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Sep 13, 2003 - Structural information on humic acids is difficult to obtain because of the heterogeneity of the acids. Herein liquid chromatography at...
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Anal. Chem. 2003, 75, 5544-5553

Initial Characterization of Humic Acids Using Liquid Chromatography at the Critical Condition Followed by Size-Exclusion Chromatography and Electrospray Ionization Mass Spectrometry Shannon L. Phillips and Susan V. Olesik*

Department of Chemistry, 100 West 18th Avenue, Columbus, Ohio 43210

Structural information on humic acids is difficult to obtain because of the heterogeneity of the acids. Herein liquid chromatography at the critical condition, LCCC, is used to provide a sorting mechanism for the diverse types of molecules contained in humic acids. The critical condition of polymers that are believed to model some subunit of the humic acid is determined. Humic acids from three different terrestrial sources (soil, compost, and peat) are then separated under these chromatographic conditions. The portion of the humic acid that has structure similar to that of the model polymer elutes at the retention volume of the critical condition of the model. Next, fractions are collected and further characterized. This detailed characterization includes high-efficiency size-exclusion chromatography and electrospray mass spectrometry. The size-exclusion chromatograms of the fractions were found to be markedly different from that of the original humic acid sample. This is strong evidence that the LCCC separation mechanism is different from size fractionation. The mass spectra of the humic acid fractions were also markedly different from those of the bulk humic acids previously reported. The mass spectra of specific fractions collected had repeating clusters of m/z values, which is more evidence that the critical condition separation is a powerful sort function. Humic substances (HS) are described as environmental degradation products of plant, fungal, and bacterial biopolymers.1 Three fractions of HS are operationally defined in terms of differences in solubility. Humins are insoluble in aqueous solutions at any pH. Humic acids are the base-soluble and acid-insoluble fraction of HS, and fulvic acids are soluble in aqueous solutions at all pH values. Historically, soluble HS (fulvic and humic acids) were believed to consist of macromolecular polyelectrolytes with heterogeneity that varies depending on their environment. Parameters such as pH, ionic strength, proximity, and type of metal species are also believed to affect the molecular weight and threedimensional structure of the humic materials.2,3 Structure elucida* To whom correspondence should be addressed. E-mail: [email protected]. (1) Leenheer, J. A.; Rostad, C. E.; Gates, P. M.; Furlong, E. T.; Ferrer, I. Anal. Chem. 2001, 73, 1461-1471.

5544 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

tion has been attempted utilizing a variety of techniques, including nuclear magnetic resonance (NMR),4 solid-state NMR,5 pyrolysis gas chromatography (GC)/mass spectrometry (MS),5 electrospray (ES), high-resolution Fourier transform ion cyclotron resonanceMS,6,7 quadrupole time-of-flight (Q-TOF)-MS,7 inductively coupled plasma (ICP)-MS,8 and size exclusion chromatography (SEC).9-12 Despite the application of these techniques for the analysis of humic substances, minimal definitive structural information has been obtained to date because of the heterogeneity of these natural oligomers or polymers. Molecular size determination of these samples is often attempted using size exclusion chromatographic (SEC) techniques. Although SEC has provided information regarding the size distribution of humic samples, the results may be suspect as a result of the lack of suitable chromatographic systems and the potential for clustering or association of the humic materials.10,13 Polymer standards that are commonly used for the SEC of humic acids are illustrated in Table 1. These model polymers were chosen as standards for humic materials because of the perceived similarity of structure between the model and some portion of the humic material or because the model polymer is believed to be a component of humic matter. Table 1 also describes expected similarities and differences between the polymer standards and (2) Hayes, M. H. B.; Graham, C. L. Humic Substances Versatile Components of Plants, Soils, and Water; Ghaabbour, E. H., Davies, G., Eds.; The Royal Society of Chemistry: Cambridge, U.K., 2000. (3) Wrobel, K.; Sadi, B. B. M.; Wrobel, K.; Castillo, J. R.; Caruso, J. A. Anal. Chem. 2003, 75, 761-767. (4) Simpson, A. J.; Kingery, W.; Hatcher, P. G. Environ. Sci. Technol. 2003, 37, 337-342. (5) Chen, Y.; Chefetz, B.; Rosario, R.; van Heemst, J. D.; Romaine, C. P.; Hatcher, P. G. Compost Sci. Util. 2000, 8, 347-359. (6) Fievre, A.; Solouki, T.; Marshall, A. G.; Cooper, W. T. Energy Fuels 1997, 11, 554-560. (7) Kujawinski, E. B.; Hatcher, P. G.; Freitas, M. A. Anal. Chem. 2002, 74, 413-419. (8) Amarasiriwardena, D.; Siripinyanond, A.; Barnes, R. M. In Humic Substances Versatile, Components of Plants, Soil and Water; Ghabbour, E. H., Davises, G., Eds.; The Royal Society of Chemistry: Cambridge, U.K., 2000. (9) Perminova, I. V.; Frimmel, F. J.; Kovalevskii, D. V.; Abbt-Braun, G.; Kudryavtsev, A. V.; Hesse, S. Wat. Res. 1998, 32, 872-881. (10) Perminova, I. V. Soil Sci. 1999, 164, 834-840. (11) Conte, P.; Piccolo, A. Environ. Sci. Technol. 1999, 33, 1682-1690. (12) Chin, Y.-P.; Aiken, G.; O’Loughlin, E. Environ. Sci. Technol. 1994, 28, 18531858. (13) Piccolo, A.; Nardi, S.; Concheri, G. Chemosphere 1996, 33, 595-602. 10.1021/ac0344891 CCC: $25.00

