Application of diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) for the identification of potential diagenesis and crystallinity changes in teeth

Microchemical Journal 76 (2004) 141–149

Application of diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) for the identification of potential diagenesis and crystallinity changes in teeth Ethan F. Greene, Socheata Tauch, Ellen Webb, Dulasiri Amarasiriwardena* School of Natural Science, Hampshire College, Amherst, MA 01002, USA Received 6 October 2003; accepted 9 November 2003

Abstract During burial, diagenetic alteration can invalidate the paleodietary signature, which the hydroxyapatite (Ca5 (PO4 )3 OH) matrix in incremental tissues, such as tooth enamel, provides. Thus, analytical methods that can evaluate diagenetic changes are crucial in anthropological and archaeological investigations. Modern deciduous tooth enamel (exfoliated) from Solis, Mexico and Kalama, Egypt, as well as Bronze Age (circa 2200 B.C.E.) adult enamel from (present-day) Tell Abraq, U.A.E. and adult enamel from the New York African Burial Ground (NYABG) in lower Manhattan, were analyzed using diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). The samples were compared to synthetic hydroxyapatite powder and bone ash samples. The DRIFTS spectra of tooth enamel yielded similar infra red finger print pattern to previous pellet-based FTIR spectra in both absorbance and Kubelka–Munk units. The study demonstrates that DRIFTS is a convenient alternative to pellet-based transmission FTIR in testing diagenetic changes in hard tissue for archaeological investigations. Tooth enamel samples contained a higher carbonate–phosphate ratio than synthetic hydroxyapatite and bone ash standard samples. Correlations are reported between Crystallinity Index (CIAb ) and carbonate–phosphate ratio, strontium–calcium ratio, and fluoride peak appearance. Crystallinity indexes (CIAb) were in the range of 2.6–3.8 (in absorbance units) and Kubelka–Munk Crystallinity indexes (CIKM ) were in the range of 3.1–4.9. 䊚 2003 Elsevier B.V. All rights reserved. Keywords: DRIFTS; Diagenesis; Hydroxyapatite; Enamel; Crystallinity Index; Kubelka–Munk function

1. Introduction The chemistry of incremental tissues, specifically tooth enamel, provides remarkable insight into ancient subsistence practices and paleoenvironments. During interment, tooth enamel, primarily composed of crystalline hydroxyapatite (Ca5(PO4)3OH), is more resistant to deterioration than bone w1x, and may be said to preserve a ‘paleodietary signature.’ Although many cation and anion substitutions can be made to the hydroxyapatite matrix without significantly disrupting its structure w2x, the precise mechanisms for these substitutions remain to be determined. Further, the extent to which ion exchange with soil and groundwater may alter premortem elemental concentrations, and subsequently, *Corresponding author. Tel.: q1-413-559-5561; fax: q1-413-5595448. E-mail address: [email protected] (D. Amarasiriwardena).

invalidate the paleodietary signature, is not widely understood. In order to draw reliable dietary inferences, petrographic, crystallographic, andyor analytical chemical criteria must be developed to determine if a tooth sample has been significantly altered. 1.1. Bioapatite and diagenesis Diagenetic alteration can occur as (a) uptake of environmental carbon and other elements from groundwater or soil, andyor (b) partial dissolution or recrystallization of biogenic apatite w3x. The calcium rich bioapatite structure (Ca10(PO4)6OH2 ) is related to the mineral dahllite w2,3x. Ion exchange between groundwater and hydroxyapatite molecules may occur at y Ca2q, PO3y sites w4x. In this case the 4 , andyor OH incorporation of carbonate groups, which may substitute for hydroxide-OHy (type A substitution) or phosphate-

0026-265X/04/$ - see front matter 䊚 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2003.11.006

