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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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