to the occurrence of rising damp at the bottom of the wall due to the slope of the garden soil towards the northern exterior. The moisture found in the upper area was ascribed to condensation phenomena associated with the presence of a considerable amount of soluble, hygroscopic salts. In the framework of this integrated study, IRT investigation and gravimetric methods validated portable unilateral NMR as a new analytical tool for measuring in situ and without any sampling of the distribution and amount of moisture in wall paintings.

Keywords: IR thermography; Unilateral NMR; Wall paintings; Gravimetric tests; Moisture

Development and application of a portable LIPS system for characterising copper alloy artefacts by J. Agresti; A. A. Mencaglia; S. Siano (pp. 2255-2262).

We report the development of a novel portable and low-cost laser induced plasma spectroscopy (LIPS) system and describe the application method for quantitative characterisation of quaternary copper alloy artefacts. The device was carefully calibrated and phenomenologically characterised using a set of reference samples. The reliability of the quantitative measurement of the depth profile and bulk compositions was assessed through crossed comparisons with traditional analytical techniques. Finally, the LIPS system was applied to investigate a museum figurine of unknown origin composed of several pieces, which is representative of a typical authentication problem.

Keywords: Archeometry / Fine arts; Laser spectroscopy; Laser ablation; Metals / Heavy metals; Optical sensors; UV/VIS

Development and application of a portable LIPS system for characterising copper alloy artefacts by J. Agresti; A. A. Mencaglia; S. Siano (pp. 2255-2262).

Keywords: Archeometry / Fine arts; Laser spectroscopy; Laser ablation; Metals / Heavy metals; Optical sensors; UV/VIS

Immuno-PCR-based quantification of multiple phosphorylated tau-epitopes linked to Alzheimer’s disease by David Singer; Hilkka Soininen; Irina Alafuzoff; Ralf Hoffmann (pp. 2263-2267).

Several lines of evidence suggest that quantification of phosphorylated sites in the tau-protein (phospho-τ) might be favorable for early and specific Alzheimer’s disease diagnosis. The typical setup to quantify phosphorylated τ-epitopes relies on a sandwich ELISA with a capture antibody (Ab) recognizing τ independent of its phosphorylation status and a detector Ab binding specifically to a certain phosphorylation site. Besides Ab specificities, major challenges arise from the very low τ-concentrations in cerebrospinal fluid (CSF) ranging from 100 to 2,000 pg/ml. Based on the phosphorylation degree of a given position, which can be below 10%, the corresponding phospho-τ-level might be much lower, especially for multiphosphorylated epitopes studied here. Thus, a novel, highly sensitive, and generally applicable immunoassay is described to quantify τ-versions, which are phosphorylated at pThr212/pSer214/pThr231/pSer235, down to τ-concentrations of 2 pg/ml in CSF.

Keywords: Alzheimer’s disease; Antibodies; Digoxigenin; Immunoassay; Phospho-tau

Immuno-PCR-based quantification of multiple phosphorylated tau-epitopes linked to Alzheimer’s disease by David Singer; Hilkka Soininen; Irina Alafuzoff; Ralf Hoffmann (pp. 2263-2267).

Keywords: Alzheimer’s disease; Antibodies; Digoxigenin; Immunoassay; Phospho-tau

Proteomic strategies for the identification of proteinaceous binders in paintings by Gabriella Leo; Laura Cartechini; Piero Pucci; Antonio Sgamellotti; Gennaro Marino; Leila Birolo (pp. 2269-2280).

The identification of proteinaceous components in paintings remains a challenging task for several reasons. In addition to the minute amount of sample available, complex and variable chemical composition of the paints themselves, possible simultaneous presence of several binders and contaminants, and degradation of the original materials due to aging and pollution are complicating factors. We proposed proteomic strategies for the identification of proteins in binders of paintings that can be adapted to overcome the requirements and difficulties presented by specific samples. In particular, we worked on (1) the development of a minimally invasive method based on the direct tryptic cleavage of the sample without protein extraction; (2) the use of microwave to enhance the enzymatic digestion yield, followed by the analysis of the peptide mixtures by nanoLC-MS/MS with electrospray ionization (ESI). Moreover, as an additional tool to tackle the problem of contaminating proteins, we exploited the possibility of generating an exclusion list of the mass signals that in a first run had been fragmented and that the mass spectrometer had to ignore for fragmentation in a subsequent run. The methods, tested on model samples, allowed the identification of milk proteins in a sample from paintings attributed to Cimabue and Giotto, thirteenth-century Italian masters, decorating the vaults of the upper church in the Basilica of St. Francis in Assisi, Italy.

