Acid-base characterization of molecular weight fractionated humic acid

Talanta ELSEVIER

Talanta

43 (1996)

383-390

Acid- base characterization of molecular weight fractionated humic acid Masami Fukushima”,

Shunitz Tanaka”,

Hiroshi

Nakamura”.*,

,’ Dicision of Mntrricd Sctence. Graduate School qf Encirontnmtcd Earth Scicw~, b Hokkaido Nrrtional Industrial Rrsearch Institute . 2- I7 T\ukisat,lu-hi~aski,

Saburo Itob

Hokkaido C’niwrsit~~. 060 Supporo, Toyohira-ku. Sapporo 062. Japan

Received 17 March 1995; revised 17 August 1995; accepted 25 August

Jupan

1995

Abstract Acid-base properties of molecular weight fractionated humic acids (HAS) were investigated by the acid-base potentiometric titration. The acidic group contents (C,J and the average values of apparent pK (pK:,,,) were evaluated by applying a modified HendersonPHasselbalch equation to the experimental titration curves. The average values of pK,,, of the fractionated and unfractionated HAS were about 4.1-4.4. and the distribution of pK:,,, values could be represented by the relationships between ZJand pi&,, plots in the range 2-8. The C,,, values increased with a decrease in molecular size, as did the aromaticity. This suggests that the acidic group contents are related to the aromaticity of the HA. Keywords:

Acid- base properties:

Humic acids: Aromaticity

1. Introduction Humic substances acids, and are widely

are polyelectrolytes of weak distributed in soil and aque-

ous environments [I ,2]. Knowledge of acid-base equilibria of humic substancesgives useful information to enable discussionof the complexation of humic substanceswith heavy-metal ions, their binding abilities with organic pollutants (e.g. pesticides and herbicides) [3-51 and the catalyzing degradation of such pollutants in the environment [6].

*Corresponding

author. Fax: (81) 11-716-6101.

0039-9140;96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI

0039.9140(95)01727-5

Humic substances are a mixture of weak-acid polyelectrolytes having various molecular weight moieties and functional groups. The range of molecular weight (Da) of humic substances is from

several hundred to several hundred thousand, and their chemical structures are not completely known. Therefore, in order to obtain more detailed information about humic substancesfrom the viewpoint of the heterogeneouspolyelectrolyte, it is necessary to investigate the characterization of the fractionated humic substances. Generally, humic substances are classified according to molecular size. That is, the insoluble fraction in acidified solution (i.e. the fraction of

higher molecular weight) is humic acid (HA) and the soluble fraction in acidified solution (i.e. the fraction of lower molecular weight) is fulvic acid (FA) [l]. It is known that there are differences in the structural features, acidities and metal ion bindings between HA and FA [7]. The HAS have a more aliphatic structure and a higher surface potential than the FAs, while the acidic group contents and metal binding capacities of FAs are higher than those of HAS. In contrast, a comparison between the molecular weight fractions of the FAs was performed by Wang et al. [8]. They investigated the spectroscopic and structural characterization of molecular weight fractionated FA by ultrafiltration. Their study included the evaluation of the binding behavior of the atrazine with different fractions of the FA. It suggested that the atrazine would mainly bind with the higher molecular weight fraction and that competitive binding occurred between atrazine and the lower molecular weight fraction of FA. Moreover, Green et al. [9] investigated the electrostatic properties of molecular weight fractionated HA by fluorescence quenching. They concluded that the higher molecular weight fraction had a larger apparent surface potential than the lower molecular weight fraction. Therefore, the large humic molecule seems to have the higher binding ability to metal ions. However, the binding parameters (e.g. conditional binding constants and binding capacities) of the fractions of humic substances have not been evaluated and compared with the unfractionated ones. In the present work, to investigate the acidbase properties of the molecular weight fracitonated HA, and HA was fractionated by using a Sephadex G-50 column, and the aciddbase equilibria of the fractionated HAS were evaluated by potentiometric titration with the modified HendersonHasselbalch interpretation.

