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Focus

Comparison of transmission FTIR, ATR, and DRIFT spectra:

implications for assessment of bone bioapatite diagenesis

Melanie M. Beasley a,*, Eric J. Bartelink b, Lacy Taylor c, Randy M. Miller d

a Department of Anthropology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0532, USAb Department of Anthropology, California State University, Chico, 400 West First Street, Chico, CA 95929-0400, USAc Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322, USAd Department of Chemistry, California State University, Chico, 400 West First Street, Chico, CA 95929-0210, USA

a r t i c l e i n f o

Article history:

Received 8 October 2012

Received in revised form

26 February 2014

Accepted 1 March 2014

Keywords:

Diagenesis

Bone bioapatite

Transmission FTIR

ATR

DRIFT

a b s t r a c t

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 reection (ATR), and diffuse reectance infrared Fourier transform (DRIFT). Each tech-

nique 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 modernbioapatite 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.

2014 Elsevier Ltd. All rights reserved.

1. Introduction

Diagenesis is a complex process that involves physical and

chemical postmortem alterations to bones and teeth that is directly

inuenced by the burial environment, including local geological

and groundwater conditions (Nielsen-Marsh and Hedges, 2000).Asbone collagen and bioapatite show signs of diagenesis, changes in

the molecular structure of bone can be detected by vibrational

spectroscopy (i.e., infrared and Raman) (Carden and Morris, 2000;

King et al., 2011). Anthropologists commonly use a potassium

bromide (KBr) pelleting technique to prepare samples for trans-

mission Fourier transform infrared (FTIR) spectroscopy to evaluate

diagenesis of the carbonate and phosphate components of bone

and enamel bioapatite (Garvie-Lok et al., 2004; Lee-Thorp and

Sponheimer, 2003; Lee-Thorp and van der Merwe, 1991; Weiner

and Bar-Yosef,1990; Wright and Schwarcz, 1996). FTIR spectroscopy

is considered a semi-quantitative tool that uses infrared radiation

to determine what fraction of the incident light is absorbed at aparticular wavelength. This produces a spectrum characterizing the

vibrations of the bonds within a molecule for analyzing the struc-

ture of various materials. Each spectrum acts as a chemical

ngerprint for mineral identication, and can be analyzed for

unique information about mineral structure (Ferraro and Krishnan,

1990; Grifths, 1983). Advances in instrumentation have generated

alternate sample preparation techniques for vibrational spectros-

copy that are less costly and labor-intensive; these techniques

produce the same spectra but show less variation than the method

involving the manual creation of pellets for transmission FTIR

(Bruno, 1999; Cardell et al., 2009; Fuller and Grifths, 1978;

* Corresponding author. Tel.: 1 858 534 4145; fax: 1 858 534 5946.

E-mail addresses: mbeasley@ucsd.edu(M.M. Beasley), ebartelink@csuchico.edu

(E.J. Bartelink), lacyloutaylor@yahoo.com (L. Taylor), RMMiller@csuchico.edu (R.

M. Miller).

Contents lists available at ScienceDirect

Journal of Archaeological Science

j o u r n a l h o m e p a g e : h t t p : / / w w w . e l s e vi e r . c o m / l o c a t e / j a s

http://dx.doi.org/10.1016/j.jas.2014.03.008

0305-4403/

2014 Elsevier Ltd. All rights reserved.

Journal of Archaeological Science 46 (2014) 16e22

mailto:mbeasley@ucsd.edumailto:ebartelink@csuchico.edumailto:lacyloutaylor@yahoo.commailto:RMMiller@csuchico.eduhttp://www.sciencedirect.com/science/journal/03054403http://www.elsevier.com/locate/jashttp://dx.doi.org/10.1016/j.jas.2014.03.008http://dx.doi.org/10.1016/j.jas.2014.03.008http://dx.doi.org/10.1016/j.jas.2014.03.008http://dx.doi.org/10.1016/j.jas.2014.03.008http://dx.doi.org/10.1016/j.jas.2014.03.008http://dx.doi.org/10.1016/j.jas.2014.03.008http://www.elsevier.com/locate/jashttp://www.sciencedirect.com/science/journal/03054403http://crossmark.crossref.org/dialog/?doi=10.1016/j.jas.2014.03.008&domain=pdfmailto:RMMiller@csuchico.edumailto:lacyloutaylor@yahoo.commailto:ebartelink@csuchico.edumailto:mbeasley@ucsd.edu