Keywords: Proteinaceous binders; Enzymatic digestion; LC-ESI-MS/MS; Paintings; Paraloid B72

Proteomic strategies for the identification of proteinaceous binders in paintings by Gabriella Leo; Laura Cartechini; Piero Pucci; Antonio Sgamellotti; Gennaro Marino; Leila Birolo (pp. 2269-2280).

Keywords: Proteinaceous binders; Enzymatic digestion; LC-ESI-MS/MS; Paintings; Paraloid B72

Improved procedure for protein binder analysis in mural painting by LC-ESI/Q-q-TOF mass spectrometry: detection of different milk species by casein proteotypic peptides by Angela Chambery; Antimo Di Maro; Carmen Sanges; Valeria Severino; Maura Tarantino; Annalisa Lamberti; Augusto Parente; Paolo Arcari (pp. 2281-2291).

Diagnostic techniques applied to the field of cultural heritage represent a very important aspect of scientific investigation. Recently, proteomic approaches based on mass spectrometry coupled with traditional spectroscopic methods have been used for painting analysis, generating promising results for binder’s protein identification. In the present work, an improved procedure based on LC-ESI/Q-q-TOF tandem mass spectrometry for the identification of protein binders has been developed for the molecular characterization of samples from an early-twentieth-century mural painting from the St. Dimitar Cathedral in Vidin, Bulgaria. The proteomic investigation has led to the identification of both egg white and egg yolk proteins, according to traditional old recipes for tempera paintings. In addition, beyond the egg components, the presence of caseins was also revealed, thus suggesting the use of milk as binding medium, fixative or stabilising agent. Furthermore, for the first time, the capability to discriminate the milk origin on the basis of alpha casein proteotypic peptides is reported, that are diagnostic for a given species, thus opening interesting perspectives in art and archaeological fields.

Keywords: Cultural heritage; Protein binders; Mass spectrometry; LC-ESI/Q-q-TOF; Casein; Proteotypic peptides; FT-IR spectromicroscopy

IR spectroscopy reveals effect of non-cytotoxic doses of anti-tumour drug on cancer cells by Florence Draux; Pierre Jeannesson; Cyril Gobinet; Josep Sule-Suso; Jacek Pijanka; Christophe Sandt; Paul Dumas; Michel Manfait; Ganesh D. Sockalingum (pp. 2293-2301).

Identifying early cellular events in response to a chemotherapy drug treatment, in particular at low doses that will destroy the highest possible number of cancer cells, is an important issue in patient management. In this study, we employed Fourier transform infrared spectroscopy as a potential tool to access such information. We used as model the non-small cell lung cancer cell line, Calu-1. They were exposed to cytostatic doses (0.1 to 100 nM for 24, 48 and 72 h) of gemcitabine, an anti-tumour drug, currently used in treatment of lung cancer patients. In these conditions, inhibition of cell proliferation ranges from weak (≤5%), to moderate (∼23%), to high (82–95%) without affecting cell viability. Following drug treatment as a function of doses and incubation times, the spectra of cell populations and of individual cells were acquired using a bench-top IR source and a synchrotron infrared microscope. It is demonstrated that spectral cell response to gemcitabine is detectable at sublethal doses and that effects observed on cell populations are similar to those from single cells. Using cluster analysis, spectra could be classified in two main groups: a first group that contains spectra of cells exhibiting a weak or moderate proliferation rate and a second group with spectra from cells presenting a high growth inhibition. These results are promising since they show that effects of subtoxic doses can also be monitored at the single-cell level with the clinical implications that this may have in terms of patient benefit and response to chemotherapy.

Keywords: Cell–drug interaction; FT-IR; Synchrotron; Cancer cells; Gemcitabine

Identifying cytoplasmic proteins that affect receptor clustering using fluorescence resonance energy transfer and RNA interference by Deepak Dibya; Suzanne Sander; Emily A. Smith (pp. 2303-2311).

