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Aspartic acid interaction with Ca-montmorillonite: adsorption, desorption and thermal stability
ELSEVIER
Applied Clay Science 9 (1994) 265-281
Aspartic acid interaction with Ca-montmorillonite: adsorption, desorption and thermal stability A. Naidja, P.M. Huang * Department of Soil Science, University of Saskatchewan, Saskatoon, Sask., S7N OWO,Canada
Received 18 November 1993; accepted after revision 4 July 1994
Abstract Aspartic acid, one of the most abundant amino acids in nature, was adsorbed on Ca-montmorillonite at pH 7.0. The adsorption was a fast reaction process; 78% of the aspartic acid was retained by the clay surfaces at the end of a 15 min reaction period. The maximum amount adsorbed reached 84% (56.2 #mol/g clay) after 2 h. X-ray diffraction analysis showed that aspartic acid was intercalated in Ca-montmorillonite. The doo~ spacing of the aspartate---Ca-montmorillonite complex remained constant (18.1 A) with increasing temperature from 25 to 150°C. However, the d0o~ spacing of the aspartate-Ca-montmorillonite complex decreased linearly from 18.1 A to 10.3 A with increasing temperature from 150 to 600°C. The purification treatments of the montmorillonite did not affect its ability to adsorb aspartic acid and the nature of the aspartate-Ca-montmorillonite complex formed. Adsorbed aspartate was extractable by 1.0, 0.5 or 0.05M KC1 solutions and completely desorbed after two washings with water. At pH 7.0, aspartic acid appeared weakly bound on the clay surfaces. Hence, the data indicate that aspartic acid was intercalated into the montmorillonite by the formation of an outer-sphere complex with the exchangeable Ca through a "water bridge", and an H-bonding of the protonated amino group to the structural oxygen of the siloxane surfaces.
I. Introduction About 20% of the carbon in soil organic matter is in amino acids (S~rensen, 1972), and most of the nitrogen ( 9 0 % ) in soil is present in organic forms ( B o y d and Mortland, 1990). One half of this part o f nitrogen is identified as amino acids (Stevenson, 1982). However, the largest part of amino acids is bound in proteins or on other soil constituents (Flaig, 1971). The retention of organic matter in soil is due to its ability to form a complex with clay minerals (Pinck et al., 1954). In studying the change in concentration and nature of amino * Corresponding author. 0169-1317/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDIO169- 1317(94 )00022-0
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A. Naidja, P.M. Huang / Applied Clay Science 9 (1994) 265--281
acids in the Sahel ( Sudan ), Rougeon et al. (1990) showed that the concentrations of aspartic and glutamic acids and glycine are the highest in these soils, compared to the other amino acids. Organic compounds of biological importance, such as peptides and amino acids, including aspartic acid, are adsorbed by smectites (Theng, 1974). These aluminosilicates are extremely reactive in soil environments because of their expansive nature and their negative charge (Borchardt, 1989). During the last decade, Dashman and Stotzky (1984, Dashman and Stotzky, 1985, Dashman and Stotzky 1986) investigated the adsorption and binding of peptides and amino acids, including aspartic acid, on homoionic montmorillonite and kaolinite. They showed that the adsorption isotherms were dependent on the type of amino acid, the type of clay, the nature of the exchangeable cation and the pH of the suspension. According to the reviews of Theng (1974, Theng 1979), most studies on the adsorption of amino acids, peptides and proteins by clays were carried out in a medium (acidic pH) favoring the predominance of the correspondent cationic or zwitterionic forms of the adsorbate. Thus, the interaction of aspartic acid with Ca-montmorillonite at neutral pH, favoring the predominance of the correspondent anionic form (99.77%) of the adsorbate, deserves attention. Furthermore, to shed more light on the thermal stability of amino acid-clay complexes, a gradual heating over a wide range of temperatures (up to 600°C) and its effect on the d¢~j spacing of the amino acid-clay complex needs investigation. The decrease of the do~ spacing may be correlated with the conformation and the loss of weight of the intercalated organic substances upon thermal treatments. Determinations of the dc~1 spacing of an aspartate~Ca-montmoritlonite complex at different temperatures coupled with Fourier transform infrared (FTIR) analysis may help to understand the mechanism of bonding of aspartic acid in aspartate-clay complexes. The objectives of the present study were to examine ( 1 ) the adsorption of aspartic acid on Ca-saturated montmorillonite as a function of time, (2) the thermal stability of the aspartate-Ca-montmorillonite complex, and (3) the desorption of the adsorbed aspartate by KC1 and water. The effect of the purification treatments of the mineral on the adsorption of the amino acid was also investigated.
2. Materials and m e t h o d s
2. I. Preparation of montmorillonite samples The montmorillonite sample (SWy-I Crook County, Wyoming, USA) was obtained from the Source Clay Repository of the Clay Mineral Society. (i) The < 2 /~m fraction of Ca-montmorillonite (natural): 200 g of the sample was dispersed in deionized~:tistilled-boiled water overnight using a magnetic stirrer. The < 2 /~m fraction of the clay was collected by sedimentation. Ca-montmorillonite was obtained after several washings with 0.5M CaCI 2 (Wang and Huang, 1989). (ii) The < 2 / z m fraction of Ca-montmorillonite (purified): 200 g of the sample clay was sequentially treated with IM Na-acetate (pH 5.0), Na-citrate-bicarbonate-dithionite (pH 7.3) and H202 (30%) (Jackson, 1979). The purified montmorillonite was treated with 0.5M CaCI:, and then the < 2 /xm fraction of Ca-montmorillonite was obtained by sedimentation as previously described.
A. Naidja,P.M.Huang/Applied ClayScience9 (1994)265-281
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Both natural and purified samples were freeze-dried and stored. The cation exchange capacity (CEC) of the mineral was determined according to Alexiades and Jackson ( 1965 ). The CEC values of the < 2/.tm Ca-montmorillonite (natural) and < 2/xm Ca-montmorillonite (purified) were 84 and 85 cmol kg-~, respectively.
2.2. Adsorption of aspartic acid as a function of time Aspartic acid [HOOC-CH2-CH(NH2)-COOH] of high purity grade was supplied by Sigma Chemical Company, USA. In different flasks, 25 mg of Ca-montmorillonite (natural) were suspended in 5 ml of deionized-distilled-boiled water at pH 7.0, then 5 ml of 0.66mM aspartic acid solution at pH 7.0 was added. The final concentration of aspartic acid in the suspension was 0.33mM. The suspensions were shaken in a water bath at 25.0°C for different periods. After centrifugation at 19,000g, the amount of aspartic acid remaining in solution was measured by colorimetry with ninhydrin reagent at A= 570 nm (Rosen, 1957), as modified by Moore (1968), using a Pye Unicam SP6 UV-VIS spectrophotometer.
2.3. Preparation of aspartate-Ca-montmorillonite complex Five hundred milligrams of natural or purified Ca-montmorillonite was dispersed overnight in flasks containing 10 ml of deionized-distilled-boiled water at pH 7.0. The pH of the suspension was maintained at 7.0 with continuous stirring, using a Brinkmann 682 titroprocessor. Fifteen milliliters of 66.6 mM aspartic acid solution was added (the solution was freshly prepared and maintained at pH 7.0 by addition of 0.1M NaOH). The final concentration of aspartic acid in the suspension was 40.0mM. The suspension was shaken at 25.0°C for different periods in an oscillating ( 140 strokes/min) temperature-controlled water bath.
2.4. Desorption of adsorbed aspartic acid To examine the diffusion of aspartic acid adsorbed on the clay surfaces to the external solution, aspartic acid solutions of 40.0, 4.0, 0.40 and 0.04mM were prepared at pH 7.0. After adsorption equilibrium (2 h) of the 40.0mM aspartic acid solution by Ca-montmorillonite, the complex suspension was centrifuged ( 19,000g). The supernatant was removed and replaced by an aspartic acid solution of lower concentration (4.0mM) for a second equilibration period of 2 h. The operation was then repeated by replacing the supernatant for third and fourth equilibrium periods of 2 h with 0.40 and 0.04mM of aspartic acid solutions, respectively. After each equilibrium treatment, the amino acid-clay complexes were examined by X-ray diffraction analysis.
2.5. X-ray diffraction analysis (XRD) The montmorillonite or aspartate-montmorillonite complex before and after treatments with aspartic acid solutions or washings with KCI solutions or water were mounted on glass slides and maintained at a relative humidity (RH) of 50.0% at room temperature. XRD analysis was carried out on a Rigaku D/MAX-RBX diffractometer (Rigaku Company,
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A. Naidja, P.M. Huang /Applied Clay Science 9 (1994) 265-281
Japan) with monochromatic Cu-Kc~ radiation at 50 kV and 150 mA. A separate set of the samples were heated (2 h) at different temperatures (110-600°C) and kept dry in a desiccator on silica gel before XRD analysis.