2. Experimental 2.1. Fractionation

und prepurution

of HA

The HA used in the present work was purchased from Wako Pure Chemicals (Osaka,

Japan). This was extracted with 1 M of NaOH aqueous solution and purified by a protocol of the International Humic Substances Society (IHSS) [lo]. The HA powder obtained was dissolved in 5 x lop2 M Na,HPO, solution (1 g ll’), and then 7 ml of aliquot was injected into a Sephadex G-50 column (50 mm i.d. x 460 mm). The eluent (1 x IO-’ M phosphate buffer at pH 8) was passed through the column at a flow rate of 4.5 ml min ’ by a peristaltic pump (P-l type, Pharmacia LKB Biochemistry) [1 11. The eluent was collected by a fraction collector (Pharmacia LKB Biochemistry, RediFrac) every 4 min. The HA in the eluent was detected at 254 nm by a Hitachi L-4200 UV-vis detector. An analytical column (25 mm i.d. x 500 mm) was also used to confirm a chromatographic profile, and 1 mg of the HA was injected into this column where the flow rate was 1.0 ml min’. A typical chromatogram of the HA has three peaks as shown in Fig. 1, and the molecular weight fractions of the HA were collected separately according to the three peaks in Fig. 1: the highest fraction, F,; the middle fraction, F,; the lowest fraction, F,. The unfractionated HA (UF) was used for the control experiment. The solutions containing each molecular weight fractionated HA were evaporated, and the HAS were precipitated by acidification with HCl (pH < 1). Then, these precipitates were transferred into the dialysis tube (Spectra/Pore, molecular weight cut-off 1000) to remove chloride ions, and the dialysis was performed against distilled water. Subsequently, molecular weight fractionated HA powder was obtained by freeze-drying. The results of elemental analyses for these HAS are summarized in Table 1. 2.2. Potentiotnetric

titration

The HA aqueous solution (1000 mg 1 ‘) was prepared by dissolving in 5 x 10 ’ M NaOH solution under a nitrogen atmosphere. Subsequently, 10 ml of this solution and 5 ml of 1 M NaCl aqueous solution were pippeted into a 50 ml volumetric flask and diluted up to 50 ml with decarbonated water. A portion (30 ml) of this soluiton was pipetted into the 50 ml measurement cell and allowed to stand for about 15 min while

passing nitrogen gas. This solution was titrated down to about pH 2.8 by 0.05 M HCl. The equilibration time between the addition of the titrant and recording the pH was at least 2 min. The 0.05 M HCl solution as a titrant was standardized by Na,CO,, and the NaOH was standardized by this HCl. The acetic acid was used as a model compound of the HA. The pH of the solution was measured by a pH meter (M-13 type, Horiba Ltd.) with a glass electrode (6366 type, Horiba Ltd.), and hydrogen ion concentrations at each of the titration points were corrected with respect to the activity coefficient of hydrogen ion.

0.0

0

100

200

Retention

300

volume

analyses

of the humic

0

1

2

4

I

I

6

8

I 10

0.05 M HCI / mL Fig. 2. The potentiometric titration curve of acetic acid. (a) and blank NaOH (b). Concentration of acetic acid: 4.4 x IO ~’ M; ionic strength: 0.1 M NaCl. Correction of hydrogen ion concentration was according to the following equation: [H+ concentration of hydrogen ion; I = a,, / :;,H, ; [H + ] corrected c(r, + and ;‘,,- : activity and activity coefbcient of hydrogen ion respectively.

t 3.0 -0.8

Fb

F,

UF

‘%C %H ‘AN ‘X10 ‘%ash

51 4.7 1.7 40 2.4 I.1 1.1 130 70

53 4.4 3.1 3x 1.7 I.0 I .o 140 75

55 3.1 1.9 37 2.4 0.90 0.94 1x0 X3

52 4.9 I.2 39 3.4 I.0 I .o 130 68

a Molar ratio. b Degree of unsaturation of the HAS accordi (Y4fl ing equation of Kuwatsuka et al. [2l]: 0.429(%N) + lZ(‘i%C) x 100.