Unraveling the complex, dynamic organization of the cell membrane can provide vital information about many aspects of cellular functions. Reported herein is a method for identifying cytoplasmic proteins that affect cell membrane protein organization. RNA interference (RNAi) is used to reduce the expression of select cytoplasmic proteins and a fluorescence resonance energy transfer (FRET) assay is used to measure changes in receptor microclustering. The advantage of this assay is that it does not require attaching fluorescent tags to the receptor. A change in energy transfer after reducing the expression of a cytoplasmic protein provides information about the protein’s role in altering receptor organization. As a demonstration of the method, cytoplasmic proteins involved in integrin microclustering have been identified. The cytoplasmic proteins targeted in this study include: dreadlock, integrin-linked kinase, paxillin, steamer duck, vinculin, rhea, focal adhesion kinase, and actin 42A. Reducing the expression of vinculin, paxillin, rhea, and focal adhesion kinase increased integrin microclustering, as measured by an increase in energy transfer in cells expressing αPS2CβPS integrins. No change in integrin microclustering was measured in a control cell line. Integrin mutants exhibited different microclustering properties compared to the wild-type integrins after reducing the expression of the listed cytoplasmic proteins. The results demonstrate the utility of this assay format, and provide insight into the function of cytoplasmic proteins in integrin microclustering. Figure Schematic of a method for identifying cytoplasmic proteins that affect cell membrane protein organization. RNA interference is used to reduce the expression of select cytoplasmic proteins and fluorescence resonance energy transfer assay is used to measure changes in receptor microclustering

Keywords: Bioanalytical methods; Fluorescence; Single cell analysis; Biological samples

Identifying cytoplasmic proteins that affect receptor clustering using fluorescence resonance energy transfer and RNA interference by Deepak Dibya; Suzanne Sander; Emily A. Smith (pp. 2303-2311).

Keywords: Bioanalytical methods; Fluorescence; Single cell analysis; Biological samples

Detection of microbial volatile organic compounds (MVOCs) by ion-mobility spectrometry by Carlo Tiebe; Hans Miessner; Bernhard Koch; Thomas Hübert (pp. 2313-2323).

Traces of microbial volatile organic compounds (MVOCs) in air can indicate the presence of growth of moulds in the indoor environment. Ion-mobility spectrometry is a very promising method for detection of these MVOCs, because of its high sensitivity. For development of an in-situ method for detection of MVOCs, a portable ion-mobility spectrometer (IMS) was used and test gases of 14 MVOCs and their respective mixtures were investigated. IMS spectra were recorded as a function of concentration of MVOCs in air. Drift time and mobility of reactant ions formed in positive polarity mode were determined and correlated with the mass-to-charge ratio (m/z) of the MVOCs investigated. The estimated detection limit has a specific value for each MVOC and is in the range 3 to 96 μg m⁻³ (1 to 52 ppb_V). Indoor trials show that IMS can indicate hidden mould growth.

Keywords: Trace gas analysis; MVOCs; Ion-mobility spectrometry; Mould detection

Detection of microbial volatile organic compounds (MVOCs) by ion-mobility spectrometry by Carlo Tiebe; Hans Miessner; Bernhard Koch; Thomas Hübert (pp. 2313-2323).

Keywords: Trace gas analysis; MVOCs; Ion-mobility spectrometry; Mould detection

Determination of organophosphate flame retardants in sediments by microwave-assisted extraction and gas chromatography–mass spectrometry with electron impact and chemical ionization by Hsiao-Wu Chung; Wang-Hsien Ding (pp. 2325-2334).

An efficient microwave-assisted extraction (MAE) procedure coupled to gas chromatography–mass spectrometry (GC–MS) with electron impact (EI) and chemical ionization (CI) has been developed to determine five organophosphate flame retardants (OPFRs) in marine and river sediments. The effects of various operating parameters on the quantitative extraction of the OPFRs through MAE were systematically investigated. Selected OPFRs were extracted from the sediments through MAE using 40 mL of acetone at 120 °C for 20 min. The limits of quantitation ranged from 0.1 to 0.4 ng/g (dry weight) in 2 g of the sediment samples. Moreover, as the chlorinated alkyl phosphates present no molecular ions in EI, GC–MS with furan-CI (furan-CI) was applied to confirm their determination in complex environmental samples. The recoveries of the selected OPFRs in spiked sediment samples ranged from 62% to 106% (relative standard derivation, 1−11%). The total concentrations of the selected OPFR residues in marine and river sediments ranged from 1.0 to 12.6 ng/g.

