J. Phys. Chem. 1996, 100, 2417-2421
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Adsorption of Aqueous Nucleobases, Nucleosides, and Nucleotides on Compost-Derived Humic Acid. 2. Naturally Occurring Purines1 Ahmed H. Khairy,†,‡ Geoffrey Davies,*,§ Hesham Z. Ibrahim,‡ and Elham A. Ghabbour*,| Institute of Graduate Studies and Research, Alexandria UniVersity, Alexandria, Egypt, Chemistry Department and the Barnett Institute, Northeastern UniVersity, Boston, Massachusetts 02115, and Soil Salinity Laboratory, Agricultural Research Center, 21616 Bacos, Alexandria, Egypt ReceiVed: August 9, 1995X
This paper describes adsorption of the aqueous purines adenine (10), adenosine (11), adenosine 5′monophosphate (12), guanosine (13), and guanosine 5′-monophosphate (14) on compost-derived humic acid (HA) over the solute concentration (12-1200 µM) and temperature (10.0-40.0 °C) ranges used with pyrimidine solutes in Part 1. Nucleobase 10 and nucleosides 11 and 13 are neutral under the experimental conditions, while nucleotides 12 and 14 are monoanions. All the purines interact with HA, but only adenine causes HA dissolution at pH 5-6 and temperatures above 25.0 °C, as found with cytosine and (-)nicotine. The IR spectrum of the soluble adenine-HA product indicates strong interaction with carboxylic and phenolic HA groups that is not due to simple proton transfer. Pyrimidines and purines are adsorbed in sequence on three HA sites A, B, and C. Most of the data fit the Langmuir model to generate site capacities VA, VB and VC and equilibrium constants KA, KB, and KC for each solute and site. Average site capacities increase 〈VA〉 < 〈VB〉 < 〈VC〉, while equilibrium constants at 25.0 °C decrease KA > KB > KC. Site capacities Vi are independent of temperature and solute molar mass or charge, indicating adsorption through solute nucleobase units. Adenine has unusually high average 〈VA〉 and 〈VB〉 related to its ability to dissolve HA at pH 6, but 〈VC〉 is unexceptional. The equilibrium constant data show that compost-derived HA sites have quite different solute selectivities. For example, site A discriminates poorly between the solutes thymine (4), thymidine (5), and 11 while site C has a strong preference for 4. These selectivities could influence animal, plant, soil, and sediment biochemistry. Linear correlation of the enthalpies and entropies for adsorption of every solute at each HA site points to a common underlying adsorption mechanism.
Introduction Humic substances (HS’s) are widely distributed organic macromolecules with crucial natural role as water retainers, buffers, and adsorbents. There is continuing, intense interest in how their structures and functions are related and how their properties, modification, and use can benefit agriculture and human health.1-3 A subset of HS called humic acids (HA’s, average 〈Mw〉 ca. 12 to at least 100 kDa) are versatile adsorbents that are insoluble in water below pH 8.2 Part 11 is a detailed study of adsorption of the naturally occurring pyrimidines cytosine (1), thymine (4), and uracil (7) and their respective nucleosides and nucleotides on a highly purified, homogenized HA sample (〈Mw〉 33 kDa) extracted from municipal compost. The work was undertaken for two main reasons. Firstly, HA’s have important but incompletely understood functions in animals,4 plants,5 soils,3,6 and water sediments.2,7 Although nucleobases are known to associate with HA, the nature and extent of the interactions is an open question.8 Secondly, we found that most of the pyrimidine adsorption data could be fitted to the Langmuir model, which generates an adsorption site capacity Vi and an equilibrium constant Ki (with associated thermodynamic parameters) for each of three adsorption steps A, B, and C.1 This is significant because thermodynamic data for adsorption on HA are in short supply9-17 and are needed to help us better understand HA properties and functions.2-7 †
Deceased. Alexandria University. § Northeastern University. | Agricultural Research Center. X Abstract published in AdVance ACS Abstracts, January 1, 1996. ‡
0022-3654/96/20100-2417$12.00/0
HA’s are heterogeneous in the sense that they have either different building blocks or different building block connectivity, depending on the source and HA growth conditions.2,3,17 The best way to obtain reproducible adsorption data for such materials is to use a large, homogenized sample from a single source.1 We studied the adsorption of pyrimidines over wide solute concentration and temperature ranges with three objectives.1 Firstly, we could sort the data into subsets that fit the Langmuir model or not. Secondly, we could confirm or deny that adsorption site capacities Vi are positive and independent of temperature.17,18 Thirdly, we could confirm that the Ki’s are true adsorption equilibrium constants by obtaining linear plots of log Ki Vs 1/T and calculating the enthalpies and entropies of adsorption.1 Analyis of pyrimidine solute data that fit the Langmuir adsorption model17,18 led to the following conclusions.1 Firstly, compost-derived HA has at least three sites A, B, and C11,17,19 with different site capacities that increase VA < VB < VC. Secondly, the Vi values are, within experimental error, independent of solute molar mass and charge, indicating adsorption through pyrimidine nucleobase units. Thirdly, reaction of HA with cytosine, but not with the other pyrimidines, results in HA dissolution even at pH 5-6, as found with (-)nicotine.19 Fourthly, adsorption of pyrimidines on compost-derived HA is endothermic, thermoneutral, or exothermic, depending on the adsorption site and solute. Lastly, linear correlation of ∆H with ∆S for each detected adsorption step points to a common underlying adsorption mechanism.1 Purines and their respective nucleosides and nucleotides are the other class of nucleic acid constituents.20 Their nucleobases © 1996 American Chemical Society
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Khairy et al.