2.6. Fourier transform infrared ( FTIR ) absorption spectrometry The FTIR spectra of the montmorillonite or aspartate-montmorillonite complex before and after washings with KCI solutions or water were recorded on KBr disks that contained 1% of sample by weight, using a BioRad-Digilab-3240 SPS spectrophotometer. The spectra were then referenced against the spectra of pure KBr (Spectrosol, BDH, England).
3. Results
3.1. Adsorption of aspartic acid as a function of time The adsorption of aspartic acid on natural Ca-montmorillonite was fast (Fig. 1). A substantial amount (78%) of aspartic acid was adsorbed on the clay mineral surfaces after 15 min, and the equilibrium was reached after 2 h (56.2 mmol/g clay). This fast adsorption (Fig. 1) was attributed, in part, to the preswelling of the clay in water overnight, giving a high degree of dispersion of the particles (Puri and Keen, 1925). Dashman and Stotzky (1982) showed that the adsorption isotherm of aspartic acid on Ca-montmorillonite at pH 70
60
E "0 e n
40
30 0
E 20
<
10
0
2
4
6
8
10
12
Time (h) Fig. I. Adsorption ot'aspartic acid (0.33mM) by natural Ca-montmorillonite (25 rag) as a function of time at pH 7.0 and 25.0°C.
A. Naidja, P.M. Huang/Applied Clay Science 9 (1994) 265-281
269
20
19 18
i
m
m
17
~ 16 0 0 '0
14
13 12 11 10
,
i
2
,
i
4
,
i
6
,
i
8
,
i
10
,
i
12
Time (11) Fig. 2. The doo~spacingof montmorillonite(natural) and aspartate--Ca-montmorillonite(natural) complexas a function of time at pH 7.0 and 25.0°C. ( • ) Ca-montmoriUoniteand ( • ) aspartate~a-montmorillonitecomplex, 8.21 was of S-class (high affinity of adsorbate to the adsorbent). There was an abrupt increase of dool spacing of the natural Ca-montmorillonite from 14.9 to 18.1 ,~ after 30 rain of reaction with aspartic acid (Fig. 2). Purified Ca-montmorillonite upon interaction with aspartic acid yielded the same data (not shown). This rapid expansion was evidently the result of the intercalation of aspartic acid in the Ca-montmorillonite structure. Barshad (1952) showed an expansion of the structure of Ca-vermiculite to 17.6/~ after adsorption of glycine. 3.2. FTIR analysis
FTIR analysis of the aspartate-Ca-montmorillonite complex (Fig. 3 and Table 1) confirmed the adsorption of the amino acid on the clay mineral surfaces. Fig. 3 shows the shifting of some bands after adsorption of aspartic acid on Ca-montmorillonite. The broad band at 3116 cm -~ (Figs. 3c and 3d) corresponds to the NH~" group (Cross and Alan Jones, 1969; Bellamy, 1975). The band at 1618 c m - l (Figs. 3c and 3d), which was much stronger than the band (Fig. 3b) corresponding to the deformation of the OH group of hygroscopic water ( 1620-1630 cm 1) in untreated montmorillonite, was attributed to the C O 0 - stretching vibration (Nakagawa et al., 1965; Orlov, 1985). The shoulder at 1478 c m - ~ might be due to the symmetric deformation of the NH~- group weakly bound on the mineral surface (Nakagawa et al., 1965; Orlov, 1985). The band of high intensity, at 1407
270
A. Naidja, P,M. Huang/ Applied Clay Science 9 (1994) 265-281
..£2
(b)
~
~ ~11,
me4
m
o~i l/
~
1
~" ~ooo
(o) 3~0o
3ooo
2 5 o o 2o0o
Wavenumber
~soo
(crn-~)
Fig. 3. Infrared spectra of the aspartate-Ca-montmorillonite (natural) comple×; (a) pure aspartic acid, (b) Camonlmorillonite, (c) aspartate-Ca-monlmorillonite complex (0.5 h of conlact) and (d) aspartate-Ca-montmorillomte complex (24 h of contact).
cm r indicates the symmetric stretching vibration of ionized CO() group at the end of the chain; this band appears at 1416 cm ~ in intercalated lysine on Ca-montmorillonite (Nakamoto et al., 1961: Jang and Condrate, 1972). The bands at 1351, 1309 and 1224 cm t (Figs. 3c and 3d) represent C O 0 , CH deformation and CO stretching vibration (Cross and Alan Jones, 1969; Jang and Condrate, 1972). The increase in the intensity of these absorption bands after 24 h of contact (Fig. 3d) indicated that the adsorption equilibrium was not reached after thirty minutes of contact. Purified Ca-montmorillonite upon interaction with aspartic acid yielded the same data (not shown). 3.3. EffEct of temperature on the do.: spacing of the aspartate-Ca-montmorillonite complex The natural and purified Ca-montmorillonite samples and the aspartate-Ca-montmorilIonite complex were heated at different temperatures. Fig. 4 shows the variation of the d~,~ spacing with temperature. For the natural Ca-montmorillonite before reaction with aspartic
A. Naidja, P.M. Huang/Applied Clay Science 9 (1994) 265-281
271
Table 1 FTIR bands assignmentof the aspartate-Ca-montmorillonitea complexafter 0.5 h of reaction at pH 7.0 and 25.0°C. Values are wavenumbers (cm - 1 ) Aspb -
3021 2960 1693 1646 1605 1517 1424 1351 1309 1249
Ca-Mte c
Asp--Ca-Mte
Assignment~
3634 3416 1627
3634 3416 3116 1618 1478 1407 1351 1309 1224
vOH, Mte vOH, H20 vNH3+ vCH v~CO,COOH vaCOO8OH, H20 vaCOOv~NH3+ vsCOOvsCOO6CH vCO
-
" Natural clay h Asp: aspartic acid, only the main bands are given. c Mte: montmorillonite. d v, vs and v,: stretching vibrations, symmetric and asymmetric; & deformation. acid, the dooL spacing decreased drastically from 14.9 ,~ (room temperature) to 10.2 ,~ at 150°C (Fig. 4), indicating the loss of water molecules which occur mostly as interlayer water between the silicate sheets (Grim, 1968). Upon further heating to 600°C, the dool spacing slowly decreased until the interlayers were completely collapsed (dool = 9.6 .~). Similar data were obtained with the purified clay (Fig. 4). For the aspartate-Ca-montmoriilonite (natural clay) complex, the interlayer spacing remained constant (doo~ = 18.1 ,~) upon heating to 150°C and slowly decreased to 17.2 A at 170°C (Fig. 4), indicating a slight dehydration of the aspartate-Ca-montmorillonite complex. Upon heating from 170 to 600°C, the doo~ spacing of the complex decreased linearly ( r 2 = 0.997 and p --- 1 0 - 9), at a rate of 0.017/~/°C, with increasing temperature to 10.3 ,~. This may correspond to a thermal desorption of the decomposition products, as suggested by M c A t e e and H a w t h o m e ( 1 9 6 4 ) and Chou and McAtee (1969) for some organo-ammonium compounds adsorbed by montmorillonite. The aspartate-Ca-montmorillonite complex (purified clay) showed the same trend (Fig. 4). In the presence of aspartic acid, the interlayer space did not completely collapse, even after heating at 500°C (dnol = 12.1 .~ with natural and 12.0 .~ with purified clay). Dashman and Stotzky (1985) found that the dool spacing of an aspartate-Ca-montmorillonite complex decreased to 10.0 ,~ after heating at 150°C (48 h under vacuum). Heat treatment above 170°C affected the stability of the aspartate-Ca-montmorillonite complex (Fig. 5 ). Heating at 200°C affected the adsorbed amino acid and its transformation (oxidation and decomposition). The FTIR spectra (Fig. 5) showed the disappearance of the main bands o f the amino acid adsorbed on Ca-montmorillonite surface after heating at 200°C and the appearance of a strong band at 1078 c m - 1, which was observed after heating at 170°C and different from that assigned to the S i - O group at 1047 c m - ~ in the aluminos-
272
A. Naidja. P.M. Huang/Applied Clay Science 9 (1994)265-281
2o!
•o
14
Temperature (°C) Fig. 4. Effect of heating on the d(x~]spacing of the aspartate-Ca-montmorillonitecomplex: untreated clay system and purified clay system. (0) Ca-montmorilloniteand (11) aspartate-Ca-montmorillonitecomplex (untreated clay): (©) Ca-montmorilloniteand ([]) aspartate-Ca-montmorillonitecomplex (purified clay). ilicate lattice. The band at 1078 cm t, which persisted with heating at 600°C, might be attributed to the skeleton ~ 2 ~ - (Nakagawa et al., 1965) remaining after oxidation or destruction of the adsorbed amino acid. Tettenhorst (1962) assumed that the band at 1078 c m - ~ indicates that some interlayer cations migrate to the silicate skeleton. On the other hand, Farmer and Russell (1964) ascribed this band at 1078 c m - ' to the irregularity in the structure or to the ionic substitution in the lattice caused by the increase of heating.
