Infrared Splitting Factor Research Articles

Preservation of bone organic fraction is not predictive of the preservation of bone inorganic fraction when assessing stable isotope analysis sample quality control measures

Stable isotope analysis of carbon (C), nitrogen (N), hydrogen (H), and oxygen (O) of archaeological bone has become an increasingly common research method for interpreting human behavior in the past. However, diagenesis of skeletal material can invalidate stable isotope ratios, thereby compromising interpretations. We examine patterns of bone diagenesis using infrared spectroscopy of bone bioapatite samples in relation to indicators of organic preservation quality indicators. We assess crystallinity, an indicator of bioapatite preservation, using the infrared splitting factor (IR-SF) and carbonate content (carbonate to phosphate ratio, C/P) calculated from infrared spectra. We then test the assumption that if the organic fraction of bone is preserved, the mineral fraction will also be unaffected by postmortem chemical alteration. We analyzed 454 bone bioapatite and extracted bone organic sample pairs from modern, historic, and prehistoric humans. Consistent with previous studies, we observed a strong, statistically significant negative linear relationship between IR-SF and C/P (r = −0.855, p < 0.001). Modern bone bioapatite samples unaltered by diagenesis have low IR-SF and high C/P values. There was no significant association between the collagen yield or atomic C:N ratio and IR-SF or C/P values. The range of variation in IR-SF and C/P values for samples with organic yields between 0 and >20 percent spanned the range of modern bone bioapatite unaltered by diagenesis as well as bone bioapatite significantly affected by diagenesis. The lack of predictive patterning between bone inorganic and organic diagenesis suggests that the depositional context and site formation history play critical and independent roles in the organic and mineral fraction of bone. Thus, the preservation of the organic fraction of bone is not predictive of the preservation of the inorganic fraction (e.g., bioapatite).

Multifactorial approach to describe early diagenesis of bones: The case study of the Merovingian cemetery of Saint-Linaire (France)

The excavation of the Merovingian cemetery of Saint-Linaire (France) was an opportunity to describe the completeness of the tombs preserved from soil erosion. An anthropobiological study was carried out on the osteological material and the different categories of graves. On the basis of a complete archaeo-anthropological corpus we have undertaken an analysis of the differential preservation of the bones according to the different archaeobiological parameters such as the type of bone, the individual age but also the type of grave such as earthen graves, coffin, or sarcophagus.In order to establish whether and how funerary practices had impacted the early diagenesis of the bones, FTIR was used to investigate the carbonate and fluoride content of the bones, and the infra-red splitting factor (SF), while XRD analysis was performed to estimate the crystallinity index (CI). In addition, the micro, macro and total porosity of the bones were measured by water adsorption, and bone carbon and nitrogen amounts were obtained by EA-IRMS. These diagenetic variables were analysed with regards to environmental (grave filling: humus, calcareous, clay), cultural (type of grave) and biological (mature versus immature individuals) factors. While there is a tendency of a better preservation of the skeletons in humus, and of juvenile bones to show less post-mortem alteration than the adult skeletons, the most important factor which determines the fate of the skeleton is the type of bone, with a preferential preservation of the ribs at Saint-Linaire.

Valorization of animal bone into phosphorus biofertilizer: effects of animal species, thermal processing method, and production temperature on phosphorus availability

ABSTRACT Thermal processing has been promoted to recycle phosphorus (P) contained within animal bone. There is a dearth of information on the mechanisms that control P solubility and availability from thermally treated bones. Chemical extraction, XRD, and FTIR were used to elucidate P solubility, and incubation and pot experiments to evaluate P bioavailability from different animal bones, thermal processing methods (pyrolysis vs. combustion), and production temperatures. Pyrolysis resulted in higher formic P than combustion, particularly at temperatures >500°C. Chicken bone showed the highest water-soluble P content (p< 0.001), irrespective of processing methods and temperatures. In contrast, pig bone exhibited the lowest water-soluble P despite its high total P content. The XRD and FTIR studies confirmed higher degree of crystallization for those produced from pig bone, combustion, and higher temperatures (>700°C). Infrared splitting factor and width at 85% of the height of the 604 cm−1 peak were used to assess bioapatite crystallinity, and confirmed a negative correlation between crystallinity and P availability (p< 0.001), indicating the attribution of bioapatite crystallization for low soluble P during combustion and higher temperatures. In low pH soil (pH~4), the addition of thermally treated bones increased Olsen-P and plant P uptake by two- to five-folds compared with unamended soils; however, no significant variation was observed in higher pH soil (pH~7). The finding suggested processing bones below temperatures of 700°C resulted in less crystallinity, thus higher P solubility. The P-fertilizing value of thermally treated bones was more pronounced in lower pH soil.

