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The infra-red absorption spectra of carbonate in calcified tissues
Arch. oralBlol.Vol.7,pp.671-683, 1962.Pergamon Press Ltd.Printed inGt.Britain.
THE INFRA-RED ABSORPTION SPECTRA OF CARBONATE IN CALCIFIED TISSUES W. H. EMERSONand E. E. FISCHER Tufts University School of Dental Medicine, Boston, Mass., U.S.A. Abstract-Infrared absorption spectra of calcified tissues have been obtained in the region 5-15 ~1, using potassium bromide and mineral oil as suspending media. A method of preparing the specimens as powders is described. It was found that whereas the absorption bands of a pure carbonate mineral (calcite) showed the expected inverse relation of absorption to particle size, no such relation was observed for bone, and only limited relation was observed with enamel. Thus at very small particle sixes the intensity of the us and “a modes of carbonate in these substances was not consistent with the amount of CO, present. Studies of NaSCOS in KBr as mechanical and fused mixtures revealed that in the mechanical mixture the out of plane mode near 1I.5 p (v,) consisted of a single peak, but in the fused mixture two separate peaks were seen indicating that the CO3 ion was present in two different environments. When powdered enamel, dentine and bone were examined in the same region using high resolution, it was found that two absorption bands were also present, one at 11.38 ~1and one at Il.45 p. Annealing studies of enamel have revealed that three bands apparently arise from C-O stretching modes of the carbonate ion at 6.5, 6.9 and 7.1 p. During the annealing process the bands at 6.5, 6.9 and 1I.38 p become more intense. In view of the known vibrational characteristics of the carbonate ion these findings lead to the conclusion that in the calcified tissues this ion is present in two nonequivalent sites. INTRODUCTION ALTHOUGHconsiderable
work has been done in the study of naturally occurring minerals and inorganic compounds (HUNT, WISHERDand BONHAM, 1950; MILLER and WILKINS, 1952; HUNT and TURNER, 1953; TUDDENHAM and LYON, 1960). few detailed investigations have been made of the i&a-red absorption characteristics of biological minerals. In 1954 and 1955 there were two independent studies of these substances. POSNERand DUYKAERTS(1954) compared the infra-red spectra of enamel, dentine, bone and francolite with those of calcium and magnesium carbonate. The hard tissue spectra showed absorption bands coinciding with the major bands seen in the synthetic carbonates. UNDERWOOD,TORIBARAand NEUMAN (1955) compared the absorption of bone and enamel with various synthetic carbonates. They concluded that carbon dioxide was present only as carbonate. Their investigation was limited to a study of the band at 11.5 CL. The purpose of the present study was to examine the spectra of calcified tissues in more detail and to develop standardized methods of sample preparation. Since it has been shown that infra-red absorption spectra of complex ions are sensitive to perturbations arising from the crystal field, such studies might contribute additional information concerning the environment of the carbonate ion. 671
672
W. H. EMERSONAND E. EXPERIMENTAL
E. FISCHER
METHODS
Sample preparation and particle size studies
It is generally accepted that particle size is important when studying the infra-red absorption spectra of powdered solids. This has been demonstrated by DUYKAERTS (1959) using calcite, and similar results have been reported by TUDDENHAMand LYON (1960) with quartz. Both studies have demonstrated that as the particle size of the powder diminishes, the intensity of the absorption bands increases. Preparation of bone and tooth enamel appeared to offer special problems; whereas naturally occurring minerals can be triturated in a mortar and pestle, enamel and especially bone not only possess extreme hardness, but are bound by a protein matrix which makes pulverization difficult. After experimenting with several different methods of preparation the following procedure was chosen. Samples of bone and enamel were ground with a diamond wheel driven by a dental engine and the grindings collected in a Millipore filter connected to a vacuum pump. It was found that this method could easily and quickly produce large amounts of powdered material. Microscopic examination showed the particles to have a mean diameter of 10 p with a range of 1-15 TV. Using this method, samples of the following were obtained: human enamel and dentine, cod bone (Gadus morhua), carp bone (Cytrinus carpio), Iamb bone and canine enamel. Protein was removed from portions of the pulverized bone and dentine samples by refluxing with ethylene diamine. The enamel was purified by the flotation method of MANLYand HODGE(1939). Spectra of these samples were obtained between 5 and 15 p using potassium bromide as a suspending medium. l-4 mg of sample were homogenized with either 200 or 400 mg of KBr in a dental amalgamator. All spectra were obtained on a Perkin-Elmer Model 21 spectrophotometer. Spectra were also obtained from samples that had received additional hand grinding under alcohol in an agate mortar and pestle. This procedure was originally suggested by TUDDENHAM and LYON(1960) and was found to give the most reproducible absorption values for the materials they studied. Using this method, the mean particle size of bone and enamel could be reduced to about 2 II. Comparison of the spectra revealed that the additional grinding apparently did not produce any increase in intensity of the various absorption bands. Since this finding was at variance with the results of DUYKAERTS and of TUDDENHAM and LYON,the relation of particle size and absorbance in the hard tissues was studied by two approaches. (1) A single crystal of optical grade calcite (Ward’s Natural Science Establishment, Rochester, New York) was first ground with a diamond wheel and then further ground to dryness with alcohol, once, twice, three and six times. The absorbance was determined at 7 p and at 11.45 p by the base line method. A similar procedure was carried out on protein-free cod and lamb bone. (2) Since differences in hardness and cleavage properties between calcite, enamel and bone might cause different rates in particle size reduction, the absorption of these materials was compared over the same particle size range. Samples were obtained by grinding and then sedimenting in absolute alcohol. The mean particle size for each
THE INFRA-RELI
ABSORPTION
SPECTRA
OF CARBONATE
IN CALCIFIED
TISSIJB
613
sample was estimated using a microscope equipped with a calibrated Whipple grid. It was realized that in sedimenting bone and enamel slight differences in density of the particles might cause preferential settling. Although this might be important in the case of geologic mineral aggregates (CHAMOTand MASON,1958), it was decided that local variations of density in bone or enamel were not great enough (LITTLEand BRUDEVOLD, 1958) to cause significant variation in sedimentation rates. For these studies the ratios employed were: 2 mg of calcified tissue in 200 mg potassium bromide and O-27 mg of calcite in 400 mg of potassium bromide. This amount of calcite corresponded to a concentration of 3 % COa in the bone and enamel. The use of these ratios allowed measurements at both 7 and 11.5 TV.The samples were weighed on a semi-micro balance. At least two determinations were made for each grinding interval or particle size. The thickness of the disks was measured with a micrometer caliper. In a series of ten disks selected at random it was determined that the range of thickness was 0.0015 in. Studies of absorbance as related to path length showed that for bone there was no deviation from Lambert’s law for disk thickness between 0.020 and 0.045 in. At 11.5 p the variation observed in path length corresponded to a range of O-020 absorbance units. High resolution studies of the carbonate band at 11.5 to (1) Small amounts of sodium carbonate were fused in potassium bromide in a platinum crucible. The melt was chilled and from this melt a disk was prepared. Disks were also prepared from mixtures of the carbonate and the halide prepared in the usual manner. For both preparations the sample ratio was 0+27 mg in 400 mg of potassium bromide. The absorption of the carbonate ion was then studied for both mixtures in the 1l-5 p region. (2) Specimens of powdered enamel, dentine and bone were scanned between 11 and 1l-80 p in both potassium bromide and Nujol mulls under conditions allowing maximum resolution. The dentine and bone specimens were protein-free. In the preparation of the mineral oil mulls, 20 mg of the sample were triturated with four drops of Nujol and the mull placed between two polished salt plates separated by a spacer having a thickness of O-075 mm. From studies of the absorption maxima of a polystyrene film in the same region the error of wave length measurement was determined to be ho.02 p. Annealing studies Previous investigation in this laboratory indicated that when enamel was heated slowly to 900°C not all the CO, was evolved over the same temperature range. This finding suggested the presence of more than one phase of carbonate. It seemed pertinent to determine if the earlier studies could be verified by heating enamel over the same temperature ranges and noting if changes occurred in the relative intensities of the carbonate absorption bands. About 100 mg of enamel were spread on a platinum tray and heated in a closed electric muffle from 100°C slowly up to 800°C. Samples were taken for analysis at 100 deg. intervals. The heating rate was 7.5 deg./min and was linear. After the final