3.4. Desorption of adsorbed aspartic acid Diffusion of adsorbed aspartic acid Fig. 6 shows that after equilibrating with 4.0mM aspartic acid, the initial peak at 18.1 corresponding to the d(×)~ spacing of the aspartate-Ca-montmorillonite complex, which was formed upon interaction with 40.0mM aspartic acid, changed to 17.9 A,, and a new peak at 12.8 A appeared, indicating that interlayer aspartic acid diffused to the diluted aspartic acid solution. The concentration of aspartic acid at 4.0mM seemed to be critical for the coexistence of two layers, namely the 17.9 .~ phase, corresponding to the aspartic acid molecule intercalated montmorillonite, and the 12.8 ,~ phase, corresponding to the hydrated Nasaturated montmorillonite (Fig. 6c). Aspartic acid was transformed to the sodium salt when the pH of the aspartic acid solution was adjusted from 2.9 to 7.0 by the addition of 0.1M NaOH. In the presence of lower concentrations of aspartic acid (0.40 and 0.040mM), only
A. Naidja, P.M. Huang /Applied Clay Science 9 (1994) 265-281
273
c
o
4000
3500
3000
2500
Wavenumber
2000
1500
1000
( c m -t )
Fig. 5. Infraredspectraof the aspartate-Ca-montmorillonite(natural) complexafter heating at differenttemperatures (2 h). the peaks at 12.2-12.3 A, characteristic of Na-montmorillonite, were observed. The diffusion of adsorbed aspartic acid to the bulk solution, which contained aspartic acid at the lower concentrations, apparently resulted from competition with water molecules for the adsorption sites, which is in accord with the complete removal of adsorbed aspartate after the second washing with water.
Desorption by water After the first washing with water (pH 7.0), the strong peak at 18.1 A changed to a weak peak at 17.9 ]~ (Fig. 7c), indicating the desorption of most of the interlayer aspartic acid molecules. In addition to the peak at 17.9 ,~, a peak at 12.2 ]~ was observed after the first washing with water. The dool spacing of the aspartate-Ca-montmorillonite complex collapsed to 11.6 ,~ (Fig. 7d) after the second washing with water, which was the result of a complete desorption of the amino acid, as confirmed by FTIR spectroscopy (Fig. 8d).
274
A. Naidja. P.M. Huang /Applied Clay Science 9 (1994) 265-281 o(I o)
J L_,~(b) 0
ao
V~(c)
J
Y
J
~_(e)
I
I
I
I
2.0
5.0
10.0
15.0
*20, Cu Kc~ Fig. 6. X-raydiffractionpatternsof the aspartate-Ca-montmorillonite( natural) complexalter contacts ( 2 h) with a series of diluted aspartic acid solutions: (a) Ca-montmorillonite;(b) aspartate-Ca-montmorillonitecomplex formedin the presenceof asparticacid at an initialconcentrationof 40.OmM;and the aspartate-Ca-montmoriUonite complex after subsequentcontactwith4.0mM aspartic acid (c), 0.40mMasparticacid (d) and 0.04 mM aspartic acid re). Extraction by KCl After 1 h of contact of the aspartate~Ca-montmorillonite complex with 1M and 0.5M KC1, the do(, spacing of the aspartate-Ca-montmorillonite complex decreased to l 1.8 and 11.9 A, respectively, which are virtually the same as the d~)~ spacing of the unreacted K-montmorillonite (Figs. 9c and 9d), indicating that the aspartic acid initially adsorbed was extracted by KC1, as confirmed by FTIR spectroscopy (Fig. 8f). The negative charge of the clay layers was balanced by K + cations after the KC1 extraction. Greenland et al. (1965a) showed that histidine and arginine were retained against a 20 h extraction with
A. Naidja, P.M. Huang/Applied Clay Science 9 (1994) 265-281
275
o,1¢
~'.o ~.o "20,
1o'.o
~5.0
Cu Kot
Fig. 7. X-ray diffraction patterns of the aspartate-Ca-montmorillonitecomplexbefore and after several washings with water: Ca-montmorillonite (a); aspartate-Ca-montmorillonite complex (b); the aspartate-Ca-montmorillonite complexafter the first washing (c), after the secondwashing (d). after the third washing (e); and unreacted Na-montmorillonite (f). I M KC1 to an extent ranging from 28 to 58% of the amount initially present, whereas lysine was almost completely extracted from the highly expanded complex formed with montmorillonite. Hence, the extraction by KC1 of adsorbed amino acids on clay minerals depends on the nature of the amino acid and the type of the complex formed with the interlayer exchangeable cation. Greenland et al. (1965b) stated that aspartic acid, which has a large charge and a small hydrocarbon chain, is dissolved in KC1. When the concentration of the KCI solution was decreased to 0.05M, the dool spacing of the aspartate-Ca-montmorillonite complex decreased to 14.9 ,~., which was the phase corresponding to Ca-saturated montmorillonite before adsorption (Fig. 9e), suggesting that only the adsorbed aspartic acid was removed and the exchangeable Ca remained intact.
4. Discussion 4. I. Thermal stability o f aspartate-Ca-montmorillonite complex The thermal stability of clay minerals has been extensively studied (Russell and Farmer, 1964; MacEwan and Wilson, 1980; Harris et al., 1992) to determine the mechanisms of the
276
A. Naidja, P.M. Huang / Applied Clay Science 9 (1994) 265-281
(1) u e--
4000 3soo
3000 2soo
2000
15oo
looo
Wavenumber (cm-1) Fig. 8. Infrared spectra of the aspartate-Ca-montmorillonite (natural) complex after washing with water and extraction with KCI for I h. (a) Ca-montmorillonite;aspartate-Ca-montmorillonitecomplexbefore washing with water (b), after the first washing with water (c), after the second washing with water (d), after the third washing with water (e), and after extraction with 1.0M KCI (f). hydration/dehydration processes and subsequently the structure and the classification of such minerals in soil. However, despite the significant occurrence of amino acids in soil (Stevenson, 1982), detailed work on the thermal stability of amino acid--clay mineral complexes still remains lacking (Cloos et al., 1966). Upon a gradual heating over a wide range of temperatures ( 110 to 600°C), the aspartate~2a-montmoritlonite complex was not dehydrated after heating to 150°C. The dehydration of adsorbed monocarboxylic amino acids occurred around 140°C (Cloos et al., 1966). Kanamaru and Vand (1970) assumed that water molecules might have an important role in stabilizing the interlayer structure by hydrogen bonding with the oxygen in the C = O group and the nitrogen in the NH~- group. In the case of the aspartate-Ca-montmorillonite complex, the loss of interlayer water molecules coordinated to the exchangeable Ca cation and the aspartic acid apparently started after a temperature higher than 150°C, and the do~l spacing decreased linearly with increasing temperature (Fig. 4). Therefore, interlayer aspartic acid molecules, which were apparently linked to the Ca through water bridges and also bound to the silicate tetrahedral sheet through hydrogen bonding as discussed below, were destabilized after dehydration at a temperature higher than around 170°C (Fig. 4). Adsorbed aspartic acid was transformed (oxidized and decomposed) after heating at 200°C (Fig. 5).
4.2. Adsorption/desorption of aspartic acid The adsorption of amino acids, peptides and proteins on clay minerals has been investigated by numerous researchers (Walker and Garrett, 1961; Fripiat et al., 1966; Harter and
A. Naidja, P.M. Huang / Applied Clay Science 9 (1994) 265-281
277
L_2°) 0
j 2'.0
;.o "2~,
' 10.0
' 15.0
Cu K(x
Fig. 9. X-mydiffraction patterns of the aspartate-Ca-montmorillonitecomplexafterextraction for 1 h with KCI salt solution: (a) Ca-montmorillonite;(b) the aspartate-Ca-montmorillonitecomplexbeforeextraction;aspartateCa-montmoriUoniteafter extraction with 1.0M KC1 (c), 0.5M KCl (d), 0.05M KCI (e); and unreacted Kmontmorillonite(f). Stotzky, 1973; Dashman and Stotzky, 1985, Dashman and Stotzky, 1986; Naidja and Siffert, 1989; Siffert and Naidja, 1992). The present study showed that the adsorption of aspartic acid by Ca-montmorillonite is a rapid reaction process (Fig. 1). At pH 7.0, aspartic acid appeared to be weakly adsorbed on the montmorillonite surfaces, including the interlayer spaces. The intercalation of aspartic acid in the montmorillonite was evidently responsible for the interlayer expansion of Ca-saturated montmorillonite (Fig. 2). pH is an important factor governing the interaction between aluminosilicates and amino acids (Theng, 1974), as amino acids are amphoteric, and their retention by the silicate layers is more effective when they are positively charged. The positive charge is important in enabling aspartic acid to approach the negatively charged clay. The influence of pH on the speciation of aspartic acid is described below:
A. Naidja, P.M. Huang /Applied Clay Science 9 (1994) 265-281
278
_n +
HOOC-CH2-~H-COOH
_..