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PO3y (type B substitution) in the hydroxyapatite matrix 4 is of particular interest. Carbonate dissolves into groundwater through leaching from calcium-carbonate rich bedrock and decayed flora w3x and is then incorporated into the hydroxyapatite matrix of enamel during burial. Carbonated apatite tends to be more poorly crystallized and more water soluble w2,6x than non-substituted apatite. Thus, we expect stronger crystals to contain less carbonate. Aside from carbonate substitution, other exchanges, such as fluoride (Fy) substitution for hydroxide (OHy), and strontium (Sr2q) and other trace cations (Zn2q, Pb2q) substitution for calcium (Ca2q), are also of interest. Hydroxyl groups in the hydroxyapatite matrix have been shown to lie perpendicular to trigonal planar units of calcium atoms w2x. They are displaced from the ˚ and all center of the triangle by approximately 0.3 A, of the hydroxyl dipoles in a column of hydroxyapatite molecules must face the same direction w2x. Fluoride, however, may present in the center of each triangle of calcium atoms, reducing crystal strain. Thus, fluoridation strengthens the crystal hardness and improves crystallinity of hydroxyapatite w7x. We expect a distinct FTIR peak at ;1096 cmy1 w8x to accompany greater crystal strength. Further, the incorporation of fluoride ion has been shown to promote crystal growth w9x, so the presence of a distinct 1096 cmy1 peak may indicate the formation of a new mineral phase during burial w3x. Previous studies w3,8x have posited that the presence of a distinct fluoride peak, accompanied by a greater degree of crystallinity and a lower carbonate content, may indicate post-burial recrystallization, and the selective dissolution of poorly crystallized, carbonated apatite. Strontium’s effect on apatitic tissues during interment is of somewhat lesser agreement among researchers. Strontium, which substitutes for calcium in hydroxyapatite matrices w10x, has been shown to expand the hydroxyapatite crystal on both a and c-axes w6,11x, as could be predicted from strontium’s greater atomic radius. After strontium substitution was shown in various epidemiological and animal studies w12,13x to have a cariostatic effect on teeth, it was deduced that strontium incorporation would increase crystallinity and reduce solubility of enamel hydroxyapatite w12x. However, LeGeros et al. showed w6,11x that strontium substitution promoted crystal strain and, consequently, greater solubility. This reduction in crystallinity was greatly minimized by the simultaneous substitution of fluoride, suggesting a synergistic relationship between fluoride and strontium in tooth enamel w6,14x. 1.2. Infrared spectroscopy Beginning in 1966 with Fowler et al. w15x, and Termine and Posner w5x, Fourier transform infrared (FTIR) spectroscopy has been widely used to analyze

the chemical makeup of hydroxyapatite and to evaluate crystallization of hydroxyapatite in bone tissues. After the magnitude of an infrared absorbance peak was confirmed to be proportional to the concentration of its corresponding analyte w16x, investigators w8,17x began using infrared spectroscopy to identify the presence and to determine the approximate quantity of each chemical moiety in apatitic substances. In conjunction with substitution patterns, various studies w2,8x have used the ratio of the triply degenerate asymmetric PO3y band 4 splitting between 560 and 605 cmy1 as a measure of the degree of crystallinity wi.e. Crystallinity Index, or CIs(A565qA605)yA595)x of the apatite matrix using dispersive IR w5x; and KBr pellet-based FTIR w3,9,18x spectroscopy. Crystallinity Index (CI) indicates the relative sizes of crystals and the degree to which atoms in the lattice are ordered w3,5,18x. A higher crystallinity index may indicate the presence of greater sized andyor ordered crystals, or, when accompanied by evidence of interaction with water, the selective dissolution of smaller, less ordered crystals w8,18x. From the development of functional group quantification and crystallinity index concepts, infrared spectroscopy has emerged as an invaluable tool in assessing the degree of post burial crystal development and degradation in apatitic materials. At the same time, however, solid sample FTIR has become well known for its tedium. This method has traditionally required either the dispersion of a solid sample in a mineral oil or mulling agent, or the preparation of a KBr disk pellet–usually recognized as both time consuming and arduous processes w19x. Furthermore, a relatively large quantity of sample has been necessary for analysis using traditional FTIR equipment. Diffuse reflectance, a technique perfected by Griffiths and Fuller w19x, reduces sample size and eliminates the traditional pellet or liquid FTIR sample preparation process. Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) requires only 1–3 mg of sample (i.e. coal) w20x and in some cases microgram amounts, and no more than 15 min preparation time per trial. Subsequently, DRIFTS allows for more trials and a much greater degree of sample preservation. Furthermore, DRIFTS spectra lack the fallaciously intense OH interference peaks originating from the contamination with moisture which is common to spectra of FTIR samples which have undergone the KBr pellet preparation process w21x. DRIFTS method has successfully applied for variety of applications including humic and fulvic acids in peat w21x, characterization of humic substances in peat and compost and characterization w22x, identification of components separated by thin layer chromatography (TLC) w19x, characterization of degree of esterification in pectin w23x, quantitative determination of humic substances and organic matter in sediments w24x and identification of pollen w25x. These diverse applications demonstrate DRIFTS method