Keywords: Organophosphate flame retardants; Microwave-assisted extraction; GC–MS; Sediment analysis

Comparison of single-reflection near-infrared and attenuated total reflection mid-infrared spectroscopies to identify and monitor hydrocarbons spilled in the marine environment by G. Pérez-Caballero; J. M. Andrade; S. Muniategui; D. Prada (pp. 2335-2347).

The capability of single-reflection near-IR (NIR) spectroscopy to differentiate, characterize and monitor the fate of a set of hydrocarbons spilled in the marine environment was compared with that of multiple-reflection horizontal attenuated total reflection mid-IR (ATR-MIR) spectroscopy. Multivariate pattern recognition techniques [principal component analysis (PCA), multivariate polynomial regression, cluster analysis and potential curves] were applied to unravel the major trends of the weathering processes of four generic types of crude oils and two heavy fuel oils spilled under controlled conditions for almost 4 months. A chemical interpretation of the NIR spectra related the weathering processes and the PCA loadings, which had not already been done in the literature. Weathering for both light and heavy products was characterized by a contrast among the linear aliphatic structures (more volatile and easy to degrade) and the branched and aromatic structures (more recalcitrant). Potential curves were applied to model each product and determine objectively whether unknown samples could be classified correctly. Polynomial regression on the PCA scores was employed to evaluate the time elapsed from the oil spillage to its sampling; this represents a new approach to assess the age of a hydrocarbon lump. In general, NIR spectroscopy yielded good results when light crude oils were studied, whereas ATR-MIR spectroscopy led to satisfactory results for both light and heavy products.

Keywords: Oil spills; Single-reflection near-infrared spectroscopy; Attenuated total reflection mid-infrared spectroscopy; Crude oil; Fuel oil

Optimization and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, cotinine, trans-3′-hydroxycotinine and norcotinine in human oral fluid by Diaa M. Shakleya; Marilyn A. Huestis (pp. 2349-2357).

An analytical procedure was developed and validated for the simultaneous identification and quantification of nicotine, cotinine, trans-3′-hydroxycotinine, and norcotinine in 0.5 mL of human oral fluid collected with the Quantisal™ oral fluid collection device. Solid phase extraction and liquid chromatography-tandem mass spectrometry with multiple reaction monitoring were utilized. Endogenous and exogenous interferences were extensively evaluated. Limits of quantification were empirically identified by decreasing analyte concentrations. Linearity was from 1 to 2,000 ng/mL for nicotine and norcotinine, 0.5 to 2,000 ng/mL for trans-3′-hydroxycotinine, and 0.2 to 2,000 ng/mL for cotinine. Correlation coefficients for calibration curves were >0.99 and analytes quantified within ±13% of target at all calibrator concentrations. Suitable analytical recovery (>91%) was achieved with extraction efficiencies >56% and matrix effects <29%. This assay will be applied to the quantification of nicotine and metabolites in oral fluid in a clinical study determining the most appropriate nicotine biomarker concentrations differentiating active, passive, and environmental nicotine exposure.

Keywords: Nicotine; Cotinine; trans-3′-Hydroxycotinine; Norcotinine; Oral fluid; Tobacco biomarkers; LCMSMS; Saliva

Determination of low-level ink photoinitiator residues in packaged milk by solid-phase extraction and LC-ESI/MS/MS using triple-quadrupole mass analyzer by Dong-xu Shen; Hong-zhen Lian; Tao Ding; Jin-zhong Xu; Chong-yu Shen (pp. 2359-2370).