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Haberhauer and Gerzabek, 1999; Yan et al., 1999). Two of these

sample preparation techniques, attenuated total reection (ATR)

and diffuse reectance infrared Fourier transform (DRIFT), have

been used for spectral analysis as an alternative to transmission

FTIR for decades in other elds, including chemistry, medicine,

biology, and geology. These reectance techniques operate with

different optical properties, which do not require the traditional

KBr pelleting preparation used for transmission FTIR spectroscopy.

Correction equations can be used to account for the differences in

how the infrared light beam is absorbed by the sample, so each

spectra produced by the three techniques should be comparable

(Ferraro and Krishnan, 1990; see Fig. 1). We compare the three

techniques in this study to assess whether values of C/P and IR-SF

(measures calculated from spectra) obtained from KBr pelleting

for transmission FTIR correspond to the values obtained from

spectra produced by ATR and DRIFT.

One reason to explore alternatives to the KBr pelleting method

is that studies have shown the preparation technique for producing

KBr pellets for transmission FTIR can introduce variation in

diagenesis indicators due to differences in sintering pressures and

times, KBr concentration, and individual preparation experience

with sample preparation (Surovell and Stiner, 2001). Only a few

studies have used ATR in an anthropological context, includingstudies on burned bone (Thompson et al., 2009) and diagenesis

(Hollund et al., 2013; Stathopoulou et al., 2008); one study used

DRIFT to characterize archaeological bone (Cardell et al., 2009). A

pilot study comparing the diagenesis measures from transmission

FTIR, ATR, and DRIFT spectra concluded that the three techniques

produce similar spectra (i.e., identify the same positions of ab-

sorption bands for a sample), but show different relative peak in-

tensities resulting in different values for calculations of diagenesis

measurements (i.e., C/P and IR-SF) (Beasley and Carman, 2009). The

purpose of this study is to analyze these measures from spectra

produced by these three preparation techniques to evaluate

whether the data are comparable for evaluating diagenesis in

archaeological samples.

2. Bone diagenesis

Bone is a biphasic material composed of an organic component

(predominately collagen) and an inorganic carbonated calcium

phosphate mineral (bioapatite) fraction. Bone mineral crystallites

and collagen bers create a matrix that forms the structure of bone.

Stable carbon and nitrogen isotope values from the collagen frac-

tion primarily track dietary protein. Early studies using the car-

bonate component of bone mineral (bioapatite), however, were

initially rejected because biogenic signatures can be altered by

diagenesis (Schoeninger and DeNiro, 1982; Sullivan and Krueger,

1983). Subsequent research on bioapatite suggested that contami-

nants, such as exogenous carbonates, could be successfully

removed by pretreatment of bone samples with dilute acetic acid

(Garvie-Lok et al., 2004; Koch et al., 1997; Lee-Thorp and van der

Merwe, 1991; Nielsen-Marsh and Hedges, 2000; Yoder and Barte-

link, 2010). Thus, stable carbon isotope analysis of bone bioapatite

is commonly used to complement data from bone collagen as an

additional measure of dietary composition of modern and archae-

ological bone (seeFroehle et al., 2010).