have more potential points or regions of attachment to HA than the pyrimidines. We have used the strategy of Part 11 to investigate the adsorption of adenine (10),21 adenosine (11), adenosine 5′-monophosphate (12), guanosine (13), and guanosine 5′-monophosphate (14) on the same homogenized sample of compost-derived HA as in Part 1.1 We report that adenine behaves like cytosine and (-)nicotine in causing HA dissolution even at pH 6. The adsorption data for solutes 10-14 amplify our previous conclusions1 and give new insights concerning HA adsorption processes.
Figure 1. KBr disk IR spectra of (a) compost-derived HA; (b) adenine (10); (c) HA-adenine product S; (d) adenine hydrochloride.
Experimental Section Solutes 10-14 and guanine were used as supplied by Sigma. Water was distilled from an all-glass still. The HA sample, experimental procedures, and data analysis methods of this study were identical in all respects to those used in Part 1.1 Results and Discussion We could not study the adsorption of guanine on HA because it is insoluble in water at the pH’s employed previously.1 However, the solubilities of solutes 10-14 were sufficient to allow measurements over the same concentration (12-1200 µM) and temperature (10.0-40.0 °C) ranges as before.1 All solutes 10-14 were found to interact with HA. Equilibrium interaction with the nucleobases and nucleosides gave solutions with pH 5.9-6.6, while the pH with nucleotides 12 and 14 was 3.43.7. The nucleobase and nucleotide solutes are neutral molecules under these conditions, but the nucleotides are monoanions.22 Dissolution of HA by Adenine. Like other nitrogenous bases,23-26 (-)nicotine is strongly adsorbed by HA.19 Addition of excess nicotine causes fragmentation and dissolution of HA at pH values where HA normally is insoluble. Part 11 reported that addition of 10 mL of 1200 µM cytosine to 10 mg of compost-derived HA results in 80% HA dissolution to give a residue R and a solid S obtained by supernatant solution evaporation at 40 °C. The IR spectra of R and S implicated HA carboxylic functional group involvement in this interesting and potentially diagnostic reaction.19,27,28 None of the other pyrimidine solutes of Part 1 increased the solubility of HA in water at pH’s below 8. We found that 10 mL of 1200 µM adenine (10), but none of the other purine solutes, readily causes 10 mg of HA to dissolve completely in water at 30 °C or higher temperatures and pH ≈ 6 to give brown solutions. As before,1 we measured the IR spectrum of solid S obtained by evaporation of the brown
Figure 2. Isotherms for the adsorption of adenine (10), adenosine (11), and adenosine 5′-monophosphate (12) on compost-derived HA at 25.0 °C.
solutions. The spectra shown in Figure 1 have the following features. The adenine-HA product S has none of the characteristic bands of 10, and its spectrum is quite unlike that of adenine hydrochloride. It appears that dissolution of HA in the presence of 10 is not due to protonation of adenine by HA. Formation of HA product S results in marked reduction of the HA bands at 2500 cm-1 (H-bonded COOH functions) and at 1725-1750 cm-1 (carboxyl, carbonyl).2 Absorption at 1640 cm-1 (COOin HA; CdC, CdN in adenine) is a doublet in product S. More intense absorption at 1450-1420 cm-1 (COO-) is evident, and phenolic bands at 1270-1230 cm-1 are diminished. It appears that carboxylic acid and phenol groups1-3,17 of HA are involved in the HA-solubilizing reaction with adenine. We stirred solid S in water at pH 6 and 30.0 °C but could find little evidence for adenine in the supernatant solution. This indicates that adenine reacts very strongly with HA at 30.0 °C or higher temperatures in what appears to be a chemical reaction with HA carboxylic acid and/or phenol groups. Adsorption Characteristics of Purine Solutes. Representative isotherms for adsorption of solutes 10-14 are shown in Figures 2 and 3. Here, A has units of millimole of solute per gram of HA and c is the solute concentration at equilibrium. Except with high concentrations of adenine at temperatures
Adsorption on Compost-Derived Humic Acid
J. Phys. Chem., Vol. 100, No. 6, 1996 2419
Figure 3. Isotherms for the adsorption of guanosine (13) and guanosine 5′-monophosphate (14) on compost-derived HA at 20.0 °C.
