Analytical constraints on acidic functional groups in humic substances

Organic Geochemistry 39 (2008) 783–799

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Analytical constraints on acidic functional groups in humic substances Jason D. Ritchie a,*, E. Michael Perdue b a b

Department of Natural Sciences, Shorter College, Rome, GA, USA School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta, GA, USA

a r t i c l e

i n f o

Article history: Received 5 October 2007 Received in revised form 27 February 2008 Accepted 5 March 2008 Available online 18 March 2008

a b s t r a c t Four standard fulvic acids and two standard humic acids from the International Humic Substances Society (IHSS) were analyzed to quantify the in situ generation of new acidity during direct titrations, as observed by hysteresis between tandem forward and reverse titration curves. The resulting concentrations of carboxyl and phenolic groups were then corroborated by analytical constraints imposed by elemental data and 13C NMR data. Collectively, carboxyl groups in the IHSS samples, as determined by rapid forward direct titrations, consistently accounted for 69% (±6) of the maximum possible carboxyl-like structures that were estimated by 13C NMR spectra. The concentrations of carboxyl groups in the samples inczreased for two separate reverse titrations initiated at 30 min and 24 h after the completion of the forward titrations, representing 73% (±7) and 84% (±8) of the maximum carboxyl-like groups, respectively. Conversely, the concentrations of phenolic groups were invariant between forward and reverse titrations. Elemental constraints coupled with titration data predicted that the IHSS samples have, on average, one acidic hydroxyl group per every two phenyl rings, as constrained by 13C NMR estimates of aromatic carbon. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Understanding the acid–base properties of humic acids (HAs), fulvic acids (FAs), and natural organic matter (NOM) in natural waters is a prerequisite for understanding and accurately modeling metal binding reactions in soil and aquatic environments (Burch et al., 1978). Indirect titrations with calcium acetate and barium hydroxide (Blom et al., 1957; Brooks and Sternhell, 1957; Schnitzer and Gupta, 1965) are still widely used for determining concentrations of carboxyl and phenolic acid groups, in spite of the potential flaws associated with them (Dubach et al., 1964; Van Dijk, 1966; Holtzclaw and Sposito, 1979; Perdue et al., 1980; Perdue, 1985; Bonn and Fish, 1991; Sierra et al., 2004). Direct titration methods also have potential errors (Fish and Morel, 1985; Marshall et al., 1995; Santos et al., 1999); however, direct titrations yield more comparable and consistent estimates of the concentrations of car-

* Corresponding author. Tel.: +1 706 233 7263; fax: +1 706 236 1509. E-mail address: [email protected] (J.D. Ritchie). 0146-6380/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.orggeochem.2008.03.003

boxyl and phenolic groups than indirect titrations (Ritchie and Perdue, 2003). A major complication associated with direct titration methods is the downward drift in pH during titrations from acidic to alkaline pH and the observed hysteresis between tandem forward and reverse direct titrations. Hysteresis has been reported for about a dozen terrestrial and aquatic HAs and FAs (Børggaard, 1974; Sposito et al., 1977; Davis and Mott, 1981; Paxeus and Wedborg, 1985; Bowles et al., 1989; Antweiler, 1991; Marshall et al., 1995; Santos et al., 1999; Cooke et al., 2007), and even for an XAD-4 isolate (Santos et al., 1999). Over the years, some authors have reported that hysteresis does not occur in titrations of fulvic acids. Good examples from the older and more recent literature of very careful experiments where hysteresis was not observed are given by Plechanov et al. (1983) and by Cooke et al. (2007). The exact mechanism(s) responsible for pH drift and hysteresis is/are not known. Børggaard (1974) demonstrated that pH drift could be prevented if samples were sterilized and de-oxygenated. That observation strongly hints at a biological source of hysteresis, in which aerobic

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respiration of the sample at neutral to alkaline pH produces CO2 and/or additional organic acids. A second hypothesis invokes conformational changes of solutes that are induced by electrostatic repulsion between neighboring dissociated acidic functional groups (Sposito et al., 1977; Varney et al., 1983; Paxeus and Wedborg, 1985; Marshall et al., 1995; Tombacz, 1999). In this hypothesis, the gradual accumulation of negative charge on individual molecules with increasing pH eventually culminates in sufficient intramolecular electrostatic repulsion to overcome hydrogen bonding between neutral polar groups and protonated carboxyl groups. When this point is reached, affected solutes adapt new conformations that minimize electrostatic repulsion, and in so doing, some previously protected carboxyl groups are able to dissociate, albeit at a pH that may be much greater than the pKa of the carboxyl group. As charge is gradually decreased in reverse (acidimetric) titrations, intramolecular hydrogen bonds between non-acidic polar groups and carboxyl groups only form (and the original conformation is re-assumed) after carboxylate ions are reprotonated at pH values that are close to the pKa of the acid, thus producing the observed hysteresis. A third hypothesis is base-catalyzed hydrolysis of esters and amides (Bowles et al., 1989; Antweiler, 1991; Leenheer et al., 1995a; Sierra et al., 2004). New carboxylic acids form slowly from esters at neutral-to-alkaline pH, at which point they are immediately neutralized. Hysteresis will be observed if the chemical equilibrium between esters, carboxylic acids, and alcohols is partially or completely reversible on the experimental time scale, so that esters can re-form at acidic pH during reverse (acidimetric) titrations. Despite different hypotheses that attempt to explain pH drift and hysteresis, three general observations are common in all of the aforementioned papers: (1) a downward drift in pH is first observed during the forward direct titration at an approximate threshold of pH 6; (2) the carboxyl acidity of the reverse titration is always greater than carboxyl acidity of the previous forward titration; and (3) the concentrations of phenolic groups in reverse titrations tend to be equal to or less than those in the previous forward titrations. Those observations apply to aquatic and terrestrial HAs, FAs, XAD-4 acids (Santos et al., 1999), and whole peat samples (Cooke et al., 2007). There are simply too many similarities between hysteretic titration curves for samples of different origin, average solute size, solubility, and solution preparation (background electrolyte and sample concentration) (Santos et al., 1999) to conclude that hysteresis is an artifact for one class of samples (e.g., fulvic acids) and a real phenomenon for humic acids and organic rich materials (see e.g., Cooke et al., 2007). The preponderance of evidence in the literature supports the statement that pH drift and hysteresis are common characteristics of acid–base titrations of humic substances. If these phenomena are the result of new acidity that is generated during the measurement itself, then the apparent concentration of carboxyl groups in a sample will reflect the inclusion or exclusion of that newly generated acidity. Gamble (1970, 1972) and Leenheer et al. (1995a,b) discuss carboxyl groups in humic substances as strong and

weak. For the purposes of this paper, it is more convenient to classify carboxyl groups as either titratable carboxyl groups or as latent carboxyl groups. Titratable carboxyl groups are true Brønsted acids in aqueous solution, their rates of protonation and deprotonation are virtually instantaneous, and their solution chemistry can be described completely using equilibrium thermodynamics (i.e., their pKa values). Latent carboxyl groups are those that exist in a non-acidic, protected form at acidic pH (e.g., as esters or as strongly hydrogen bonded intramolecular complexes) and are converted into their free acid forms in a kinetically slow step at neutral to alkaline pH. Once liberated, they behave as Brønsted acids, thus dissociating at pH values that are well above their pKa values. The extent of liberation of new acidic groups from latent groups is a function of the maximum pH to which the forward titration was performed (Huizenga and Kester, 1979; Antweiler, 1991; Santos et al., 1999) and the length of time between the completion of the forward titration and the beginning of the reverse titration (Marshall et al., 1995; Santos et al., 1999). Hysteresis will affect the perceived concentrations of carboxyl groups and will ultimately impact any models that describe pH dependent thermodynamics involving humic substances. Both elemental and structural constraints can be used to validate or reject the results of indirect and direct titrations, as well as set limits to the maximum possible concentrations of carboxyl and phenolic groups with respect to hysteresis. Because carboxyl and phenolic groups contain oxygen, hydrogen, and unsaturation (rings and/or pbonds), their concentrations within humic substances should be reflected in and constrained by elemental composition and by estimates of the distribution of carbon among its possible chemical forms, as obtained using 13C nuclear magnetic resonance (NMR) spectroscopy. In fact, concentrations of acidic functional groups, elemental analyses, and mole percentages of various forms of organic carbon from 13C NMR spectra are all weighted-average bulk properties of humic substances, and the results of those analyses should corroborate each other (Perdue, 1998). This paper will analyze hysteresis in direct titrations of selected standard and reference fulvic and humic acid samples from the International Humic Substances Society (IHSS). A unique aspect of the work is the use of elemental composition, molecular weight (MW), and 13C NMR spectra to guide the interpretation of titration results and provide analytical constraints on the concentrations of acidic functional groups in humic substances. To this end, a limited analysis of the internal consistency (or lack thereof) between analytical constraints that arise from elemental analysis and those that arise from 13C NMR spectroscopy is included for the IHSS samples and for a broader set of published results.