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robustness and its ability to analyze previously stubborn samples. The Kubelka–Munk function, which factors sample concentration and scattering factor of the crushed sample particles into intensity value, provides quantitative analysis capabilities in DRIFTS studies w24,26,27x. According to The Kubelka–Munk (KM) theory for an infinitely thick layer, the KM function is defined as follows: fŽR`.sŽ1yR`.2 y2R`sKM

(1)

where R` is the absolute reflectance of the infinitely thick sample. KMskys

(2)

where KM is the spectrum in Kubelka–Munk (KM) units, ‘s’ is ‘scattering factor,’ and ‘k’ is the absorption coefficient that is defined as ks2.303ac. Substituting the expression, ks 2.303ac, into Eq. (2) yields KMs2.303acys

(3)

143

day Tell Abraq, U.A.E., and adult enamel from the New York African Burial Ground (NYABG) in lower Manhattan, were analyzed (all teeth samples were kindly provided by Prof. Alan H. Goodman, Hampshire College, Amherst, MA). Calcium hydroxyapatite powder (Aldrich Chemical Company, Inc., Milwaukee, WI, Catalog No. 23,093-6) and National Institute of Standards and Technology-NIST, Bone Ash standard reference material (SRM 1400) samples were analyzed. Before enamel removal, tooth samples were soaked in distilled deionized (DDI) water for 10 min, and then in 1% (wyw) papain (Sigma EC 噛232-627-2) solution for 10 min to remove lipids and proteins. Next, the samples were rinsed with DDI water and soaked in 3% (vyv) hydrogen peroxide for 30 s. The samples were rinsed again with DDI water and allowed to dry before filing. Each sample (1–3 mg) was collected by carefully scraping the outer enamel surface with a steel file. The sample was then ground to a fine powder with an agate mortar and pestle. FT-IR grade potassium bromide (9799 mg, Thermo Spectra-Tech, Franklin, MA, CAS 噛7758-02-3) was ground to a fine powder in a separate agate mortar and pestle. The two powders (100 mg total) were then combined and mixed with a spatula. An additional 100 mg of KBr was ground into a fine powder, and used to obtain background spectra.

where ‘a’ is absorptivity related to analyte species, ‘c’ is the analyte concentration w26,27x. Kubelka–Munk peak patterns are analogous to those of absorbance spectra w21x, and KM intensity values are directly proportional to the concentration if one controls the scattering factor, which is dependent on sample preparation w27x. Although conversion from absorbance to Kubelka–Munk units tends to amplify strong absorption bands–a tendency which has led investigators to question the accuracy of functional group concentration calculations from KM spectra w21,22x peak intensity ratios have been shown w20,22x to be reliable for quantitative determinations of functional groups. Further, in 2001 Ragain and Johnston w28x verified that Kubelka–Munk theory accurately predicts the reflectance of enamel and dentin under various particle thickness conditions, suggesting that variations in sample preparation may affect DRIFTS spectra (under Kubelka– Munk conversion) less than originally thought. Both Kubelka–Munk and absorbance units are used in this study. While investigators have traditionally used pelletbased transmission FTIR spectroscopy for diagenetic analysis on hard tissues in archeological investigations, the purpose of this study is to evaluate the applicability of DRIFTS as a potentially more efficient method.

The sample was transferred into a sample cup to overflowing, and a cover slip was dragged across the top of the cup to remove excess powder and smoothed the sample surface in order to maintain uniform distribution of particle size. The process was then repeated with pure KBr in a separate sample cup. The sample cups were then loaded into a MIDAC Series Model M 2010 FT-IR spectrometer (Midac Corporation, Irvine, CA) fitted with Spectros DRIFTS accessory (Spectros Associates, Shrewsbury, MA). Absorbance and Kubelka–Munk infrared spectra were obtained between 4000 and 400 cmy1 wavenumber using Gramsy386 version 3.0 for Windows software (Galactic Industries, Salem, NH). Resolution was set at 16 cmy1 and 100 scans were taken and averaged for each spectrum. The pure KBr background spectrum was subtracted from the sample spectrum. Duplicate analyses were made for each sample.