The diagenetic pathways affecting bone preservation are very

complex, and are inuenced by factors such as microbial attack,

temperature, humidity, hydrology, pH, and conditions of the burial

environment (see review Ttken and Vennemann, 2011). The

mineral phase of bone is thermodynamically metastable and in vivo

crystal growth inhibitors regulate the bone structure during life;

however, once the inhibitors are removed, bone crystallites will

spontaneously recrystallize and increase in size (Berna et al., 2004).Recrystallization appears to be related to collagen decomposition,

carbonate loss, and possibly uorine uptake (Berna et al., 2004;

Surovell and Stiner, 2001). While the process of bone diagenesis

is complex, changes in crystal size have been used as a proxy

measure for evaluating diagenesis. Crystallinity is a measure of

structural order within bioapatite that is directly related to the

mean crystal length. The crystallinity of a sample is evaluated by a

measure known as the infrared splitting factor (IR-SF) or crystal-

linity index (CI) (Surovell and Stiner, 2001; Wright and Schwarcz,

1996). The splitting factor refers to the double peak in the nger-

print region of the FTIR spectra that becomes increasingly sepa-

rated with increasing crystallinity. The carbonate content ratio (C/

P) is another measure of diagenesis that reects the carbonate

(CO3) to phosphate (PO4) content in a bone sample (Sponheimerand Lee-Thorp, 1999; Wright and Schwarcz, 1996). These mea-

sures can be calculated through the use of X-ray diffraction (XRD)

or FTIR and have been used to assess whether the pretreatment

method has successfully removed the diagenetic effects of exoge-

nous calcium phosphate (Garvie-Lok et al., 2004; Yoder and

Bartelink, 2010).

3. FTIR spectroscopy

Infrared (IR) spectroscopy uses infrared radiation to measure

what fraction of the incident radiation is absorbed at a particular

wavelength, which can be used to establish semi-quantitative

measures of bone composition (Carden and Morris, 2000; Ferraroand Krishnan, 1990; Grifths, 1983; Thompson et al., 2009). Pho-

tons of IR radiation transmit through a sample and excite the

molecules in bioapatite to higher rotational or vibrational states.

This results in some wavelengths of light being absorbed, whereas

other wavelengths pass through unaffected. The molecular struc-

ture determines the wavelengths that are transmitted or absorbed,

and the absorbed wavelengths promote atomic bonds to enter

excited vibrational states that can be interpreted in the spectra

output collected by a detector. FTIR uses a mathematical algorithm,

Fourier transform (FT), to convert the raw wavelength data

collected by a detector into the spectra. Therefore, a spectrum is the

product of the vibrations of the bonds within a molecule that are

produced after passing an IR beam through a sample and collecting

the resulting wavelength information. The observed absorbance

Fig. 1. Comparison of modern bone spectra produced from each FTIR accessory:

transmission FTIR (KBr pellet), ATR, and DRIFT (for one sample).

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bands can be ascribed to the internal vibrations of particular mo-

lecular groups, such as carbonate (CO3) and phosphate (PO4).

Traditionally, powdered samples are mixed with KBr and a

pellet is made with a hydraulic press (Ferraro and Krishnan, 1990).

KBr is used for sample preparation because it has a wide spectral

range, it has no signicant wavelengths in the middle-IR (MIR)

region, and it produces a smooth, transparent disk when mixed

with a powdered solid (Ferraro and Krishnan, 1990). This spec-

troscopy technique has been shown to be sufciently sensitive for

the analysis of bone samples to identify the carbonate content, loss

of carbon dioxide, identication of hydroxyl groups, and changes in

lattice parameters due to substitutions during diagenesis (Garvie-

Lok et al., 2004; Lee-Thorp and Sponheimer, 2003; Lee-Thorp and

van der Merwe, 1991). Changes in the molecular structure of bone

bioapatite are detected by FTIR because the different ionic radii of

the substituents cause changes in the environments of the molec-

ular groups, which result in alterations to the characteristic vibra-

tion modes that are reected in their infrared absorption spectra

(LeGeros, 1981).

