Influence of Borate Buffers on the Electrophoretic Behavior of Humic

Depending on the molarity of borate ions in the separation buffer, the humic acids exhibit electropherograms with sharp peaks consistently extending f...
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Anal. Chem. 1998, 70, 3798-3808

Influence of Borate Buffers on the Electrophoretic Behavior of Humic Substances in Capillary Zone Electrophoresis Ph. Schmitt-Kopplin,*,† N. Hertkorn,† A. W. Garrison,‡ D. Freitag,† and A. Kettrup†

GSFsNational Research Center for Environment and Health, Institute for Ecological Chemistry, Ingolstaedter Landstrasse 1, D-85764 Neuherberg, Germany, and National Exposure Research Laboratory, U.S. Environmental Protection Agency, 960 College Station Road, Athens, Georgia 30605-2720

The influence of tetrahydroxyborate ions on the electrophoretic mobility of humic acids was evaluated by capillary electrophoresis (CE). Depending on the molarity of borate ions in the separation buffer, the humic acids exhibit electropherograms with sharp peaks consistently extending from a “humic hump”. The variations in the migration times of these peaks depend on the concentration of borate ions in the separation buffer. The complexation of borate ions and humic acid fractions was also analyzed with 11B and 1H NMR spectroscopy as well as UV spectrophotometry in solutions of the same composition as the CE separation buffers. Supplementary studies with model compounds (flavonoids, phenolic and sugar acids) indicate reaction mechanisms that include the formation of bidentate esters (monocomplexes) as well as spiranes (tetradentate esters or dicomplexes) within the humic substructure. Special attention must be given to the interpretation of CE electropherograms while fingerprinting humic substances with borate buffers since observed peaks do not necessarily indicate distinct humic components but may be artifacts caused by the interaction of the borate ions with the humic substances.

Because the pKa values of the carboxylic acid functional groups of natural humic substances (HS) are in the range of 3-5,5 the HS are polyanionic and migrate in a CZE system with a separation buffer exceeding a pH of about 3 or 3.5. Regardless of the apparent match between HS analytes and CE technology, CE has only scarcely been applied for the characterization of HS. Polydisperse polymeric materials investigated by capillary electrophoresis include HS,6-12 coal extracts,13 and lignin-related compounds.14,15 The obtained electropherograms are mainly interpreted as fingerprints of the analyzed humic substances. CZE has found only a few limited applications in studies of the interactions of humic substances with organic pollutants or metal ions.16-18 In these affinity studies, it is especially important to avoid artifacts due to the presence of species reacting with the HS in the separation buffers. Our previous work9 indicated that borate buffers interact with humic acids relative to other buffer systems at the same pH, drastically changing the electropherograms while only slightly altering the pH. We hypothesized that these changes were caused by complexing two hydroxy groups of boric acid (or borate ions) with cis-diol groups in the humic acids.9

* Corresponding author: (tel) 08161 987720; (fax) 08161 81612; (e-mail) [email protected]. † Institute for Ecological Chemistry. ‡ U.S. Environmental Protection Agency. (1) Guzman, N. A.; Moschera, J.; Iqbal, K.; Malick, A. W. J. Chromatogr., A 1992, 608, 191-204. (2) Brumley, W. C. LC-GC 1995, 13, 556-568. (3) Garrison, A. W.; Schmitt, Ph.; Kettrup, A. J. Chromatogr., A 1994, 688, 317-327.

(4) Schmitt, Ph.; Garrison, A. W.; Freitag, D.; Kettrup, A. J. Chromatogr., A 1996, 723, 169-177. (5) Leenheer, J. A.; Wershaw, R. L.; Reddy, M. M. Environ. Sci. Technol. 1995, 29, 393-405. (6) Kopacek, K.; Kaniansky, D.; Hejzlar, J. J. Chromatogr., A 1991, 545, 461470. (7) Schmitt, Ph.; Kettrup, A. GIT Fachz. Lab. 1994, 12, 1312-1318. (8) Rigol, A.; Lopez-Sanchez, J. F.; Rauret, G. J. Chromatogr. 1994, 664, 301305. (9) Garrison, A. W.; Schmitt, Ph.; Kettrup, A. Water Res. 1995, 29, 2149-2159. (10) Schmitt, Ph.; Freitag, D.; Sanlaville, Y.; Lintelmann, J.; Kettrup, A. J. Chromatogr., A 1995, 709, 215-225. (11) Ciavatta, C.; Govi, M.; Sitti, L.; Gessa, C. Commun. Soil. Sci. Plant Anal. 1995, 26, 3305-3313. (12) Pompe, S.; Heise, K. H.; Nitsche, H. J. Chromatogr., A 1996, 723, 215218. (13) Wright, B. W.; Ross, G. A.; Smith, R. D. Energy Fuel 1989, 3, 428-430. (14) Sjo ¨holm, E.; Nilvebrant, N. O.; Colmsjo¨, A. J. Wood Chem. Technol. 1993, 13 (4), 529-544. (15) Dahlman, O.; Ma˚nsson, K. J. Wood Chem. Technol. 1996, 16 (1), 47-60. (16) Schmitt, Ph.; Garrison, A. W.; Freitag, D.; Kettrup, A. Fresenius J. Anal. Chem. 1996, 354, 915-920. (17) Norden, M.; Dabeck-Zlotorzynska, E. J. Chromatogr., A 1996, 739, 421429. (18) Schmitt, Ph.; Trapp, I.; Garrison, A. W.; Freitag, D.; Kettrup, A. Chemosphere 1997, 35 (1/2), 55-75.