Using bone bioapatite yield for quality control in stable isotope analysis applications

Since the late 1970s, stable isotope analysis of bone has become a routine method in archaeological science for reconstructing an individual’s life history. There is published guidance for assessing bone quality prior to stable isotope analysis, including measurement of bioapatite molecular structure, collagen yield, and collagen element content. This study investigated bioapatite yield as an additional quality indicator. Bioapatite and collagen were extracted from 182 modern (20th Century) and 272 prehistoric (5420–230 cal BP) human skeletal elements. As expected, modern remains were well-preserved, with ranges of 3–28% for collagen yield and 3.1–3.4 for the atomic carbon-to-nitrogen (C:N) ratio; bioapatite yield ranged from 21% to 63%. There was a significant, but only fair, negative correlation between bioapatite and collagen yields of modern samples (r = -0.31, p < 0.0001). Conversely, no correlation was observed between bioapatite and collagen yields of prehistoric samples, suggesting preservation of one is not indicative of preservation of the other. Prehistoric sample condition was evaluated by measuring the infrared splitting factor (IR-SF) to evaluate bioapatite crystallinity and the carbonate-to-phosphate (C/P) ratio to measure carbonate concentration; yield and C:N ratio were used to evaluate collagen condition. Prehistoric samples in the best condition had bioapatite yields between 29% and 62%, within the range of modern samples. In contrast, prehistoric samples in poor condition had significantly higher bioapatite yields, suggesting diagenetic addition of material. The range of bioapatite yields from modern bone samples, 21–63%, is proposed as the least conservative acceptable threshold for screening bone prepared for stable isotope analysis.

Screening archaeological bone for palaeogenetic and palaeoproteomic studies.

The recovery and analysis of ancient DNA and protein from archaeological bone is time-consuming and expensive to carry out, while it involves the partial or complete destruction of valuable or rare specimens. The fields of palaeogenetic and palaeoproteomic research would benefit greatly from techniques that can assess the molecular quality prior to sampling. To be relevant, such screening methods should be effective, minimally-destructive, and rapid. This study reports results based on spectroscopic (Fourier-transform infrared spectroscopy in attenuated total reflectance [FTIR-ATR]; n = 266), palaeoproteomic (collagen content; n = 226), and palaeogenetic (endogenous DNA content; n = 88) techniques. We establish thresholds for three different FTIR indices, a) the infrared splitting factor [IRSF] that assesses relative changes in bioapatite crystals’ size and homogeneity; b) the carbonate-to-phosphate [C/P] ratio as a relative measure of carbonate content in bioapatite crystals; and c) the amide-to-phosphate ratio [Am/P] for assessing the relative organic content preserved in bone. These thresholds are both extremely reliable and easy to apply for the successful and rapid distinction between well- and poorly-preserved specimens. This is a milestone for choosing appropriate samples prior to genomic and collagen analyses, with important implications for biomolecular archaeology and palaeontology.

Bone diagenesis in a Mycenaean secondary burial (Kastrouli, Greece)

This paper presents the characteristics of bone diagenesis in a secondary commingled Mycenaean burial in Kastrouli (Phocis, Greece) through the histological (light microscopy), physical (FTIR-ATR), and biochemical (collagen) analysis of seventeen human (including two petrous bones) and seven animal bones. Post-mortem modifications in bone microstructure, bioapatite, and collagen were characteristic of burial environments with seasonal groundwater and temperature fluctuations. The two human petrous bones displayed a lack of microscopic focal destruction (MFD) sites and a generally good histological preservation, but although a small sample size, did not show any better bioapatite and collagen preservation compared with human femora. Intra-site variations were defined by three main diagenetic patterns that display differences in histological modifications, crystallinity changes, and collagen degradation. These different patterns were either related to different microenvironment conditions and/or influenced by possible differences in the early taphonomic histories experienced by bones prior to secondary deposition. Further, this study highlights the importance of infrared splitting factor (IRSF), carbonate/phosphate ratio (C/P), and general histological index (GHI) for the qualitative assessment of archaeological bone, and the potential use of amide/phosphate ratio (Am/P) as a collagen predictor.