2. Experimental methods 2.1. Published chemical data Published data for ten of the IHSS standard and reference HAs and FAs are shown in Table 1. The data in Table

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1 were gathered from four sources, and each individual analysis for all samples was performed by the same research group under the same experimental conditions at the same time. Elemental analysis was conducted at Huffman Laboratory (Golden, CO) in the summer of 1996 using methods that are described by Huffman and Stuber (1985), and results were reported directly to the IHSS for public distribution. The number-average MWs (Mn) were estimated by flow field-flow fractionation (FFFF) (Dycus et al., 1995). The concentrations of carboxyl and phenolic acidic functional groups were determined by forward direct titration (Ritchie and Perdue, 2003). The integrated 13 C NMR spectra were determined by solution-state 13 C NMR (Thorn et al., 1989). Other literature data were collected in which integrated 13 C NMR spectra were reported with corresponding elemental data and titration data. First, obviously moist samples (O/C > 0.85 and H/C > 1.6 mol mol1) were removed from the dataset. Next, the integrated 13C NMR spectra for the remaining data were re-interpreted, if necessary, in terms of five major classes (aliphatic: 0–50 ppm, oxygenated alkyl: 50–110 ppm, aromatic: 110–160 ppm, carboxyl-like structures: 160–190 ppm, and carbonyl: 190–220 ppm) according to the procedure outlined in Perdue and Ritchie (2004). Values of Mn by either colligative (vapor pressure osmometry) or non-colligative methods (size-exclusion chromatography, flow field-flow fractionation, or light scattering) were used as reported. If MW data were not provided, values of Mn were arbitrarily set to 1000 for aquatic and terrestrial FAs, 2000 for aquatic HAs, and 4000 for terrestrial HAs. All concentrations of acidic functional groups by indirect and direct titrations were used as reported (mmol g1). 2.2. Analytical constraints on 13C NMR data by elemental data Recent breakthroughs in advanced solid state 13C NMR use special spectral editing, dipolar dephasing, and chemical shift anisotropy (ASC) filtering techniques to deconvolute the underlying and overlapping contributions of specific C–C, C–H, C–O, C–N, aromatic, protonated and un-protonated carbon, and carbonyl signals within one

dimensional solid state 13C NMR spectra (Keeler and Maciel, 2000; Mao et al., 2001, 2002; Smernik and Oades, 2001; Mao and Schmidt-Rohr, 2002, 2004a,b; Schmidt-Rohr and Mao, 2002a,b,c; Schmidt-Rohr et al., 2004). Other works have shown that variations of magnetic field strength, pulse contact time, relaxation time, acquisition time, and moisture will disproportionately alter the intensity of signals in some 13C NMR shift regions compared to other shift regions (Preston and Blackwell, 1985; Frund and Ludemann, 1989; Hatcher and Wilson, 1991; Malcolm, 1992; Monteil-Rivera et al., 2000; Smernik and Oades, 2001; Keeler and Maciel, 2003; Peuravuori et al., 2003; Dria et al., 2004). Commonly, 13C NMR spectra differ for the same HA or FA sample when 13C NMR is performed by different research groups using variations of the technique. Most 13C NMR spectra have been, and continue to be, integrated and reported as qualitative one dimensional spectra between chemical shifts of 0–220 ppm with four, five, or six defined structural regions. 13C NMR spectra can indirectly provide the relative distributions of oxygen and hydrogen across the major structural groups in humic substances, and the weighted sums of O and H should hypothetically equal the elemental analysis if the elemental and NMR analyses are unbiased. Schmidt-Rohr and colleagues (Mao et al., 1998, 2000), and to some extent Conte et al. (2002), have demonstrated that their solid state NMR techniques could account for the nearly exact distribution of hydrogen and oxygen in soil HAs. The elemental analysis of a FA or HA sample yields the concentrations of C, H, O, N, and S (in wt%, mol%, mmol/g, etc.). When such information is coupled with the proportions of various carbon containing structural subunits, as determined using 13C NMR spectroscopy, the distributions of oxygen, hydrogen, and other properties among those structural subunits can be estimated. The general equation takes the form: " # X  C i P  PNMR ¼ C total C i C total i "X      # %C EA 1000 Ci P ¼ ð1Þ 12:011 C 100 C total i i

Table 1 Chemical properties of the International Humic Substances Society (IHSS) standard and reference fulvic and humic acids Elemental analysisa

Suwannee FA Soil FA Peat FA Nordic FA Suwannee HA Soil HA Peat HA Leonardite HA Nordic HA Summit Hill HA a b c d

MWb

Direct titrationc

13

%C

%H

%N

%O

Mn

COOH

ArOH

%CAlk

C NMRd %COAlk

%CAr

%CCOOH

%CC@O

52.44 50.12 50.45 52.31 52.55 58.13 56.37 63.97 53.33 54.00

4.31 3.82 3.52 3.98 4.40 3.68 3.82 3.60 3.97 4.84

0.72 3.75 2.56 0.68 1.19 4.14 3.69 1.25 1.16 5.13

42.20 42.61 45.47 45.12 42.53 34.08 37.34 31.62 43.09 37.90

1119 819 840 1666 2247 3252 3020 3062 2374 3146

6.00 6.40 6.72 5.84 5.04 4.81 5.08 4.77 4.83 3.86

1.52 1.14 1.17 1.66 2.22 1.09 1.08 1.48 1.72 1.31

33.0 22.0 20.0 18.0 21.0 16.0 19.0 14.0 15.0 25.0

16.0 10.0 12.0 19.0 16.0 10.0 9.0 5.0 18.0 18.0

24.0 30.0 34.0 31.0 37.0 50.0 47.0 58.0 38.0 30.0

20.0 25.0 28.0 24.0 19.0 18.0 20.0 15.0 19.0 19.0

7.0 12.0 7.0 10.0 8.0 6.0 5.0 8.0 10.0 7.0

Elemental analysis reported as dry, ash-free % weight (Courtesy of IHSS and Huffman Laboratory, Golden, CO). Number-average MW (g mol1) estimated by flow field-flow fractionation (Dycus et al., 1995). Concentrations of carboxyl and phenolic groups (mmol g1) determined by rapid forward direct titration (Ritchie and Perdue, 2003). The distribution of carbon structures (mol mol1) determined by solution state 13C NMR (Thorn et al., 1989).