674
W. H.
EMERSON
AND
E. E. FISCHER
sample was removed at 8OOC, the remaining enamel was held at 900°C for 30 min. Spectra of these samples were then obtained between 5 and 15 p using potassium bromide as a suspending medium. RESULTS
AND DISCUSSION
Absorption spectra between 5 and 15 p for some of the materials are shown in Figs. 1 and 2. All ofthe spectra shown were obtained from materials that had received additional hand grinding. In the extracted samples three major absorption bands
-_,i 6
!
a
4
10
WAVflfNClH
I
II
I?
13
14
4
1s
MICRONS)
FIG. I
were seen. Two areas of absorption are due to carbonate, one at 7 p and the other near 11.5 p. The broad band stretching between 8 and 11 p is due to absorption by orthophosphate groups. These findings are in agreement with the observations made by P~XNER and DUYKAERTS (1954) and UNDERWOODet al. (1955). In the unextracted samples additional areas of absorption occur at 6.05 p and 6.6 II. These probably arise from A-H (where A is C or N) bending modes and carbonyl groups in the organic matrix. With both human and canine enamel a pronounced doublet was consistently
THE INFRA-RED ABSORPTION SPECTRA OF CARBONATE IN CALCIPJED TISSUES
675
observed near the 7 to region. One peak occurred at 6.9 ~1,the other at 7.1 ~1. A very weak band was also seen at 6.5 CL.The significance of these bands will be discussed further on.
I
6
1
7
a
9
10
II
1
12
I
I i
13
11
15
WAVELtNCTH (MICRONS)
FIGS. 1 and 2. Absorption spectra of mineralized tissues between 5 and 15 I*. Materials that have had protein extracted are labelled (e), whole tissues (w). Human calculus was pulverized directly in the agate mortar. In the extracted tissues the peak near 6.05 TVresults from small amounts of water in the KBr. Ration of sample to KBr was 1 mg/400 mg.
The results of the grinding and particle size studies are presented in Figs. 3 and 4. As the amount of grinding is increased the absorbance of calcite at 11.45 CLincreases, whereas no change occurs for bone at either 7 or 11.5 CL.The absorbance of calcite increases most markedly between 0 and 3 times ground. The shape of this curve is probably a reflexion of the manner in which the distribution of particle size is altered by grinding. It should be noted that the symbol 0 at the origin refers to samples that were ground only by the diamond wheel. That the difference in behaviour between calcite and bone is not the result of differences in hardness or cleavage properties is proven by the results of the particle size studies (Fig. 4). As the particle size of calcite falls below 10 II, there is a marked
W. H. EMSWONAND E. E. FISCHER
676
increase in absorption. No change occurs for bone over the particle size range studied. The values for enamel increase in the vicinity of 10 TV,but reach a limiting value coincident with that of bone near 8 CL.
O-600
.
. 11111~~1111~1~11~~~_~~~~~~~~~~~.
.
.
lamb 7y
oml
0.400 w Y * z z m N
0.300
0200
/.
.~~llr..l~lrll~~~~~~~~~~~~~~~~~~~, lamb . . ~,“,,,,,,‘,““,‘,““““I”“,“,’ ,”,,,tllllllllllllx,,11,,*,*,111,, II,< ,,,,, *,,,,,,,1,*~1,,, ,~,,,~//.,~,,,,,,,,,,,,,,,,,,,,,, ,,,,., ,,,,,,,,,,,,,,,,,,,,,,,,,,/,1,,,,1,,, L eod ll*fY
0.100
i
-;
; TIMES
6
CROUNO
FIG. 3. Comparison of the effect of hand grinding on absorbance of cod bone, lamb bone and calcite. Specimens were initially obtained by grinding with a diamond wheel. With increased grinding the absorbance of bone does not change, but the absorbance of calcite increases markedly.
The behaviour of calcite was as expected and agrees with the findings of The increase in absorption is explained as due to reduction in energy losses from scattering and reflexion as particle size falls below the dimensions of the wavelength at which absorption occurs, and to the increased dispersion of particles in the beam. The failure of the intensity of the carbonate absorption bands in bone to increase with diminishing particle size is surprising. No reports of similar behaviour for other minerals could be found in the literature. These findings would certainly be of significance where quantitative studies of carbon dioxide were anticipated, since from these results it would appear that after initial grinding with the diamond wheel, no further intensity of the carbonate absorption bands would occur with further grinding. For enamel, additional trituration would be desirable. DUYKAERTS.
l’HE
INFRA-RED
ABSORPTION
SPECTRA
OF (‘ARBONATE
1N CALCIFI7ZD
TISSUES
677
11 has been suggested that the failure of absorbance to relate to the particle size of bone may possibly arise from the “adsorption” of carbonate on the surfaces of the hydroxyapatite crystals, and because the dimensions of the bone crystallites lie far below the size level approached in the grinding process (A. S. POSNER, personal communication; NEUMANet al., 1958). It has been demonstrated that the surface area of bone as determined by nitrogen adsorption is also not related to particle size
0.100
Il-CalCltt
0.300
C--0-kll8 8-
rrantl
z = 0~200 z =: =
O*lOO
FIG.4. Relation of particle size and absorbance at 11.5 p for enamel, cod bone and calcite. As the particle size of calcite falls below IO p the absorbance increases. No change occurs for cod bone. The absorbance values for enamel increase between I7 and 10 p but reach a limiting value coincident with that of bone. Point b, represents absorbance values for bone using a sample: KBr ratio of 4 mg/400 KBr.