© 2003 American Chemical Society Published on Web 09/13/2003

Table 1. Polymer Standards Utilized for SEC Analysis of Humic Acid polymer standard globular proteins polysaccharides sodium polystyrene sulfonates poly(acrylic acid)s

similarity to humic acid

difference from humic acid

water-soluble water-soluble component of HA coil configuration acidity

humic acids.9,11,12 Obviously, there is not one polymer standard that comprehensively models the characteristics of humic acids. Furthermore, the most appropriate polymer standard for the SEC analysis of humic species may differ as a result of structural differences found in humic material from different terrestrial or aquatic environments. Liquid chromatography at the critical condition (LCCC) is a chromatographic technique that allows for the isolation of one aspect of a polymer system so that heterogeneities within the polymer system can be probed by SEC or adsorption chromatography. For example, if the desired result is to obtain functionality distribution information on a hydroxylated polystyrene polymer, then the critical condition (CC) of the backbone polystyrene is first determined. At the critical condition, the retention volume of the polystyrene of differing molecular weights is the same (i.e., ∆G of transfer is zero for polystyrene). Next, when functionalized polystyrene is separated at the CC of polystyrene, a separation based entirely on the functionality distribution will result. Critical chromatography, CC, is typically used for the characterization of synthetic polymers and copolymers that are primarily soluble in organic solvents. Examples of useful applications of CC include determination of functional group distribution, determining block size for block copolymers,14 differentiating blocks of star and comb copolymers,15 and separating polymers with the same molecular weight by differing in that one is linear and the other is branched.16 We have previously illustrated the use of enhancedfluidity liquids to improve the performance and the ease of finding the critical condition when enhanced-fluidity liquids are used as the mobile phase solvents.16-18 Enhanced-fluidity liquids are organic solvents to which high proportions of liquified gases have been added. These solvents have solvent strengths that can be controlled by using small changes in pressure or temperature. In addition, because enhanced-fluidity liquids have low viscosities, long columns could be used to generate LCCC separation with nearly 100 000 theoretical plates.16 Recently, the scope of application of critical chromatography was further broadened to include the characterization of water- soluble copolymers.19 In aqueousbased solvents, the critical condition is reached by variation in ionic strength and pH. In addition, Brun20 recently illustrated that critical chromatography was also a viable choice for characterizing the components within statistical copolymers. Herein the aqueous-based LCCC is used to begin a systematic characterization of the composition of humic acids. Making the (14) Lee, W.; Cho, D.; Chang, T.; Hanley, K. J.; Lodge, T. P. Macromolecules 2001, 34, 2353-2358. (15) Pasch, H.; Esser, E.; Klonger, C.; Iatrou, H.; Hadjichristidis, N. Macromol. Chem. Phys. 2001, 202, 1424-1429. (16) Yun, H.; Olesik, S. V.; Marti, E. J. Microcolumn Sep. 1999, 11, 53-61. (17) Yun, H.; Olesik, S. V. Anal. Chem. 1998, 70, 3298-3303. (18) Souvignet, I.; Olesik, S. V. Anal. Chem. 1996, 69, 66-71. (19) Phillips, S.; Olesik, S. V. Anal. Chem. 2003, submitted. (20) Brun, Y. J. Liq. Chromatogr. Relat. Technol. 1999, 22, 3066-3090.