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where PNMR is the property of interest in the bulk sample, %CEA is the weight percent of carbon in the sample as determined by elemental analysis, (Ci/Ctotal) is the mole fraction of the ith structural subunit as determined by 13 C NMR spectroscopy, and (P/C)i is the ratio of the intensive value of a property P to carbon in each structural subunit (mmol g1). This general equation will be used to evaluate the internal consistency (or lack thereof) between NMR results and elemental analyses. 2.2.1. Maximum unsaturation (U) Unsaturation (U) is the deficiency of hydrogen caused by the presence of rings and p-bonds in an organic compound. This property is sometimes known as double bond equivalents. The molecular formula of any organic compound consisting of only C, H, O, N, and S can be written as: H2ð1UÞ ðCH2 ÞC OO ðNHÞN SS ; where U, C, O, N, and S are in units of mol mol1. C must be greater than zero, and U, O, N, and S must be greater than or equal to zero. The total number of moles of H in this compound is H = 2(1  U) + 2C + N, which can be rearranged to calculate unsaturation as: U¼C

H N þ þ 1; 2 2

ð2Þ

where C, H, and N represent the number of carbon, hydrogen, and nitrogen atoms in the compound. Benzoic acid (C7H6O2), for example, has 5 mol mol1 of unsaturation: four p-bonds (three aromatic and one carboxyl) and one ring. Perdue (1984) translated the calculation of maximum unsaturation to complex mixtures such as humic substances using bulk elemental composition and numberaverage molecular weight (Mn), U EA ¼ C 

H N 1000 þ þ 2 2 Mn

ð3Þ

where C, H, and N are the bulk mole quantities of each respective element (mmol g1) and Mn is the number-average MW of the humic substance (g mol1). The dependence of UEA on Mn is greatest when Mn is small (6 1000) such as for terrestrial and aquatic FAs (Perdue, 1984). The value of UEA calculated from Eq. (3) applies to the entire mixture. Substituting the unique (U/C)i molar ratios of the structural subunits in Table 2 into the general Eq. (1) yields Eq. (4), which can be used to calculate total unsaturation of a sample from its NMR spectrum.          %C EA 1000 C Alk 1 C OAlk 1  þ U NMR ¼ 12:011 10 6 100 C total C total          C Ar 4 C COOH 1 C CO 1 þ þ þ ð4Þ 6 1 1 C total C total C total As given in Table 2, carboxyl and carbonyl groups have 1 mol mol1 of unsaturation per carbon atom (p-bond). Aromatic rings have 4 mol mol1 of unsaturation per six carbon atoms (three p-bonds + one ring), assuming that all aromatic carbons are in phenyl rings (i.e., polycyclic aromatic rings and black carbon are negligible). The (U/C)i ratio of 1/10 for alkyl structures assumes a minor occurrence of aliphatic rings (i.e. lipids, pigments, and

Table 2 The ratios of unsaturation (U), oxygen (O), and hydrogen (H) with carbon associated with structural subunits in 13C NMR spectra Structure

Aliphatic O-Alkyl Aromatic Carboxyl Carbonyl

Shift d (ppm)

Alk O-Alk Ar COOH C@O

0–50 50–110 110–160 160–190 190–220

Ratios (mol mol1) Ui/C

Oi/C

Hi/C

1/10 1/6 4/6 1 1

0 5/6 0 2 1

1.5 11/6 2.5/6 6/10 7/10

terpenes). The (U/C)i ratio of 1/6 for O-alkyl groups assumes that aldohexoses and ketohexoses are much more abundant than other types of sugars and ethers. 2.2.2. Estimations of oxygen content The bulk organic oxygen content of a humic substance is most often calculated as the mass deficit between the total organic mass and the measured C, H, N, S, and P content (on dry, ash-free basis). Using the (O/C)i ratios of the five structural subunits in 13C NMR spectra (see Table 2), the oxygen content of a humic substance may also be predicted from NMR results.          %C EA 1000 C Alk 0 C OAlk 5  þ ONMR ¼ 12:011 1 6 100 C total C total          C Ar 0 C COOH 2 C CO 1 þ þ þ  0:85N 6 1 1 C total C total C total ð5Þ 1

N in Eq. (5) is the concentration of nitrogen (mmol g ) from elemental analysis. The 0.85 coefficient for N was taken from the works of Almendros et al. (1991), Knicker et al. (1993, 1995), and Knicker and Ludemann (1995), whose research using 15N NMR showed that approximately 80-90% of nitrogen atoms in soil-derived humic substances, composts, and forest litter are amides. It is noteworthy that the (O/C)i ratio for aromatic carbon in Table 2 and Eq. (5) is set to 0. Except in the case of phenolic hydroxyl groups and aryl–aryl ethers, all aryl oxygen is bound to another type of carbon (ester, ether, etc.) and is already accounted for in such assignments. It is thus assumed implicitly that alkyl ethers and esters are much more abundant than phenols. The concentrations of polyhydroxy benzenes, such as gallic acid in condensed tannins (Hernes et al., 2001), should be very minor (Burdon, 2001). 2.2.3. Estimations of hydrogen content Hydrogen is liberated and directly measured as liberated H2O during elemental analysis by high-temperature combustion (Huffman and Stuber, 1985). The numbers of hydrogen atoms on functional groups that contain oxygen (alcohols, aldehydes, and carboxyl groups) are relatively easy to calculate. The uncertainty lies in alkyl and aromatic structures. The Hi/C of aromatic groups depends on the number of non-hydrogen atom substituents attached directly to phenyl rings. The (H/C)i ratios in Eq. (6) and Table 2 assume that the majority of alkyl carbons (0–50 ppm) are secondary and tertiary carbons. Phenyl rings are designated to have three and four non-hydrogen atom substitu-

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ents, as is suggested by pyrolysis-mass spectrometry and oxidative degradation methods (Green and Steelink, 1962; Liao et al., 1982; Hatcher, 1988; Saiz-Jimenez, 1994; Krasner et al., 1996). The 0.3 coefficient for nitrogen in Eq. (6) accounts for the two hydrogen atoms on terminal amine groups (2  0.15).       %C EA 1000 C Alk 1:5  12:011 1 100 C total          C OAlk 11 C Ar 2:5 C COOH 6 þ þ þ 6 6 10 C total C total C total    C CO 7 þ þ 0:3N ð6Þ 10 C total

HNMR ¼

2.3. Analytical constraints on acidic functional groups 2.3.1. Carboxyl groups There have been numerous reports that the estimates of ‘‘carboxyl-like” carbon in solid and solution-state 13C NMR (chemical shift of 160–190 ppm) are 1.2–2 times greater than carboxylic acid concentrations that were determined by indirect titrations with calcium acetate (Hatcher et al., 1981; Schnitzer and Chan, 1986; Wilson et al., 1987; Rasyid et al., 1992; Barancikova et al., 1997). Both methods have known biases, so it is difficult to know which one is more reliable. The concentration of carboxyl-like groups estimated by 13C NMR should always be greater than carboxylic acid groups determined by the calcium acetate titration method because the 160–190 ppm shift region of 13C NMR spectra also accounts for ester and amide signals (Leenheer et al., 1995a). There is a tendency for the carboxyl contents of highly soluble humic substances, especially an aquatic FA or aquatic NOM sample, to be overestimated by the calcium acetate method. This problem has been attributed to the failure to remove all of the humic substance from the equilibrated reaction mixture (Perdue et al., 1980). Undissociated functional groups at the equilibrium pH of the reaction mixture of calcium acetate and humic substances (pH 6.8–7.4) consume base as NaOH is added to reach the titration endpoint of pH 9.8. To the extent that some of the reacting groups may be phenols, protonated amines, or thiols, the concentration of carboxyl groups in the sample will be overestimated. Even so, the equilibrium pH of the reaction mixture is too low to cause significant base-catalyzed hydrolysis of esters or amides during the 24-h equilibration time, and at least some of the sample is removed by filtration before titration to pH 9.8, so this method most likely does not detect latent carboxyl groups. There are, however, relatively few reports of direct titration data with corroborating 13C NMR data. If a forward direct titration is performed fast enough, the kinetically-slow reactions that liberate and dissociate latent carboxyl groups are minimal or negligible, and the resulting titration curve only characterizes the concentrations and thermodynamic properties of titratable carboxyl and phenolic groups. In theory, the difference between carboxyl groups determined by a rapid forward direct titration and carboxyl-like groups from 13C NMR spectra represents the maximum possible concentration of latent

carboxyl groups that could cause hysteresis under total alkaline hydrolysis conditions. The hydrolysis of esters to form carboxylic acids and alcohols and the hydrolysis of amides to form carboxylic acids and amines in aqueous solutions are generally irreversible under alkaline conditions because neither an alcohol nor an amine will react with the carboxylic acid anion that exists at alkaline pH. Under acidic conditions, the reverse reaction of an alcohol and a carboxylic acid is generally favorable, and the entire ester–acid–alcohol system may often reach chemical equilibrium. In contrast, amides do not re-form under acidic conditions, because the amine (the potential nucleophile) exists as a protonated cation that has no tendency to act as a nucleophile. In anticipation of the results that will follow, only esters will be considered as latent carboxyl groups. The maximum quantity of carboxyl-like structures (COOHmax) in a humic substance constrained by 13C NMR is calculated by Eq. (7), COOHmax ¼ ð%C COOH Þ