2. Experimental

3. Results and discussion

2.1. Samples and sample preparation

3.1. Data analysis

Deciduous exfoliated tooth samples from Solis, Mexico and Kalama, Egypt, as well as Bronze Age (approx. 2200 B.C.E.). Magan adult tooth samples from present-

The Kubelka–Munk function was defined previously in equation 1 w26,27x. All spectra were converted to Kubelka–Munk units in Gramsy386 for Windows. All

2.2. DRIFTS spectra collection

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Fig. 1. DRIFTS absorbance spectrum of synthetic hydroxyapatite powder between 500 and 800 cmy1. Crystallinity Index, CI is given by (aqc)yb.

relevant peaks were baseline corrected in accordance with the antisymmetric phosphate splitting method pioneered by Termine and Posner w5x. The ratio between type B carbonate absorbance (;1419 cmy1; y3 CO3) and symmetric phosphate absorbance (;1034 cmy1, y3 PO4) is used to normalize carbonate data against variations in sample loading. Since 1034 cmy1 band is the strongest phosphate peak in apatite this can be used for normalize the intensity of the carbonate band in order to correct for differences in sample preparation. Thus, we can rely on this carbonate (C) to phosphate(P) intensity ratio (CyP) value for an approximation of the extent of type B carbonate substitution w3,29x. The Kubelka–Munk CyP ratio value is analogous to absorbance CyP ratio; i.e. the ratio of carbonate 1419 cmy1 Kubelka–Munk intensity to phosphate 1035 cmy1. Kubelka–Munk intensity. Likewise, the degree of type A carbonate substitution, which is indicated by a peak at ;1445 cmy1, can be approxi-

Fig. 2. (a) DRIFTS absorbance spectrum for synthetic hydroxyapatite powder. (b) The corresponding DRIFTS Kubelka–Munk spectrum.

mated in this fashion. Type A substitution appears to be the more prevalent form in some Kalama samples. Crystallinity Index (CI) is defined here as CIAbs (A602qA563)yA586, where Ax is the absorbance at wave number x (see Fig. 1) w5,8x. In this study, we have defined the Kubelka–Munk Crystallinity Index as CIKMs(KM602qKM563)yKM586. Its applicability is evaluated in this investigation. All strontium–calcium ratio data were collected by Webb et al. w30x. 3.2. DRIFTS spectra The collected DRIFTS spectra were well resolved and, in both absorbance (see Fig. 2a) and Kubelka–

Table 1 Comparison of wavenumbers of major functional groups determined in hydroxyapatite matrix by pellet-based FTIR and DRIFT spectroscopy Functional Group

Publisheda pellet-based FTIR peak (cmy1)

Observed DRIFTS peak (cmy1) (absorbance and Kubelka–Munk)

Antisymmetric phosphate Symmetric phosphate Carbonate (type A) Carbonate (type B) Fluoride

565-577-605 1045 1450 1412 1096

563-586-603 1034 1442 1419 1095

a

See Ref. w8x.

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occurred at ;1041 cmy1 wavenumber region, while antisymmetric bending vibration (n4) doublets of orthophosphate were observed at ;563 and 602 cmy1, with a valley at ;586 cmy1. The carbonate (n3) peak was found at ;1419 cmy1 (type B substitution, according to Shemesh) w8x or ;1442 cmy1 (type A substitution, according to Shemesh) w8x. Various fluoride peaks, categorized as Minimal Contour Change, Shoulder, Emerging Peak, or Distinct Peak, were observed at ;1096 cmy1. The Kubelka–Munk spectra contained analogous yet sharp peak patterns (Fig. 3b). Rapid sample preparation in the DRIFTS process facilitated duplicate analyses of each sample, between which negligible sample-to-sample variation was observed. The Kubelka–Munk spectra also showed little variation between sample trials. Subsequently, DRIFTS proved to be an very practical laboratory method with high sample throughput, and errors in sample preparation or loading could be easily corrected. Just as Merry et al. w31x showed in their 2001 DRIFT based rapid soil analysis study using mid infrared range (MIR) demonstrates the capability for large-scale studies that would be impossible with the preparation time necessary for analysis of pellet-based FTIR samples. 3.3. Diagenetic implications

Fig. 3. (a) Representative DRIFTS absorbance spectra of dental enamel samples from each dataset. (b) The corresponding representative DRIFTS Kubelka–Munk spectra.