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Various modes of capillary electrophoresis (CE) are now widely applied to the separation of biomolecules such as carbohydrates, amino acids, peptides, proteins, nucleic acids, oligonucleotides, and DNA sequence products. Capillary zone electrophoresis (CZE) has proven very useful for the separation of proteins and peptides; complete resolution can often be obtained for analytes differing by only a single amino acid.1 CE is simple in concept yet provides a higher separation efficiency than either GC or HPLC for a much broader range of organic and inorganic analytes. Organic ions in environmental samples, for example, are particularly amenable to separation by CZE .2-4

© 1998 American Chemical Society Published on Web 08/14/1998

In this work, we intend to describe the interactions of borate ions with humic acids (HA) by utilizing capillary electrophoresis as well as 11B NMR spectroscopy and UV-visible spectrophotometry in identical buffer systems. Reproducible peak sharpening and other changes in the mobility of HA depending on the borate concentration allowed deductions concerning the type of binding as well as the presence and spatial arrangement of specific diol groups within these humic acids. MATERIALS AND METHODS Instrumentation. Capillary electrophoresis instrumentation consisted of a Beckman P/ACE 2100 and P/ACE 5000 series HPCE with Beckman System Gold Chromatography Software and a BioFocus 3000 capillary electrophoresis system from Bio-Rad Laboratories. The uncoated fused-silica CE columns (75-µm i.d., 375-µm o.d., 50-cm length to the detector, total length 57 cm) were obtained from Beckman Instruments, Inc. CZE conditions for the separation of the HA fractions were as follows: temperature, 30 °C; voltage, 20 kV; detector wavelength, 254 nm; hydrodynamic injection, 5 or 10 s. The two buffer stock solutions were a 100 mM borate buffer (pH 9.1) and a 10 mM carbonate buffer (pH 9.1); the concentration in borate ions was adjusted by mixing adequate amounts of stock solutions while keeping the pH constant at 9.1; the total volume was 2 times 500 µL required for the separation buffer vials. The CZE gave good reproducibility of migration times (a standard deviation of 0.06 mins0.89% relative standard deviation; n ) 5) with Scheyern humic acids. The concentration in HS of the sample had no significant influence on the average electrophoretic mobility (AEM). Day-to-day changes in migration times occurring because of relative changes in the electroosmotic flow (different capillary surface conditions) could be controlled by washing the capillary with 0.1 M NaOH for 2 min between each run. For CE analysis, the humic fractions were dissolved in 0.1 M sodium hydroxide to a concentration of ∼1 mg/mL-1. The lowest measurable concentration under these conditions was 50 µg/mL-1. UV-visible spectra were recorded on a Cary 1G UV-visible spectrophotometer from 190 to 600 nm; 50 µL of the waterdissolved model compounds was diluted from the stock solution (1 mg/mL) in 3 mL of buffer and measured against the corresponding buffer-only reference. The UV-visible spectra of the model compounds were compared in the two buffer systems (10 mM carbonate buffer and the 40 mM borate), identical to the CE separation buffers. 11B NMR spectra were acquired (aq ) 340 ms) with 90-deg pulses (19 µs) and a relaxation delay of 1 s at 160.46 MHz with a 10-mm broad-band probe at 303 K on a Bruker DMX 500 spectrometer. Suprasil NMR tubes (Wilmad; 733-5PQ) were not sufficient to reduce boron background resonances still originating from the probehead and the HS-derived spectra had to be baseline corrected with Bruker standard software (X-Win-NMR) procedures. Neat BF3‚OEt2 was used as an external reference (δ ) 0.0 ppm). Samples (15 mg) of HS were dissolved in 3 mL of buffer solutions prepared as for CE separation with different borate molarities (0.1, 1, 10, 20, 40, 80, and 150 mM) with D2O instead of H2O. Catechin and rutin were dissolved in the same buffer solutions at concentrations of 4.59 and 2.05 mM, respectively. Humic Substances. The Scheyern soil (Ap Horizon, 0-20 cm) was sampled from a cultivated loamy brown soil from the