12:011  0:85N 100  C EA

ð7Þ

where %CCOOH is the percentage of carboxyl-like structures from NMR analysis (d 160–190 ppm) in the sample, and CEA and N have the same meaning as in Eq. (4). 2.3.2. Phenolic groups Phenolic groups are constrained by the estimated aromaticity (chemical shift region of 110–160 ppm). The maximum concentration of phenyl groups in a humic substance, hmax (mmol g1), is calculated as hmax ¼

ð%C Ar Þ 12:011  6 100  C EA

ð8Þ

where %CAr is the percentage of aromatic carbon from NMR (d 110–160 ppm) in the sample. Phenol monomers that are derived from lignin have one acidic hydroxyl group and zero (cinnamyl), one (vanillyl), or two (syringyl) ortho-methoxy ethers per phenyl ring. Low MW phenolic acids, such as phenol, cresol, and resorcinol have relatively high vapor pressures and low melting points, and most likely are spirited away during sample freeze drying. Even though gallic acid and other polyhydroxy benzenes have two and three acidic hydroxyl groups per phenyl ring (Hernes et al., 2001), they tend to be much lower in concentration in refractory organic matter compared to partially-degraded lignin oligomers (Burdon, 2001). Therefore, the bulk ratio of acidic hydroxyl groupsto-hmax in a humic substance should always be 61.0, and the ratio of acidic hydroxyl-to-aromatic carbon should be 61/6. If significant quantities of black carbon and fused aromatic structures are present, those ratios should be even lower. 2.4. Comparisons of elemental,

13

C NMR, and titration data

The calculated U, O, and H from NMR spectra will be compared to those derived from elemental analysis. Titration data will be evaluated against COOHmax and hmax. If calculated U, O, and H from NMR data are in near 1:1 relationships with those calculated by bulk elemental

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composition, then the distribution of structural subunits in the humic substance by NMR, including carboxyl and phenolic groups, are probably correct. Deviations from the 1:1 relationship, either in favor of NMR or elemental data, will indicate potential biases by either method. When titration data are compared to NMR data, COOHmax should always be greater than carboxyl groups determined by indirect titrations and by forward direct titrations in the absence of hysteresis. Phenolic group concentrations by titration methods should also be lower than hmax, a maximum of one acidic hydroxyl group per phenyl ring. 2.5. Hysteretic titrations 2.5.1. Preparation of solutions A stock solution containing seven organic acids (SOA) was prepared in 100.0 ml of degassed Fisher HPLC-grade water with 0.10 M NaNO3. The SOA solution contained 1  103 M benzoic acid; 1  103 M 4-hydroxybenzoic acid; 1  103 M salicylic acid; 8.0  104 M 1,2,4,5-benzenetetracarboxylic acid; 1.0  103 M potassium hydrogen phthalate; 2.0  103 M vanillin; and 1.7  103 M oxalic acid. The SOA sample was used to validate the experimental protocol. Separate stock solutions of the IHSS samples were prepared by dissolving 15 mg (peat (1S103F) and soil (1S102F) standard FAs) or 20 mg (Suwannee River standard FA (1S101F) and HA (1S101H) and the Nordic reference FA (1R105F) and HA (1R105H)) of freeze-dried, vacuum-oven dried sample into 25 ml of degassed Fisher HPLC-grade water with 0.10 M NaNO3 as the background electrolyte. The headspace of each stock solution bottle was purged with N2(g) and then vigorously shaken for 24 h in darkness at room temperature (20–22 °C). After 24 h, the total organic carbon (TOC) of all stock solutions was measured by wet-oxidation on the Sievers TOC 800 analyzer, from which mass concentrations were determined. Prior to TOC analysis and titration, each stock solution was inspected visually to ensure that each sample was dissolved and dispersed. Stock solutions were also shaken continuously between titrations to keep samples thoroughly mixed. In our previous work with the same suite of IHSS samples (Ritchie and Perdue, 2003), only stock solutions of the terrestrial HA samples (soil, peat, leonardite, and Summit Hill soil) required adjustment to pH 6– 7 to ensure dissolution and dispersion prior to titration work. The Suwannee River and Nordic HA samples as prepared in Ritchie and Perdue (2003), albeit stock solutions were approximately 1/2 the concentrations of stock solutions prepared in this work, did not require the pH adjustment. 2.5.2. Titration protocol An Orion combination pH electrode was calibrated with pH 3.00, 4.00, 7.00 and 10.00 standardized buffers prior to each set of titrations. Standardized 0.100 M NaOH (verified by titration with KHP) and 0.125 M HCl (verified by titration with standardized 0.100 M NaOH) solutions were used as titrants. Ten-ml aliquots of SOA, FA, and HA stock solutions were used in all titrations. Samples were stirred and equilibrated at 25.00 ± 0.02 °C with a water-saturated

atmosphere of N2(g) for 10 min prior to the onset of, and during, each titration. Unless otherwise stated, the titrants (0.100 M NaOH and 0.125 M HCl) were added in increments of 6–10 ll using a calibrated Gilmont microburette, and pH was recorded 15 s after each addition of titrant. Forward titrations (NaOH) were conducted to a maximum pH of 10.5, and reverse titrations (HCl) were conducted to a minimum pH of 3.0. At the end of each forward or reverse titration, samples were stirred under N2(g) for 30 min before reversing the direction of the titration. These titrations will be discussed as the R-30m titrations. The SOA sample was included to validate this experimental protocol. None of the acids in the SOA sample would be expected to cause any hysteresis between forward and reverse titrations. It was anticipated that forward and reverse titration curves for SOA would be completely superimposable. By using exactly the same protocol for this mixture and for the FA and HA samples, the absence of hysteresis in the titration curves of SOA and its occurrence in titration curves of FA and/or HA would eliminate the possibility that hysteresis is an artifact of the experimental protocol. A variant of the standard protocol was used to determine the effect of longer equilibration time between the forward and reverse titrations. The forward titration of each FA and HA sample was replaced by a single addition of 0.100 M NaOH, the volume of which equaled the volume used to reach pH 10.5 in the standard forward titration of that sample. The headspace of the titration vessel was purged with N2 for 2 min, and a gas tight cap was quickly placed onto the vessel. The alkaline solutions were shaken for 23 h in darkness at room temperature (20–22 °C), after which the vessel was placed into the temperature-controlled water bath at 25.00 ± 0.02 °C for 1 h, and then the standard reverse titration was conducted. These titrations will be discussed as the R-24h titrations. All titration work was completed within 48 h after preparation of the stock solutions.

2.5.3. Calculations The experimental titration data were processed according to the procedure in Ritchie and Perdue (2003), where activity coefficients (cH and cOH), molar concentrations of H+ and OH, and ionic strength were iteratively calculated at each pH during the course of a forward or reverse titration. The resultant titration data were then plotted as charge density (QpH) vs. pH, and modeled by the modified Henderson–Hasselbalch model. The modified Henderson– Hasselbalch equation (Eq. (9)) describes two classes of overlapping proton binding sites (carboxyl and phenolic), where C1 and C2 are the concentrations of carboxyl and phenolic groups (mmol g1), K1 and K2 are mean protonbinding equilibrium constants, and n1 and n2 are width parameters that describe the distribution of log K values for carboxyl and phenolic binding sites. Q pH ¼

C1 1 þ ð½Hþ K 1 Þ1=n1

þ

C2 1 þ ð½Hþ K 2 Þ1n2

ð9Þ

The modified Henderson–Hasselbalch model is a continuum model of carboxyl and phenolic binding sites, and uses no correction for internal electrostatic interactions

J.D. Ritchie, E.M. Perdue / Organic Geochemistry 39 (2008) 783–799

or counterion binding——as is applied the NICA–Donnan model (Kinniburgh et al., 1996; Benedetti et al., 1996; Milne et al., 2001) and Model VI (Tipping, 1998)——other than iterative calculations of the ionic strength of the bulk solution. Because the background electrolyte and the titrants used in this work are 0.1 M, the iteratively-calculated ionic strength is expected to remain at or very close to 0.1, and electrostatic effects are equalized throughout the titrations. Concentrations of carboxyl and phenolic groups were also estimated by the model-independent method of assigning arbitrary pH cutoffs as endpoints for titration of carboxyl and phenolic groups (Bowles et al., 1989). The concentration of carboxyl groups was set equal to QpH at pH 8.0, and the concentration of phenolic groups was set as two times the increase in QpH between pH 8.0 and 10.0. This method of assigning the concentration of phenolic groups from titration data assumes implicitly that the mean log K for proton binding by phenolic groups is 10.0. Model and model-independent estimates of acidic functional groups yield slightly different concentrations of carboxyl and phenolic groups (Santos et al., 1999; Ritchie and Perdue, 2003). Discussions of acidic functional group concentrations or titrations in this work will be based upon the model-independent pH method, unless otherwise noted. The estimates of carboxyl and phenolic groups for the forward titrations, reverse titrations at 30 min, and reverse titrations at 24 h will be compared to the estimates of COOHmax and hmax.