(HENDRICKS and HILL, 1950). The behaviour of enamel, Fig. 4, might substantiate this suggestion, since it is known that the crystallites of enamel are larger than those of bone (FRANK et al., 1960), and that surface area is related to particle size. In other
words, from both the grinding and particle size studies it would seem that no change occurs in “apparent particle size” of bone, whereas some change does occur for enamel between 10 and 8 CL.However, when the bone particles diminkh in size from 20 to 1 p, their number increases by a factor of 8000. Therefore it would seem that this increased
678
W. H. EMERSON AND E. E. FISCHER
dispersion should have more effect than is observed. That the difference in intensity at minimum particle size did not arise from differences in path length was shown by adjusting the ratio of sample to KBr so that the number of carbonate ions was approximately the same for bone and for calcite. The mean of two determinations is
V Mechanical
Wavelength
7.r Fused
(microns)
FIG. 5. Absorption characteristics of the out of plane mode of the carbonate ion in Na,COI (anhydrous) suspended in KBr. The overall intensity in the fused sample is reduced due to vibration at two different frequencies.
represented in Fig. 4 by the symbol b,. These values were obtained using 4 mg of bone in 400 mg of KBr. Although the bone certainly contains at least 3 % C02, its intensity falls below the range for calcite at similar particle size. It would thus appear that some factor or factors were operating to diminish the intensity of the band at this wavelength. A mechanism which might partly explain the reduced intensity of the band at 11.5 p in the calcified tissues has been demonstrated using fused and mechanical mixtures of sodium carbonate in potassium bromide (Fig. 5). It will be noticed that although the carbonate concentration is the same in both samples, the overall intensity in the fused mixture is reduced, and two separate absorption bands occur. This is interpreted as indicating the presence of the carbonate ion in two different environments, one of them probably Na,CO,, since the low frequency peak corresponds exactly with that of the peak in the mechanical mixture. Examination of the disk under a polarizing microscope revealed discrete birefringent crystals. No statement can be made concerning the relative amounts of carbonate present in the different sites, since changes in environment may also induce changes in the intrinsic intensity of the band. However, it would appear that the division of a given amount of
THEINFBA-BED ABSORPTION SPECTRA OFCARBONATEIN
CALCIFJEDTISWES
679
carbonate ion among several sites results in a reduction of overall intensity due to absorption at slightly different frequencies.
1
’
i2p
wavelength
FIG. 6. High resolution spectra of the out of plane mode of the carbonate ion in enamel, dentine and lamb bone. In all three substances the band actually consists of two separate peaks occurring at 1 I.38 and 11.45 P. KBr disk, 2 mg sample in 200 mg KBr. In the process of reproduction the original tracings were enlarged 2 x .
When the 11.5 TVband in the calcified tissues was examined under high resolution, it was found that indeed the band actuahy consisted of two separate peaks (Fig. 6). One band occurred at 1I.38 p and the other at 1I .45 p. No change in wavelength was induced by differences in suspending media. High resolution studies on a grating spectrometer by the Perkin-Elmer Corporation have confirmed the presence of this doublet. It seems pertinent to observe that the wavelengths of the two bands were the same for all materials studied. Similar results have been obtained (J. C. ELLIOT, personal communication) using thin sections of whole enamel. Fig. 7 represents absorption spectra of enamel annealed over various temperature ranges. It will be noted that the control enamel, annealed at lOO”C,is identical to the curve in Fig. 1. The sample removed from the muffle at 800°C shows marked differregion it will be observed that two bands ences in the carbonate bands. First, in the 7 TV
680
W. H.
EMERSON AND E. E. FISCHER
have undergone intensification and decrease in half width. The band at 7.1 TVhas diminished. In the 1 I.5 p region a similar intensification is noticed. High resolution studies showed that this change in band shape was due to intensification of the peak at 1I.38 CL.After 30 min at 900°C the sample contained only a trace of carbonate.
control
enamel
WlVIllNCTN (MICRONSI
FIG. 7. Absorption curves of enamel showing variation in intensity of carbonate bands with various degrees of annealing. After treatment at 800°C bands at 6.5, 6.9 and Il.38 p have become more prominent. The band at 7.1 p has diminished. After prolonged heating at 900°C (t hr) only a trace of carbonate remains.
From the results of the high resolution and annealing studies some inferences may be drawn concerning the nature of the environment of the carbonate ion in bone and enamel. In the region 5-l 5 p the carbonate ion has three infra-red active modes (HERZBERG, 1960; WV. 1939). Near 7 TV the oxygen atoms vibrate about the central carbon in the plane of the ion. From structural considerations two different vibrations will occur at the same frequency, and the mode is said to be doubly degenerate. In structures where the ion is relatively unperturbed, i.e. carbonate compounds with the calcite structure, these C-0 stretching modes occur at essentially one frequency (RAWLINS
THE
INFRA-RED
ABSORPTION
SPECTRA
OF CARBONATE
IN CALCIFIED
-MSSUES
681
and TAYLOR, 1928) and only a single absorption band is observed. It has been predicted that in the event that the symmetry of the ion is disturbed, the two modes will occur at different frequencies (HORNIG, 1948). When this happens, two separate frequencies or bands will be observed, and “splitting” or separation of the bands will occur. The symbol for this mode is vs. A second mode occurs near 115 p (~2) and consists of a vibration of the carbon atom along the threefold axis of the ion. This mode is not degenerate. It is frequently referred to as the “out of plane mode”. A third mode has been observed at 14 TVin the anhydrous carbonates (Ye),but has never been detected by us in any spectra of the calcified tissues. It has been shown that these absorption frequencies of the carbonate ion are sensitive to alterations in environment. HUNT and TURNER(1953) observed that the frequency of the out of plane mode (1 l-5 p) was displaced to successively Iongel wavelengths for the series magnesium, calcium-magnesium (dolomite), iron, strontium and bismuth carbonates. This finding was later confirmed by MILLERand WILKINS (1952). More recently KETELAARand VANDERELSKEN(1959) studied the changes in frequency and intensity of complex ions dissolved in alkali halides. They concluded in part that a shift to higher frequency occurred with decreasing lattice parameter of the solvent, but that frequency was also related to the nature of the ions in the halide lattice. Thus in the calcified tissues the presence of two separate peaks representing the out of plane mode suggests that the carbonate ion exists in two different sites in these substances. Although this has been suggested by earlier investigators (LOGAN and TAYLOR,1938) on the basis of chemical studies, the infra-red results would seem to be the first direct evidence of a non-random distribution of carbonate ion. If all carbonate ions existed in equivalent sites, we would expect to observe only one absorption peak occurring in this region, as in the spectra of the anhydrous carbonates, i.e. calcite, magnesite, etc. That this doublet was not observed by POSNER or UNDERWOOD can be explained by the fact that it is not apparent under normal scanning conditions. Furthermore, UNDERWOODreported using a slit width of I.25 mm. It is doubtful if such narrowly separated bands would be resolved under these conditions. it must be emphasized that these findings cannot, at this time, be used to determine the exact position of the CO, ion in relation to the crystallites of bone or enamel. For example, they should not be construed to indicate that the CO; ion occupies surface positions or lattice positions or both. The experiment involving sodium carbonate fused in KBr suggests that if a given amount of carbonate is distributed between two different sites, there results a reduction in the overall intensity of the out of plane mode of the two bands. This observation would partly explain the reduced intensity in the biologic minerals. No direct comparison regarding structure should be made between the results obtained with the fused halides and the presence of a doublet in the calcified tissues. Similar results might be obtained with mechanical mixtures of different carbonates. When enamel was first studied it was thought that the peak at 6-5 P arose from A-H bending modes in the protein fraction. The results of the annealing studies
W. H. EMHWN AND E. E. FISCHER
682
lead to the tentative conclusion that this band represents one of the C-O stretching modes of carbonate. If all the carbonate ions in enamel occupied identical sites, we would expect to observe only two bands in this region if splitting occurred. The presence of three bands can be accounted for by assuming that the ug mode is split in either one or both of two sites. In the latter instance, if two bands coincided or were very narrowly separated, the result would also be observed as three separate peaks. Splitting of the v) mode has been observed by us in complex synthetic carbonates such as 3 MgCOsMg (OH),nH,O with bands occurring at 6.7 and 7 p. Thus in enamel the configuration of bands in the 7 p region would further suggest that the carbonate ion is present in two uniquely different sites. A summary of carbonate absorption bands is presented in Table 1. TABLE 1. SUMMARYOF CARBONATEABSORPTION BANDSIN M~NERALIZEJJ TISSUES
Absorption bands of calcite have been included for comparison. v3 Substance Enamel Calcite Cod bone Protein free
(cm-l) 1538 1449 1408 1430 1538* 1460 1420
%
*b
(P)
(cm-l)
(P)
6.5 6.90 7.10 7.00 6.50 6.85 7.05
880
11.38
(cm-l)
Not observed
873 873 880 873
11.45 11.45 11.38 11.45
712 14.05 Not observed
(cc)
* Very weak band. Although splitting of the V~mode is observed in all extracted bone samples, the peaks are never as well defined as in enamel, and show slight differences in frequency.