A confirmatory and quantitative method based on liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) has been developed for simultaneous determination of seven photoinitiator residues: benzophenone, (1-hydroxycyclohexyl)phenylketone (Irgacure 184), isopropylthioxanthone (ITX), 2-ethylhexyl-(4-dimethylamino)benzoate (EHA or EHDAB), 2-methyl-1-[4-(methylthio)phenyl]-2-(4-morpholinyl)-1-propanone (Irgacure 907), (2,4,6-trimethylbenzoyl)diphenylphosphine oxide (TPO) and 2-benzyl-2-(dimethylamino)-1-(4-morpholinophenyl)-1-butanone (Irgacure 369) in packaged milk and related packaging materials. Residues of photoinitiators were extracted from milk using acetonitrile, and further enriched and purified on HLB solid-phase extraction cartridges prior to being analyzed by LC-ESI/MS/MS with selected reaction monitoring mode, while photoinitiators in packaging materials were extracted using the same solvent. Satisfactory recovery (from 80 to 111%), intra- and inter-day precision (below 12%), and low limits of quantification (from 0.1 to 5.0 μg kg⁻¹) were evaluated from spiked samples at three concentration levels (5.0, 10.0 and 25.0 μg kg⁻¹ for Irgacure 184 and 2.5, 5.0 and 25.0 μg kg⁻¹ for others). These excellent validation data suggested the possibility of using the LC-ESI/MS/MS method for simultaneous determination of low-level photoinitiator residues migrating from printed food-packaging materials into milk. The method has been successfully applied to the analysis of real samples of different fat contents ranging from 8 to 30 g L⁻¹. The photoinitiator residues were revealed to be higher in milk with higher fat content and the most important contaminations were benzophenone and ITX in concentration ranges of 2.84–18.35 and 0.83–8.87 μg kg⁻¹, respectively. Figure Recoveries of photoinitiators dealt with different kinds of elution solvents in solid-phase extraction process

Keywords: Photoinitiator; Milk; Packaging materials; SPE; Triple-quadrupole mass spectrometry

Solid-contact pH-selective electrode using multi-walled carbon nanotubes by Gastón A. Crespo; Derese Gugsa; Santiago Macho; F. Xavier Rius (pp. 2371-2376).

Multi-walled carbon nanotubes (MWCNT) are shown to be efficient transducers of the ionic-to-electronic current. This enables the development of a new solid-contact pH-selective electrode that is based on the deposition of a 35-µm thick layer of MWCNT between the acrylic ion-selective membrane and the glassy carbon rod used as the electrical conductor. The ion-selective membrane was prepared by incorporating tridodecylamine as the ionophore, potassium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate as the lipophilic additive in a polymerized methylmethacrylate and an n-butyl acrylate matrix. The potentiometric response shows Nernstian behaviour and a linear dynamic range between 2.89 and 9.90 pH values. The response time for this electrode was less than 10 s throughout the whole working range. The electrode shows a high selectivity towards interfering ions. Electrochemical impedance spectroscopy and chronopotentiometry techniques were used to characterise the electrochemical behaviour and the stability of the carbon-nanotube-based ion-selective electrodes.

Keywords: Potentiometric ion-selective electrodes; Solid-state sensors; pH; Multi-walled carbon nanotubes

Solid-contact pH-selective electrode using multi-walled carbon nanotubes by Gastón A. Crespo; Derese Gugsa; Santiago Macho; F. Xavier Rius (pp. 2371-2376).

Keywords: Potentiometric ion-selective electrodes; Solid-state sensors; pH; Multi-walled carbon nanotubes

Sensitive fluorescent probes for determination of hydrogen peroxide and glucose based on enzyme-immobilized magnetite/silica nanoparticles by Qing Chang; Lihua Zhu; Guodong Jiang; Heqing Tang (pp. 2377-2385).

Sensitive fluorescent probes for the determination of hydrogen peroxide and glucose were developed by immobilizing enzyme horseradish peroxidase (HRP) on Fe₃O₄/SiO₂ magnetic core–shell nanoparticles in the presence of glutaraldehyde. Besides its excellent catalytic activity, the immobilized enzyme could be easily and completely recovered by a magnetic separation, and the recovered HRP-immobilized Fe₃O₄/SiO₂ nanoparticles were able to be used repeatedly as catalysts without deactivation. The HRP-immobilized nanoparticles were able to activate hydrogen peroxide (H₂O₂), which oxidized non-fluorescent 3-(4-hydroxyphenyl)propionic acid to a fluorescent product with an emission maximum at 409 nm. Under optimized conditions, a linear calibration curve was obtained over the H₂O₂ concentrations ranging from 5.0 × 10⁻⁹ to 1.0 × 10⁻⁵ mol L⁻¹, with a detection limit of 2.1 × 10⁻⁹ mol L⁻¹. By simultaneously using glucose oxidase and HRP-immobilized Fe₃O₄/SiO₂ nanoparticles, a sensitive and selective analytical method for the glucose detection was established. The fluorescence intensity of the product responded well linearly to glucose concentration in the range from 5.0 × 10⁻⁸ to 5.0 × 10⁻⁵ mol L⁻¹ with a detection limit of 1.8 × 10⁻⁸ mol L⁻¹. The proposed method was successfully applied for the determination of glucose in human serum sample. Figure Sensitive fluorescent probes for the determination of H₂O₂ and glucose were developed by immobilizing enzyme horseradish peroxidase on Fe₃O₄/SiO₂ magnetic core-shell nanoparticles.