3. Results and discussion 3.1. Evaluation of analytical constraints Elemental analysis is the most commonly performed chemical method in the humic sciences, and it is a robust method when samples have minimal inorganic ash and are thoroughly dried prior to analysis (Huffman and Stuber, 1985). Indirect titrations are regularly performed on humic substances, especially for soil HAs and FAs, but not as routinely as elemental analysis. Reports of direct titration data for soil HA and FA samples are significantly fewer than indirect titration data (Ritchie and Perdue, 2003), and vice versa for aquatic HA and FA samples (Perdue and Ritchie, 2004). NMR is the most versatile and widely-used method for characterizing average structure and concentrations of functional groups in humic substances (see reviews by Wilson, 1987; Preston, 1996; Kögel-Knabner, 1997; Cardoza et al., 2004; Cook, 2004). Two prominent studies have shown that bulk elemental compositions (Rice and MacCarthy, 1991) and CPMAS spectra (Mahieu et al., 1999) for hundreds of HAs and FAs are statistically very similar. Among the peer-reviewed journal articles published between 1980 and 2006 on 13C NMR analyses of humic substances that were collected for this study, approximately 50% reported the elemental compositions of those samples. Less than 25% of those articles reported concentrations of carboxyl and phenolic groups, and indirect titration meth-

789

ods were used in all but two of those studies. Solid-state NMR (e.g., CPMAS and DPMAS) can be used on humic substances that contain non-paramagnetic inorganic matter (Knicker et al., 1995); however, elemental analyses and titrations of such samples may yield unreliable results if they contain high quantities of inorganic matter. 3.1.1. Elemental vs. NMR data Because NMR results will be used to constrain the interpretation of titration data and the possible contribution of latent carboxyl groups to the titratable acidity of a sample, it is appropriate to ask whether the NMR results are sufficiently quantitative. This question has been addressed using the best data sets that are currently available for the IHSS standard and reference FAs and HAs (see Section 2.1). Estimates of total unsaturation (U), oxygen (O), and hydrogen (H) from NMR data were calculated using Eqs. (4)–(6) and were compared with values of U, O, and H from elemental analyses, where U was calculated from elemental data using Eq. (3). The NMR based estimates are plotted vs. the actual elemental analyses in Fig. 1, and, as a guide, contour lines representing deviations of ±10%, ±20%, ±30%, and >±30% from the 1:1 trend line are superimposed on the plot. The calculated U, O, and H from 13C NMR for the 10 IHSS samples were in a near 1:1 relationship (within ±10%) with those calculated from elemental data and Mn. The agreement between elemental and NMR data suggests that the distributions of structures as predicted from 13 C NMR spectra (Thorn et al., 1989), most importantly the distributions of oxygen and unsaturation, are likely correct. The only outlier was for the calculated O for the Nordic FA (NFA) sample where the elemental-to-NMR ratio was 0.89. Interestingly, the aforementioned assignment of (O/C) = 0 for aromatic carbon, assuming that phenols are negligible in comparison with aryl ethers and esters, gave slightly better agreement between elemental and NMR data than (O/C) = 1/12 (1 phenol per 2 phenyl rings) and much better than (O/C) = 1/6 (1 phenol per phenyl ring). Based solely on inferences made from NMR data constrained by elemental data, one may speculate that there is approximately one phenolic hydroxyl group per every two phenyl rings in the IHSS samples. The comparisons of U, O, and H from 200 other literature reports of HAs and FAs that included elemental analysis and 13 C NMR are also shown in Fig. 1. The other data were categorized by NMR methodology (CPMAS and solution-state) and by the type of sample. Although there is much scatter in these data sets, it is perhaps constructive to rank the various predictions into three groups: underpredicted (error < 10%), correct (10% 6 error 6 +10%), and overpredicted (+10% < error). When predictions are counted in this manner, the results for predictions of unsaturation are: underpredicted (132), correct (54), overpredicted (14). The results of predictions of hydrogen content are: underpredicted (34), correct (60), overpredicted (106). The results of predictions of oxygen content are: underpredicted (66), correct (119), overpredicted (15). Only nine of the 200 literature reports had all three values of U, O, and H that lay within ±10% of the 1:1 line. So as a generality, reported 13C NMR spectra tend to underpredict U, overpredict H, and correctly predict O. The lower U, greater H, and

790

J.D. Ritchie, E.M. Perdue / Organic Geochemistry 39 (2008) 783–799

Maximum unsaturation 40

+ 30%

IHSS

+ 20%

+ 10%

40

1:1

+ 30%

Others

+ 20%

+ 10%

1:1 - 10%

35

- 10% - 20%

30

- 30%

- 20%

30 - 30%

20

25

20

10

Elemental Analysis Property (mmol g-1)

20

25

30

35

10

40

20

30

40

Oxygen content 35

+ 30%

IHSS

+ 20%

+ 10%

40

1:1

+ 30%

Others

+ 20%

+ 10%

- 10%

- 10%

30

1:1

- 20%

30 - 20%

- 30%

25 - 30%

20

20

15

10

15

20

25

30

35

10

20

30

40

Hydrogen content 50

IHSS

+ 30%

+ 20%

+ 10%

45

80

1:1

+ 30%

Others

+ 20%

+ 10%

1:1

70

- 10%

60

- 20%

- 10%

- 30%

40

- 20%

35

- 30%

50 40 30

30

20 30

35

40

45 13

50

20

30

40

50

60

70

80

-1

C NMR Predicted Property (mmol g )

Fig. 1. Comparison of maximum unsaturation (U), oxygen (O), and hydrogen (H) content for 10 of IHSS FAs and HAs, and for 200 other reports of HAs and FAs calculated from 13C NMR spectra and elemental composition. Data are plotted by domains with ±10%, ± 20%, ± 30% and >±30% deviations from the 1:1 trend line. () CPMAS FA; () Soln-state FA; (N) CPMAS HA; (M) Soln-state HA. Others: Aiken et al. (1992), Arshad et al. (1988), Barancikova et al. (1997); Belzile et al. (1997), Boerschke et al. (1996), Chen et al. (2002), Chen and Pawluk (1995); Conte and Piccolo (1999), Cook and Langford (1998), Cozzolino et al. (2001), Dereppe et al. (1980), Frund et al. (1989), Gondar et al. (2005), Gonzalex-Perez et al. (2004), Gonzalez-Vila et al. (2001), Hedges et al. (1992), Kaiser (2003), Malcolm and MacCarthy (1986), Mao et al. (2000), Mauice et al. (2002), Monteil-Rivera et al. (2000), Novak and Smeck (1991), Peuravuori et al. (2001), Preston and Schnitzer (1987), Rasyid et al. (1992), Schnitzer and Preston (1986), Stearman et al. (1989), Thorn et al. (1992), Ussiri and Johnson (2003), Watt et al. (1996), Westerhoff et al. (1999).

similar O can all be explained by a systematic underprediction of carbonyl (d 190–220 ppm) and carboxyl (d 160–190 ppm) carbon with an equivalent overprediction of oxygenated alkyl (d 50–110 ppm) carbon (Keeler and Maciel, 2003). Aromatic carbon is probably not significantly underpredicted or overpredicted.