The increased intensity after annealing is assumed to result from crystal perfection and release of strain. It is known that changes in crystal perfection may cause alterations in band width and intensity. Although most studies have demonstrated decreases in intensity with excessive grinding or pressure (DUYKAERTS, 1959; TIJDDENHAM and LYON, 1959), it has also been shown that recrystallization from a relatively disordered state will result in band intensification (BAKER, 1957). 0. R. TRAUTZ (personal communication) has noted relations between crystal perfection as determined by X-ray diffraction and the infra-red spectra of apatites precipitated in solutions containing carbonate. We have observed that when synthetic defect apatites were treated with carbonate-containing solutions, the doublet at 11-5 p cannot be resolved with present techniques. This suggests that in these materials the crystals are in a more amorphous state than in the biologic minerals; a relative lack of perfection in the latter might also account in part for reduced intensity and failure of absorption to increase with diminishing particle size. Further studies are in progress to determine the relation of annealing to band intensity and to determine if the results of the infrared studies can be expressed in terms of the crystal environment of the carbonate ion.
THBINFRA-RED ABSORPTIONSPECTRAOFCARBONAT!~INCALCIFIED
TISSUES
683
Acknowledgements-The authors wish to express their appreciation to Dr. AARON S. POSNER,National Institute of Dental Research, and Professor M. KENT WILSON, Chairman of the Department of Chemistry at Tufts University, for their cooperation in the initial stages of the investigation and for many helpful suggestions during the preparation of the manuscript. The samples of fish bone were kindly supplied by Dr. MELVINMoss, College of Physicians and Surgeons, New York. The authors wish to express their thanks to Mr. JOHN B. PLANKof the PerkinElmer Corporation for arranging these studies. This study was supported by U.S. Public Health Service Grant, D-1357, National Institutes of Health, REFERENCES BAKER, A. W. 1957. Solid state anomaliesin infra-red spectroscopy. J. phys. Chem. 61, 45&458. CHAMOT,E. M. and MASON, C. W. 1958. Handbook of Chemical Microscopy, Vol. 1 (3rd Ed.), p. 143. Wiley, New York. DUYKAERTS,G. 1959. The infra-red analysis of solid substances. Anaiysr 84, 201, 212-213. FRANK, R. M., SOGNNAES,R. F. and KERN, R. 1960. Calcification in Biological Systems, p. 187. American Association for the Advancement of Science, Washington. HENDRICKS,S. B. and HILL, W. L. 1950. The nature of bone and phosphate rock. Proc. Nat. Acad. Sci., Wash. 36, 731-737.
1960. Molecular Spectra and Molecular Structure, Spectra of Polyatomic Molecules. Van Nostrand, Princeton.
HERZBERG,G.
Vol. II. lnfra-rrd
and Kumon
HORNIG,D. F. 1948. The vibrational spectra of molecules and complex ions in crystals- I. General theory. J. them. Phys. 12, 1063-1076. HUNT, J. M. and TURNER,D. S. 1953. Determination of mineral constituents of rocks by infra-red spectroscopy. Analyt. Chem. 25, 1169-I 174. HUNT, J. M., WISHERD,M. and BONHAM,L. C. 1950. Infra-red absorption spectra of minerals and other inorganic compounds. Analyt. Chem. 22, 1478-1497. KETELAAR,J. A. A. and VAN DER ELSKEN, J. 1959. Frequency shifts in the infra-red absorption spectrum of complex ions in solid solution. J. chern. Phys. 30,336-337. LITTLE, M. F. and BRUDEVOLD,F. 1958. A study of the inorganic carbon dioxide in intact human enamel. J. dent. Res. 37, 994-995. LOGAN,M. A. and TAYLOR,H. L. 1938. Solubility of bone salt. J. b&l. Chetrr. K&391-397. MANLY, R. S. and HODGE, H. 1939. Density and refractive index studies of dental hard tissues. J. dent. Res. 18, 133-141. MILLER,F. A. and WILKINS,C. H. 1952. Infra-red spectra and characteristic frequencies of inorganic ions. Analyr. Chem. 24, 1253-1294. NEUMAN,W. F. and NEIJMAN,M. W. 1958. The Chemical Dynamics o,/ Bone Minrral. pp. 95-96. University of Chicago Press. POSNER, A. S. and DUYKAERTS,G. 1954. lnt’ra-red study of the carbonate in bone, teeth and francolite. Experienfia 10,424425. RAWLINS, F. I. G. and TAYLOR,A. M. 1928. The fnfia-red Analysi.sof Molecular Structurr. Cambridge University Press. TUDDENHAM,W. M. and LYON, R. J. P. 1959. Relation of infra-red spectra and chemical analysis for some chlorite minerals. Analyt. Chem. 31, 337-380. TUDDENHAM, W. M. and LYON,R. J. P. 1960. Infra-red techniques in the identification and measurement of minerals. Anulyt. Chenr. 32, 1630-I 634. UNDERWOOD,A. L., TORIBARA,T. Y. and NEUMAN,W. F. 1955. An infra-red study of the nature of bone carbonate. J. Amer. them. Sot. 77, 317-319. Wu, TA-YOU. 1939. Infra-red Spectra ofPolyaromic Molecules, p. 207. China Science Foundation, Shanghai.