Forschungsverbund Agraro¨kosystem Mu¨nchen (FAM) in Scheyern, Germany (sampling points 290/190 and 270/190). The Bouzule soil (wet prairie pseudogley) was obtained from Prof. M. Schiavon (EÄ cole Nationale Supe´rieure d′Agronomie et des Industries Alimentaires, Nancy, France). These soils and the humic substances extracted from them were described elsewere.19,20 The humic acids were extracted and isolated from the Scheyern and Bouzule soils according to the procedures of the International Humic Substances Society (IHSS) as already described in Schmitt et al.16 Standard water HA (Suwannee River HA) (U.S. Geological Survey Open File Report 87-557) was obtained from the IHSS. Chemicals. The phenolic acids were obtained from Sigma Chemicals (St. Louis, MO) (all p.a. grade), the flavonoids from Fluka Chemie Biochemika (Buchs, Switzerland), and the chemicals from Merck (Darmstadt, Germany). All aliphatic acids and sugar acids were a gift from Dr. K. Fischer, from the GSF-IO ¨ C, Munich, Germany (oxalic, pyruvic, malonic, citric, fumaric, succinic, tartronic, formic, adipic, tartaric, acetic, propionic, glucaric, glycolic, butyric, lactic, glyoxylic, valerianic, glyceric, levulinic, threonic, gluconic, 2-keto-gluconic, galactaric, and 5-keto-gluconic acids). There are no particular safety precautions necessary for research with the used chemicals; e.g., the neat standards should only be handled in a hood. Dilute solutions such as used in this research should be handled like any other standard solution. BACKGROUND 1. Borate Binding Sites. The equilibrium between boric acid (B(OH)3, 1) and tetrahedral borate (B(OH)4-, 2) is very sensitive to pH as indicated by large variations in the chemical shift of a single averaged 11B NMR resonance representing the two rapidly interconverting species.21 Equilibrium 1 corresponds

(1)

to the ionization of boric acid to borate (tetrahydroxyborate) ions at a pH around the pKa. Both 1 and 2 are known to complex diol groups of the proper geometry in cyclic and noncyclic polyhydroxy compounds.22 Fivemembered-ring complexes with 1,2-diols (n ) 0; 3) or sixmembered rings with 1,3-diols (n ) 1; 4) as shown in eq 2 give bidentate esters (5, 6), also called the monocomplex (BL-), as well as the spiranes (7-9) or tetradentate esters (dicomplex BL2-). Depending on the pH of the reaction solution, hydroxycarboxylic acids and dicarboxylic acids can be involved in the formation of the complexes as well as diols. The “charge rule” predicts that the highest stability of the complex is reached at the pH where the sum of the charges of the esterifying species is (19) Schmitt, Ph.; Garrison, A. W.; Freitag, D.; Kettrup, A. Water Res. 1997, 31 (8), 2037-2049. (20) Akim, L. G.; Schmitt, Ph.; Bailey, G. W. Org. Geochem. 1998, 28 (5), 325336. (21) How, M. J.; Kennedy, G. R.; Mooney, E. F. Chem. Commun. 1969, 50, 267268. (22) Bo ¨eseken, J. Adv. Carbohydr. Chem. 1949, 4, 189-210. (23) van Duin, M.; Peters, J. A.; Kieboom, A. P. G.; van Bekkum, H. Tetrahedron 1984, 40 (15), 2901-2911.

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(2)

higher polymeric polyols such as agar or poly(vinyl alcohol),42,43 considering the dependence of the borate/ligand ratio on the pH of the solution. Cis 1,2-diols are preferred in complexation to trans 1,2-diols and a higher stability of the complex 5 was found when the hydroxyls of the 1,2-diol are in the threo position.44 1,2-Diol complexes are more stable than 1,3-diol complexes, and stability constants K are higher in the order tridentate > bidentate > monodentate45 as well as with increasing numbers of hydroxyl groups in the polyol.46 With an excess of borate, the (BL2-) ester is preferred.47

(4) equal to the charge of the ester.23 According to this charge rule, complexes formed with boric acid according to eq 2 are less stable at higher pH than the complexes formed with tetraborate ions.24 Tridentate complexes can be formed from polyols of proper geometry such as scyllo-quercitol ( eq 3).25

(3)