%fM?\r-

-0.6

-0.4

-0.2

0.0

0.2

0.4

-log (laa) Fig. 3. Henderson tb).

acids

F.,

Unsaturatedb

I

2’

/ mL

Parameter

lWO1” IN1+ lOl:lCl [WW

6

400

Fig. I. Size exclusion chromatogram of the HA. Column size: 25 mm i.d. x 500 mm: sample volume: I ml aliquot of the Ig I- ’ HA; eluent: I x IO-’ M phosphate butler (pH 8): flow rate: I ml min’, UV detection w,av*elength: 254 nm. Table I Elemental

____-______

%*

Hasselbalch

plot of acetic acid (a) and HA

The titration was performed under a nitrogen atmosphere at 25 + O.l”C. The distilled water used in the experiment was decarbonated by boiling and nitrogen bubbling. This water was stored in a polyethylene bottle with a soda-lime tube to protect from carbon dioxide absorption. 2.3. Spa-troscopir meusurements The UVVis. fluorescence and FTIR spectra of the HAS were recorded by a spectrophotometer (Shimadzu UV-2200), a spectrofluorophotometer

386

M. Fukushirnu

et rd. I Tulunru

(Shimadzu RF-510) and a Fourier transform infrared spectrophotometer (Perkin Elmer 1720-X type) using a KBr disk method respectively. The excitation wavelength at 330 nm was adopted in the measurement of fluorescence spectra. The fluorescence intensities were not corrected for the sensitivity of the spectrometer. Therefore, in order to correct the fluorescence intensity to consider absorption of excitation light by the inner filter effect, the relative quantum yield (Q,,,,) of the fluorescence was calculated by the following equation:

where r,,, Fobs and Abs,, represent absorptivity at the excitation wavelength (330 nm), fluorescence intensity observed and absorbance at the excitation wavelength respectively. The fluorescence intensity with correction of the inner filter effect is proportional to the Qre,, values.

3. Results and discussion 3.1. Titrution compound

curce

of acetic ucid us N model

The acetic acid in excess alkaline aqueous solution with NaOH was titrated with HCl (Fig. 2). Although excess NaOH is consumed by HCl in the higher pH region, ionized species of acetic

43 (1996) Table 2 Acidic group

383-390

contents

HA

C,,(meq

F‘, F, FC UF

5.32 6.01 6.99 4.76

+ f k f

and average g-’

0.07 0.02 0.04 0.13

C)

pKilpp values Average 4.15 4.38 4.19 4.05

(n = 3)

pK,,,,

F0.02 * 0.07 ) 0.01 * 0.04

acid are protonated after the end-point of the strong acid-base equilibrium. Thus the concentration of acetic acid was evaluated by subtracting the end-point of the titration curve for NaOH without acetic acid (Fig. 2b) from that for acetic acid (Fig. 2a). In general, the experimental data can be illustrated by the dependence on the degree of ionization (2) of the apparent acid dissociation constant, as defined by a modified HendersonHasselbalch equation [ 12,131: PH = pk,,,

- log &

where pK,,, denotes apparent acid dissociation constant. The HendersonHasselbalch plot (pH vs. - log[r/( 1 - a)]) for acetic acid is shown in Fig. 3a. The pH at -log[a/( 1 - z)] = 0 corresponds to the acid dissociation constant of acetic acid. This value is 4.89 and is in good agreement with the reported value (4.80) [14]. This shows that the titration with excess alkaline solution is not influenced by carbon dioxide contamination. Therefore, the pK>,,, values and the concentrations of acidic groups (C,4t) of the HAS were evaluated by the same titration manner as for acetic acid.

7-

3.2. Titration $5%

i

.

I9 0.0

0 Fa ’ Fb 0 Fc n

0.2

0.4

0.6

UF 0.8

a Fig. 4. r vs. pK,,,

plots

for HAS.