NH3 +
(+ 1)
~.
-OOC-CH2-~H-COOH
+H +
NH3 +
pKl = 2.10
(0)
p l = 2.98
(isoelectric point) /IN
-H+ ][+H + pK2= 3.86
q|
_n +
"OOC-CH2-~H-COONH2 (-2)
"~
./ +H+ pK3 = 9.82
"OOC-CH2-ffH-COO NH3+ (- 1)
At pH 7.0, the predominant species, determined from the dissociation constants (Weast, 1980), have net negative charge, as described below, and accounts for 99.77% of the aspartic acid concentration : -OOC-CH2-~H-COONH3 ÷ In solution, aspartic acid is chelated to alkaline earth metal cations as a bidentate (Lumb and Martell, 1953). However, the mechanism of bonding of aspartic acid with exchangeable Ca as a complex in the interlayer space of montmorillonite, as observed in this study, may differ from that in solution, as suggested by Lumb and Martell (1953). The easy removal (desorption) of intercalated aspartate from the clay in dilute aspartic acid solution (Fig. 6), water (Figs. 7 and 8) and KCI solutions (Figs. 8 and 9) indicated that aspartic acid was weakly bound to the clay silicate layers at pH 7.0. Cloos et al. (1966) assumed that, in acidic medium (pH ~ 3), the adsorption of glycine, glycylglycine and /3-alanine hydrochlorides by sodium and calcium montmorillonite occurs essentially through ion exchange or proton transfer mechanisms. Subsequently, various other mechanisms have been proposed to interpret the adsorption of amino acids by clays (Mortland, 1970, Mortland, 1986; Stotzky, 1986). The function of the exchangeable cation seemed to be paramount in the different interactions, including direct coordination of polar groups (carboxyl, carbonyl or amino) to the exchangeable cation or indirect coordination to the exchangeable cation through a "water bridge". In addition, the protonated amino group (--lqH3) is an excellent hydrogen-bond donor (Marsh and Donohue, 1967), and it can form a hydrogen-bond with the structural oxygen of siloxane surfaces (Raussel-Colom and Salvador, 1971 ). The complete removal of the weakly adsorbed aspartate by the second washing with water (Figs. 7d and 8d) or by IM, 0.5M and 0.O5M KCI solutions (Figs. 9c, 9d and 9e) substantiates the reasoning that the formation of an H-bonding may be the predominant binding force in the intercalation of aspartate in montmorillonite, as shown below in the proposed scheme. The carboxylate groups of aspartate were apparently bound to the hydrated Ca by a water bridge through an H-bonding in an outer-sphere complex. In addition, the protonated amino group (--lqH3) also appeared to interact with the structural oxygen of the siloxane surfaces through an H-bonding (Raussel-Colom and Salvador, 1971). Stotzky (1986) stated that, it is probable that several mechanisms function simultaneously in the surface interaction of clay and the biological entities. In the present system, the cation exchange mechanism was apparently not involved, since the positive charge of the proton-
A. Naidja, P.M. Huang / Applied Clay Science 9 (1994) 265-281
279
ated amino group is compensated by the intra-molecular interaction in the net negatively charged aspartate.
I
H I
y H20
_Q.X,,+~H2
H 2 0 . O--H" • • ~ - - - - C H
cla /
I .
H
.
.
.
0
5. Conclusions The aspartate-Ca-montmorillonite complex was resistant to dehydration with increasing temperature to 150°C. Heating at higher temperatures ( > 200°C) caused an alteration of the aspartate-Ca-montmorillonite complex and degradation ( oxidation and decomposition) of the intercalated amino acid, which led to a linear decrease in the doo~ spacing of the complex with increasing temperature. The intercalated aspartate was virtually removed after the second washing with water. It was easily extractable by one washing with 1.0M, 0.5M or 0.05M KC1. Therefore, at neutral pH and in its corresponding anionic form, aspartate was weakly intercalated in montmorillonite layers. The intercalation appeared to proceed through the formation of an outer-sphere complex with exchangeable Ca, and an H-bonding of the protonated amino group to the structural oxygen of siloxane surfaces. Consequently, in the terrestrial environment with the pH near neutrality, the fraction of the aspartic acid not mineralized by microbes would be readily transported from the soil to ground and surface waters through diffusion and leaching.
Acknowledgements This study was supported by Grant GP 2383-Huang of the Natural Sciences and Engineering Research Council of Canada.
References Alexiades, C.A. and Jackson, M.L., 1965. Quantitative determination of vermiculite. Soil Sci. Soc. Am. Proc., 29: 522-529.
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Barshad, 1., 1952. Factors affecting the interlayer expansion of vermiculite and montmorillonite with organic substances. Soil Sci. Soc. Am. Proc., 16: 176-182. Bellamy, L.J., 1975.The Infra-red Spectra of Complex Molecules. 3rd ed. Chapman and Hall, London. Borchardt, G.A., 1989. Smectites. In: J.B. Dixon and S.B. Weeds (Editors), Minerals in Soil Environments. Soil Sci. Soc. America, Madison, WI, 2nd ed., pp. 675-727. Boyd, S.A. and Mortland, M.M., 1990. Enzyme interactions with clays and clay-organic matter complexes. In: J.M. Bollag and G. Stotzky (Editors), Soil Biochemistry, 6. Marcel Dekker, New York, pp. 1-28. Chou, C.C. and McAtee, J.L., Jr., 1969. Thermal decomposition of organo-ammonium compounds exchanged onto montmorillonite and hectorite. Clays Clay Miner., 17: 339-346. Cloos, P., Calicis, B., Fripiat, J.J. and Makay, K., 1966. Adsorption of amino-acids and peptides by montmorillonite. I Chemical and X-ray diffraction studies. Proc. Int. Clay Conf. (Jerusalem), pp. 233-245. Cross, A.D. and Alan Jones, R., 1969. An Introduction to practical Infra-red Spectroscopy. 3rd ed. Butterworths, London. Dashman, T. and Stotzky, G., 1982. Adsorption and binding of amino acids on homoionic montmorillonite and kaolinite. Soil Biol. Biochem, 14: 447-456. Dashman, T. and Stotzky, G., 1984. Adsorption and binding of peptides on homoionic montmorillonite and kaolinite. Soil Biol. Biochem., 16:51-55. Dashman, T. and Stotzky, G., 1985. Physical properties of homoionic montmorillonite and kaolinite complexed with amino acids and peptides. Soil Biol. Biochem., 17: 189-195. Dasbman, T. and Stotzky, G., 1986. Microbial utilization of amino acids and a peptide bound on homoionic montmorillonite and kaolinite. Soil Biol. Biochem., 18: 5-14. Farmer, V.C. and Russell, J.D., 1964. The infra-red spectra of layer silicates. Spectrochim. Acta, 20:1149-1176. Flaig, W., 1971. Organic compounds in soil. Soil Sci., I11: 19-33. Fripiat, J.J., Cloos, P., Calicis, B. and Makay, K., 1966. Adsorption of amino-acids and peptides by montmorillonite. II. Identification of adsorbed species and decay products by infrared spectroscopy. Proc. Int. Clay Conf. (Jerusalem), pp. 223-232. Greenland, D.J., Laby, R.H. and Quirk, J.P., 1965a. Adsorption of amino-acids and peptides by montmorillonite and illite. I. Cation exchange and proton transfer. Trans. Farad. Soc., 61: 2013-2023. Greenland, D.J., Laby, R.H. and Quirk, J.P., 1965b. Adsorption of amino-acids and peptides by montmorillonite and illite. I1. Physical Adsorption. Trans. Farad. Soc., 61: 2024-2035. Grim, A., 1968. Clay Mineralogy. McGraw Hill, New York. Harris. W.G., Hollien, K.A., Bates, S.R. and Acree, W.A., 1992. Dehydration of hydroxy-interlayered vermiculite as a function of time and temperature. Clays Clay Miner., 20: 335-340. Harter, D. and Stotzky, G., 1973. X-ray diffraction, electron microscopy, electrophoretic mobility, and pH of some stable smectite-protein complexes. Soil Sci. Soc. Am. Proc., 37 : 116-123. Jackson, M.L., 1979. Soil Chemical Analysis - - Advanced Course. 2nd ed. Published by the author, Dept. of Soil Science, Univ. of Wisconsin, Madison, Wl. Jang, S.D. and Condrate, R.A., St., 1972. The IR spectra of lysine adsorbed on several cation-substituted montmorillonites. Clays Clay Miner., 20: 79-82. Kanamaru, F. and Vand, V., 1970. The crystal structure of clay organic complex of 6-amino hexanoic acid and vermiculite. Am. Miner., 55: 1550-1560. Lumb, R.F. and Martell, A.E., 1953. Metal chelating tendencies of glutamic and aspartic acids. J. Phys. Chem., 57: 690-693. MacEwan, D.M.C. and Wilson, M.J., 1980. Interlayer and intercalation complexes of clay minerals. In: G.W. Brindley and G. Brown (Editors), Crystal Structures of Clay Minerals and their X-ray Identification. Mineralogical Society, London, pp. 197-248. M~u'sh, R.E. and Donohue, J.. 1967. Crystal structure studies of amino acids and peptides. Adv. Protein Chem., 22: 235-256. McAtee, J.L., Jr. and Hawthorne J.M., 1964. Heating-oscillating X-ray diffraction studies of some organomontmorillonites. Am. Miner.. 49: 247-257. Moore, S., 1968.Amino acids analysis: aqueous dimethylsulfoxide as solvent for the uinhydriu reaction. J. Biol. Chem., 243: 6282-6283. Mot/land, M.M., 1970. Clay-organic complexes and interactions. Adv. Agron., 23:75-117