At higher borate concentration in the aqueous phase, tri- to pentameric polyanionic species such as B3O3(OH)52-, B3O3(OH)4-, B4O5(OH)42-, and B5O6(OH)4- are present. The distribution of these polyborates as a function of concentration and pH has been studied in detail by 11B NMR.26-28 These species can react with single hydroxyl groups of molecules to form charged complexes, and this property was used by Revilla et al.29 to separate R- from β-naphthol by CZE at high concentrations of borate buffers (0.4 M). 11B NMR spectroscopy allows distinction between all of the above-mentioned complexes occurring either in natural isolates30,31 or in aqueous solutions containing low-molecular-weight aliphatic acids,32-34 polyhydroximes,35 phenols,36 carbohydrates,37-41 or (24) Smith, J. T.; El Rassi, Z. J. Chromatogr., A 1994, 685, 131-143. (25) Bell, C.; Beauchamp, R. D.; Short, E. L. Carbohydr. Res. 1986, 147, 191203. (26) Salentine, C. G. Inorg. Chem. 1983, 22, 3920-3924. (27) Momii, R. K.; Nachtrieb, N. H. Inorg. Chem. 1967, 6, 1189-1192. (28) Smith, H. D.; Wiersema, R. J. Inorg. Chem. 1972, 11, 1152-1154. (29) Revilla, A. L.; Havel, J.; Jandik, P. J. Chromatogr., A 1996, 745, 225-232. (30) Kaneko, S.; Ishii, T.; Matsunaga, T. Phytochemistry 1997, 44 (2), 243-248. (31) Ishii, T.; Matsunaga, T. Carbohydr. Res. 1996, 284, 1-9. (32) Pizer, R.; Ricatto, P. J. Inorg. Chem. 1994, 33, 2402-2406. (33) Pizer, R.; Ricatto, P. J. Inorg. Chem. 1994, 33, 4985-4990. (34) Bessler, E.; Weidlein, J. Z. Naturforsch. 1982, 37B, 1020-1025. (35) van Heveren, J.; Peters, J. A.; Batelaan, J. G.; van Bekkum, H. Inorg. Chim. Acta 1992, 192, 261-270.

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Borate complexation induces changes in the charge-to-mass ratios of the ligands; this property was already used in the early 1950s to determine the configuration of carbohydrates22,48,49 or to selectively separate proteins50 by means of electrophoretic techniques (zone and gel electrophoresis). The routine analysis of carbohydrates and oligosaccharides with CZE is also based on these properties; borate not only induces the formation of charged and mobile complexes from uncharged carbohydrates, thus increasing the selectivity of their separation,51 but also significantly enhances the UV absorbance, facilitating on-line UV detection52 and the spectrophotometric quantification of sugars,53 catechols, and catecholic proteins.54 Six-membered hexoses also can form 1,3-diols of the type 6a as confirmed with 11B NMR55 and illustrated in eq 4. The chelation (36) Pasdeloup, M.; Brisson, C. Org. Magn. Reson. 1981, 16 (2), 164-167. (37) Chapelle, S.; Verchere, J. F. Tetrahedron 1988, 44 (14), 4469-4482. (38) Chapelle, S.; Verchere, J. F. Carbohydr. Res. 1989, 191, 63-70. (39) Yoshimura, K.; Miyazaki, Y.; Sawada, S.; Waki, H. J. Chem. Soc., Faraday Trans. 1996, 92 (4), 651-656. (40) Gey, C.; Noble, O.; Perez, S.; Tavarel, F. R. Carbohydr. Res. 1988, 173, 175-184. (41) Noble, O.; Tavarel, F. R. Carbohydr. Res. 1988, 184, 236-243. (42) Sinton, S. W. Macromolecules 1987, 20, 2430-2441. (43) Jasinski, R.; Redwine, D.; Rose, G. J. Polym. Sci.: Part B: Polym. Phys. 1996, 34, 1477-1488. (44) Munoz, A.; Lamande, L. Carbohydr. Res. 1991, 225, 113-121. (45) Van Duin, M.; Peters, J. A.; Kieboom, A. P. G.; Van Bekkum, H. Tetrahedron 1985, 41 (16), 3411-3421. (46) Dawber, J. G.; Green, S. I. E. J. Chem. Soc., Faraday Trans. 1986, 82, 34073413. (47) van den Berg, R.; Peters, J. A.; van Bekkum, H. Carbohadr. Res. 1994, 253, 1-12. (48) Foster, A. B. J. Adv. Carbohydr. Chem. 1957, 12, 81-115. (49) Foster, A. B.; Stacey, M. J. Chem. Soc. 1955, 1778-1781. (50) Lerch, B.; Stegemann, H. Anal. Biochem. 1969, 29, 76-83. (51) Honda, S.; Iwase, S.; Makino, A.; Fujiwara, S. Anal. Biochem. 1989, 176, 71-77. (52) Hoffstetter-Kuhn, S.; Paulus, A.; Gassmann, E.; Widmer, H. M. Anal. Chem. 1991, 63, 1541-1547. (53) Honda, S.; Takahashi, M.; Nishimura, Y.; Kakehi, K.; Ganno, S. Anal. Biochem. 1981, 118, 162-167. (54) Waite, J. H. Anal. Chem. 1984, 56, 1935-1939. (55) Kennedy, G. R.; How, M. J. Carbohydr. Res. 1973, 28, 13-19.