Attenuated total reectance (ATR) is a rapid technique that is a

useful initial step to characterize minerals with minimal sample

preparation. The advantages of ATR is that sample preparation is

less labor-intensive, spectra variation due to sample preparation isminimal, and the impact of sample preparation due to KBr grinding

and particle size differences on results is greatly reduced

(Thompson et al., 2009). ATR is based on the phenomenon of total

internal reection (Bruno, 1999), and measures changes that occur

in an internally reected infrared beam that comes into contact

with the sample through a zinc selenide (ZnSe) crystal or diamond

(Bruno, 1999; Stathopoulou et al., 2008). When a sample is placed

in contact with the ATR crystal, the resulting evanescent wave is

attenuated in the regions of the IR spectrum where the sample

absorbs energy (Bruno, 1999). Instead of mixing the bone powder

sample with KBr as in transmission FTIR, the sample is placed

directly on the sampling plate of the device over the optic window

with the ZnSe crystal; it is then held in place by a micrometer-

controlled compression clamp to ensure good contact betweenthe sample and the crystal.

The DRIFT preparation technique is commonly used in chem-

istry to prepare powder and solid samples. When samples are

penetrated with an IR beam, there are two types of reected energy

generated, specular and diffuse reectance (Bruno, 1999). Specular

reectance occurs at the sample surface and has no absorptive

interaction with the sample, while diffuse reectance results from

penetration into the sample interacting with the sample particles

(Bruno, 1999). The diffuse reectance contains the spectral infor-

mation of IR absorption. The DRIFT accessory optimizes the

collection of the diffuse reected energy while minimizing the

specular reected energy (Bruno, 1999). The DRIFT preparation

technique still requires samples to be mixed with KBr, but avoids

the need for pelleting with a hydraulic press. DRIFT results can beadversely affected by particle size differences and incident IR

wavelengths. To offset these issues, samples are mixed with KBr to

obtain accurate DRIFT spectra in the 1200e400 cm1 region of the

middle-IR (MIR) region. The DRIFT technique can result in

increased resolution of the spectra and reduced interference from

water bands compared to transmission techniques (Haberhauer

and Gerzabek, 1999).

4. Materials and methods

4.1. Bone samples

A total of nine sets of bone bioapatite samples (n 452) from

prehistoric, historic, and modern contexts were used in this

research (Table A.1 Supplement Material). The prehistoric sample

(n 405) consisted of samples spanning 5000e1000 B.P. from

various shell and earthen mound archaeological sites surrounding

the San Francisco Bay and the Central California Delta region

(Bartelink et al., 2010; Beasley, 2008). The historic sample (n 22)

consisted of nine human samples and one canid (Canis familiaris)

sample from a late 19th Century Nevada cemetery and 12 human

samples from a mid-19th Century historic Virginia slave cemetery.

The modern sample (n 25) consisted of 21 faunal bones from the

California State University, Chico Zooarchaeological Laboratory and

four donated human bones from the CSU, Chico Human Identi-

cation Laboratory. The historic bone samples failed to show evi-

dence of alteration based on the IR-SF and C/P values, therefore for

the purpose of comparing the three FTIR sample techniques, these

samples are grouped with the modern samples because there were

no signicant differences between the groups.

4.2. FTIR sample preparation

All of the bone bioapatite samples were prepared for stable

isotope analysis prior to diagenesis assessment using FTIR

(following commonly accepted anthropological practice; Garvie-

Lok et al., 2004; Yoder and Bartelink, 2010). The bones were

ground into a powder with a steel mortar and pestle, and then

sieved through a mesh-screen (234 mm). The bone powder was

then treated in a 1.5% sodium hypochlorite solution for 48 h

(replaced once at 24 h) following a 0.04 ml solution/mg sample

ratio to remove the organic component of bone ( Koch et al., 1997).

Next the powder samples were treated in a 1.0 M solution of dilute

acetic acid, buffered with NaOH to a pH of 4.5 in the same sample-

to-solution ratio for 24 h (replaced once at 12 h) to remove

contaminants.