Munk units (see Fig. 2b), closely mimicked the pelletbased FTIR spectra reported by previous studies (see Table 1). As shown in Fig. 3a, in all dental enamel spectra the strongest absorbance peak of (n3) PO3y 4

As shown in Fig. 4, the enamel samples contained higher carbonate to phosphate ratios (CyP) than the synthetic hydroxyapatite standard and bone ash samples (NIST 1400). This is to be expected, as the synthetic hydroxyapatite material is reportedly pure Ca5(PO4)3OH, and bone ash standard should contain significantly less carbonate as a result of the ashing process. In both absorbance (see Fig. 4a) and Kubelka– Munk (see Fig. 4b) units, CyP (I1459 yI1041) intensity ratio varied negatively with increasing Crystallinity Index (CI). The co-relationship, calculated for enamel samples only (excluding synthetic hydroxyapatite and bone ash NIST 1400), was significantly stronger with Kubelka–Munk spectra (r 2s0.8814, ns19) than with absorbance spectra (r 2s0.6883, ns19), possibly demonstrating the greater degree of accuracy under Kubelka–Munk conversion. The negative correlation between carbonate–phosphate ratio and crystallinity index shown here is in accordance with a similar relationship observed in Mayan skeletal remains by Wright and Schwarcz w3x. Most of the Kalama teeth samples had relatively high carbonate-phosphate ratios and low CI, while the Solis teeth samples had low carbonate–phosphate ratios and high crystallinity indexes (see Fig. 4). The excavated teeth samples generally showed CyP and CI in between the Kalama and Solis ranges. Tell Abraq teeth samples had greater variation in carbonate–phosphate ratio and crystallinity index than NYABG samples. While further

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Fig. 4. (a) The relationship between carbonate-phosphate ratio and Crystallinity Index (CIAb ) of all samples; R 2 value is calculated for enamel samples only (excluding synthetic hydroxyapatite and bone ash NIST 1400 standard); (b) the corresponding relationship and R 2 calculation with Kubelka–Munk Crystallinity Index (CIKM) (excluding synthetic hydroxyapatite and bone ash NIST 1400 standard).

environmental and dietary enamel studies would have to be conducted to make any concrete deductions regarding diagenesis from these trends, the wider variation in CI and carbonate-phosphate intensity ratio among Tell Abraq samples may reflect that sample group’s longer duration of interment. However, the NYABG samples have higher CIKM and slightly lower Kubelka–Munk carbonate–phosphate (CyP) intensity ratios than five out of six of the Tell Abraq samples. Following Wright and Schwarcz’ supposition w3x that the degree of diagenetic alteration is more contingent on soil hydrology than on burial duration, this might suggest a greater degree of tooth preservation in Tell Abraq–a more arid environment than New York City–despite a much longer period of interment. As shown in Fig. 5a, among Tell Abraq samples, strontium to calcium concentration ratio (wSrx y wCax) varied negatively with crystallinity index (CI). This is in accordance with the reported literature w8x on fish remains from Pliocene to Triassic period. Interestingly,

observed trend in this study is much stronger with CI based on Kubelka–Munk units (r 2s0.8982, ns15, see Fig. 5b) than with standard absorbance units (r 2s 0.4625, ns15). Tell Abraq strontium contents were a degree of magnitude greater than those of Kalama and Solis teeth enamel samples. Further, fluoride peaks were more well defined in Tell Abraq and NYABG samples than in Kalama and Solis samples (see Tables 2 and 3). Environmental and dietary studies would be necessary to fully analyze the implications of these differences. Tell Abraq 噛142 and Tell Abraq 噛154 are the Tell Abraq dataset’s outliers in the Kubelka–Munk comparison of carbonate–phosphate ratio and CI. Tell Abraq 噛142 has the greatest strontium to calcium ratio and carbonate to phosphate ratios (measured in Kubelka– Munk, KM units), and the lowest CIKM of the Tell Abraq samples; Tell Abraq 噛154 has the lowest strontium-calcium ratio and carbonate-phosphate ratio (measured in Kubelka–Munk units), and the highest CIKM. This could suggest that both teeth underwent some