Chart 1

of boronic acids with catechols has been known for over 100 years56 and Bo¨eseken (1949)22 investigated the reactions of borate with hydroxy groups of catechol- or salycylic acid-like molecules (eqs 5 and 6):

(5)

(6)

CZE and micellar electrokinetic capillary chromatography (MECC) using these complex formation properties were employed for the characterization of catechols,57,58 catecholamines,59 and other polyols such as triiodinated X-ray contrast media.60 Azomethine H has been shown to be a good chelating agent for the analysis of boron from an artificial river water matrix with CZE;61 Azomethine H, as well as chromotropic acid, reacts with borate, forming the stable tetradentate ester (dicomplex). Azomethine H contains spatially adjacent hydroxyl groups separated by several covalent bonds (eq 7).

(7)

The controlled borate complexation of alkylglucosides for use as charged micelles is an interesting CE application in MECC for the analysis of pesticides.23 The surface charge of the alkylglucosides-borate micelles can be controlled by changing the operational separation parameters such as the borate concentra-

tion or the pH.62-64 The combined use of β-cyclodextrin (CD) and borate was shown to efficiently separate 1,2-diols by chiral capillary electrophoresis, based on both inclusion into the chiral cavity of the CD and borate complexation with the diol group.65 As described above, borates are very reactive species that can be used in separation buffers for the CZE/MECC analysis of various polyols. Variations in pH of the medium and in the concentration of borate control the complexation and induce specific changes in the charge-to-mass ratios of the analytes, enabling a broad range of selective separation conditions. 2. Analysis of Borate Binding to Humic Substances. Humic substances are generally known to include a broad range/ variety of alcoholic, phenolic, and carboxylic hydroxy groups, respectively.5 The formation of borate-HS complexes (noted HS-Bn) can be described by considering a series of reactions HS undergoes with n borate ions, as shown in Chart 1. The association constant K′i for the ith binding site can thus be defined as

K′i ) [HS-Bi]/[HS-B(i-1)][B]

(a)

The macroconstant Kn is a combined value of different association constants of borate with individual HS functional groups, each of which displays a different microconstant. For n binding sites, the resulting binding macroconstant is defined as

Kn ) K′1K′2, ..., K′i, ..., K′n or Kn )

[HS-Bn] [HS]tot[B]n

(b)

where [HS]tot is the total concentration (g/L-1) of HS; [B] is the concentration (mol/L-1) of borate; [HS-Bn] is the concentration of the HS-borate complex, with [HS]tot ) [HS] + [HS-Bn]; n is (56) Jahn, F. Arch. Pharm. 1878, 12, 212-221. (57) Wallingford, R. A.; Ewing, A. G. J. Chromatogr. 1988, 441, 299-309. (58) Wallingford, R. A.; Curry, P. D.; Ewing, A. G. J. Microcolumn Sep. 1989, 1 (1), 23-27. (59) Kaneta, T.; Tanaka, S.; Yoshida, H. J. Chromatogr. 1991, 538, 385-391. (60) Thanh, H. H. J. Chromatogr. A 1994, 678, 343-350. (61) Oxspring, D. A.; McClean, S.; O’Kane, E. O.; Smyth, W. F. Anal. Chim. Acta 1995, 317, 295-301. (62) Smith, J. T.; El Rassi, Z. J. Microcolumn Sep. 1994, 6, 127-138. (63) Cai, J.; El Rassi, Z. J. Chromatogr., A 1992, 608, 31-45. (64) Smith, J. T.; Nasabeh, W.; El Rassi, Z. Anal. Chem. 1994, 66, 1119-1133. (65) Jira, T.; Bunke, A.; Schmid, M. G.; Gu ¨ bitz, G. J. Chromatogr., A 1997, 761, 269-275.

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the number of borate ions involved in the complex; and the dissociation constant Kdn can be defined as Kdn ) 1/Kn. In CZE, the wall of the capillary will be negatively charged from the ionization of the silanol groups (the pI of fused silica is ∼1.5) and this will attract positively charged ions from the buffer, creating an electrical double layer. With a voltage applied across the capillary, the cations of the double layer will migrate to the cathode, creating a net flow of buffer solution to the negative electrode (electroosmotic flow, EOF). The analyte ion migration times do not reflect the actual electrophoretic mobilities µ of the analytes in the separation system since ion mobilities are independent of the EOF. The effective electrophoretic mobilities µ of the anionic analytes are negatively signed and are obtained by subtracting the mobility of the electroosmotic flow (µeof) from the measured electrophoretic mobilities (µmes) of the analytes. The effective electrophoretic mobility µ is directly proportional to the velocity and is used throughout this paper as absolute electrophoretic values for the evaluation of the binding constants; a decrease of µ corresponds to an increase of the ion velocity (increase of the magnitude of µ). The mobility of the complex is a weighted average of the effective electrophoretic mobilities of all the free and bound forms of the analyte in the studied system.