Keywords: Fe₃O₄/SiO₂ magnetic nanoparticles; Immobilized enzyme; Hydrogen peroxide; Glucose; Fluorometry; Catalysts; Clinical/biomedical analysis

Pesticide extraction from table grapes and plums using ionic liquid based dispersive liquid–liquid microextraction by Lidia M. Ravelo-Pérez; Javier Hernández-Borges; Antonio V. Herrera-Herrera; Miguel Ángel Rodríguez-Delgado (pp. 2387-2395).

Room temperature ionic liquids (RTILs) have been used as extraction solvents in dispersive liquid–liquid microextraction (DLLME) for the determination of eight multi-class pesticides (i.e. thiophanate-methyl, carbofuran, carbaryl, tebuconazole, iprodione, oxyfluorfen, hexythiazox, and fenazaquin) in table grapes and plums. The developed method involves the combination of DLLME and high-performance liquid chromatography with diode array detection. Samples were first homogenized and extracted with acetonitrile. After evaporation and reconstitution of the extract in water containing sodium chloride, a quick DLLME procedure that used the ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate ([C₆MIM][PF₆]) and methanol was developed. The RTIL dissolved in a very small volume of acetonitrile was directed injected in the chromatographic system. The comparison between the calibration curves obtained from standards and from spiked sample extracts (matrix-matched calibration) showed the existence of a strong matrix effect for most of the analyzed pesticides. A recovery study was also developed with five consecutive extractions of the two types of fruits spiked at three concentration levels. Mean recovery values were in the range of 72–100% for table grapes and 66–105% for plum samples (except for thiophanate-methyl and carbofuran, which were 64–75% and 58–66%, respectively). Limits of detection (LODs) were in the range 0.651–5.44 µg/kg for table grapes and 0.902–6.33 µg/kg for plums, representing LODs below the maximum residue limits (MRLs) established by the European Union in these fruits. The potential of the method was demonstrated by analyzing 12 commercial fruit samples (six of each type).

Keywords: Room temperature ionic liquids; Dispersive liquid–liquid microextraction; Pesticides; Table grapes; Plums; High-performance liquid chromatography

Determination of human urinary kanamycin in one step using urea-enhanced surface plasmon resonance light-scattering of gold nanoparticles by Xinyi Wang; Mingjian Zou; Xiao Xu; Rong Lei; Kean Li; Na Li (pp. 2397-2403).

The purpose of this study was to establish a simple, sensitive analytical method for kanamycin (KANA) in human urine. Enhancement of the plasmon resonance light-scattering (PRLS) of gold nanoparticles (AuNPs) by KANA provided the basis for this analytical method. At pH 6.7, KANA induced AuNPs aggregation with enhanced PRLS. The PRLS of the AuNPs–KANA system was further enhanced by addition of urea. The linear range and detection limit for KANA were from 20–800 nmol L⁻¹ and 2 nmol L⁻¹, respectively. Potential interfering substances present in urine had a negligible effect on the determination, thus preliminary sample separations were not necessary. Recovery of KANA from spiked human urine was 94–104%. This simple, sensitive method, using urea to enhance the PRLS of the AuNPs–KANA system, may provide a new approach for determination of compounds rich in OH groups. Figure Schematic diagram of KANA-induced aggregation of AuNPs and enhancement by urea, including magnification for illustration of N···H-O hydrogen bond between KANA and urea

Keywords: Kanamycin; Plasmon resonance light-scattering; Gold nanoparticles; Urea enhancement

Flat hydrogel substrate for atomic force microscopy to observe liposomes and lipid membranes by Akihiko Takagi; Hitomi Hokonohara; Tomoji Kawai (pp. 2405-2409).