Figure 5. Plots of eq 1 for adsorption of guanosine (13) and guanosine 5′-monophosphate (14) on compost-derived HA on (a) site A and (b) sites B and C at 20.0 °C.
Figure 4. Plots of eq 1 for adsorption of adenosine (11) and adenosine 5′-monophosphate (12) on compost-derived HA on (a) site A and (b) sites B and C at 35.0 °C.
above 25.0 °C, the electronic spectra of all supernatant solutions closely resembled those of the solutes and c was calculated from the respective solute molar absorptivity in the absence of HA.1 All the isotherms clearly exhibited evidence for a sequence of adsorption steps. The Langmuir model predicts that a plot of 1/A Vs 1/c will be linear with positive intercept 1/V and slope 1/KV,17,18 as indicated by eq 1 and illustrated in Figures 4 and 5. Parameters obtained from this fit of the data are listed in Table 1 along with adsorption enthalpies and entropies calculated from linear plots of log Ki Vs 1/T for each detectable adsorption step, where T is the absolute temperature.
1/A ) 1/V + 1/KVc
(1)
As before,1 data clearly out of line with other measurements (for example, KA ) 410 for adenosine (11) adsorption at HA site A at 30.0 °C, Table 1) were attributed to random sample
microinhomogeneity in specific experiments and omitted from the analysis. Site Capacities. Major features of the site capacity data in Table 1 are as follows. Most of the adsorption data fit eq 1. Each isotherm has three steps A, B, and C. Each step has a site capacity that is independent of temperature in the range 10.0-40.0 °C for each solute. Average site capacities 〈VA〉 and 〈VB〉 for each solute are, within experimental error, estimated from linear least squares analysis with eq 1, independent of solute molar mass and charge for solutes 11-14. The similarity of the average site capacities 〈VA〉 indicates adsorption of 11-14 through their respective nucleobase units, as found for the pyrimidines.1 The same is true for 〈VB〉 (Table 1). The exceptions to these statements refer to adenine (10), whose 〈VA〉 and 〈VB〉 are considerably larger than those for the other solutes. As noted earlier, adenine is the only naturally occurring purine that increases the solubility of HA in water. HA dissolution and adenine-HA product S solubility depend on temperature: we successfully fitted the isotherms for 10 at 20.0 and 25.0 °C but not at higher temperatures where adenineHA product S dissolves. Average site capacities 〈VC〉 for purine solute adsorption at site C are larger than for steps A and B and cover the range 0.19-1.1 mmol of solute/g of HA. The site capacity VC for solute 10 is within this range, so adsorption of adenine at HA site C is unexceptional (Table 1). This finding suggests that HA dissolution in the presence of adenine primarily is due to
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TABLE 1: Langmuir Parameters for Adsorption of Purines on Compost-Derived HAa sites A solute
temp
b
K
adenine
20.0 25.0
8.5 44
adenosine
10.0 20.0 25.0 30.0 35.0 40.0
6.5 7.4 19 410c 25 S-curve
10.0 20.0 25.0 30.0 35.0 40.0
14 34 36 45 85 S-curve
10.0 20.0 25.0 30.0 40.0
34 17 100 520 360
10.0 20.0 25.0 30.0-40.0
62 5.7 76 S-curve
adenosine 5′-MP
guanosine
guanosine 5′-MP
V
B ∆H, ∆S