1.0

curl’es of’ the humic acids

A HendersonHasselbalch plot of the unfractionated HA is not linear, as shown in Fig. 3b. This suggests that the HAS include different types of ionic groups in their structure [15,16]. The CA, values of the HAS could be evaluated by the same manner as for acetic acid, and the aveage values of PK,,, could be evaluated by the relationships between pK,,, and z shown in Fig. 4. The relationship between pK,,, and x shows the distribution of pK,,, values of the acidic group in the

a)

b)

-

Fa

-F

b

-F

C

-

UF

. 2

0.6

0.4

0.2

330

430

530

630

730

0.0 400

Wavelength / nm

450

500

550

600

Wavelength / nm

Fig. 5. UV-Vis absorption and fluorescence spectra of HAS: (a) UV-Vis spectra; (b) fluorescence spectra. Carbon contents in the HAS (mg C 1-l): UF, II; F,. 10; F,. II: F,. 11. The pH was 8 (0.1 M HEPES burer); excitation wavelength, 330 nm; excitation and emission bandwidths IOnm. Absorbance of the HA at 330 nm was used to calculate the CD,,, value.

HA. The pK,,, values distribute in the range 2-8, value at x = 0.5 corresponds to the and the p&, are sumaverage pK,,, The average pK,,, values marized in Table 2. In other previous reports, these average pK,, values were at about 4 w 5 [14- 181. In our work, the average values of pK,,, are at about 4.0-4.4, and these values correspond to those of other reports. In contrast, the CAt values of humic substances were in the range of 5 A 7 meq gP I of C and these values were consistent with the reported values (3 h 10 meq gg’ C) [7,19]. The CA, values increased with a decrease in molecular size, and the largest C.&, value was found in the lowest molecular weight fraction, F,. For example, FA, which is the lower molecular weight fraction of humic Table 3 Absorbance E -‘ofCinHA) Parameter

parameters

ezxo --

at 280 nm (I cm-’

3.3. Elmentul

mal?‘sr3

HA F,

~400!‘%41

and absorptivity

substances, is known as the further humification product of HA [l], and the acidic group contents of FAs are higher than those of HAS 173. Moreover, Swift et al. [20] proposed that the aromaticity and degree of unsaturation increased with the decrease in molecular weight and that the low molecular weight components were final products of the humification process. This shows that the proceedings of the humification process brings about the higher contents of carboxylic groups in the FAs. The results obtained in the present work also show a similar tendency to the results of Swift et al. [20] in the comparison between HA and FA. If the lower molecular weight fraction relates to the degree of humification of HA, the structural features (i.e. aromaticity and unsaturation) would be the important factor to compare the C,,, values of the fractionated HAS.

6.1 63

Fb 8.4 68

FL

UF

8.7 70

5.8 41

The C. H, N and ash contents in the fractionated HAS are summarized in Table 1, From these values, the molar ratio of 0 to C ([O]/[C]), the molar ratio of polar elements (mainly 0) to C ([N] + [O]/[C]), the molar ratio of C to H ([Cl/

388

hf. Fukushima

et ul. / Talunla

[H]), and the degree of unsaturation were also calculated. The acidic groups in humic substances are related to the polar element contents (N and 0). The values decreased with a decrease in molecular size, and this seems to be contrary to the order of the CA, values of the molecular weight fractions. Although the oxygen atom relates to the carboxylic and phenolic hydroxyl groups in humic substances, the oxygen atom also relates to ether and alcholic hydroxyl groups which are not concerned with the acidity of HA. The higher molecular weight fraction contains aliphatic moieties rather than the lower molecular weight fraction [7,20]. Therefore, the increase of [O]/[C] and ([N] + [O])/[C] with increasing molecular size suggests a contribution from ether and alcoholic hydroxyl groups in the higher molecular weight fractions. However, it is known that the [CJ/[H] values and the degree of unsaturation increase with proceeding humification [21]. The degree of unsaturation may also be referred to the [C]/[H] values of the HAS. The [C]/[H] values and the degree of unsaturation of the HAS in the present study increased with the decrease in molecular weight: F, > F,, > F,. Since the dehydrogenation was proceeded in the process of the degradation of humic substances (i.e. humification), the amounts of unsaturated carbon contents in the smaller fraction of HA were larger than those of other fractions [21]. Therefore, this suggests that the lower molecular weight fraction contains the larger content of unsaturated carbons such as aromatic moieties. 3.4, LTV- Vis und Juorescence