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Mortland, M.M., 1986. Mechanisms of adsorption of nonhumic organic species by clays. In: P.M. Huang and M. Schnitzer, (Editors), Interactions of Soil Minerals with Natural Organics and Microbes. Soil Sci. Soc. Am., Madison, WI, pp. 305--429. Naidja, A. and Siffert, B., 1989. Glutamic acid deamination in the presence of montmorlllonite. Clay Miner., 24: 649-661. Nakagawa, 1., Hooper, R.J., Walter, J.L. and Laneo, T.J., 1965. Infrared absorption spectra of metal-amino acid complex. III. The infrared spectra and normal vibrations of metal-valine chelates. Spectrochim. Acta, 21: 114. Nakamoto, K., Morimoto, Y. and Martell, A., 1961. Infrared spectra of addition compounds of metallic acetylacetonates. J. Am. Chem. Soc., 83: 4533-4536. Orlov, D.S., 1985. Humus Acids of Soils. Balkema, Rotterdam. Pinck, L.A., Dyal, R.S. and Allison, F.E., 1954. Proteins-montmorillonite complexes, their preparation and the effects of soil microorganisms on their decomposition. Soil Sci., 78:109-118. Purl, A.N. and Keen, B., 1925. The dispersion of soil in water under various conditions. J. Agfic. Sci., 15: 146161. Raussel-Colom, J.A. and Salvador, P.S., 1971. Complexes vermiculite-aminoacides. Clay Miner., 9:139-149. Rosen, H., 1957. A modified ninhydrin colorlmetrlc analysis for amino acids. Arch. Biochem. Biophys., 67: 1015. Rougeon, D., Rougeon, C., Levieux, J. and Trichet, J., 1990. Variation in the amino-acid content in zebu dung in the Sahel during nesting by dung-beetles (Coleoptera, Carabaeidae). Soil Biol. Biochem., 22: 217-223. Russell, J.D. and Farmer, V.C., 1964. Infra-red spectroscopic study of the dehydration of montmorlllonite and saponite. Clay Miner. Bull., 5: 443-464. Siffert, B. and Naidja, A., 1992. Stereoselectivity of montmorillonite in the adsorption and deamination of some amino acids. Clay Miner., 27:109-118. Scrensen, L.H., 1972. Role of amino acid metabolites in the formation of soil organic matter. Soil Biol. Biochem., 4: 245-255. Stevenson, F.J., 1982. Origin and distribution of nitrogen in soil. In: F.J. Stevenson, (Editor), Nitrogen in agricultural soils. Soil Sci. Soc. Am., Madison, WI, pp. 1~,2. Stotzky, G., 1986. Influence of soil mineral colloids on metabolic processes, growth, adhesion, and ecology of microbes and viruses. In: P.M. Huang and M. Schnitzer, (Editors), Interactions of Soil Minerals with Natural Organics and Microbes. Soil Sci. Soc. Am., Madison, W1, pp. 305--429. Tettenhorst, R., 1962. Cation migration in montmorlllonites. Am. Miner., 47: 769-773. Theng, B.K.G., 1974. The Chemistry of Clay Organic Reactions. Wiley, New York. Theng, B.K.G., 1979. Formation and Properties of Clay-Polymer Complexes. Elsevier, Amsterdam. Walker, G.F. and Garrett, W.G., 1961. Complexes of vermiculite with amino-acids. Nature, 191 : 1389. Wang, M.C. and Huang, P.M., 1989. Pyrogallol transformations as catalyzed by nontronite, bentonite, and kaolinite. Clays Clay Miner., 37: 525-531. Weast, C., 1980. Handbook of Chemistry and Physics. CRC Press, Boca Raton, FL.
Applied Clay Science 9 (1994) 265-281
Aspartic acid interaction with Ca-montmorillonite: adsorption, desorption and thermal stability A. Naidja, P.M. Huang * Department of Soil Science, University of Saskatchewan, Saskatoon, Sask., S7N OWO,Canada
Received 18 November 1993; accepted after revision 4 July 1994
Abstract Aspartic acid, one of the most abundant amino acids in nature, was adsorbed on Ca-montmorillonite at pH 7.0. The adsorption was a fast reaction process; 78% of the aspartic acid was retained by the clay surfaces at the end of a 15 min reaction period. The maximum amount adsorbed reached 84% (56.2 #mol/g clay) after 2 h. X-ray diffraction analysis showed that aspartic acid was intercalated in Ca-montmorillonite. The doo~ spacing of the aspartate---Ca-montmorillonite complex remained constant (18.1 A) with increasing temperature from 25 to 150°C. However, the d0o~ spacing of the aspartate-Ca-montmorillonite complex decreased linearly from 18.1 A to 10.3 A with increasing temperature from 150 to 600°C. The purification treatments of the montmorillonite did not affect its ability to adsorb aspartic acid and the nature of the aspartate-Ca-montmorillonite complex formed. Adsorbed aspartate was extractable by 1.0, 0.5 or 0.05M KC1 solutions and completely desorbed after two washings with water. At pH 7.0, aspartic acid appeared weakly bound on the clay surfaces. Hence, the data indicate that aspartic acid was intercalated into the montmorillonite by the formation of an outer-sphere complex with the exchangeable Ca through a "water bridge", and an H-bonding of the protonated amino group to the structural oxygen of the siloxane surfaces.
I. Introduction About 20% of the carbon in soil organic matter is in amino acids (S~rensen, 1972), and most of the nitrogen ( 9 0 % ) in soil is present in organic forms ( B o y d and Mortland, 1990). One half of this part o f nitrogen is identified as amino acids (Stevenson, 1982). However, the largest part of amino acids is bound in proteins or on other soil constituents (Flaig, 1971). The retention of organic matter in soil is due to its ability to form a complex with clay minerals (Pinck et al., 1954). In studying the change in concentration and nature of amino * Corresponding author. 0169-1317/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDIO169- 1317(94 )00022-0
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acids in the Sahel ( Sudan ), Rougeon et al. (1990) showed that the concentrations of aspartic and glutamic acids and glycine are the highest in these soils, compared to the other amino acids. Organic compounds of biological importance, such as peptides and amino acids, including aspartic acid, are adsorbed by smectites (Theng, 1974). These aluminosilicates are extremely reactive in soil environments because of their expansive nature and their negative charge (Borchardt, 1989). During the last decade, Dashman and Stotzky (1984, Dashman and Stotzky, 1985, Dashman and Stotzky 1986) investigated the adsorption and binding of peptides and amino acids, including aspartic acid, on homoionic montmorillonite and kaolinite. They showed that the adsorption isotherms were dependent on the type of amino acid, the type of clay, the nature of the exchangeable cation and the pH of the suspension. According to the reviews of Theng (1974, Theng 1979), most studies on the adsorption of amino acids, peptides and proteins by clays were carried out in a medium (acidic pH) favoring the predominance of the correspondent cationic or zwitterionic forms of the adsorbate. Thus, the interaction of aspartic acid with Ca-montmorillonite at neutral pH, favoring the predominance of the correspondent anionic form (99.77%) of the adsorbate, deserves attention. Furthermore, to shed more light on the thermal stability of amino acid-clay complexes, a gradual heating over a wide range of temperatures (up to 600°C) and its effect on the d¢~j spacing of the amino acid-clay complex needs investigation. The decrease of the do~ spacing may be correlated with the conformation and the loss of weight of the intercalated organic substances upon thermal treatments. Determinations of the dc~1 spacing of an aspartate~Ca-montmoritlonite complex at different temperatures coupled with Fourier transform infrared (FTIR) analysis may help to understand the mechanism of bonding of aspartic acid in aspartate-clay complexes. The objectives of the present study were to examine ( 1 ) the adsorption of aspartic acid on Ca-saturated montmorillonite as a function of time, (2) the thermal stability of the aspartate-Ca-montmorillonite complex, and (3) the desorption of the adsorbed aspartate by KC1 and water. The effect of the purification treatments of the mineral on the adsorption of the amino acid was also investigated.