overestimation of MW charge density cross-linking, branching, aromatic carbon content branching

assumption that a humic acid is a mixture of primary structures or supramolecular aggregates that may contain repeat units, such as peptides or amino acid linkages, carbohydrate,s and polyacids, and that some of these repeat units may be expected to be common to humic acids from similar terrestrial sources,21,22 critical chromatography should be useful to determine which synthetic polymer represents subunits of humic acids well and which do not. The work described herein is the beginning of a test of these hypotheses. By finding the critical condition of a polymer that is believed to represent a portion of the humic acid and then injecting the humic acid into the chromatographic system using those critical conditions, the resultant separation should place the portion of the humic acid that is similar to that model polymer at the retention time corresponding to the critical condition of the model polymer. The molecular weight variation of that portion of the humic acid will not contribute to the separation. Therefore, a uniquely selective separation should be possible. Fractions of that separation could be collected and characterized further using a combination of analysis techniques. The initial polymers chosen for this test are those most used for SEC standards for humic studies, because they are believed to either have subunits similar to humics or to be a component of humics. This work starts with the study of poly(acrylic acid) (PAA) and sodium polystyrene sulfonate (PSS) as models. Humic acids derived from three different terrestrial environments sources are studied. The isolation of humic fractions based on functionality and molecular weight is demonstrated with the LCCC technique. Subsequent SEC and ESI-MS analysis of these fractions is also described. EXPERIMENTAL CONDITIONS Materials. An Elliot soil humic acid standard (1S102H) and a Pahokee peat sample were obtained from the International Humic Substances Society (IHSS, Golden, CO). This will allow others to compare their results to ours. The compost humic acid was derived from a compost prepared from brewer’s grains, poultry manure, gypsum, and horse manure-straw bedding with a detailed analysis by Chen et al.5 Humic acids were derived from the peat and compost samples utilizing a modified version of Swift’s procedure.23 The sample was mixed in a 1:10 ratio with a 0.1 M NaOH solution under nitrogen for 20 h. The humic and fulvic acids were separated from the solution using centrifugation (4000g), and the supernatant was collected and acidified to a pH of 1 with 6 M HCl. Once the solution stood overnight, the fulvic acid fraction was removed using centrifugation (9000g). The peat and compost humic acid (21) Kappler, A.; Ji, R.; Brune, A. Soil Biol. Biochem. 2000, 32, 1271-1280. (22) Stenson, A.; Cooper, W. T. Abstracts of Papers, 225th ACS National Meeting, New Orleans, LA, March 23-27, 2003. (23) Swift, R. In Chemical Methods; Sparks, D. Ed.; Part 3; SSSA: Madison, WI, 1996; pp 1011-1069.

Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

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Table 2. Molecular Weight Moments for Bulk Humic Acid Samples sample

appearance

Mn (KDa)

MW (KDa)

Mz (KDa)

Pa

peat (well-humified)b compost (lignin rich)b soil

dark brown, coarse light brown, fine dark brown, fine

7.3 5.7 3.8

14.9 12.9 7.9

20.4 18.9 11.1

2.1 2.3 2.1

a Polydispersivity. b NMR studies and elemental analysis showed that the peat sample had a greater amount of aromatic carbon and a greater amount of nitrogen than the compost sample.