spectru

The LJV-Vis spectra of the HAS are shown in Fig. 5. The abosrbance parameters can be generally calculated by dividing the absorptivity at 400 nm by that at 600 nm [22]. The parameters are known to be correlated with (i) molecular size, (ii) free radical concentration and (iii) chromophore and auxochrome concentration [23]. The relationships between the humification and absorbance parameters were investigated by Kumada [24], in which the absorbance parameter increased with the humification. Moreover, Chin et al. [25] reported that there were relationships between the

43 (1996)

383-390

absorptivity at 280 nm and the aromaticity of humic substances. The absorbance parameters of the HAS and the absorptivity at 280 nm are summarized in Table 3. The lower molecular weight fractions (Fb and F,) showed larger values of the absorbance parameters and absorptivity at 280 nm. These suggest that the lower molecular weight fractions contain larger amounts of the unsaturated groups such as aromatic moieties in the structure. 3.5. Comparison of jimctionul group contents between the moiecuiur weight fkactionuted and unjiiactionated humic acids The C,, value of the unfractionated HA was not the arithmetic mean value of the fractionated HA but the lowest value of the four. Since the unfractionated HA is regarded as a mixture of molecular weight fractions of HA, it is expected that the acidic sites in the humic molecule are changed by intermolecular interaction between different molecular weight fractions. That is, the conformational change with intermolecular interactions brings about the change of acidity. In order to clarify intermolecular interaction, we investigated UV-Vis and fluorescence spectra of the unfractionated and fractionated HAS. The UV Vis absorbance spectrum of UF (Fig. 5a) was not the arithmetic mean of the spectra of the fractions but the lowest absorptivity. Moreover, the fluorescence spectrum of UF is clearly different from those of the fractionated HAS (Fig. 5b). These data suggest a donor-acceptor interaction including inter-molecular aggregation as described by Wang et al. ]8]. The FTIR spectra and their peak assignments are shown in Fig. 6 and Table 4. In the UF, the peak at about 3200 cm- ’ was remarkably broad and strong, and this showed the O-H stretching of carboxylic acid with the hydrogen bond. Moreover, if the hydrogen bond of carboxylic groups is concerned with the intermolecular aggregation of the HA, it would be expected that the peaks at about 1720 cm- ’ of CO stretching would shift to the lower wavenumber. Although the wavenumbers of the fractionated HAS were at about 1720 cm ‘, the wavenumber of the unfractionated one

UF

4000

3200

2400

2000

1600

1200

uuu

^_^ _^. >uo

4000

3200

4000

3200

2400

2000

1600

1200

800

4000

3200

600

wavenumber Fig. 6. FTIR

spectra

I cm-’

of the HAS with

was at 1705 cm-‘, as shown in Table 4. These results indicate the presence of a hydrogen bond of carboxylic acid in the case of UF. These data suggest that the intermolecular interactions occur in the unfractionated HA. Such interactions may influence the acidic sites of HA.

4. Conclusions The acid-base equilibria of the fractionated and unfractionated HAS could be evaluated by the modified Henderson-Hasselbalch interpretation. The distribution of pK,,, values of the HAS

2400

a KBr

,

2400

2000

2000

1600

1600

1200

000

GO0

1200

1300

600

disk method

were in the range 2-8, and the average values were 4.0-4.4. The largest amounts of acidic groups were found in the lowest molecular weight fraction, F,. Moreover, the lowest molecular weight fraction showed the higher [C]/[H] value, degree of unsaturation and e,,,/e,,, value. This suggests that the structural feature (the degree of unsaturation and the aromaticity) relates to the functional group contents of the HAS. The CA, value of UF was not the arithmetical mean value but the lowest value, and this might be due to the intermolecular interaction accompanying the conformational changes which influenced the acidic site of the HA.