µ)

[HS]

µ0 + [HS]tot

∑[HS - B ]µ i

ci

i

[HS]tot

(c)

where µo is the electrophoretic mobility of the free analyte for [B] ) 0 and µci is the electrophoretic mobility of the formed complex with i borate molecules. By increasing the concentration of borate, different binding sites of the high-molecular-weight humic molecules are successively bound but the fitting of the experimental data in terms of multiple site binding is a difficult task. The appearance/disappearance and changes in mobility of the sharp peaks in the electropherograms by increasing borate concentration is thus described as 1:1 complexes between borate and low-molecularweight HS fractions; in agreement with most of the metalcomplexing studies involving humic substances, the fitting of the “humic hump” data to the model was also done by assuming the approximation of 1:1 complexes. Under these approximations (n ) 1), the combination of eqs c with b gives eq d and the

µ ) (µ0 + K[B]µc)/(1 + K[B])

(d)

experimental data can be fitted to eq d for the description of the reaction mechanisms assuming the 1:1 complexes. RESULTS AND DISCUSSION Fingerprinting of Humic Substances by CZE and Ionic Strength Effects. Acetate, phosphate, borate, and carbonate have been proposed by the authors to be good CZE buffers for fingerprinting HS.9 HS are high-molecular-weight polyelectrolytes composed of polyaromatic, heteroatom-substituted, and aliphatic subunits. Compared to the FA, the HA are of higher molecular weight and lower total acidity. The degree of ionization of these macromolecules is governed by the pH-dependent ionization of 3802 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998

Figure 1. Corrected electropherograms (-µeff versus UV absorption at 254 nm) of the IHSS soil standard humic acid at different NaCl concentrations in the separation buffer (carbonate buffer (pH 9.1), 30 °C, 20 kV, 254 nm). Inset: changes in AEM as a function of NaCl concentration.

the phenolic and carboxylic groups of the humic core (which is a function of buffer pH). The measured electropherograms (UV adsorption versus migration time scale) can be converted to electropherograms showing the real distribution of the effective mobilities of the polyanions by plotting the UV signals versus the negative effective mobility -µeff (Figure 1). This representation of the electropherograms of HS shows clearly a homogeneous “humic hump” in the anionic region with a “Gaussian” distribution around an average electrophoretic mobility. The mobilities of the humic acids are lower in magnitude than those of the corresponding fulvic acids, indicating a lower charge-to-mass ratio of the former, which is governed by total proton-exchange capacity and molecular size. Cations have never been detected in our CZE studies of humic substances. Figure 1 shows the variation in AEM of the IHSS soil humic acid standard as a function of the ionic strength of the separation buffer (ionic strength of the 10 mM carbonate buffer, pH 9.1, as adjusted by NaCl); AEM is decreased slightly in magnitude with increase in ionic strength. Changes in the Electrophoretic Behavior of HS in the Presence of Borate Ions. The typical changes in peak shapes and mobilities are illustrated in Figure 2 with the Scheyern soil humic acid measured with increasing borate ion concentration in a carbonate buffer system. A homogeneous humic hump is obtained with the 100% carbonate buffer (mobility distribution as in Figure 1). With a borate concentration of only 0.5 mM a new peak (*) appears in the high charge-to-mass region and moves to lower mobilities on increasing borate concentration. This can be interpreted as a lowering of the charge-to-mass ratio of peak * as the borate concentration increases. The mobility of * is diminished until it reaches a stable value at a concentration of 15 mM borate; excess borate causes its intensity to decrease. Peak O appears simultaneously at a borate concentration of 15 mM and moves to higher mobilities. A nearly identical evolution of peaks could be observed when the IHSS Suwannee river humic acid and the Bouzule soil humic acid are employed. Borate ions strongly influence the electropherograms of the soil humic acids with the intensity and the mobility of the

Figure 2. Changes in the electropherograms of the Scheyern humic acid as a function of the borate concentration in the separation buffer (pH 9.1, 30 °C, 20 kV, 254 nm). Peaks O and * correspond to BL2- and BL-, respectively; x-axis is time scale.

produced peaks governed by the concentration in borate ions rather than the composition of the HS. A pH dependence was already shown in previous studies,9 and the complex formation was minimized at pH below 8.3. The peaks * and O are therefore best explained to be artifacts produced by borate complexation with fractions of the HS mixture that possess suitable functional groups in proper geometry to react with borate.