THE INFRA-RED ABSORPTION SPECTRA OF CARBONATE IN CALCIFIED TISSUES W. H. EMERSONand E. E. FISCHER Tufts University School of Dental Medicine, Boston, Mass., U.S.A. Abstract-Infrared absorption spectra of calcified tissues have been obtained in the region 5-15 ~1, using potassium bromide and mineral oil as suspending media. A method of preparing the specimens as powders is described. It was found that whereas the absorption bands of a pure carbonate mineral (calcite) showed the expected inverse relation of absorption to particle size, no such relation was observed for bone, and only limited relation was observed with enamel. Thus at very small particle sixes the intensity of the us and “a modes of carbonate in these substances was not consistent with the amount of CO, present. Studies of NaSCOS in KBr as mechanical and fused mixtures revealed that in the mechanical mixture the out of plane mode near 1I.5 p (v,) consisted of a single peak, but in the fused mixture two separate peaks were seen indicating that the CO3 ion was present in two different environments. When powdered enamel, dentine and bone were examined in the same region using high resolution, it was found that two absorption bands were also present, one at 11.38 ~1and one at Il.45 p. Annealing studies of enamel have revealed that three bands apparently arise from C-O stretching modes of the carbonate ion at 6.5, 6.9 and 7.1 p. During the annealing process the bands at 6.5, 6.9 and 1I.38 p become more intense. In view of the known vibrational characteristics of the carbonate ion these findings lead to the conclusion that in the calcified tissues this ion is present in two nonequivalent sites. INTRODUCTION ALTHOUGHconsiderable
work has been done in the study of naturally occurring minerals and inorganic compounds (HUNT, WISHERDand BONHAM, 1950; MILLER and WILKINS, 1952; HUNT and TURNER, 1953; TUDDENHAM and LYON, 1960). few detailed investigations have been made of the i&a-red absorption characteristics of biological minerals. In 1954 and 1955 there were two independent studies of these substances. POSNERand DUYKAERTS(1954) compared the infra-red spectra of enamel, dentine, bone and francolite with those of calcium and magnesium carbonate. The hard tissue spectra showed absorption bands coinciding with the major bands seen in the synthetic carbonates. UNDERWOOD,TORIBARAand NEUMAN (1955) compared the absorption of bone and enamel with various synthetic carbonates. They concluded that carbon dioxide was present only as carbonate. Their investigation was limited to a study of the band at 11.5 CL. The purpose of the present study was to examine the spectra of calcified tissues in more detail and to develop standardized methods of sample preparation. Since it has been shown that infra-red absorption spectra of complex ions are sensitive to perturbations arising from the crystal field, such studies might contribute additional information concerning the environment of the carbonate ion. 671
672
W. H. EMERSONAND E. EXPERIMENTAL
E. FISCHER
METHODS
Sample preparation and particle size studies
It is generally accepted that particle size is important when studying the infra-red absorption spectra of powdered solids. This has been demonstrated by DUYKAERTS (1959) using calcite, and similar results have been reported by TUDDENHAMand LYON (1960) with quartz. Both studies have demonstrated that as the particle size of the powder diminishes, the intensity of the absorption bands increases. Preparation of bone and tooth enamel appeared to offer special problems; whereas naturally occurring minerals can be triturated in a mortar and pestle, enamel and especially bone not only possess extreme hardness, but are bound by a protein matrix which makes pulverization difficult. After experimenting with several different methods of preparation the following procedure was chosen. Samples of bone and enamel were ground with a diamond wheel driven by a dental engine and the grindings collected in a Millipore filter connected to a vacuum pump. It was found that this method could easily and quickly produce large amounts of powdered material. Microscopic examination showed the particles to have a mean diameter of 10 p with a range of 1-15 TV. Using this method, samples of the following were obtained: human enamel and dentine, cod bone (Gadus morhua), carp bone (Cytrinus carpio), Iamb bone and canine enamel. Protein was removed from portions of the pulverized bone and dentine samples by refluxing with ethylene diamine. The enamel was purified by the flotation method of MANLYand HODGE(1939). Spectra of these samples were obtained between 5 and 15 p using potassium bromide as a suspending medium. l-4 mg of sample were homogenized with either 200 or 400 mg of KBr in a dental amalgamator. All spectra were obtained on a Perkin-Elmer Model 21 spectrophotometer. Spectra were also obtained from samples that had received additional hand grinding under alcohol in an agate mortar and pestle. This procedure was originally suggested by TUDDENHAM and LYON(1960) and was found to give the most reproducible absorption values for the materials they studied. Using this method, the mean particle size of bone and enamel could be reduced to about 2 II. Comparison of the spectra revealed that the additional grinding apparently did not produce any increase in intensity of the various absorption bands. Since this finding was at variance with the results of DUYKAERTS and of TUDDENHAM and LYON,the relation of particle size and absorbance in the hard tissues was studied by two approaches. (1) A single crystal of optical grade calcite (Ward’s Natural Science Establishment, Rochester, New York) was first ground with a diamond wheel and then further ground to dryness with alcohol, once, twice, three and six times. The absorbance was determined at 7 p and at 11.45 p by the base line method. A similar procedure was carried out on protein-free cod and lamb bone. (2) Since differences in hardness and cleavage properties between calcite, enamel and bone might cause different rates in particle size reduction, the absorption of these materials was compared over the same particle size range. Samples were obtained by grinding and then sedimenting in absolute alcohol. The mean particle size for each
THE INFRA-RELI
ABSORPTION
SPECTRA
OF CARBONATE
IN CALCIFIED
TISSIJB
613
sample was estimated using a microscope equipped with a calibrated Whipple grid. It was realized that in sedimenting bone and enamel slight differences in density of the particles might cause preferential settling. Although this might be important in the case of geologic mineral aggregates (CHAMOTand MASON,1958), it was decided that local variations of density in bone or enamel were not great enough (LITTLEand BRUDEVOLD, 1958) to cause significant variation in sedimentation rates. For these studies the ratios employed were: 2 mg of calcified tissue in 200 mg potassium bromide and O-27 mg of calcite in 400 mg of potassium bromide. This amount of calcite corresponded to a concentration of 3 % COa in the bone and enamel. The use of these ratios allowed measurements at both 7 and 11.5 TV.The samples were weighed on a semi-micro balance. At least two determinations were made for each grinding interval or particle size. The thickness of the disks was measured with a micrometer caliper. In a series of ten disks selected at random it was determined that the range of thickness was 0.0015 in. Studies of absorbance as related to path length showed that for bone there was no deviation from Lambert’s law for disk thickness between 0.020 and 0.045 in. At 11.5 p the variation observed in path length corresponded to a range of O-020 absorbance units. High resolution studies of the carbonate band at 11.5 to (1) Small amounts of sodium carbonate were fused in potassium bromide in a platinum crucible. The melt was chilled and from this melt a disk was prepared. Disks were also prepared from mixtures of the carbonate and the halide prepared in the usual manner. For both preparations the sample ratio was 0+27 mg in 400 mg of potassium bromide. The absorption of the carbonate ion was then studied for both mixtures in the 1l-5 p region. (2) Specimens of powdered enamel, dentine and bone were scanned between 11 and 1l-80 p in both potassium bromide and Nujol mulls under conditions allowing maximum resolution. The dentine and bone specimens were protein-free. In the preparation of the mineral oil mulls, 20 mg of the sample were triturated with four drops of Nujol and the mull placed between two polished salt plates separated by a spacer having a thickness of O-075 mm. From studies of the absorption maxima of a polystyrene film in the same region the error of wave length measurement was determined to be ho.02 p. Annealing studies Previous investigation in this laboratory indicated that when enamel was heated slowly to 900°C not all the CO, was evolved over the same temperature range. This finding suggested the presence of more than one phase of carbonate. It seemed pertinent to determine if the earlier studies could be verified by heating enamel over the same temperature ranges and noting if changes occurred in the relative intensities of the carbonate absorption bands. About 100 mg of enamel were spread on a platinum tray and heated in a closed electric muffle from 100°C slowly up to 800°C. Samples were taken for analysis at 100 deg. intervals. The heating rate was 7.5 deg./min and was linear. After the final