Bone diagenesis in a Mycenaean secondary burial (Kastrouli, Greece)

This paper presents the characteristics of bone diagenesis in a secondary commingled Mycenaean burial in Kastrouli (Phocis, Greece) through the histological (light microscopy), physical (FTIR-ATR), and biochemical (collagen) analysis of seventeen human (including two petrous bones) and seven animal bones. Post-mortem modifications in bone microstructure, bioapatite, and collagen were characteristic of burial environments with seasonal groundwater and temperature fluctuations. The two human petrous bones displayed a lack of microscopic focal destruction (MFD) sites and a generally good histological preservation, but although a small sample size, did not show any better bioapatite and collagen preservation compared with human femora. Intra-site variations were defined by three main diagenetic patterns that display differences in histological modifications, crystallinity changes, and collagen degradation. These different patterns were either related to different microenvironment conditions and/or influenced by possible differences in the early taphonomic histories experienced by bones prior to secondary deposition. Further, this study highlights the importance of infrared splitting factor (IRSF), carbonate/phosphate ratio (C/P), and general histological index (GHI) for the qualitative assessment of archaeological bone, and the potential use of amide/phosphate ratio (Am/P) as a collagen predictor.

Petrous bone diagenesis: a multi-analytical approach

The discovery of petrous bone as an excellent repository for ancient biomolecules has been a turning point in biomolecular archaeology, especially in aDNA research, but excessive and uncontrolled sampling could result in loss of this valuable resource for future research.This study reports on the histological (optical microscopy), physical (FTIR-ATR), elemental (CHN) and biochemical (collagen and DNA analysis) preservation of 15 human petrous bones spanning from the c. 2100 BCE to 1850 CE. Through the combined application of a number of diagenetic parameters (general histological index; infrared splitting factor; carbonate/phosphate ratio; amide/phosphate ratio; col wt%; % C, % N and C/N of whole bone and collagen; % endogenous DNA), we provide new insights into petrous bone micromorphological characteristics and diagenesis, and new evidence to enhance screening practices for aDNA and collagen analysis.

Preparation of bone powder for FTIR-ATR analysis: The particle size effect

Fourier transform infrared (FTIR) spectroscopy using attenuated total reflection (ATR) is commonly used for the examination of bone. During sample preparation bone is commonly ground, changing the particle size distribution. Although previous studies have examined changes in crystallinity caused by the intensity of grinding using FTIR, the effect of sample preparation (i.e. particle size and bone tissue type) on the FTIR data is still unknown.This study reports on the bone powder particle size effects on mid-IR spectra and within sample variation (i.e. periosteal, mesosteal, trabecular) using FTIR-ATR. Twenty-four archaeological human and faunal bone samples (5 heated and 19 unheated) of different chronological age (Neolithic to post-Medieval) and origin (Belgium, Britain, Denmark, Greece) were ground using either (1) a ball-mill grinder, or (2) an agate pestle and mortar, and split into grain fractions (>500 μm, 250–500 μm, 125–250 μm, 63–125 μm, and 20–63 μm).Bone powder particle size has a strong but predictable effect on the infrared splitting factor (IRSF), carbonate/phosphate (C/P) ratio, and amide/phosphate (Am/P) values. The absorbance and positions of the main peaks, the 2nd derivative components of the phosphate and carbonate bands, as well as the full width at half maximum (FWHM) of the 1010 cm−1 phosphate peak are particle size dependent. This is likely to be because of the impact of the particle size on the short- and long-range crystal order, as well as the contact between the sample and the prism, and hence the penetration depth of the IR light. Variations can be also observed between periosteal, cortical and trabecular areas of bone. We therefore propose a standard preparation method for bone powder for FTIR-ATR analysis that significantly improves accuracy, consistency, reliability, replicability and comparability of the data, enabling systematic evaluation of bone in archaeological, anthropological, paleontological, forensic and biomedical studies.