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Table 2 Overall data in order of ascending absorbance Crystallinity Index (CIAb) Dental enamel sample

CIAba

CyPb

wSrxywCaxc (=10y4)

Fluoride peak (;1095 cmy1)

Tel Abraq 噛15 Tel Abraq 噛142 Kalama 噛30197 Kalama 噛40056 Kalama 噛120089 Kalama 噛10047 Tel Abraq 噛156 Bone Ash NIST 1400 Kalama 噛10052 Tel Abraq 噛151 NYABGe B16 Tel Abraq NYABG B4B Tel Abraq 噛154 Hydroxyapatite Standard Solis 噛6171132 NYABG B10 Kalama 噛70004 Solis 噛50980834

2.59 2.77 2.88 2.93 3.06 3.08 3.11 3.14 3.31 3.34 3.41 3.52 3.52 3.54 3.56 3.67 3.72 3.74 3.74

0.417 0.481 0.494d 0.515 0.461 0.425 0.370 0.165 0.352 0.379 0.315 0.357 0.363 0.365 0.117 0.265 0.339 0.303 0.324

65.6 95.8 5.15 6.16 6.21 6.22 67.2 7.39 7.63 67.2 NAf 56.1 NA 46 NA 1.51 NA 4.13 1.65

Shoulder Shoulder Shoulder Shoulder Shoulder Minimal contour change Shoulder Distinct peak Shoulder Emerging peak Shoulder Shoulderyemerging peak SyEPyEPg Distinct peak Distinct peak Shoulderyemerging peak Shoulderyemerging peak Emerging peak Emerging peak

a

Crystallinity Index (see text and Figs. 1 and 2). Carbonate–phosphate intensity ratio (see w3x). c As reported by Webb et al. w30x. d Italics denotes type A carbonate substitution peaks (;1442 cmy1). e NYABG, New York African Burial Ground. f NA, not available. g One trial displayed Shoulder (s)yemerging peak (EP), and the other Emerging peak. b

Table 3 Overall data in order of ascending Kubelka–Munk Crystallinity Index (CIKM) Dental enamel sample

CIKMa

K-M CyPb

wSrxywCaxc (=10y4)

Fluoride peak (;1095 cmy1)

Kalama 噛30197 Tel Abraq 噛142 Kalama 噛10047 Kalama 噛40056 Kalama 噛120089 Kalama 噛10052 Tel Abraq 噛15 Tel Abraq 噛156 Tel Abraq 噛153 Tel Abraq 噛151 NYABG B16e NYABG B10 Bone Ash NIST 1400 Solis 噛50980834 Kalama 噛70004 NYABG B4B Hydroxyapatite Standard Solis 噛6171132 Tel Abraq 噛154

3.14 3.24 3.33 3.33 3.38 3.75 3.92 4.20 4.25 4.27 4.37 4.39 4.40 4.51 4.52 4.52 4.66 4.66 4.89

0.369d 0.242 0.327 0.309 0.276 0.266 0.210 0.178 0.184 0.180 0.161 0.182 0.027 0.184 0.152 0.168 0.027 0.116 0.097

5.15 95.9 6.22 6.16 6.21 7.63 65.6 67.2 56.2 67.2 NAf NA 7.39 1.65 4.13 NA NA 1.51 46

Minimal contour change Shoulder Minimal contour change Shoulder Shoulder Shoulder Shoulder Distinct peak Shoulder Shoulder Shoulderyemerging peak Shoulder Distinct peak Shoulderyemerging peak Emerging peak Shoulder Distinct peak Shoulder Shoulder

a

Kubelka–Munk Crystallinity Index (see text and Fig. 2). Kubelka–Munk carbonate–phosphate intensity ratio (see w6,22x). c As reported by Webb et al. w30x. d Italics denotes type A carbonate substitution peaks (;1442 cmy1). e NYABG, New York African Burial Ground. f NA, not available. b

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Fig. 5. (a) Strontium–calcium concentration ratio (=10y4 ) vs. Crystallinity Index (CIAb ) for enamel samples from Tell Abraq, Kalama, and Solis (NYABG wSrxywCax values were unavailable), and bone ash NIST 1400 as reported by Webb et al. w30x; (b) the corresponding relationship with Kubelka–Munk Crystallinity Index (CIKM).