674
W. H.
EMERSON
AND
E. E. FISCHER
sample was removed at 8OOC, the remaining enamel was held at 900°C for 30 min. Spectra of these samples were then obtained between 5 and 15 p using potassium bromide as a suspending medium. RESULTS
AND DISCUSSION
Absorption spectra between 5 and 15 p for some of the materials are shown in Figs. 1 and 2. All ofthe spectra shown were obtained from materials that had received additional hand grinding. In the extracted samples three major absorption bands
-_,i 6
!
a
4
10
WAVflfNClH
I
II
I?
13
14
4
1s
MICRONS)
FIG. I
were seen. Two areas of absorption are due to carbonate, one at 7 p and the other near 11.5 p. The broad band stretching between 8 and 11 p is due to absorption by orthophosphate groups. These findings are in agreement with the observations made by P~XNER and DUYKAERTS (1954) and UNDERWOODet al. (1955). In the unextracted samples additional areas of absorption occur at 6.05 p and 6.6 II. These probably arise from A-H (where A is C or N) bending modes and carbonyl groups in the organic matrix. With both human and canine enamel a pronounced doublet was consistently
THE INFRA-RED ABSORPTION SPECTRA OF CARBONATE IN CALCIPJED TISSUES
675
observed near the 7 to region. One peak occurred at 6.9 ~1,the other at 7.1 ~1. A very weak band was also seen at 6.5 CL.The significance of these bands will be discussed further on.
I
6
1
7
a
9
10
II
1
12
I
I i
13
11
15
WAVELtNCTH (MICRONS)
FIGS. 1 and 2. Absorption spectra of mineralized tissues between 5 and 15 I*. Materials that have had protein extracted are labelled (e), whole tissues (w). Human calculus was pulverized directly in the agate mortar. In the extracted tissues the peak near 6.05 TVresults from small amounts of water in the KBr. Ration of sample to KBr was 1 mg/400 mg.
The results of the grinding and particle size studies are presented in Figs. 3 and 4. As the amount of grinding is increased the absorbance of calcite at 11.45 CLincreases, whereas no change occurs for bone at either 7 or 11.5 CL.The absorbance of calcite increases most markedly between 0 and 3 times ground. The shape of this curve is probably a reflexion of the manner in which the distribution of particle size is altered by grinding. It should be noted that the symbol 0 at the origin refers to samples that were ground only by the diamond wheel. That the difference in behaviour between calcite and bone is not the result of differences in hardness or cleavage properties is proven by the results of the particle size studies (Fig. 4). As the particle size of calcite falls below 10 II, there is a marked
W. H. EMSWONAND E. E. FISCHER
676
increase in absorption. No change occurs for bone over the particle size range studied. The values for enamel increase in the vicinity of 10 TV,but reach a limiting value coincident with that of bone near 8 CL.
O-600
.
. 11111~~1111~1~11~~~_~~~~~~~~~~~.
.
.
lamb 7y
oml
0.400 w Y * z z m N
0.300
0200
/.
.~~llr..l~lrll~~~~~~~~~~~~~~~~~~~, lamb . . ~,“,,,,,,‘,““,‘,““““I”“,“,’ ,”,,,tllllllllllllx,,11,,*,*,111,, II,< ,,,,, *,,,,,,,1,*~1,,, ,~,,,~//.,~,,,,,,,,,,,,,,,,,,,,,, ,,,,., ,,,,,,,,,,,,,,,,,,,,,,,,,,/,1,,,,1,,, L eod ll*fY
0.100
i
-;
; TIMES
6
CROUNO
FIG. 3. Comparison of the effect of hand grinding on absorbance of cod bone, lamb bone and calcite. Specimens were initially obtained by grinding with a diamond wheel. With increased grinding the absorbance of bone does not change, but the absorbance of calcite increases markedly.
The behaviour of calcite was as expected and agrees with the findings of The increase in absorption is explained as due to reduction in energy losses from scattering and reflexion as particle size falls below the dimensions of the wavelength at which absorption occurs, and to the increased dispersion of particles in the beam. The failure of the intensity of the carbonate absorption bands in bone to increase with diminishing particle size is surprising. No reports of similar behaviour for other minerals could be found in the literature. These findings would certainly be of significance where quantitative studies of carbon dioxide were anticipated, since from these results it would appear that after initial grinding with the diamond wheel, no further intensity of the carbonate absorption bands would occur with further grinding. For enamel, additional trituration would be desirable. DUYKAERTS.
l’HE
INFRA-RED
ABSORPTION
SPECTRA
OF (‘ARBONATE
1N CALCIFI7ZD
TISSUES
677
11 has been suggested that the failure of absorbance to relate to the particle size of bone may possibly arise from the “adsorption” of carbonate on the surfaces of the hydroxyapatite crystals, and because the dimensions of the bone crystallites lie far below the size level approached in the grinding process (A. S. POSNER, personal communication; NEUMANet al., 1958). It has been demonstrated that the surface area of bone as determined by nitrogen adsorption is also not related to particle size
0.100
Il-CalCltt
0.300
C--0-kll8 8-
rrantl
z = 0~200 z =: =
O*lOO
FIG.4. Relation of particle size and absorbance at 11.5 p for enamel, cod bone and calcite. As the particle size of calcite falls below IO p the absorbance increases. No change occurs for cod bone. The absorbance values for enamel increase between I7 and 10 p but reach a limiting value coincident with that of bone. Point b, represents absorbance values for bone using a sample: KBr ratio of 4 mg/400 KBr.