2. Materials and m e t h o d s
2. I. Preparation of montmorillonite samples The montmorillonite sample (SWy-I Crook County, Wyoming, USA) was obtained from the Source Clay Repository of the Clay Mineral Society. (i) The < 2 /~m fraction of Ca-montmorillonite (natural): 200 g of the sample was dispersed in deionized~:tistilled-boiled water overnight using a magnetic stirrer. The < 2 /~m fraction of the clay was collected by sedimentation. Ca-montmorillonite was obtained after several washings with 0.5M CaCI 2 (Wang and Huang, 1989). (ii) The < 2 / z m fraction of Ca-montmorillonite (purified): 200 g of the sample clay was sequentially treated with IM Na-acetate (pH 5.0), Na-citrate-bicarbonate-dithionite (pH 7.3) and H202 (30%) (Jackson, 1979). The purified montmorillonite was treated with 0.5M CaCI:, and then the < 2 /xm fraction of Ca-montmorillonite was obtained by sedimentation as previously described.
A. Naidja,P.M.Huang/Applied ClayScience9 (1994)265-281
267
Both natural and purified samples were freeze-dried and stored. The cation exchange capacity (CEC) of the mineral was determined according to Alexiades and Jackson ( 1965 ). The CEC values of the < 2/.tm Ca-montmorillonite (natural) and < 2/xm Ca-montmorillonite (purified) were 84 and 85 cmol kg-~, respectively.
2.2. Adsorption of aspartic acid as a function of time Aspartic acid [HOOC-CH2-CH(NH2)-COOH] of high purity grade was supplied by Sigma Chemical Company, USA. In different flasks, 25 mg of Ca-montmorillonite (natural) were suspended in 5 ml of deionized-distilled-boiled water at pH 7.0, then 5 ml of 0.66mM aspartic acid solution at pH 7.0 was added. The final concentration of aspartic acid in the suspension was 0.33mM. The suspensions were shaken in a water bath at 25.0°C for different periods. After centrifugation at 19,000g, the amount of aspartic acid remaining in solution was measured by colorimetry with ninhydrin reagent at A= 570 nm (Rosen, 1957), as modified by Moore (1968), using a Pye Unicam SP6 UV-VIS spectrophotometer.
2.3. Preparation of aspartate-Ca-montmorillonite complex Five hundred milligrams of natural or purified Ca-montmorillonite was dispersed overnight in flasks containing 10 ml of deionized-distilled-boiled water at pH 7.0. The pH of the suspension was maintained at 7.0 with continuous stirring, using a Brinkmann 682 titroprocessor. Fifteen milliliters of 66.6 mM aspartic acid solution was added (the solution was freshly prepared and maintained at pH 7.0 by addition of 0.1M NaOH). The final concentration of aspartic acid in the suspension was 40.0mM. The suspension was shaken at 25.0°C for different periods in an oscillating ( 140 strokes/min) temperature-controlled water bath.
2.4. Desorption of adsorbed aspartic acid To examine the diffusion of aspartic acid adsorbed on the clay surfaces to the external solution, aspartic acid solutions of 40.0, 4.0, 0.40 and 0.04mM were prepared at pH 7.0. After adsorption equilibrium (2 h) of the 40.0mM aspartic acid solution by Ca-montmorillonite, the complex suspension was centrifuged ( 19,000g). The supernatant was removed and replaced by an aspartic acid solution of lower concentration (4.0mM) for a second equilibration period of 2 h. The operation was then repeated by replacing the supernatant for third and fourth equilibrium periods of 2 h with 0.40 and 0.04mM of aspartic acid solutions, respectively. After each equilibrium treatment, the amino acid-clay complexes were examined by X-ray diffraction analysis.
2.5. X-ray diffraction analysis (XRD) The montmorillonite or aspartate-montmorillonite complex before and after treatments with aspartic acid solutions or washings with KCI solutions or water were mounted on glass slides and maintained at a relative humidity (RH) of 50.0% at room temperature. XRD analysis was carried out on a Rigaku D/MAX-RBX diffractometer (Rigaku Company,
268
A. Naidja, P.M. Huang /Applied Clay Science 9 (1994) 265-281
Japan) with monochromatic Cu-Kc~ radiation at 50 kV and 150 mA. A separate set of the samples were heated (2 h) at different temperatures (110-600°C) and kept dry in a desiccator on silica gel before XRD analysis.
2.6. Fourier transform infrared ( FTIR ) absorption spectrometry The FTIR spectra of the montmorillonite or aspartate-montmorillonite complex before and after washings with KCI solutions or water were recorded on KBr disks that contained 1% of sample by weight, using a BioRad-Digilab-3240 SPS spectrophotometer. The spectra were then referenced against the spectra of pure KBr (Spectrosol, BDH, England).
3. Results
3.1. Adsorption of aspartic acid as a function of time The adsorption of aspartic acid on natural Ca-montmorillonite was fast (Fig. 1). A substantial amount (78%) of aspartic acid was adsorbed on the clay mineral surfaces after 15 min, and the equilibrium was reached after 2 h (56.2 mmol/g clay). This fast adsorption (Fig. 1) was attributed, in part, to the preswelling of the clay in water overnight, giving a high degree of dispersion of the particles (Puri and Keen, 1925). Dashman and Stotzky (1982) showed that the adsorption isotherm of aspartic acid on Ca-montmorillonite at pH 70
60
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2
4
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Time (h) Fig. I. Adsorption ot'aspartic acid (0.33mM) by natural Ca-montmorillonite (25 rag) as a function of time at pH 7.0 and 25.0°C.