For KBr pelleting sample preparation, a hydraulic press is used

to make KBr pellets of the bone powder. For the samples analyzed

in this study, 1.5 mg of bioapatite powder was ground with 200 mg

of KBr in an agate mortar and pestle. The mixture was then pressed

into a 3 mmdisc in a hydraulic press at 10,000 psifor 2 min toform

a pellet. For ATR, a few milligrams of bioapatite powder was placed

on the optic window with a ZnSe crystal and the compression

clamp engaged to 6 psi to ensure good contact between the sample

and the crystal. For DRIFT, approximately 2 mg of bone powder was

ground with 200 mg of KBr in an agate mortar and then transferred

to the sample holder cup. The mixture lled the DRIFT accessory

sample holder cup and the top was leveled off. Each sample prep-

aration technique using the corresponding accessory attachment

on a Nicolet Magna 500 FTIR analyzer from 4000 to 400 cm1 using

100 scans at a resolution of 8 cm1. The spectra were analyzed using

the OMNIC (v7.0) software program. Each spectra was baseline

corrected to measure the heights at the approximate peaks and

trough for the calculations described below.

4.3. IR-SF

Over the past several years, there have been multiple techniques

to measure IR-SF reported in the literature. Currently the most

common technique used in archaeology is the method of Weiner

and Bar-Yosef (1990)that measures the heights at the absorption

bands at 565 and 605 cm1 and the height of the minimum trough

between the split peaks (Fig. 2). The equation is expressed as:

IR SF 565ht 605ht

590ht

Modern fresh bone values for IR-SF have been reported to be

between 2.5 and 3.25 (Berna et al., 2004; Thompson et al., 2009).

The IR-SF value represents the degree of order within the crystal

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matrix of the mineral component of bone, so high IR-SF values

indicate large crystal size and a more regularly organized lattice,

whereas low IR-SF values are consistent with modern bone that

have smaller-sized crystals with an irregular lattice structure

(Surovell and Stiner, 2001). Archaeological bone samples that

contain a measurable amount of collagen tend to have IR-SF values

less than 3.3, while samples that exhibit alteration frequently have

IR-SF values greater than 3.4.

4.4. C/P

FTIR spectra of bone have carbonate bands that appear at 870,

1415, and 1470 cm1 and phosphate bands that appear at 565(575),

605, and 1035 cm1 (Featherstone et al., 1984;Fig. 2). In the liter-

ature, various methods have been used to calculate the C/P ratio.

For the purpose of this study, the C/P ratio was calculated following

Wright and Schwarcz (1996).Wright and Schwarcz (1996) argued

that the phosphate peak at 1035 cm1 should be used for the

calculation because it was the main phosphate absorbance peak

and it was not a peak affected by the phosphate peak splitting at

565 and 605 cm1 (region used for IR-SF calculation). The resulting

C/P equation is expressed as:

C=P 1415ht=1035ht

Modern bone C/P values that have been previously reported fallbetween 0.23 and 0.34 using the KBr pellet preparation (Garvie-Lok

et al., 2004; Nielsen-Marsh and Hedges, 2000; Wright and

Schwarcz, 1996). Diagenetically altered bone bioapatite will result

in either an elevated or depleted C/P ratio in comparison to modern

bone values.

4.5. Statistical analyses

Comparisons between the preparation techniques were statis-tically analyzed by means of the repeated measures ANOVA test to

examine the differences in the IR-SF and C/P measurements

calculated from the KBr pellet, ATR and DRIFT spectra. A previous

KolmogoroveSmirnov test indicated a normal distribution for all

variables analyzed. Statistical analyses were computed using Sta-

tistical Package for the Social Sciences (SPSS, v. 18.0) and the sig-

nicance level was set at a 0.05.

Duplicate sample preparations for additional FTIR spectra were

performed on a subsample for each of the three sample preparation

techniques to determine the repeatability of the IRSF and C/P

measurements. Paired-samplet-tests were performed on replicate

sets of data from each of the subsample preparation techniques to

evaluate instrument repeatability. No signicant differences were

found for sample replicates for IR-SF and C/P values; however,

signicant differences were found in IR-SF values from the DRIFT

preparation (t 2.46,p 0.019). The mean difference of repeated

measurements for IR-SF and C/P values are the same for KBr pel-

leting (n 20; IR-SF 0.1, C/P 0.0) and ATR(n 29; IR-SF 0.1, C/

P 0.0), while the DRIFT preparation results in greater variation

(n 20, IR-SF 0.8, C/P 0.2). The duplicate sample measurements

suggest that the KBr pellet and ATR techniques produce spectra

with a greater repeatability in the IR-SF and C/P measurements.