(HENDRICKS and HILL, 1950). The behaviour of enamel, Fig. 4, might substantiate this suggestion, since it is known that the crystallites of enamel are larger than those of bone (FRANK et al., 1960), and that surface area is related to particle size. In other
words, from both the grinding and particle size studies it would seem that no change occurs in “apparent particle size” of bone, whereas some change does occur for enamel between 10 and 8 CL.However, when the bone particles diminkh in size from 20 to 1 p, their number increases by a factor of 8000. Therefore it would seem that this increased
678
W. H. EMERSON AND E. E. FISCHER
dispersion should have more effect than is observed. That the difference in intensity at minimum particle size did not arise from differences in path length was shown by adjusting the ratio of sample to KBr so that the number of carbonate ions was approximately the same for bone and for calcite. The mean of two determinations is
V Mechanical
Wavelength
7.r Fused
(microns)
FIG. 5. Absorption characteristics of the out of plane mode of the carbonate ion in Na,COI (anhydrous) suspended in KBr. The overall intensity in the fused sample is reduced due to vibration at two different frequencies.
represented in Fig. 4 by the symbol b,. These values were obtained using 4 mg of bone in 400 mg of KBr. Although the bone certainly contains at least 3 % C02, its intensity falls below the range for calcite at similar particle size. It would thus appear that some factor or factors were operating to diminish the intensity of the band at this wavelength. A mechanism which might partly explain the reduced intensity of the band at 11.5 p in the calcified tissues has been demonstrated using fused and mechanical mixtures of sodium carbonate in potassium bromide (Fig. 5). It will be noticed that although the carbonate concentration is the same in both samples, the overall intensity in the fused mixture is reduced, and two separate absorption bands occur. This is interpreted as indicating the presence of the carbonate ion in two different environments, one of them probably Na,CO,, since the low frequency peak corresponds exactly with that of the peak in the mechanical mixture. Examination of the disk under a polarizing microscope revealed discrete birefringent crystals. No statement can be made concerning the relative amounts of carbonate present in the different sites, since changes in environment may also induce changes in the intrinsic intensity of the band. However, it would appear that the division of a given amount of
THEINFBA-BED ABSORPTION SPECTRA OFCARBONATEIN
CALCIFJEDTISWES
679
carbonate ion among several sites results in a reduction of overall intensity due to absorption at slightly different frequencies.
1
’
i2p
wavelength
FIG. 6. High resolution spectra of the out of plane mode of the carbonate ion in enamel, dentine and lamb bone. In all three substances the band actually consists of two separate peaks occurring at 1 I.38 and 11.45 P. KBr disk, 2 mg sample in 200 mg KBr. In the process of reproduction the original tracings were enlarged 2 x .
When the 11.5 TVband in the calcified tissues was examined under high resolution, it was found that indeed the band actuahy consisted of two separate peaks (Fig. 6). One band occurred at 1I.38 p and the other at 1I .45 p. No change in wavelength was induced by differences in suspending media. High resolution studies on a grating spectrometer by the Perkin-Elmer Corporation have confirmed the presence of this doublet. It seems pertinent to observe that the wavelengths of the two bands were the same for all materials studied. Similar results have been obtained (J. C. ELLIOT, personal communication) using thin sections of whole enamel. Fig. 7 represents absorption spectra of enamel annealed over various temperature ranges. It will be noted that the control enamel, annealed at lOO”C,is identical to the curve in Fig. 1. The sample removed from the muffle at 800°C shows marked differregion it will be observed that two bands ences in the carbonate bands. First, in the 7 TV
680
W. H.
EMERSON AND E. E. FISCHER
have undergone intensification and decrease in half width. The band at 7.1 TVhas diminished. In the 1 I.5 p region a similar intensification is noticed. High resolution studies showed that this change in band shape was due to intensification of the peak at 1I.38 CL.After 30 min at 900°C the sample contained only a trace of carbonate.
control
enamel
WlVIllNCTN (MICRONSI
FIG. 7. Absorption curves of enamel showing variation in intensity of carbonate bands with various degrees of annealing. After treatment at 800°C bands at 6.5, 6.9 and Il.38 p have become more prominent. The band at 7.1 p has diminished. After prolonged heating at 900°C (t hr) only a trace of carbonate remains.
From the results of the high resolution and annealing studies some inferences may be drawn concerning the nature of the environment of the carbonate ion in bone and enamel. In the region 5-l 5 p the carbonate ion has three infra-red active modes (HERZBERG, 1960; WV. 1939). Near 7 TV the oxygen atoms vibrate about the central carbon in the plane of the ion. From structural considerations two different vibrations will occur at the same frequency, and the mode is said to be doubly degenerate. In structures where the ion is relatively unperturbed, i.e. carbonate compounds with the calcite structure, these C-0 stretching modes occur at essentially one frequency (RAWLINS
THE
INFRA-RED
ABSORPTION
SPECTRA
OF CARBONATE
IN CALCIFIED
-MSSUES
681
and TAYLOR, 1928) and only a single absorption band is observed. It has been predicted that in the event that the symmetry of the ion is disturbed, the two modes will occur at different frequencies (HORNIG, 1948). When this happens, two separate frequencies or bands will be observed, and “splitting” or separation of the bands will occur. The symbol for this mode is vs. A second mode occurs near 115 p (~2) and consists of a vibration of the carbon atom along the threefold axis of the ion. This mode is not degenerate. It is frequently referred to as the “out of plane mode”. A third mode has been observed at 14 TVin the anhydrous carbonates (Ye),but has never been detected by us in any spectra of the calcified tissues. It has been shown that these absorption frequencies of the carbonate ion are sensitive to alterations in environment. HUNT and TURNER(1953) observed that the frequency of the out of plane mode (1 l-5 p) was displaced to successively Iongel wavelengths for the series magnesium, calcium-magnesium (dolomite), iron, strontium and bismuth carbonates. This finding was later confirmed by MILLERand WILKINS (1952). More recently KETELAARand VANDERELSKEN(1959) studied the changes in frequency and intensity of complex ions dissolved in alkali halides. They concluded in part that a shift to higher frequency occurred with decreasing lattice parameter of the solvent, but that frequency was also related to the nature of the ions in the halide lattice. Thus in the calcified tissues the presence of two separate peaks representing the out of plane mode suggests that the carbonate ion exists in two different sites in these substances. Although this has been suggested by earlier investigators (LOGAN and TAYLOR,1938) on the basis of chemical studies, the infra-red results would seem to be the first direct evidence of a non-random distribution of carbonate ion. If all carbonate ions existed in equivalent sites, we would expect to observe only one absorption peak occurring in this region, as in the spectra of the anhydrous carbonates, i.e. calcite, magnesite, etc. That this doublet was not observed by POSNER or UNDERWOOD can be explained by the fact that it is not apparent under normal scanning conditions. Furthermore, UNDERWOODreported using a slit width of I.25 mm. It is doubtful if such narrowly separated bands would be resolved under these conditions. it must be emphasized that these findings cannot, at this time, be used to determine the exact position of the CO, ion in relation to the crystallites of bone or enamel. For example, they should not be construed to indicate that the CO; ion occupies surface positions or lattice positions or both. The experiment involving sodium carbonate fused in KBr suggests that if a given amount of carbonate is distributed between two different sites, there results a reduction in the overall intensity of the out of plane mode of the two bands. This observation would partly explain the reduced intensity in the biologic minerals. No direct comparison regarding structure should be made between the results obtained with the fused halides and the presence of a doublet in the calcified tissues. Similar results might be obtained with mechanical mixtures of different carbonates. When enamel was first studied it was thought that the peak at 6-5 P arose from A-H bending modes in the protein fraction. The results of the annealing studies
W. H. EMHWN AND E. E. FISCHER
682
lead to the tentative conclusion that this band represents one of the C-O stretching modes of carbonate. If all the carbonate ions in enamel occupied identical sites, we would expect to observe only two bands in this region if splitting occurred. The presence of three bands can be accounted for by assuming that the ug mode is split in either one or both of two sites. In the latter instance, if two bands coincided or were very narrowly separated, the result would also be observed as three separate peaks. Splitting of the v) mode has been observed by us in complex synthetic carbonates such as 3 MgCOsMg (OH),nH,O with bands occurring at 6.7 and 7 p. Thus in enamel the configuration of bands in the 7 p region would further suggest that the carbonate ion is present in two uniquely different sites. A summary of carbonate absorption bands is presented in Table 1. TABLE 1. SUMMARYOF CARBONATEABSORPTION BANDSIN M~NERALIZEJJ TISSUES
Absorption bands of calcite have been included for comparison. v3 Substance Enamel Calcite Cod bone Protein free
(cm-l) 1538 1449 1408 1430 1538* 1460 1420
%
*b
(P)
(cm-l)
(P)
6.5 6.90 7.10 7.00 6.50 6.85 7.05
880
11.38
(cm-l)
Not observed
873 873 880 873
11.45 11.45 11.38 11.45
712 14.05 Not observed
(cc)
* Very weak band. Although splitting of the V~mode is observed in all extracted bone samples, the peaks are never as well defined as in enamel, and show slight differences in frequency.