A. Naidja, P.M. Huang/Applied Clay Science 9 (1994) 265-281
269
20
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Time (11) Fig. 2. The doo~spacingof montmorillonite(natural) and aspartate--Ca-montmorillonite(natural) complexas a function of time at pH 7.0 and 25.0°C. ( • ) Ca-montmoriUoniteand ( • ) aspartate~a-montmorillonitecomplex, 8.21 was of S-class (high affinity of adsorbate to the adsorbent). There was an abrupt increase of dool spacing of the natural Ca-montmorillonite from 14.9 to 18.1 ,~ after 30 rain of reaction with aspartic acid (Fig. 2). Purified Ca-montmorillonite upon interaction with aspartic acid yielded the same data (not shown). This rapid expansion was evidently the result of the intercalation of aspartic acid in the Ca-montmorillonite structure. Barshad (1952) showed an expansion of the structure of Ca-vermiculite to 17.6/~ after adsorption of glycine. 3.2. FTIR analysis
FTIR analysis of the aspartate-Ca-montmorillonite complex (Fig. 3 and Table 1) confirmed the adsorption of the amino acid on the clay mineral surfaces. Fig. 3 shows the shifting of some bands after adsorption of aspartic acid on Ca-montmorillonite. The broad band at 3116 cm -~ (Figs. 3c and 3d) corresponds to the NH~" group (Cross and Alan Jones, 1969; Bellamy, 1975). The band at 1618 c m - l (Figs. 3c and 3d), which was much stronger than the band (Fig. 3b) corresponding to the deformation of the OH group of hygroscopic water ( 1620-1630 cm 1) in untreated montmorillonite, was attributed to the C O 0 - stretching vibration (Nakagawa et al., 1965; Orlov, 1985). The shoulder at 1478 c m - ~ might be due to the symmetric deformation of the NH~- group weakly bound on the mineral surface (Nakagawa et al., 1965; Orlov, 1985). The band of high intensity, at 1407
270
A. Naidja, P,M. Huang/ Applied Clay Science 9 (1994) 265-281
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cm r indicates the symmetric stretching vibration of ionized CO() group at the end of the chain; this band appears at 1416 cm ~ in intercalated lysine on Ca-montmorillonite (Nakamoto et al., 1961: Jang and Condrate, 1972). The bands at 1351, 1309 and 1224 cm t (Figs. 3c and 3d) represent C O 0 , CH deformation and CO stretching vibration (Cross and Alan Jones, 1969; Jang and Condrate, 1972). The increase in the intensity of these absorption bands after 24 h of contact (Fig. 3d) indicated that the adsorption equilibrium was not reached after thirty minutes of contact. Purified Ca-montmorillonite upon interaction with aspartic acid yielded the same data (not shown). 3.3. EffEct of temperature on the do.: spacing of the aspartate-Ca-montmorillonite complex The natural and purified Ca-montmorillonite samples and the aspartate-Ca-montmorilIonite complex were heated at different temperatures. Fig. 4 shows the variation of the d~,~ spacing with temperature. For the natural Ca-montmorillonite before reaction with aspartic
A. Naidja, P.M. Huang/Applied Clay Science 9 (1994) 265-281
271
Table 1 FTIR bands assignmentof the aspartate-Ca-montmorillonitea complexafter 0.5 h of reaction at pH 7.0 and 25.0°C. Values are wavenumbers (cm - 1 ) Aspb -
3021 2960 1693 1646 1605 1517 1424 1351 1309 1249
Ca-Mte c
Asp--Ca-Mte
Assignment~
3634 3416 1627
3634 3416 3116 1618 1478 1407 1351 1309 1224
vOH, Mte vOH, H20 vNH3+ vCH v~CO,COOH vaCOO8OH, H20 vaCOOv~NH3+ vsCOOvsCOO6CH vCO
-
" Natural clay h Asp: aspartic acid, only the main bands are given. c Mte: montmorillonite. d v, vs and v,: stretching vibrations, symmetric and asymmetric; & deformation. acid, the dooL spacing decreased drastically from 14.9 ,~ (room temperature) to 10.2 ,~ at 150°C (Fig. 4), indicating the loss of water molecules which occur mostly as interlayer water between the silicate sheets (Grim, 1968). Upon further heating to 600°C, the dool spacing slowly decreased until the interlayers were completely collapsed (dool = 9.6 .~). Similar data were obtained with the purified clay (Fig. 4). For the aspartate-Ca-montmoriilonite (natural clay) complex, the interlayer spacing remained constant (doo~ = 18.1 ,~) upon heating to 150°C and slowly decreased to 17.2 A at 170°C (Fig. 4), indicating a slight dehydration of the aspartate-Ca-montmorillonite complex. Upon heating from 170 to 600°C, the doo~ spacing of the complex decreased linearly ( r 2 = 0.997 and p --- 1 0 - 9), at a rate of 0.017/~/°C, with increasing temperature to 10.3 ,~. This may correspond to a thermal desorption of the decomposition products, as suggested by M c A t e e and H a w t h o m e ( 1 9 6 4 ) and Chou and McAtee (1969) for some organo-ammonium compounds adsorbed by montmorillonite. The aspartate-Ca-montmorillonite complex (purified clay) showed the same trend (Fig. 4). In the presence of aspartic acid, the interlayer space did not completely collapse, even after heating at 500°C (dnol = 12.1 .~ with natural and 12.0 .~ with purified clay). Dashman and Stotzky (1985) found that the dool spacing of an aspartate-Ca-montmorillonite complex decreased to 10.0 ,~ after heating at 150°C (48 h under vacuum). Heat treatment above 170°C affected the stability of the aspartate-Ca-montmorillonite complex (Fig. 5 ). Heating at 200°C affected the adsorbed amino acid and its transformation (oxidation and decomposition). The FTIR spectra (Fig. 5) showed the disappearance of the main bands o f the amino acid adsorbed on Ca-montmorillonite surface after heating at 200°C and the appearance of a strong band at 1078 c m - 1, which was observed after heating at 170°C and different from that assigned to the S i - O group at 1047 c m - ~ in the aluminos-
272
A. Naidja. P.M. Huang/Applied Clay Science 9 (1994)265-281
2o!
•o
14
Temperature (°C) Fig. 4. Effect of heating on the d(x~]spacing of the aspartate-Ca-montmorillonitecomplex: untreated clay system and purified clay system. (0) Ca-montmorilloniteand (11) aspartate-Ca-montmorillonitecomplex (untreated clay): (©) Ca-montmorilloniteand ([]) aspartate-Ca-montmorillonitecomplex (purified clay). ilicate lattice. The band at 1078 cm t, which persisted with heating at 600°C, might be attributed to the skeleton ~ 2 ~ - (Nakagawa et al., 1965) remaining after oxidation or destruction of the adsorbed amino acid. Tettenhorst (1962) assumed that the band at 1078 c m - ~ indicates that some interlayer cations migrate to the silicate skeleton. On the other hand, Farmer and Russell (1964) ascribed this band at 1078 c m - ' to the irregularity in the structure or to the ionic substitution in the lattice caused by the increase of heating.
3.4. Desorption of adsorbed aspartic acid Diffusion of adsorbed aspartic acid Fig. 6 shows that after equilibrating with 4.0mM aspartic acid, the initial peak at 18.1 corresponding to the d(×)~ spacing of the aspartate-Ca-montmorillonite complex, which was formed upon interaction with 40.0mM aspartic acid, changed to 17.9 A,, and a new peak at 12.8 A appeared, indicating that interlayer aspartic acid diffused to the diluted aspartic acid solution. The concentration of aspartic acid at 4.0mM seemed to be critical for the coexistence of two layers, namely the 17.9 .~ phase, corresponding to the aspartic acid molecule intercalated montmorillonite, and the 12.8 ,~ phase, corresponding to the hydrated Nasaturated montmorillonite (Fig. 6c). Aspartic acid was transformed to the sodium salt when the pH of the aspartic acid solution was adjusted from 2.9 to 7.0 by the addition of 0.1M NaOH. In the presence of lower concentrations of aspartic acid (0.40 and 0.040mM), only
A. Naidja, P.M. Huang /Applied Clay Science 9 (1994) 265-281
273
c
o
4000
3500
3000
2500
Wavenumber
2000
1500
1000
( c m -t )
Fig. 5. Infraredspectraof the aspartate-Ca-montmorillonite(natural) complexafter heating at differenttemperatures (2 h). the peaks at 12.2-12.3 A, characteristic of Na-montmorillonite, were observed. The diffusion of adsorbed aspartic acid to the bulk solution, which contained aspartic acid at the lower concentrations, apparently resulted from competition with water molecules for the adsorption sites, which is in accord with the complete removal of adsorbed aspartate after the second washing with water.
Desorption by water After the first washing with water (pH 7.0), the strong peak at 18.1 A changed to a weak peak at 17.9 ]~ (Fig. 7c), indicating the desorption of most of the interlayer aspartic acid molecules. In addition to the peak at 17.9 ,~, a peak at 12.2 ]~ was observed after the first washing with water. The dool spacing of the aspartate-Ca-montmorillonite complex collapsed to 11.6 ,~ (Fig. 7d) after the second washing with water, which was the result of a complete desorption of the amino acid, as confirmed by FTIR spectroscopy (Fig. 8d).
274
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10.0
15.0
*20, Cu Kc~ Fig. 6. X-raydiffractionpatternsof the aspartate-Ca-montmorillonite( natural) complexalter contacts ( 2 h) with a series of diluted aspartic acid solutions: (a) Ca-montmorillonite;(b) aspartate-Ca-montmorillonitecomplex formedin the presenceof asparticacid at an initialconcentrationof 40.OmM;and the aspartate-Ca-montmoriUonite complex after subsequentcontactwith4.0mM aspartic acid (c), 0.40mMasparticacid (d) and 0.04 mM aspartic acid re). Extraction by KCl After 1 h of contact of the aspartate~Ca-montmorillonite complex with 1M and 0.5M KC1, the do(, spacing of the aspartate-Ca-montmorillonite complex decreased to l 1.8 and 11.9 A, respectively, which are virtually the same as the d~)~ spacing of the unreacted K-montmorillonite (Figs. 9c and 9d), indicating that the aspartic acid initially adsorbed was extracted by KC1, as confirmed by FTIR spectroscopy (Fig. 8f). The negative charge of the clay layers was balanced by K + cations after the KC1 extraction. Greenland et al. (1965a) showed that histidine and arginine were retained against a 20 h extraction with
A. Naidja, P.M. Huang/Applied Clay Science 9 (1994) 265-281
275
o,1¢
~'.o ~.o "20,
1o'.o
~5.0
Cu Kot
Fig. 7. X-ray diffraction patterns of the aspartate-Ca-montmorillonitecomplexbefore and after several washings with water: Ca-montmorillonite (a); aspartate-Ca-montmorillonite complex (b); the aspartate-Ca-montmorillonite complexafter the first washing (c), after the secondwashing (d). after the third washing (e); and unreacted Na-montmorillonite (f). I M KC1 to an extent ranging from 28 to 58% of the amount initially present, whereas lysine was almost completely extracted from the highly expanded complex formed with montmorillonite. Hence, the extraction by KC1 of adsorbed amino acids on clay minerals depends on the nature of the amino acid and the type of the complex formed with the interlayer exchangeable cation. Greenland et al. (1965b) stated that aspartic acid, which has a large charge and a small hydrocarbon chain, is dissolved in KC1. When the concentration of the KCI solution was decreased to 0.05M, the dool spacing of the aspartate-Ca-montmorillonite complex decreased to 14.9 ,~., which was the phase corresponding to Ca-saturated montmorillonite before adsorption (Fig. 9e), suggesting that only the adsorbed aspartic acid was removed and the exchangeable Ca remained intact.