Changes in the Electrophoretic Behavior of Phenolic Acids and Flavonoids in the Presence of Borate Ions. To elucidate the CZE migration of the humic polyanionic mixtures in borate buffer systems in terms of formation of borate complexes, we studied model compounds of known borate affinities under the same separation conditions. Vanillic acid, syringic acid, o-, m-, and p-coumaric acid, and m- and p-hydroxybenzoic acid Analytical Chemistry, Vol. 70, No. 18, September 15, 1998

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Figure 3. (left) Changes in µeff of syringic acid (noninteracting and therefore not corrected), salicylic acid, methylcatechol, caffeic acid, and rutin depending on the borate concentration in the separation buffer. (middle) Corrected µeff of the same data set. (right) UV-visible spectra in 10 mM carbonate buffer (dotted line) and 40 mM borate buffer (bold line) at pH 9.1 where shifts of maximum and changes in absorbances indicate borate complexations.

did not present any changes in their UV-visible spectra when measured in 40 mM borate buffer as compared to 10 mM carbonate buffer (Figure 3). Borate complexation of these 3804 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998

phenolic acids is unlikely under these conditions (pH 9.1) because they do not possess diols of adequate geometry and at this pH the negatively charged carboxylate groups decrease the formation

Figure 4. 11B NMR spectra of rutin in the shift range 1.2-9.4 ppm at increasing concentrations of borate (mM) showing the existence of the borate complex with the aglycon (BL(1,2)-) at low borate concentrations and the appearance of the mannose-borate complex (BL(1,2)-) and the 1,3-diol borate complex (BL(1,3)-) at higher borate concentrations (20 and 150 mM, respectively). The spectra were baseline corrected; the spectrum at 0.1 mM borate was identical with that at 10 mM borate.

of borate esters as a result of electric repulsion between the negatively charged COO- and BO4- moieties.45,66 However, their effective electrophoretic mobility increased in magnitude when the borate concentration in the separation buffer was increased from 0 to 160 mM (Figure 3). This can be interpreted in terms

of an ionic strength effect (ionic shielding) which has already been demonstrated in Figure 1 for the IHSS soil humic acids. Curve fitting for all these supposedly unreacting species using eq d gave nearly perfect matches (with values of K ) 350 mol-1 and n ) 1); these parameters were used for the ionic strength corrections of the effective electrophoretic mobility. Figure 3 gives the evolution of µeff and the corresponding corrected µeff with increasing borate concentration in the separation buffer of syringic acid, salicylic acid, methylcatechol, caffeic acid, and rutin. The formation of borate complexes induced shiftings of the absorption maximums and increased UV-visible absorbances of the four compounds containing diols in 1,2- and 1,3-positions. Rutin is a flavonoid presenting diols both in 1,2- and 1,3-positions on the aglycon (catechin) as well as in the mannose part of the glucoside. The evolution of the corrected µeff with increasing borate concentrations is best interpreted in terms of borate complexation. The corrected µeff of salicylic acid continuously increases in magnitude on borate addition to the separation buffer indicative of forming a BL- complex (6b), K ) 700 mol-1 with eq d. Methylcatechol showed an initial rapid increase of the velocities (BL- monocomplex of type 5) followed by an decrease in magnitude of µeff, which is best explained by the formation of the tetradendate ester (BL2dicomplex of type 7) of lower charge-to-mass ratio. The BLcomplex also has a lower charge-to-mass ratio than the uncomplexed salicylic acid. Caffeic acid and rutin undergo the same reactions as methylcatechol but with a lower affinity to borate; the decrease in magnitude of µeff is thus not so pronounced. Catechin and rutin were analyzed with 11B NMR spectroscopy in buffers identical to the CZE separation buffers at various borate concentrations. The chemical shift of 8.48 ppm of “catechinborate” observed across the entire range of borate concentration could be attributed to the BL- complex of the diols when compared to the shift δ(11B) ) 8.4 ppm of “1,2-dihydroxybenzeneborate” (5a).36 At higher concentration (>80 mM borate), a

Figure 5. Proposed three-step reaction between the tetrahydroxyborate ions and humic fractions. Curves represent the experimental data of Scheyern humic acids (cf. Figure 2); the binding constants calculated from eq d are given in Table 3.

Analytical Chemistry, Vol. 70, No. 18, September 15, 1998

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Figure 6. Changes in the average electrophoretic mobility (“humic hump”) of the aquatic (Suwannee river) and soil (Scheyern, Bouzule) humic acids with increasing borate concentrations.