0.17 65.1, 0.09 226 〈V〉 0.13 (0.04) 0.04 0.04 0.02 10.5, 0.05 40 0.02 〈V〉 0.03 (0.01) 0.02 0.02 0.03 11.9, 0.02 47 0.03 〈V〉 0.02 (0.005) 0.02 0.04 0.03 18.9, 0.03 73 0.01 〈V〉 0.03 (0.01) 0.003c 0.06 nonlinear 0.02 〈V〉 0.04 (0.02)
K 0.08 0.33 2.6 4.4 5.3 7.0 7.3 47c 1.6 7.8 15 32 3.8c 56 7.9 3.2 23c 21c 1.2
C V
∆H, ∆S
7.9 2.9 〈V〉 5.4 (2.5) 0.11 0.08 0.08 0.47c 0.06 0.03 〈V〉 0.07 (0.03) 0.10 0.04 0.05 0.03 0.08 0.06 〈V〉 0.06 (0.03) 0.07 0.16 0.08 0.15 0.27c 〈V〉 0.12 (0.04) 0.03 0.08 0.07
K
55.9, 186
4.8 2.0
7.6, 29
0.3c 1.7 1.4 1.1 0.8 0.7
21.3, 77
0.05 0.16 0.24 0.47 0.70 1.4
-10.9, -35
0.10 0.20 0.30 1.0 negative V
3.2 2.8 -1.3, 2.9 -2 H-curves or chemisorption 〈V〉 0.06 (0.02)
0.4 0.7 1.2
V
∆H, ∆S
0.33 -33.9, 0.52 -112 〈V〉 0.43 (0.1) 0.69c 0.16 0.18 -8.6, 2.4c -28 0.29 0.21 〈V〉 0.21 (0.05) 2.3 1.0 1.3 19.9, 0.6 64 0.3 0.20 〈V〉 0.95 (0.7) 4.0c 1.4 1.2 23.6, 0.70 77 〈V〉 1.1 (0.3) 0.13 0.29 10.9, 0.16 37 〈V〉 0.19 (0.07)
Units: Ki ) equilibrium constant for adsorption at HA site i; Vi in mmol/g of HA; 〈Vi〉 ) average value of Vi over experimental temperature range with average error in parentheses; ∆H in kcal mol-1 (typical error (1 kcal mol-1); ∆S in cal deg-1 mol-1 (typical error (3 cal deg-1 mol-1). b °C. c Excluded from analysis, see text. a
reactions at HA sites A and/or B that give soluble HA-adenine products above 25.0 °C. We now compare the site capacity data for pyrimidine solutes 1-9 from Part 11 with those for purines 10-14 in Table 1. We noted before1 that the 〈VA〉 values for cytosine (1), cytidine (2), thymine (4), and thymidine (5) are 0.06-0.08 mmol/g of HA. The 〈VA〉 values for cytidine 5′-monophosphate (3), thymidine 5′-monophosphate (6), and uridine 5′-monophosphate (9) are lower (0.02-0.04 mmol/g of HA) and those for uracil (7) and uridine (8) are lowest (0.01 mmol/g of HA). Table 1 shows that the 〈VA〉 values for all purines except adenine fall in the range for the pyrimidine nucleotides 3, 6, and 9. Only the pyrimidines uracil (7) and uridine (8) showed clear evidence for adsorption step B, with 〈VB〉 values of 0.04 and 0.05 mmol/g of HA, respectively.1 By contrast, step B is detectable for adsorption of all the purine solutes (Table 1), but the purine 〈VB〉 values (range 0.06-0.12 mmol/g of HA, Table 1) are larger than those for 7 and 8 and considerably larger when 〈VB〉 ) 5.4 mmol/g of HA for adenine (10) is included in the comparison. Average site capacities 〈VC〉 for pyrimidines were subdivided as follows.1 Cytosine, which like adenine and (-)nicotine19 causes HA dissolution at pH 5-6, has the largest 〈VC〉 (3.3 mmol/g of HA). The average 〈VC〉 for pyrimidine solutes 2, 3, 5, 6, 7, and 9 is 0.31 ( 0.07 mmol/g of HA, while the 〈VC〉 values for thymine (4) and uridine (8) are outside this range and anomalously low. Table 1 indicates that adenosine 5′monophosphate (12) and guanosine (13) resemble cytosine in having larger than average 〈VC〉 values. Adsorption Equilibrium Constants. Adsorption of purine solutes on HA sites A, B, and C has equilibrium constants KA,
KB, and KC, respectively (Table 1). At 25.0 °C, KA > KB > KC, as found for pyrimidines.1 The orders of increasing KA, KB, and KC at 25.0 °C for the solutes we have investigated are as follows.
Site A: 6 < 1 < 2 < 3 < 11 ≈ 4 ≈ 5 < 8 < 12 < 10 < 14 < 13 < 9 < 7 Site B: 10 < 8 ≈ 14 < 7 < 11 < 12 < 13 Site C: 12 < 13 ≈ 1 ≈ 8 < 6 < 7 < 11 ≈ 14 ≈ 5 < 10 < 4