5. Results

Table 1 presents the descriptive statistics and correlation co-

efcients between IR-SF and C/P for each preparation technique.

Table 2presents the statistical comparisons for each of the threepreparation techniques by subsample grouping. The data produced

Fig. 2. Phosphate and carbonate vibrational modes of bone apatite infrared spectra.

Band peak heights and baselines are drawn for calculating IF-SF and C/P. The equations

for the FTIR measured diagenesis indicators are: IR-SF (565ht 605ht)/590htand C/P 1415ht/1035ht.

Table 1

Descriptive statistics and regression relationship for each of the three sample preparation methods.

Sample type Preparation method N IR-SF C/P Correlation

Mean SD MineMax Mean SD MineMax Pearsons r pvaluea

Modern bone KBr pellet 47 3.20 0.13 3.00e3.60 0.22 0.05 0.09e0.31 0.69

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by the three different FTIR preparation techniques were compared

and found to be signicantly different (p < 0.001).

5.1. Modern bone

The IR-SF and C/P values calculated from the FTIR spectra

compared for the three preparation techniques were signicantly

different (IR-SF:F 60.44, p < 0.001; C/P:F 59.61,p < 0.001). A

Bonferroni post-hoc test indicated that all three preparation tech-

niques were signicantly different from one another for the IR-SF

values. The Bonferroni test for the C/P values indicated that the

ATR technique produced signicantly different values compared to

the KBr pellet (p < 0.001) and DRIFT (p < 0.001) techniques, but the

KBr pellet and DRIFT techniques were not signicantly different

from one another (p 0.504).

5.2. Prehistoric bone

The IR-SF and C/P values compared for the three preparation

techniques were signicantly different (IR-SF: F 104.04,

p < 0.001; C/P:F 3.011,p 0.050). A Bonferroni test for the IR-SF

values indicated that each of the three preparation techniques are

Fig. 3. Comparison of (a) IR-SF and (b) C/P values of modern bone samples calculated for the three preparation techniques. (Error bars represent one standard deviation).

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signicantly different from one another for each comparison

(p < 0.001). The Bonferroni test indicated that all three preparation

techniques were not signicantly different from oneanother for the

C/P values.

6. Discussion and conclusion

This study demonstrates that while the three FTIR techniquesidentify the same chemical properties of a sample (based on the

similar peak locations of carbonate and phosphate), the differences

in resolution in the spectra result in different C/P and IR-SF values

for each technique. Thus, the alternative ATR and DRIFT accessories

do not result in C/P or IR-SF values that are comparable to the KBr

pellet transmission FTIR technique (Fig. 3). Variation in values for

each technique would be of no consequence if the correlation be-

tween C/P and IR-SF were the same or the distribution of values

yielded a similar relationship between the three preparation

techniques; however, the data indicate that the different tech-

niques are not directly comparable to one another. Therefore,

different criteria are required for each technique to assess the

quality of a sample in order to avoid misclassication of altered and

unaltered bone bioapatite samples during stable isotope analysis.

Surovell and Stiner (2001) found that intensive grinding of

samples with KBr results in a net decrease in the IR-SF values. The

challenging aspects of infrared analyses in terms of KBr pellet

preparation and spectral reproducibility can be potentially avoided

with the use of the ATR accessory. DRIFT spectra can result in an

increase in resolution, but can be subject to variation in the spectra

due to sample particle size differences, similar to the problems that

occur during pellet production (Cardell et al., 2009; Fuller and

Grifths, 1978). ATR spectra do not have the same level of resolu-

tion as the DRIFT technique, but avoids the inuence of grinding

(Yan et al., 1999).