Figure 8. 11B NMR spectra of Scheyern, Bouzule, and Suwannee river humic acids (spectra baseline corrected, only high-field section >10 ppm shown). Figure 7. UV difference spectra of the Scheyern and Suwannee river humic acids from measurements in 40 mM borate and 10 mM carbonate buffers. Table 1. Experimental 11B NMR Chemical Shifts (ppm) of Model Compounds and Humic Acids BL(1,2)aglycon

BL(1,3)aglycon

BL(1,2)mannose

8.4 8.4

1.4 1.4

5.7

rutin catechin HA

BL2-

Scheyern Bouzule Suwannee river

13.2 13.5 13.6

B2L- or BLof 1,2-diol complexes

8.6

6.9 6.9

6.2 6.2 6.5

BL- of 1,3-diol complexes 1.4

3.2

1.9

1.6 1.6

resonance at δ(11B) ) 1.4 ppm also appeared, probably due to boron in a six-membered ring (BL- complex of the 1,3-diol).67 Rutin showed corresponding signals (Figure 4) at δ(11B) ) 8.4 and 1.4 ppm, with an additional resonance at 5.8 ppm appearing at borate concentrations exceeding 20 mM. The later was attributed to the BL- complexation of the 1,2-diol in the mannose moiety; borate complexes of mannose units gave shifts from 5.5 to 7.5 ppm in polysaccharides of higher molecular weight.43 Due to the lower affinity of the 1,2-hydroxyl groups in the glucoside fraction, the 1,2-hydroxyl groups of the aglycon will complex preferentially at low borate concentration; this is in agreement with previous CE studies of borate complexation with flavonoids (66) Frahn, J. L. J. Chromatogr. 1984, 314, 167-181. (67) Henderson, W. G.; How, M. J.; Kennedy, G. R.; Mooney, E. F. Carbohydr. Res. 1973, 28, 1-12.

3806 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998

of different structures.68,69 CE analysis at higher borate concentrations (0.2 M) allows selective separation of flavonoids differing only in their sugar moiety.70,71 Following the Borate Binding to HS. The binding of borate ions to Scheyern humic acids can be deduced from CE measurements with eq d taking into account three steps of the interaction (Figure 5). The migration of peak * can first be interpreted as a charge decrease of a reactive highly charged humic fraction due to the complexation with one tetrahydroxyborate ion (step 1, n ) 1, bidentate monoesters BL-), followed by the formation of a tetradentate diester (step 2, BL2-; peak O) through intermolecular complexation leading to decrease of the charge-to-mass ratio as illustrated in Figure 5. The 11B NMR spectra of the same humic acids suggests the existence of a BL2- complex even at low borate concentrations (signal at 13.2 ppm in 20 and 40 mM borate buffers). The third step of the reaction is a further complexation of the spirane with borate ions, forming bidentate monoesters (confirmed by 11B NMR resonances at 6.2 ppm with 40, 80, and 160 mM borate buffers). Referring to the insignificant changes of the area of the humic hump in the electropherogram in carbonate buffer without and with high borate concentration (Figure 2), the fraction of humic substances involved in these three steps of the complexation reactions may account for only a small percentage of the total dissolved humic material. Because of the long migration times of the HS fractions involved in the borate complexes relative to the humic hump, we consider these to be of high charge-to-mass ratio, i.e., of low molecular weight (68) Pietta, P.; Mauri, P.; Bruno, A.; Gardana, C. Electrophoresis 1994, 15, 13261331. (69) Morin, Ph.; Villard, F.; Dreux, M. J. Chromatogr. 1993, 628, 161-169. (70) Morin, Ph.; Villard, F.; Dreux, M. J. Chromatogr. 1993, 628, 153-160. (71) Morin, Ph.; Dreux, M. J. Liq. Chromatogr. 1993, 16 (17), 3735-3755.

Table 2. 11B NMR Chemical Shifts (ppm) of Model Compounds and Related Association Constants from the Literature potential borate complexing in humic substances

δ(11B) shift BLcomplex (ppm)

assoc const K (mol-1)

δ(11B) shift BL2complex (ppm)

Glycol 1

6.0

9.8

C3-C6 Polyols, Polyhydroxycarboxylates threo: 6.0 to 7.25 ppm 15-1200 9.4 to 12.1 erythro: 4.9 to 5.6 ppm 2.2-140 9.4 to 12.1

assoc const K (mol-1)

ref

0.16

23

3.2-79 0.36-4.3

45 45

Pentose and Related Sugars 6.7-7.5 6.2-9 6.7

1.9-1.3

1,2-bidentate 1,3-bidentate 1,3,5-tridentate a

10.7 to 11.9 Different Mono- and Disaccharides 3-45000 Glyceric Acid 6.2

37

9.5 to 11.4

nda

47

11.1

0.62

23

0.05

45

C3-C6 Polyols, Polyhydroxycarboxylates 0.31-54

7.9

Glycolic Acid 140000

11.42

20

23

7.0

Glyceric Acid 1000000

10.4

25

23

5.4

Oxalic Acid 1.7 × 108