The increased intensity after annealing is assumed to result from crystal perfection and release of strain. It is known that changes in crystal perfection may cause alterations in band width and intensity. Although most studies have demonstrated decreases in intensity with excessive grinding or pressure (DUYKAERTS, 1959; TIJDDENHAM and LYON, 1959), it has also been shown that recrystallization from a relatively disordered state will result in band intensification (BAKER, 1957). 0. R. TRAUTZ (personal communication) has noted relations between crystal perfection as determined by X-ray diffraction and the infra-red spectra of apatites precipitated in solutions containing carbonate. We have observed that when synthetic defect apatites were treated with carbonate-containing solutions, the doublet at 11-5 p cannot be resolved with present techniques. This suggests that in these materials the crystals are in a more amorphous state than in the biologic minerals; a relative lack of perfection in the latter might also account in part for reduced intensity and failure of absorption to increase with diminishing particle size. Further studies are in progress to determine the relation of annealing to band intensity and to determine if the results of the infrared studies can be expressed in terms of the crystal environment of the carbonate ion.
THBINFRA-RED ABSORPTIONSPECTRAOFCARBONAT!~INCALCIFIED
TISSUES
683
Acknowledgements-The authors wish to express their appreciation to Dr. AARON S. POSNER,National Institute of Dental Research, and Professor M. KENT WILSON, Chairman of the Department of Chemistry at Tufts University, for their cooperation in the initial stages of the investigation and for many helpful suggestions during the preparation of the manuscript. The samples of fish bone were kindly supplied by Dr. MELVINMoss, College of Physicians and Surgeons, New York. The authors wish to express their thanks to Mr. JOHN B. PLANKof the PerkinElmer Corporation for arranging these studies. This study was supported by U.S. Public Health Service Grant, D-1357, National Institutes of Health, REFERENCES BAKER, A. W. 1957. Solid state anomaliesin infra-red spectroscopy. J. phys. Chem. 61, 45&458. CHAMOT,E. M. and MASON, C. W. 1958. Handbook of Chemical Microscopy, Vol. 1 (3rd Ed.), p. 143. Wiley, New York. DUYKAERTS,G. 1959. The infra-red analysis of solid substances. Anaiysr 84, 201, 212-213. FRANK, R. M., SOGNNAES,R. F. and KERN, R. 1960. Calcification in Biological Systems, p. 187. American Association for the Advancement of Science, Washington. HENDRICKS,S. B. and HILL, W. L. 1950. The nature of bone and phosphate rock. Proc. Nat. Acad. Sci., Wash. 36, 731-737.
1960. Molecular Spectra and Molecular Structure, Spectra of Polyatomic Molecules. Van Nostrand, Princeton.
HERZBERG,G.
Vol. II. lnfra-rrd
and Kumon
HORNIG,D. F. 1948. The vibrational spectra of molecules and complex ions in crystals- I. General theory. J. them. Phys. 12, 1063-1076. HUNT, J. M. and TURNER,D. S. 1953. Determination of mineral constituents of rocks by infra-red spectroscopy. Analyt. Chem. 25, 1169-I 174. HUNT, J. M., WISHERD,M. and BONHAM,L. C. 1950. Infra-red absorption spectra of minerals and other inorganic compounds. Analyt. Chem. 22, 1478-1497. KETELAAR,J. A. A. and VAN DER ELSKEN, J. 1959. Frequency shifts in the infra-red absorption spectrum of complex ions in solid solution. J. chern. Phys. 30,336-337. LITTLE, M. F. and BRUDEVOLD,F. 1958. A study of the inorganic carbon dioxide in intact human enamel. J. dent. Res. 37, 994-995. LOGAN,M. A. and TAYLOR,H. L. 1938. Solubility of bone salt. J. b&l. Chetrr. K&391-397. MANLY, R. S. and HODGE, H. 1939. Density and refractive index studies of dental hard tissues. J. dent. Res. 18, 133-141. MILLER,F. A. and WILKINS,C. H. 1952. Infra-red spectra and characteristic frequencies of inorganic ions. Analyr. Chem. 24, 1253-1294. NEUMAN,W. F. and NEIJMAN,M. W. 1958. The Chemical Dynamics o,/ Bone Minrral. pp. 95-96. University of Chicago Press. POSNER, A. S. and DUYKAERTS,G. 1954. lnt’ra-red study of the carbonate in bone, teeth and francolite. Experienfia 10,424425. RAWLINS, F. I. G. and TAYLOR,A. M. 1928. The fnfia-red Analysi.sof Molecular Structurr. Cambridge University Press. TUDDENHAM,W. M. and LYON, R. J. P. 1959. Relation of infra-red spectra and chemical analysis for some chlorite minerals. Analyt. Chem. 31, 337-380. TUDDENHAM, W. M. and LYON,R. J. P. 1960. Infra-red techniques in the identification and measurement of minerals. Anulyt. Chenr. 32, 1630-I 634. UNDERWOOD,A. L., TORIBARA,T. Y. and NEUMAN,W. F. 1955. An infra-red study of the nature of bone carbonate. J. Amer. them. Sot. 77, 317-319. Wu, TA-YOU. 1939. Infra-red Spectra ofPolyaromic Molecules, p. 207. China Science Foundation, Shanghai.