The IR-SF and C/P values based on KBr pellets and ATR prepa-

ration techniques distinguish the modern bone from the prehistoric

bone samples when compared in a bivariate plot (Fig. 4). This

suggests that the ATR technique is a valid tool with sufcientspectral resolution to distinguish modern from possibly altered

prehistoric bioapatite. The two indices calculated from ATR spectra

can be used to assess diagenesis, but the modern bone values have

different ranges compared to values obtained from transmission

FTIR. The mean values of IR-SF do not overlap within one standard

deviation for either technique when distinguishing modern from

prehistoric bone; however, the C/P values do. Therefore, when

assessing diagenesis both indices should be used together. How-

ever, the DRIFT preparation shows overlapping values at one

standard deviation between the modern and prehistoric bone

samples (Fig. 4). While the raw calculated values vary signicantly

based on the preparation technique, the trend in values from the

ATR preparation technique more closely approximates the trend

observed using the KBrpellet preparation. It is possible that ATR is a

better technique compared to transmission FTIR for distinguishing

altered samples because the resolution of the ATR spectra results in

better separation of the two bone groups when the indices are

plotted (Fig. 4).

Beasley and Carman (2009)found that sample particle size did

notsignicantlyalter the spectra collected using the ATR technique.

Previous studies have concluded that ATR is ideal for obtaining IR

spectra from powder samples because it minimizes distortions due

to optical saturation and dispersion, and at the same time is free of

potential chemical alterations, such as water absorption and ion-

exchange, that can occur as a result of mixing samples with KBr(Stathopoulou et al., 2008).Thompson et al. (2009)used ATR and

KBr pellets to evaluate burned bone and concluded that ATR was a

preferable FTIR preparation technique, but that IR-SF values were

affected by the sample preparation technique. It is still debated

whether indices, such as IR-SF and C/P, accurately assess diagenesis

in bioapatite samples (Lee-Thorp and Sealy, 2008; Stathopoulou

et al., 2008; Trueman et al., 2008). However, practitioners

continue to employ these techniques in the absence of better more

accessible measures of diagenesis in order to evaluate sample

quality in isotopic studies. The aim of this article is to highlight the

fact that indices calculated from spectra produced by different

vibrational spectroscopy techniques may not be directly

interchangeable.

Further research with a larger sample of modern bone samplesto establish baseline data for non-diagenetically altered bone is

needed to validate the range of IR-SF and C/P values produced by

Fig. 4. Comparison of C/P and IR-SF values calculated for the three preparation techniques. (Error bars represent one standard deviation).

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modern bone using the ATR technique. Additionally, C/P and IR-SF

values for enamel samples need to be established for the ATR and

DRIFT preparation techniques. At this point, ATR appears to be a

preferable, more cost-efcient alternative to the traditional KBr

pellet transmission FTIR technique, while DRIFT does not appear to

discriminate between altered and unaltered samples. FTIR is best

used as a gross indicator of assessing diagenesis, but multiple lines

of evidence should be used to determine if in vivo stable isotope

signatures are being attained from archaeological samples.

Acknowledgments

We would like to express our gratitude to Dr. Cassady Yoder,

Randy Wiberg, Ramona Garibay, Alan Leventhal, Rosemary Cambra

and Dr. Frank Bayham for allowing access to bone samples. The

majority of prehistoric bone bioapatite samples were from previous

studies that sampled skeletons from the archaeological collections

at the Phoebe A. Hearst Museum of Anthropology. A special thanks

goes to Dr. Tim White, Natasha Johnson and the staff at the Phoebe

A. Hearst Museum of Anthropology for allowing access to the

collection and for the assistance in sampling. Thank you to Dr.

Margaret Schoeninger, Andrew Somerville, Clinton Carman, and

other anonymous reviewers for their helpful comments and dis-

cussion of early drafts. This research was presented at the 2011

annual meeting of the Association of American Physical Anthro-

pologists in Minneapolis, MN.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.jas.2014.03.008 .

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