In order to avoid denaturation of biomolecules due to strong adsorption on solid surfaces, a soft substrate has to be used for atomic force microscopy (AFM) observation. We propose a hydrophilic agarose gel surface as a soft substrate for AFM to observe liposomes and lipid membranes. Although our simple method does not require any delicate control at the molecular level, an agarose gel surface can be simply flattened to 0.3 nm in roughness using an atomically flat solid surface during gelation. The AFM images revealed that liposomes were unruptured on the gel surface at low liposome density, whereas an unruptured state was difficult to obtain on a solid surface like mica. This indicates that the weak interaction between the liposome and the soft surface inhibits the liposome from rupturing, and also that the surface rougher than the solid surface prevents lateral diffusion of the liposomes along the surface to be fused. Increasing the liposome density resulted in a lipid membrane at various thicknesses forming on the hydrogel surface by the fusion and rupture of liposomes. Using the soft substrate, it can be expected to promote investigations of structures and functions of biomolecules at the nanometer scale under physiological conditions with AFM.

Keywords: AFM (atomic force microscopy); Biopolymer/lipid; Liposome; Hydrogel

Flat hydrogel substrate for atomic force microscopy to observe liposomes and lipid membranes by Akihiko Takagi; Hitomi Hokonohara; Tomoji Kawai (pp. 2405-2409).

Keywords: AFM (atomic force microscopy); Biopolymer/lipid; Liposome; Hydrogel

Rapid data analysis method for differential mobility cytometry by Randall D. Reif; Kelong Wang; Dimitri Pappas (pp. 2411-2413).

Differential mobility cytometry (DMC) has recently been established as a powerful method to capture cells and study adhesion processes. DMC uses an oscillation system and cell affinity chromatography to monitor cells as they adhere to a surface. In the past, differential images had to be created individually which limited the throughput of the method. A new method to create differential images is presented. The method involves the subtraction of short movies from each other to create a stack of differential images that can be easily analyzed. In the future, this method will make DMC more accessible and improve throughput.

Keywords: Differential mobility cytometry; Cell affinity chromatography; Cell adhesion

Rapid data analysis method for differential mobility cytometry by Randall D. Reif; Kelong Wang; Dimitri Pappas (pp. 2411-2413).

Keywords: Differential mobility cytometry; Cell affinity chromatography; Cell adhesion

Droplet-based magnetically activated cell separation: analysis of separation efficiency based on the variation of flow-induced circulation in a pendent drop by Youngho Kim; Sang Ho Lee; Byungkyu Kim (pp. 2415-2421).

Under the assumption that separation efficiencies are mainly affected by the velocity of flow-induced circulation due to buffer injection in a pendent drop, this paper describes an analysis of the separation efficiency of a droplet-based magnetically activated cell separation (DMACS) system. To investigate the velocity of the flow-induced circulation, we supposed that numerous flows in a pendent drop could be considered as a “theoretically normalized” flow (or conceptually normalized flow, CNF) based on the Cauchy–Goursat theorem. With the morphological characteristics (length and duration time) of a pendent drop depending on the initial volume, we obtained the velocities of the CNF. By measuring the separation efficiencies for different initial volumes and by analyzing the separation efficiency in terms of the velocity of the CNF, we found that the separation efficiencies (in the case of a low rate of buffer injection; 5 and 15 μl·min^-1) are mainly affected by the velocity of the CNF. Moreover, we confirmed that the phenomenological features of a pendent drop cause a fluctuation of its separation efficiencies over a range of specific volumes (initial volumes ranging from 40 to 80 µl), because of the “sweeping-off” phenomenon, that is, positive cells gathered into the positive fraction are forced to move away from the magnetic side by flow-induced circulation due to buffer injection. In addition, from the variation of the duration time, that is, the interval between the beginning of injection of the buffer solution and the time at which a pendent drop detaches, it could also be confirmed that a shorter duration time leads to decrease of the number of positive cells in negative fraction regardless of the rate of buffer injection (5, 15, and 50 μl·min^-1). Therefore, if a DMACS system is operated with a 15 μl·min^-1 buffer injection flow rate and an initial volume of 80 μl or more, we would have the best efficiency of separation in the negative fraction.

Keywords: Cell separation; Droplet-based magnetically activated cell separation; Flow-induced circulation; Pendent drop; Magnet

Erratum to: Immuno-PCR-based quantification of multiple phosphorylated tau-epitopes linked to Alzheimer’s disease by David Singer; Hilkka Soininen; Irina Alafuzoff; Ralf Hoffmann (pp. 2423-2423).

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Analytical and Bioanalytical Chemistry (v.395, #7)

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Analytical and Bioanalytical Chemistry (v.395, #7)