3.1.2. Comparisons of titration and NMR data The comparisons of COOHmax and hmax from 13C NMR spectra to the concentrations of carboxyl and phenolic groups in the IHSS and other samples are shown in Fig. 2. COOHmax and hmax for all 10 IHSS samples were greater than carboxyl and phenolic concentrations by direct

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J.D. Ritchie, E.M. Perdue / Organic Geochemistry 39 (2008) 783–799

Phenolic-to-hmax ratios for the IHSS samples ranged from 0.27 to 0.89 (roughly one phenolic hydroxyl group per every 1.5–4 phenyl rings). Although the terrestrial samples had greater aromaticity and greater concentrations of phenyl rings, the aquatic samples (Suwannee River and Nordic HAs and FAs) had greater phenolic hydroxylto-hmax ratios (0.61–0.89) than terrestrial HAs and FAs (0.27–0.60). Larger aromatic structures, such as partiallydegraded lignin, commonly have aromatic-to-aliphatic ether bridging via the phenolic hydroxyl oxygen to the b-carbon of the para-position aliphatic side chain on the adjacent lignin monomer (Hatcher, 1988). If greater average MWs of humic substances are indicative of greater numbers of lignin-like aromatic groups forming the ‘‘humic backbone” (Stevenson, 1994), more phenyl rings should be bridged by ethers via the phenolic hydroxyl group oxygen. Collectively, there was approximately one phenolic hydroxyl group per every two phenyl rings for the IHSS samples (0.57 ± 0.23), as was initially suggested by elemental constraints on the NMR spectra. Ninety-nine data sets from our literature search included both NMR data and measurements of concentrations of

titrations, and the ratios of carboxyl-to-COOHmax and phenol- to-hmax are shown in Table 3. The most striking point seen in Fig. 2 and made in Table 3 is that 69% (±6) of COOHmax in the IHSS samples were titrated during rapid forward titrations. Although the dataset for the IHSS samples discussed in this work was small (6 HAs and 4 FAs), there was no statistical difference between HAs (70% ± 6) and FAs (69% ± 7). It is our opinion that the approximate 0.70 ratio of ‘‘titratable” carboxyl-to-COOHmax was neither coincidental nor an analytical bias with either the NMR or titration method. A relatively constant ratio of titratable-to-latent carboxyl groups might exist if the formation of esters and carboxyl groups that are associated in strong hydrogen bonds is in chemical equilibrium. If the above statement is correct, it would support the argument that XAD-8 isolates are indeed predestined to yield specific concentrations of carboxylic acid groups (Shuman, 1990), and possibly other types of oxygen-rich structures. Because relatively few whole aquatic and soil NOM samples have been characterized by both direct titration and 13C NMR, the 0.70 ratio may or may not apply to natural organic matter that has been isolated without adsorption on XAD resins.

Carboxyl Concentration 12

+ 30%

COOH Titr. (mmol g-1)

IHSS

+ 20% + 10%

12

1:1

10

+ 30%

Others

- 10%

+ 20% + 10%

10

- 20%

- 20%

8

1:1 - 10%

8

- 30%

6

6

4

4

2

- 30%

2 2

4

6

8

10

12

2

4

6

8

10

12

-1

COOHmax (mmol g ) Phenolic Concentration 8

+ 30%

ArOH Titr. (mmol g-1)

IHSS

+ 20% + 10%

8

1:1

- 20%

6

+ 30%

Others

- 10%

+ 20% + 10%

1:1 - 10% - 20%

6

- 30%

- 30%

4

4

2

2

0

0

0

2

4

6

8

0

2

4

6

8

θ max (mmol g-1) Fig. 2. Comparison of titration data for the IHSS FAs and HAs and for 99 other reports of HAs and FAs with maximum carboxyl and phenyl ring content calculated from 13C NMR spectra. Data are plotted by domains with ±10%, ±20%, ±30% and >±30% deviations from the 1:1 trend line. (d) CPMAS FA; (s) Solution-state FA; (N) CPMAS HA; (M) Solution-state HA. Others: Barancikova et al. (1997), Frund et al. (1989), Gondar et al. (2005), Kaiser (2003); Lobartini et al. (1991); Monteil-Rivera et al. (2000), Novak and Smeck (1991), Preston and Schnitzer (1987); Rasyid et al. (1992), Schnitzer and Preston (1986); Stearman et al. (1989); Ussiri and Johnson (2003).

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J.D. Ritchie, E.M. Perdue / Organic Geochemistry 39 (2008) 783–799

Table 3 The comparison of chemical properties for the IHSS humic and fulvic acids by elemental analysis (Huffman Laboratory, Golden, CO) and direct titration (Ritchie and Perdue, 2003) with properties calculated from 13C NMR spectra (Thorn et al., 1989) Unsaturation (mmol g1)

Suwannee FA Soil FA Peat FA Nordic FA Suwannee HA Soil HA Peat HA Leonardite HA Nordic HA Summit Hill HA

Oxygen (mmol g1)

EA

NMR

E:N

EA

NMR

E:N

EA

NMR

E:N

23.4 25.3 26.7 24.7 22.8 31.9 29.6 36.2 25.5 23.1

21.4 25.4 25.9 26.0 24.7 29.3 28.0 34.0 26.1 23.2

1.09 1.00 1.03 0.95 0.92 1.09 1.06 1.06 0.98 1.00

26.4 26.6 28.4 28.2 26.6 21.3 23.3 19.8 26.9 23.7

25.9 27.1 29.1 31.7 25.2 21.8 22.4 21.7 27.3 23.9

1.02 0.98 0.98 0.89 1.06 0.98 1.04 0.91 0.99 0.99

46.4 37.9 34.9 39.5 43.7 36.5 37.9 35.7 39.4 48.0

52.6 36.2 36.3 40.9 39.7 36.7 36.5 34.4 38.7 44.6

0.91 1.05 0.97 1.02 1.10 0.98 1.04 1.04 1.02 1.08

Average SD

1.02 0.06

0.98 0.05 Phenolic content

COOH content

Suwannee FA Soil FA Peat FA Nordic FA Suwannee HA Soil HA Peat HA Leonardite HA Nordic HA Summit Hill HA

Hydrogen (mmol g1)

1.01 0.05

Titr.

COOHmaxa

T:N

Titr.

hmaxb

T:N

6.00 6.40 6.72 5.84 5.04 4.81 5.08 4.77 4.83 3.86

8.3 8.2 10.2 10.0 7.6 6.2 6.8 7.2 7.7 5.4

0.72 0.78 0.66 0.58 0.67 0.78 0.75 0.66 0.63 0.71

1.52 1.14 1.17 1.66 2.22 1.09 1.08 1.48 1.72 1.31

1.7 2.1 2.4 2.3 2.7 4.0 3.7 5.1 2.8 2.2

0.89 0.54 0.49 0.72 0.82 0.27 0.29 0.29 0.61 0.60

Average SD

0.69 0.06

0.57 0.23

Values of maximum unsaturation (U) for elemental and NMR data were calculated using Eqs. (2) and (3). Oxygen and hydrogen (O and H) content for NMR data were calculated using Eqs. (4) and (5). E:N is the ratio of elemental-to-NMR properties. T:N is the ratio of carboxyl and phenolic groups by direct titration to NMR predicted COOHmax and hmax. a Maximum carboxyl-like structures (carboxyl + ester groups) minus amide. b Maximum phenyl rings predicted by aromatic carbon/6.