A universal curve of apatite crystallinity for the assessment of bone integrity and preservation

The reliable determination of bioapatite crystallinity is of great practical interest, as a proxy to the physico-chemical and microstructural properties, and ultimately, to the integrity of bone materials. Bioapatite crystallinity is used to diagnose pathologies in modern calcified tissues as well as to assess the preservation state of fossil bones. To date, infrared spectroscopy is one of the most applied techniques for bone characterisation and the derived infrared splitting factor (IRSF) has been widely used to practically assess bioapatite crystallinity. Here we thoroughly discuss and revise the use of the IRSF parameter and its meaning as a crystallinity indicator, based on extensive measurements of fresh and fossil bones, virtually covering the known range of crystallinity degree of bioapatite. A novel way to calculate and use the infrared peak width as a suitable measurement of true apatite crystallinity is proposed, and validated by combined measurement of the same samples through X-ray diffraction. The non-linear correlation between the infrared peak width and the derived ISRF is explained. As shown, the infrared peak width at 604 cm−1 can be effectively used to assess both the average crystallite size and structural carbonate content of bioapatite, thus establishing a universal calibration curve of practical use.

Comparison of transmission FTIR, ATR, and DRIFT spectra: implications for assessment of bone bioapatite diagenesis

Evaluation of diagenesis in bioapatite samples is an important step for screening bone and tooth samples for stable isotope analysis to ensure in vivo signatures are obtained. Fourier transform infrared (FTIR) spectroscopy is one tool used to evaluate diagenesis by anthropological geochemists and commonly employs calculating the infrared splitting factor (IR-SF) and carbonate-to-phosphate ratio (C/P). There are three commonly used sample preparation techniques for vibrational spectroscopy: transmission FTIR, attenuated total reflection (ATR), and diffuse reflectance infrared Fourier transform (DRIFT). Each technique characterizes the internal vibrations of particular molecular groups, such as carbonate (CO3) and phosphate (PO4), using different optical properties to detect absorbance bands. Spectra are correlated between techniques using correction equations that account for differences in optical properties. Traditionally, anthropologists have used spectra produced by transmission FTIR to assess diagenesis, most commonly using two indices (IR-SF and C/P); however, recently the ATR and DRIFT techniques have been used as an alternative to transmission FTIR. The spectra produced by the three techniques are thought to be interchangeable in calculating the indices used to assess diagenesis. In this study, we evaluated the interchangeability of the three FTIR techniques by analyzing 452 prehistoric and modern bioapatite samples. Results indicate that IR-SF and C/P values are not equivalent between the three techniques. However, ATR produced more reliable results and was comparable to transmission FTIR. The DRIFT method showed much lower resolution, and did not distinguish between modern and prehistoric bioapatite samples as clearly.

Association between ancient bone preservation and dna yield: A multidisciplinary approach

Ancient molecular typing depends on DNA survival in archaeological bones. Finding valuable tools to predict DNA presence in ancient samples, which can be measured prior to undertaking a genetic study, has become an important issue as a consequence of the peculiarities of archaeological samples. Since the survival of DNA is explained by complex interrelations of multiple variables, the aim of the present study was to analyze morphological, structural, chemical, and biological aspects of a set of medieval human bones, to provide an accurate reflection of the state of preservation of the bony components and to relate it with DNA presence. Archaeological bones that yielded amplifiable DNA presented high collagen content (generally more than 12%), low racemization values of aspartic acid (lesser than 0.08), leucine and glutamic acid, low infrared splitting factor, small size of crystallite, and more compact appearance of bone in the scanning electron micrographs. Whether these patterns are characteristic of ancient bones or specific of each burial site or specimen requires further investigation.