390 Table 4 Comparison

M. Fukushinm

of FTIR

spectra

of the fractionated

et (11. ! Talanta

and unfractionated

43 (1996)

383-390

HAS (cm-‘)

Assignment

F,

Fb

F‘

UF

OH stretching and NH stretching H-bonded OH stretching Aromatic CH stretching Aliphatic CH stretching C=O stretching of COOH COO asymmetric stretching or aromatic C=C stretching COO symmetric stretching, CH deformation and CO stretching of phenolic OH CO stretching or COH deformation of COOH and phenolic groups Aromatic ring stretching, NH bending and CN stretching COH bending or CO stretching of alcohols and ethers CH out-of-plane deformation of aromatic hydrocarbon

3484

3486

3466

3050 region 2923 1719 1618

3050-3150 2930 1718 1618

3050 region 2927 1718 1618

3406 2550 3100 region 2921 1705 1618

1230 region

1210 region

1230 region

1400 - 1480

1400 - 1510

1400 - 1510

1277

1273

1272

830 region

References [II

1380 region

1380 region

M. Schnitzer and S.U. Khan, Humic Substances in the Environment. Dekker, New York, 1972, pp. I-7. Organic Compounds in PI S.J. Faust and J.V. Hunter. Aquatic Environment. Dekker. New York. 1971 pp. 95 160. C.L. Grant and J.H. Weber, Anal. Chem., [31 W.T. Bresnahan, 50 (1978) 1675. [41 J.C. Dobbs, W. Susetyo. L.A. Careira and L.V. Azarrzga, Anal. Chem.. 61 (1989) 1519. Anal. Chim. PI Z.-D. Wang. D.S. Gamble and C.H. Langford, Acta, 232 (1990) 181. Environ. [61 Z.-D. Wang, D.S. Gamble and C.H. Langford, Sci. Technol., 26 (1992) 560. John [71 S.D. Killops and V.J. Killops. Organic Geochemistry, Wiley, New York. 1993, pp. 955106. PI Z.-D. Wang, B.C. Pant and C.H. Langford. Anal. Chim. Acta, 232 (1990) 43. Morel and N.V. Blough, Environ. Sci. [91 S.A. Green. F.M.M. Technol., 26 (1992) 294. Humic Substances Society, Outline of Extrac1101International tion Procedures, 1982.

830 region

[I 11 S.

1230 region

1250 region

830 region

Mori, M. Hiraide and A. Mizuike, Anal. Chim. Acta, 193 (1987) 231. and E.M. Loebel, J. Phys. [I21 H.P. Gregor. L.B. Luttinger Chem., 59 (1955) 34. Y. Kanegae, K. Yoshida, M. Katsuki and Y. [‘31 T. Miyajima. Naitoh. Sci. Total Env*iron.. 117:118 (1992) 129. D.D. Perrin, Stability Constant of Metal-Jon Complexes. [I41 Part B, Pergamon Press, New York, 1979. p. 38. Can. J. Chem.. 48 (1970) 2662. [I51 D.S. Gamble, [I61 B. Leuenberger and P.W. Schindler. Anal. Chem., 60 (1988) 1471. Can. J. Chem.. 50 (1972) 2680. [I71 D.S. Gamble, [I81 L.M. Aleixo, O.E.S. Godinho and W.F. da Costa. Anal. Chim. Acta, 257 (1992) 35. Anal. Chim. Acta, 255 (1991) 23. [I91 S.E. Cabaniss, PO1Ref. [I], pp. 66. 67. K. Tsutsuki and K. Kumada, Soil Sci. Plant PII S. Kuwatsuka, Nutr., 24 (1978) 337. P21J.F. Power and C.H. Langford, Anal. Chem., 60 (1988) 842. [23] P.M. Peid. A.E. Wilkinson. E. Tipping and M.N. Jones, Geochim. Cosmochim. Acta, 54 (1990) 131. Soil Plant Food, I (1955) 29. P41 K. Kumada, Environ. Sci. P51Y.-P. Chin, G. Aiken and E. O’Loughlin. Technol.. 28 (1994) 1853.