carboxyl and phenolic groups. In 93 of the 99 samples, indirect titration methods were used to determine the concentrations of carboxyl and phenolic groups. The results of those analyses are more or less consistent with the statistical analysis of indirect titration data by Ritchie and Perdue (2003). Direct titration methods were used for only six samples. When measured concentrations of carboxyl and phenolic groups in the 99 titration data sets are compared with COOHmax and hmax from 13C NMR spectroscopy, measured concentrations of carboxyl groups were greater than COOHmax in 49 of the 99 data sets. This theoretically impossible situation results from an unknown combination of overestimation of carboxyl groups by the calcium acetate method (Perdue et al., 1980) and underestimation of the carboxyl region of NMR spectra (d 160–190 ppm), both of which have been discussed previously (Sections 2.3.1 and 3.1.1). Sixty-three data sets had measured concentrations of phenolic groups that exceeded hmax values that were obtained by 13C NMR spectroscopy. It can be argued that hmax is an arbitrary limit that results from allowing no more than one phenolic hydroxyl group per benzene ring. In 14 of the 99 data sets, however, measured concentrations of phenolic groups were so high that the average benzene ring would have to be substituted with 2–4 hydroxyl groups. It is highly probable that the experimental mea-

surements are simply too high, and the source of this bias may well be the same chemical reactions that are often invoked to account for pH drift and hysteresis in direct titrations. As noted earlier, in nearly all of the 99 data sets, the concentrations of phenolic groups were measured using indirect titrations, specifically the barium hydroxide method. If it is true that esters can hydrolyze during direct titrations to pH values of perhaps 10.5 (Marshall et al., 1995; Santos et al., 1999), then it must certainly be the case that a 24-h incubation of humic substances in 0.1 M barium hydroxide at pH 12–13 would drive most hydrolysis reactions to completion and liberate most of the latent carboxyl groups in a sample. Such a process would lead directly to an over-estimation of phenolic content. 3.2. Hysteretic titrations No hysteresis was observed for repetitive titrations (three forward and two reverse) of a single, 10-ml aliquot of SOA mixture. The titration curves in Fig. 3 overlie each other almost perfectly. This was the expected result, because the SOA mixture contained no hydrolysable esters (Bowles et al., 1989; Antweiler, 1991), and the acids’ small sizes would have prevented the formation of strong intramolecular hydrogen bonds that has been postulated for

J.D. Ritchie, E.M. Perdue / Organic Geochemistry 39 (2008) 783–799

humic substances. The average concentrations of carboxyl groups (estimated at pH 8.0) and phenolic groups (estimated as two times the difference in charge density between pH 8.0 and 10.0) for the three forward and two reverse titrations were 19.23 ± 0.10 and 3.33 ± 0.08 mmol g C1, respectively. According to the recipe of the SOA mixture (see Section 2.5.1), the actual concentrations of carboxyl and phenolic groups in the SOA mixture were 17.7 and 5.7 mmol g C1. The total acidity (sum of carboxyl and phenolic) of the experimental titration curves differed from the actual total acidity by only 0.85 mmol g C1, which was attributed to the incomplete dissociation of the hydroxyl group on salicylic acid (pKa  13). The discrepancy between the actual carboxyl concentration and the greater experimental carboxyl concentration was due to the dissociation of the phenolic hydroxyl group in vanillin (pKa = 7.4). Using the concentrations and pKa values of the individual acids in the SOA mixture, it is possible to compare the experimental and theoretical QpH values over the entire pH range of the forward and reverse titrations, rather than simply at pH 8.0 and pH 10.0. The root mean square error for the five titrations, relative to the theoretically expected values of QpH, is 0.31 mmol g C1. Having established that the experimental protocol and data processing procedures are not themselves a source of any apparent hysteresis, the titration curves of the six IHSS HAs and FAs can be examined more closely. Because the initial pHs of the stock solutions and the starting points of the forward titrations differed depending on the type of sample and concentration (Suwannee and Nordic FA: pH 2.90 and 2.88; Suwannee and Nordic HA: pH 3.02 and 3.00; Soil and Peat FA: 3.13 and 3.16), the titration curves shown in Fig. 4 are the ‘‘best-fit” Henderson–Hasselbalch models plotted over the pH range of 2.8–10.5. The fit of the Henderson–Hasselbalch models to the titration data in this work (not shown) were comparable to fits shown in Fig. 1 of Ritchie and Perdue (2003). The lowest values of QpH at all pHs were for the forward titrations. Intermediate results were obtained in the R-30m titration curves, and the greatest QpH values were obtained in the R-24h titration curves. The R-30m and R-24h titra-

25 SOA Mixture

QpH (mmol gC-1)

20 15 10 5 0 2

3

4

5

6

7

8

9

10

11

pH Fig. 3. The overlapping titration curves (three forward and two reverse) for a single aliquot of the simple organic acid mixture (SOA) repeatedly titrated between pH 3.0 and 10.5.

793

tion curves approach convergence with the forward titration curves at pH 3.0–3.2. This indicates, at least qualitatively, that the majority of new carboxylic acids generated above pH 10 were weaker acids than those proposed by Leenheer et al. (1995a,b) for the strongest carboxyl groups with pKa < 3.0. pH was observed to drift downward between pH 5.8 and 10.5 during the forward titration, but the full extent of pH drift was not evaluated in the 15 s between additions of base titrant. It should be noted that the carboxyl and phenolic acid concentrations (by the pH method) for the forward titrations performed in this work (Table 4) were within ±5% and ±15%, respectively, of those values reported in Table 1 (taken from Ritchie and Perdue, 2003). Even though the concentrations of HAs and FAs used by Ritchie and Perdue (2003) (350–400 mg l1) were approximately one-half the concentrations used in this work (725–830 mg l1), the forward titration curves (when normalized to the dilution-corrected mass of HA or FA) in this work almost exactly overlapped with the earlier titration curves of Ritchie and Perdue (2003) between pH 3.5 and 10.0, with slight variations above pH 10.0 and below pH 3.5 (not shown). During the 30 min interval between the completion of the forward titrations and the beginning of the R-30m titrations, pH drifted downward 0.27–0.34 pH units, which translated to an overall increase in dissociated acidic functional groups of 0.24–0.42 mmol g1. The pH drifted downward 0.69–1.03 pH units from pH 10.5 during the 24 h time interval between addition of base and the beginning of the R-24h titrations, which translated to an overall increase in dissociated acidic functional groups of 0.83–1.05 mmol g1. Concentrations of carboxyl groups (estimated as QpH at pH 8.0) substantially increased for all six samples with increasing time of exposure to mildly alkaline pH (Table 4). Conversely, concentrations of phenolic groups for the R-30m titrations were equal to or slightly smaller than those for the forward titrations (Table 4). Phenolic groups could not be reliably estimated for the R-24h titrations by either the pH method or by the Henderson–Hasselbalch model because pH drifted below pH 10.0 during the 24 h period after the addition of base. For the sake of argument, the ‘‘best fit” Henderson–Hasselbalch model predictions of phenolic concentrations (Table 4) for the R-24h titrations of all six samples were within ±0.2 mmol g1 of the estimates derived from the forward titrations. Nonetheless, carboxyl concentrations clearly increased with increasing time of exposure to pH 9.5–10.5 whereas the phenolic concentrations were relatively invariant. Thus only carboxyl groups were generated by slow kinetic processes at neutral and alkaline solution conditions. The invariant phenolic concentrations may indicate that leaving groups formed by ester hydrolysis were not phenols. If the concentrations of carboxyl groups that were calculated from the forward, R-30m, and R-24h titrations are compared to the COOHmax for the six samples (Fig. 5), the forward titrations account for 59–77% (67 ± 7), the R-30m account for 65-84% (73 ± 7), and the R-24h titrations account for 75–97% (84 ± 8) of the COOHmax. Suppose that the same slow chemical reactions that are hypothesized

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J.D. Ritchie, E.M. Perdue / Organic Geochemistry 39 (2008) 783–799

10

10 Suwannee River HA

Suwannee River FA 8

8

6

6

4

4

2

2 0

0

Charge density, QpH (mmol g-1)

2

3

4

5

6

7

8

9

10

11

2

10

3

4

5

6

7

8

9

10

11

4

5

6

7

8

9

10

11

4

5

6

7

8

9

10

11

10 Nordic FA

Nordic HA

8

8

6

6

4

4

2

2

0

0 2

3

4

5

6

7

8

9

10

11

2

10

3

10 Soil FA

Peat FA

8

8

6

6

4

4

2

2

0

0 2

3

4

5

6

7

8

9

10

11

2

3

pH Fig. 4. The forward (lowermost), the reverse 30 min (middle), and reverse 24 h (uppermost) titration curves for the IHSS Suwannee River FA and HA, Nordic FA and HA, Soil FA, and Peat FA.

