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, USA
b
Department of Anthropology, California State University, Chico, 400 West First Street, Chico, CA 95929-0400, USA
c
Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322, USA
d
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 (CO
3
) and
phosphate (PO
4
), 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.
2014 Elsevier Ltd. All rights reserved.
1. Introduction
Diagenesis is a complex process that involves physical and
chemical postmortemalterations to bones and teeth that is directly
inuenced by the burial environment, including local geological
and groundwater conditions (Nielsen-Marsh and Hedges, 2000). As
bone 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 a
particular wavelength. This produces a spectrumcharacterizing 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 ournal homepage: ht t p: / / www. el sevi er. com/ l ocat e/ j as
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
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, including
studies 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 (see Froehle 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
(CO
3
) to phosphate (PO
4
) content in a bone sample (Sponheimer
and 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; Ferraro
and 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 spectrumis 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).
M.M. Beasley et al. / Journal of Archaeological Science 46 (2014) 16e22 17
bands can be ascribed to the internal vibrations of particular mo-
lecular groups, such as carbonate (CO
3
) and phosphate (PO
4
).
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 is
minimal, 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 between
the 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 be
adversely 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 cm
1
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 psi for 2 min to form
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 from4000 to 400 cm
1
using
100 scans at a resolution of 8 cm
1
. 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 cm
1
and the height of the minimum trough
between the split peaks (Fig. 2). The equation is expressed as:
IR SF 565
ht
605
ht

590
ht
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
M.M. Beasley et al. / Journal of Archaeological Science 46 (2014) 16e22 18
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 cm
1
and phosphate bands that appear at 565(575),
605, and 1035 cm
1
(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 cm
1
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 cm
1
(region used for IR-SF calculation). The resulting
C/P equation is expressed as:
C=P 1415
ht
=1035
ht
Modern bone C/P values that have been previously reported fall
between 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-sample t-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 2 presents the statistical comparisons for each of the three
preparation 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 (565
ht
605
ht
)/590
ht
and C/
P 1415
ht
/1035
ht
.
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 p value
a
Modern bone KBr pellet 47 3.20 0.13 3.00e3.60 0.22 0.05 0.09e0.31 0.69 <0.001
ATR 47 3.07 0.26 2.54e4.03 0.31 0.08 0.14e0.54 0.62 <0.001
DRIFT 47 3.54 0.36 2.87e4.71 0.21 0.09 0.07e0.50 0.74 <0.001
Prehistoric bone KBr pellet 405 3.71 0.32 2.97e4.83 0.16 0.05 0.04e0.35 0.87 <0.001
ATR 405 3.90 0.37 2.58e4.85 0.16 0.05 0.07e0.39 0.91 <0.001
DRIFT 405 4.08 0.65 2.17e6.74 0.15 0.09 0.02e0.80 0.63 <0.001
a
Statistically signicant results (p 0.05) are indicated by bold-faced type in the table.
Table 2
Bonferroni post-hoc test results for FTIR preparation method comparison.
Sample type N IR-SF C/P
p value
a
Trend p value
a
Trend
Modern KBr vs ATR 47 0.002 ATR < KBr <0.001 KBr < ATR
KBr vs DRIFT 47 <0.001 KBr < DRIFT 0.504
ATR vs DRIFT 47 <0.001 ATR < DRIFT <0.001 ATR > DRIFT
Prehistoric KBr vs ATR 405 <0.001 KBr < ATR 1.000
KBr vs DRIFT 405 <0.001 KBr < DRIFT 0.311
ATR vs DRIFT 405 <0.001 ATR < DRIFT 0.160
a
Statistically signicant results (p 0.05) are indicated by bold-faced type in the
table.
M.M. Beasley et al. / Journal of Archaeological Science 46 (2014) 16e22 19
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).
M.M. Beasley et al. / Journal of Archaeological Science 46 (2014) 16e22 20
signicantly different from one another for each comparison
(p < 0.001). The Bonferroni test indicated that all three preparation
techniques were not signicantly different fromone another for the
C/P values.
6. Discussion and conclusion
This study demonstrates that while the three FTIR techniques
identify 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 fromthe prehistoric
bone samples when compared in a bivariate plot (Fig. 4). This
suggests that the ATR technique is a valid tool with sufcient
spectral 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 KBr pellet 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
not signicantly alter 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 samples
to 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).
M.M. Beasley et al. / Journal of Archaeological Science 46 (2014) 16e22 21
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 fromprevious
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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