recrystallization during burial, but the recrystallized, weaker apatite crystals in Tell Abraq 噛154 dissolved into the groundwater. However, while previous studies have linked high CI, low carbonate-phosphate ratio, and strong fluoride peaks to post-burial recrystallization andyor selective dissolution of carbonated apatite in bones w3x, the dental enamel samples do not show enough variation in carbonatephosphate ratio and crystallinity index to strongly indicate significant post-burial alteration. Further, in Shemesh’s 1990 study w8x of crystallinity in sedimentary apatites, ‘high CI’ is defined as greater than 4.0 using absorbance data. The enamel samples in this study all displayed absorbance-based crystallinity indexes (CIAb) below 3.8 while their CIKM indexes were less than 4.9. Together with the advances in DRIFTS analyses, further chemical characterization that consider age and burial environment, as well as other modes of analysis, such as oxygen isotope and rare earth element (REE) pattern analyses, along with other archeological evi-

dences would be useful in more accurately analyzing the degree of diagenesis among the tooth samples. 4. Conclusions Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) has proved to be an efficient, accurate (especially under Kubelka–Munk conversion), and convenient method for diagenetic analysis. The DRIFTS approach facilitates the characterization of functional groups (i.e. phosphate, hydroxyl, carbonate and fluoride groups) in the dental enamel matrix, estimation of carbonate content, and the evaluation of the extent of fluoridation as well as the crystallinity of the bio apatites. Since only 1–3 mg of sample is necessary for a successful DRIFTS analysis, duplicate analyses were possible for each sample, with a great degree of sample preservation. DRIFTS holds the potential for even smaller sample quantities (‘micro-DRIFTS’) and analyses which do not require potassium bromide dilu-

E.F. Greene et al. / Microchemical Journal 76 (2004) 141–149

tion, thus allowing for further preservation of the tested enamel samples for other sorts of analyses. Although it was a challenge to draw definitive conclusions about the occurrence of diagenetic alteration during interment, the negative correlations between crystallinity index (CI), and carbonate–phosphate intensity and strontium–calcium concentration ratios, and the positive correlation between CI and the appearance of fluoride peaks, demonstrates the enormous potential of DRIFTS in future diagenetic research. Furthermore, the increase in strength of these correlations under Kubelka– Munk conversion demonstrates the quantitative potential of Kubelka–Munk DRIFTS analysis and the applicability of the Kubelka–Munk crystallinity index (CIKM) in investigating diagenetic changes in geological and archaeological materials. Acknowledgments We would like to thank the National Science Foundation (NSF)–Collaborative Research in Undergraduate Institutions Grant (NSF-CRUI, Grant 噛 DBI-9978793) for funding this research project, and the financial support of the Undergraduate Biological Sciences Education Program Grant of the Howard Hughes Medical Institute to Hampshire College (HHMI 噛71100-503803) and the Kresge Foundation. We thank Professor Alan H. Goodman (Hampshire College) for kindly providing teeth samples for this project and encouragement for this study. Special thanks also goes to Kristin Shrout and Anahita Dua for laboratory assistance, and to late Professor John Reid for inspiration and support. References w1x J. Lee-Thorp, N.J. van der Merwe, J. Archa. Sci. 18 (1991) 343–354. w2x A.S. Posner, Physiol. Rev. 49 (4) (1969) 760–791. w3x L.E. Wright, H.P. Schwarcz, J. Archa. Sci. 23 (1996) 933–944. w4x J.E. Eastoe, in: C. Long (Ed.), Biochemists Handbook, Van Nostrand, Princeton, NJ, 1961, pp. 715–720. w5x J.D. Termine, A.S. Posner, Science 153 (3743) (1966) 1523–1525. w6x R.Z. LeGeros, in: E.P. Lazzari (Ed.), Handbook of Experimental Aspects of Oral Biochemistry, CRC Press, Boca Raton, FL, 1983, pp. 159–179. w7x O.R. Trautz, in: A.E.W. Miles (Ed.), Structural and Chemical Organization of Teeth, II, Academic Press, Orlando, FL, 1967, pp. 165–199. w8x A. Shemesh, Geochim. Cosmochim. Acta 54 (1990) 2433–2438.

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