A complementary approach using analytical pyrolysis to evaluate collagen degradation and mineral fossilisation in archaeological bones: The case study of Vicenne-Campochiaro necropolis (Italy)

Spectroscopic (FTIR), diffractometric (XRD), thermogravimetric (TGA) analyses were applied along with off-line analytical pyrolysis combined with GC–MS (Py/GC–MS) in order to understand the relationships between mineral composition, degree of crystallinity of carbonated hydroxylapatite (cHA), organic content and the occurrence of collagen content in archaeological bones subjected to diagenesis. Osteological samples came from Vicenne-Campochiaro necropolis in Molise (Italy). Bones mineral composition was investigated by XRD while cHA crystallinity was assessed by the crystallinity index (CI) obtained from diffractometric analysis and the Infrared Splitting Factor (IRSF) obtained from FTIR spectra. The organic fraction of the archaeological bones was quantified by TGA and characterised by Py/GC–MS. The quantity of 2,5-diketopiperazines (DKPs) produced upon pyrolysis was used as a parameter to assess quantities of collagen inside each specimen. Attention was paid to proline (Pro) and hydroxyproline (Hyp) containing DKPs, in particular cyclo(Hyp–Pro), recognised to be specific marker of collagen pyrolysis. The relative distributions of DKPs in the pyrolysates of archaeological samples and fresh bovine bone sample were rather similar suggesting good preservation of the collagen and non-collagenous proteins in all the ancient osteological tissues. The absolute quantity of DKPs in the bone samples were different and in good accordance with the total organic matter detected by TGA and consistent with cHA high degree of crystallinity and FTIR data.

Variations in Atomic Disorder in Biogenic Carbonate Hydroxyapatite Using the Infrared Spectrum Grinding Curve Method

AbstractBiogenic carbonate hydroxyapatite crystals are inherently disordered at the atomic level due mainly to the substitutions of various ions in the crystal structure, and, in the case of the bone family of materials, to the fact that these very small crystals have a very large surface‐to‐bulk ratio. Characterization of the extent of disorder is of much interest, as this relates to the stability and hence solubility of the crystals. Here the infrared spectrometry grinding curve approach developed for calcite, is adapted to carbonate hydroxyapatites. The infrared splitting factor is plotted against the full width at half height of the strong phosphate absorption peak as a function of increased grinding of the sample. By doing so, the contribution of particle size to the shape of the peaks is better separated from the contribution of atomic disorder to peak shape. It is shown that differences in disorder exist between dentine, cementum, and bone crystals which could reflect crystal size and/or atomic defects within the crystal. It is als shown that systematic differences exist between enamel samples from different taxa, which we assume only reflects atomic disorder differences within these large crystals. The method can be used to characterize atomic disorder in natural hydroxyapatites, as well as in the many different types of synthetic hydroxyapatites used for biomedical implants.

Atomic Disorder in Fossil Tooth and Bone Mineral: An FTIR Study Using the Grinding Curve Method

Bone and tooth mineral generally undergo diagenetic changes. These changes in the carbonate hydroxyapatite structure and composition can affect the signals embedded in the mineral phase, such as migration behavior, age of the specimen and the reconstruction of past environments. Mineral preservation state can be assessed using infrared spectroscopy which provides information on crystal disorder at the atomic level and mineral composition. Here we present a new approach to evaluate carbonate hydroxyapatite atomic disorder using infrared spectroscopy and the standard KBr sample mounting method. We show that by repeated grinding of the sample and then plotting the infrared splitting factor against the width of the major phosphate absorption peak after each grinding, grinding curves with well defined trend lines can be obtained. The offsets between curves reflect differences in atomic disorder. We show that grinding curve offsets can be used to evaluate the state of preservation of bone, dentine and enamel mineral.

Why do crystallinity values fail to predict the extent of diagenetic alteration of bone mineral?

Spectroscopic indicators of bone crystallinity such as the infrared splitting factor (IRSF) are commonly used to determine the general state of preservation of ancient bone. In principle such indices might be expected to act as a proxy for alteration of bone mineral and thus could be used to screen bones (or portions of bones) for likely preservation of in vivo biogenic trace element and stable isotope signals. We tested the relationship between IRSF and bone mineral composition in two suites of well-characterised recent and Pleistocene bones. Initially, crystallinity change and trace element uptake are correlated, apparently both controlled by decomposition of the organic phase and exposure of bone crystal surfaces. This relationship breaks down in older bones where authigenic phosphate growth and mineral–pore water interactions are no longer rate-limited by the breakdown of collagen and exposure of crystal surfaces. In these conditions the extent of chemical alteration of bone will be controlled by site specific conditions, and thus while FTIR spectra of bone provide a broad indication of organic content and apatite recrystallisation, they are not reliable proxies for the degree of diagenetic alteration in terms of biogenic geochemical signals.