to cause pH drift and hysteresis in direct titrations through the generation of carboxyl groups at neutral-to-alkaline pH also occur in the barium hydroxide reaction mixtures used to measure total acidity (and phenolic content). The data in Table 4 can be used to compare apparent phenolic content, which includes the newly generated carboxylic acids, and the actual phenolic contents that are measured in direct titrations. The ratio of apparent to direct phenolic content ranges from 1.92 to 2.11 for the four fulvic acids and from 1.59 to 1.75 for the two humic acids. The overall average ratio is 1.9 ± 0.2. This means that the carboxyl groups that will be generated by slow chemical reactions in the barium hydroxide reaction mixture, where pH is even greater than in these titrations, should cause phenolic contents to be over estimated by almost a factor of two. This interpreta-

tion is very strongly supported by the extensive data set that is summarized in Table 2 of Ritchie and Perdue (2003), which includes a total of 154 direct titrations and 284 indirect titrations of FAs, HAs, and NOM. From these data, the ratios of indirect to direct estimates of phenolic content range from 1.8 to 2.1 – very close to the average ratio of 1.9 that is found in this study of the effect of hysteresis on the measurement of carboxyl content in direct titrations. The data in Table 4 indicate that the titratable carboxyl contents of Suwannee River FA and HA are very nearly the same percentage of COOHmax in the forward titrations (73% and 68%), in the R-30m titrations (80% and 76%), and in the R-24h titrations (89% and 86%). A similar trend is found for the Nordic FA and HA: forward titrations (59% and 60%),

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Table 4 Carboxyl and phenolic group concentrations and the modified Henderson–Hasselbalch model parameters for the hysteretic titrations of the IHSS samples and the mixture of simple organic acids (SOA) pH methoda

Modified Henderson–Hasselbalch modelb

COOH

ArOH

COOH

log K1

n1

ArOH

log K2

19.32 19.29 19.29 19.28 19.27

3.22 3.32 3.35 3.44 3.39

19.14 19.21 19.32 19.36 19.04

4.00 3.94 3.92 3.90 3.87

2.18 2.24 2.29 2.28 2.17

2.19 2.05 1.90 1.86 2.44

8.76 8.74 8.78 8.79 8.83

1.62 1.40 1.25 1.13 1.58

SRFA Forward R-30m R-24hc

6.05 6.61 7.38

1.44 1.41 N/A

6.28 6.76 7.68

3.91 3.97 4.16

2.96 2.61 2.72

1.30 1.12 1.15

10.09 9.83 9.90

1.44 1.49 1.00

SRHA Forward R-30m R-24hc

5.11 5.64 6.50

2.35 2.42 N/A

5.31 5.84 6.88

4.28 4.18 4.48

3.12 2.96 2.93

2.13 1.61 1.79

9.90 9.45 9.55

1.77 1.39 1.15

NFA Forward R-30m R-24hc

5.88 6.57 7.55

1.61 1.62 N/A

6.23 6.77 8.00

3.82 3.85 4.08

3.47 2.98 3.13

1.58 1.40 1.48

10.28 10.22 10.09

1.60 1.93 1.00

NHA Forward R-30m R-24hc

4.67 5.21 6.00

1.78 1.62 N/A

4.93 5.39 6.33

4.25 4.11 4.37

3.27 3.00 2.96

1.77 1.29 1.47

10.19 9.78 9.93

1.83 1.76 1.44

SFA Forward R-30m R-24hc

6.25 6.84 7.91

1.49 1.38 N/A

6.68 7.39 8.25

3.61 3.71 3.65

3.73 3.82 3.15

0.88 0.92 1.18

9.87 10.17 9.71

1.09 1.00 1.00

PFA Forward R-30m R-24hc

6.59 7.04 7.94

1.42 1.17 N/A

6.92 7.46 8.19

3.97 4.14 4.03

3.11 3.07 2.73

0.80 0.96 0.72

9.71 10.31 9.27

1.28 1.00 1.28

SOA Forward 1 Reverse 1 Forward 2 Reverse 2 Forward 3

n2

a Carboxyl group concentrations were estimated as QpH at pH 8.0. Phenolic group concentrations were estimated as two times the difference between QpH at 8.0 and 10.0. Acidic functional groups are in units of mmol g1. b Modified Henderson–Hasselbalch parameters (see Eq. (6) and discussion in Section 2.2.1). c Henderson–Hasselbalch model parameters for the phenolic groups for R-24h titrations were extrapolated by model based on experimental titration data between pH 3.0 and 9.2–9.5, and may have a high degree of uncertainty.

R-30m titrations (65% and 67%), and R-24h titrations (75% and 78%). The FA and HA samples were collected simultaneously from their respective source waters (Suwannee River, USA and Norwegian humic lake) and processed identically by the XAD-8 method. The observations in this paragraph support the hypothesis that the fulvic acids and humic acids from a common water sample contain comparable proportions of titratable and latent carboxylic acids. If this is correct, it is consistent with the contention that XAD isolation methods yield a particularly uniform product (Shuman, 1990). 4. Conclusions Data for elemental analysis, average MW, direct titrations, and 13C NMR for the IHSS standard and reference HAs and FAs were compared to elucidate the analytical constraints on quantities of carboxyl and phenolic acid groups. In all, the IHSS samples had strong internal consistencies between chemical properties associated with elemental, 13C NMR, and titration data. Comparisons of maximum unsaturation, oxygen and hydrogen from ele-

mental analysis data corresponded with those properties derived from one dimensional 13C NMR spectra within ±10% tolerance. The only outlier, 1 out of 30 points of comparison for 10 different humic substances, was for oxygen content of the Nordic FA, which had an elemental-to-NMR ratio of 0.89. This high degree of internal consistency between bulk elemental and 13C NMR data for the IHSS samples supports the view that the maximum concentrations of carboxyl groups and aromatic rings deduced from NMR data are most likely correct and can be used with confidence to constrain and interpret titration data. When similar comparisons were attempted for an additional 200 data sets from the literature, it was found that 13 C NMR estimates were systematically low for unsaturation, high for hydrogen, and often correct for oxygen. All these trends are consistent with the hypothesis that 13 C NMR measurements systematically underestimate carboxyl and carbonyl groups while over-estimating alkoxy groups. When direct titration data for the IHSS samples (Ritchie and Perdue, 2003) were compared to the maximum carboxyl-like structures in 13C NMR spectra (Thorn et al.,

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12

Carboxyl concentration (mmol g-1)

10

COOHmax R-24h R-30m Forward

8 97% 75%

89% 80%

6

84% 86%

78% 69%

65% 78%

76%

67%

4 73%

77%

59% 68%

65%

60%

2

0 SRFA

SRHA

NFA

NHA

SFA

PFA

Fig. 5. The comparison of carboxyl groups as determined by a rapid forward titration, and the increases in carboxyl group concentration observed for reverse titrations at 30 min and 24 h (R-30m and R-24h). The numbers within the bars indicate the % of carboxyl groups in COOHmax accounted for by the forward and two reverse titrations.

1989), the titratable carboxyl groups constituted 70% of COOHmax. On average, the IHSS samples had one phenolic acid group per every two phenyl rings. Forward and reverse titrations (at 30 min and 24 h after completion of the forward titration) were rapidly performed on six of the 10 IHSS samples to quantify the formation of new carboxyl groups during hysteresis. The R-24h titration had greater charge density at all pH and the greater carboxyl acidity than the forward and R-30m titrations. The 30 min and 24 h time of exposure to mildly alkaline pHs (9.5–10.5) after the completion of the forward titrations allowed for additional 0.24–0.42 and 0.83–1.05 mmol g1 of carboxyl groups to ionize, which accounted for 65–84% and 75–97% of COOHmax. Total carboxyl groups, titratable plus latent groups formed by hysteretic processes, approached COOHmax, but never exceeded COOHmax. Conversely, concentrations of phenolic groups remained more-or-less invariant, in spite of the 30 minutes and 24 hour times of exposure to pH >9.5, suggesting that phenols are not formed by slow base-catalyzed hydrolysis reactions. The conversion of latent carboxyl groups into titratable acidity has the potential to cause phenolic content to be overestimated when calculated from total acidity, as measured by barium hydroxide at very high pH. The predicted overestimation of phenolic content caused by the generation of acidity during direct titrations is very similar to the average difference between indirect and direct estimates of phenolic content in the literature. Acknowledgements We are most grateful to Dr. J.-F. Koprivjnak and the School of Earth and Atmospheric Sciences, Georgia Institute of Technology for their support and cooperation. We would like to extend a gracious thank you to the reviewers

(Dr. E. Tombacz and an anonymous reviewer) for their favorable responses to this work and their comments on how to strengthen our paper’s content. Associate Editor——Ingrid Kögel-Knabner

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