4. Discussion 4. I. Thermal stability o f aspartate-Ca-montmorillonite complex The thermal stability of clay minerals has been extensively studied (Russell and Farmer, 1964; MacEwan and Wilson, 1980; Harris et al., 1992) to determine the mechanisms of the
276
A. Naidja, P.M. Huang / Applied Clay Science 9 (1994) 265-281
(1) u e--
4000 3soo
3000 2soo
2000
15oo
looo
Wavenumber (cm-1) Fig. 8. Infrared spectra of the aspartate-Ca-montmorillonite (natural) complex after washing with water and extraction with KCI for I h. (a) Ca-montmorillonite;aspartate-Ca-montmorillonitecomplexbefore washing with water (b), after the first washing with water (c), after the second washing with water (d), after the third washing with water (e), and after extraction with 1.0M KCI (f). hydration/dehydration processes and subsequently the structure and the classification of such minerals in soil. However, despite the significant occurrence of amino acids in soil (Stevenson, 1982), detailed work on the thermal stability of amino acid--clay mineral complexes still remains lacking (Cloos et al., 1966). Upon a gradual heating over a wide range of temperatures ( 110 to 600°C), the aspartate~2a-montmoritlonite complex was not dehydrated after heating to 150°C. The dehydration of adsorbed monocarboxylic amino acids occurred around 140°C (Cloos et al., 1966). Kanamaru and Vand (1970) assumed that water molecules might have an important role in stabilizing the interlayer structure by hydrogen bonding with the oxygen in the C = O group and the nitrogen in the NH~- group. In the case of the aspartate-Ca-montmorillonite complex, the loss of interlayer water molecules coordinated to the exchangeable Ca cation and the aspartic acid apparently started after a temperature higher than 150°C, and the do~l spacing decreased linearly with increasing temperature (Fig. 4). Therefore, interlayer aspartic acid molecules, which were apparently linked to the Ca through water bridges and also bound to the silicate tetrahedral sheet through hydrogen bonding as discussed below, were destabilized after dehydration at a temperature higher than around 170°C (Fig. 4). Adsorbed aspartic acid was transformed (oxidized and decomposed) after heating at 200°C (Fig. 5).
4.2. Adsorption/desorption of aspartic acid The adsorption of amino acids, peptides and proteins on clay minerals has been investigated by numerous researchers (Walker and Garrett, 1961; Fripiat et al., 1966; Harter and
A. Naidja, P.M. Huang / Applied Clay Science 9 (1994) 265-281
277
L_2°) 0
j 2'.0
;.o "2~,
' 10.0
' 15.0
Cu K(x
Fig. 9. X-mydiffraction patterns of the aspartate-Ca-montmorillonitecomplexafterextraction for 1 h with KCI salt solution: (a) Ca-montmorillonite;(b) the aspartate-Ca-montmorillonitecomplexbeforeextraction;aspartateCa-montmoriUoniteafter extraction with 1.0M KC1 (c), 0.5M KCl (d), 0.05M KCI (e); and unreacted Kmontmorillonite(f). Stotzky, 1973; Dashman and Stotzky, 1985, Dashman and Stotzky, 1986; Naidja and Siffert, 1989; Siffert and Naidja, 1992). The present study showed that the adsorption of aspartic acid by Ca-montmorillonite is a rapid reaction process (Fig. 1). At pH 7.0, aspartic acid appeared to be weakly adsorbed on the montmorillonite surfaces, including the interlayer spaces. The intercalation of aspartic acid in the montmorillonite was evidently responsible for the interlayer expansion of Ca-saturated montmorillonite (Fig. 2). pH is an important factor governing the interaction between aluminosilicates and amino acids (Theng, 1974), as amino acids are amphoteric, and their retention by the silicate layers is more effective when they are positively charged. The positive charge is important in enabling aspartic acid to approach the negatively charged clay. The influence of pH on the speciation of aspartic acid is described below:
A. Naidja, P.M. Huang /Applied Clay Science 9 (1994) 265-281
278
_n +
HOOC-CH2-~H-COOH
_..
NH3 +
(+ 1)
~.
-OOC-CH2-~H-COOH
+H +
NH3 +
pKl = 2.10
(0)
p l = 2.98
(isoelectric point) /IN
-H+ ][+H + pK2= 3.86
q|
_n +
"OOC-CH2-~H-COONH2 (-2)
"~
./ +H+ pK3 = 9.82
"OOC-CH2-ffH-COO NH3+ (- 1)
At pH 7.0, the predominant species, determined from the dissociation constants (Weast, 1980), have net negative charge, as described below, and accounts for 99.77% of the aspartic acid concentration : -OOC-CH2-~H-COONH3 ÷ In solution, aspartic acid is chelated to alkaline earth metal cations as a bidentate (Lumb and Martell, 1953). However, the mechanism of bonding of aspartic acid with exchangeable Ca as a complex in the interlayer space of montmorillonite, as observed in this study, may differ from that in solution, as suggested by Lumb and Martell (1953). The easy removal (desorption) of intercalated aspartate from the clay in dilute aspartic acid solution (Fig. 6), water (Figs. 7 and 8) and KCI solutions (Figs. 8 and 9) indicated that aspartic acid was weakly bound to the clay silicate layers at pH 7.0. Cloos et al. (1966) assumed that, in acidic medium (pH ~ 3), the adsorption of glycine, glycylglycine and /3-alanine hydrochlorides by sodium and calcium montmorillonite occurs essentially through ion exchange or proton transfer mechanisms. Subsequently, various other mechanisms have been proposed to interpret the adsorption of amino acids by clays (Mortland, 1970, Mortland, 1986; Stotzky, 1986). The function of the exchangeable cation seemed to be paramount in the different interactions, including direct coordination of polar groups (carboxyl, carbonyl or amino) to the exchangeable cation or indirect coordination to the exchangeable cation through a "water bridge". In addition, the protonated amino group (--lqH3) is an excellent hydrogen-bond donor (Marsh and Donohue, 1967), and it can form a hydrogen-bond with the structural oxygen of siloxane surfaces (Raussel-Colom and Salvador, 1971 ). The complete removal of the weakly adsorbed aspartate by the second washing with water (Figs. 7d and 8d) or by IM, 0.5M and 0.O5M KCI solutions (Figs. 9c, 9d and 9e) substantiates the reasoning that the formation of an H-bonding may be the predominant binding force in the intercalation of aspartate in montmorillonite, as shown below in the proposed scheme. The carboxylate groups of aspartate were apparently bound to the hydrated Ca by a water bridge through an H-bonding in an outer-sphere complex. In addition, the protonated amino group (--lqH3) also appeared to interact with the structural oxygen of the siloxane surfaces through an H-bonding (Raussel-Colom and Salvador, 1971). Stotzky (1986) stated that, it is probable that several mechanisms function simultaneously in the surface interaction of clay and the biological entities. In the present system, the cation exchange mechanism was apparently not involved, since the positive charge of the proton-
A. Naidja, P.M. Huang / Applied Clay Science 9 (1994) 265-281
279
ated amino group is compensated by the intra-molecular interaction in the net negatively charged aspartate.
I
H I
y H20
_Q.X,,+~H2
H 2 0 . O--H" • • ~ - - - - C H
cla /
I .
H
.
.
.
0
5. Conclusions The aspartate-Ca-montmorillonite complex was resistant to dehydration with increasing temperature to 150°C. Heating at higher temperatures ( > 200°C) caused an alteration of the aspartate-Ca-montmorillonite complex and degradation ( oxidation and decomposition) of the intercalated amino acid, which led to a linear decrease in the doo~ spacing of the complex with increasing temperature. The intercalated aspartate was virtually removed after the second washing with water. It was easily extractable by one washing with 1.0M, 0.5M or 0.05M KC1. Therefore, at neutral pH and in its corresponding anionic form, aspartate was weakly intercalated in montmorillonite layers. The intercalation appeared to proceed through the formation of an outer-sphere complex with exchangeable Ca, and an H-bonding of the protonated amino group to the structural oxygen of siloxane surfaces. Consequently, in the terrestrial environment with the pH near neutrality, the fraction of the aspartic acid not mineralized by microbes would be readily transported from the soil to ground and surface waters through diffusion and leaching.
Acknowledgements This study was supported by Grant GP 2383-Huang of the Natural Sciences and Engineering Research Council of Canada.
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