fractions were then placed under dialysis against deionized water until the conductivity of the water outside of the dialysis tubing was that of the deionized water in order to remove any excess salt. The dialysis system consisted of a Spectra/Por Membrane, MWCO 6-8000 from Fisher Scientific. Details of each humic acid are provided in Table 2. Sodium salts of polystyrene sulfonate samples (Mw ) 1640, Mw/Mn ) 1.12; Mw ) 4950, Mw/Mn ) 1.13; Mw ) 8000, Mw/Mn ) 1.11; Mw ) 16 000, Mw/Mn) 1.13; Mw ) 34 700, Mw/Mn ) 1.16; Mw ) 57 500, Mw/Mn ) 1.11; Mw ) 126 700, Mw/Mn ) 1.17; Mw ) 262 600, Mw/Mn ) 1.20) were purchased from Scientific Polymer Products, Inc. (Ontario, NY). Sodium salts of poly(acrylic acid) (Mw ) 1300, Mw/Mn ) 1.56; Mw ) 8300, Mw/Mn ) 1.34; Mw ) 18 100, Mw/Mn ) 1.41; Mw ) 36 900, Mw/Mn) 1.60; Mw ) 83 400, Mw/Mn ) 1.74; Mw ) 131 200, Mw/Mn ) 1.67; Mw ) 695 100, Mw/Mn ) 1.41) were purchased from Polymer Standard Services (Silver Spring, MD). Sodium salts of poly(acrylic acid) (Mw ) 20 000, Mw/Mn ) 1.40) were purchased from Polysciences, Inc. (Warrington, PA). Disodium phosphate dodecahydrate was purchased from J. T. Baker (Phillipsburgh, NJ). Monobasic phosphate was purchased Mallinckrodt (Paris, KY). Acetic acid (99.8% pure) was purchased from Mallinckrodt (Paris, KY). Anhydrous sodium acetate (99.8% pure) was purchased from Jenneile Enterprises (Cincinnati, OH). HPLC-grade acetonitrile was used as received from Fisher Scientific, Inc. (Fair Lawn, NJ). Water was deionized by a NANOpure II system from Sybron/ Barnstead (Boston, MA) with a resistivity of 17.8-18.0 ΩS-cm. Chromatographic Systems. Size-Exclusion Chromatography. The chromatographic system used included an ISCO 260D syringe pump from ISCO, Inc., (Lincoln, NE) operating at a flow-rate of 0.100 mL/min and a Rheodyne 7725i 7-port high-pressure injection valve with an injection volume of 20 µL from Rheodyne (Rohnert Park, CA). A 250 × 2.0-mm-i.d. column with an Asahipak GF 310 HQ stationary phase (5-µm particle size and exclusion limits of 0.31-0.42 ( 0.02 mL) from Keystone Scientific, Inc. (Bellefonte, PA) was used. The column packing was chosen because of the compatibility of the phase with a wide range of solvent conditions. A Spectroflow 757 UV-vis absorbance spectrometer from Kratos Analytical Instruments (Ramsey, NJ) operated at a wavelength of 200 nm. A piece of 100-µm-i.d. fused-silica tubing was connected directly from the column outlet and served as a view cell for the spectrometer. The homemade view cell was made by applying a low flame to remove the polyimide coating from the fused-silica capillary (Polymicro Technologies, Inc., Phoenix, AZ). Experiments were performed at room temperature. Data acquisition was achieved with an IBM Thinkpad using EZChrom software (version 6.7) from Scientific Software, Inc. (San Ramon, CA). A 99% 5 mM phosphate-buffered water/1% acetonitrile solution was utilized as 5546 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

the mobile phase. All chromatographic conditions chosen for this study were based on Cabaniss’s detail study of optimum conditions for humic acid analysis by SEC.24 In this work, Cabaniss careful analyzed the SEC column and solvent conditions necessary to obtain molecular weight information that is minimally affected by non-SEC interactions and also minimizes humic acid clustering. Sodium Polystyrene Sulfonate Critical Chromatographic System. The chromatographic system utilized for the LCCC of sodium polystyrene sulfonates is similar to that described above for the SEC analysis. A mobile phase of 46% H2O/54% ACN with an acetate buffer concentration of 23 mM was determined to establish the critical condition for sodium polystyrene sulfonates. A 150 × 2.0-mm-i.d. column packed with the same Asahipak packing material as described above with exclusion limits of 0.18-0.26 ( 0.02 mL was utilized for this system. Poly(acrylic acid) Sodium Salt Critical Chromatographic System. The chromatographic system was identical to the system described for the SEC system except for the mobile phase conditions. A mobile phase system of 53 vol % H2O/47 vol % ACN with a phosphate buffer concentration of 17 mM was established as the critical mobile phase composition for standards of poly(acrylic acid). Procedure. Mobile phase mixtures were prepared off-line from the chromatographic system. The buffered water solution (acetate or phosphate) was prepared at the appropriate buffer concentration and mixed using a sonic bath. The pH of the buffered water solution was measured with an Accumet model 10 pH meter from Fisher Scientific (Pittsburgh, PA). The pH meter was standardized with primary pH standards of pH 4.00, potassium acid phthalate (Baxter Diagnostics, Inc., Deerfield, IL) and pH 7.00, potassium phosphate monobasic, potassium phosphate dibasic (Mallinckrodt-Baker, Inc., Paris, KY). The final mobile phase solution was prepared by mixing the buffered water with a specific amount of organic solvent (acetonitrile). Solvents were mixed and degassed using a sonic bath for ∼5 min. Solvents were filtered through a 2-µm filter before being placed into the pump. Samples were prepared in the mobile phase solvent at a concentration of 0.5-1.5 mg/mL. Humic acid samples utilized for collection consisted of a solution of the humic acid sample in the range of 5 mg/mL in the mobile phase solution in order to collect a suitable sample concentration. All humic acid samples were filtered through a 2-µm polypropylene filter (Whatman, Inc., Clifton, NJ). The pH of the chromatographic eluent was monitored during sample elution using Hydrion pH paper (Fisher Scientific, Pittsburgh, PA). The pH of the chromatographic eluent remained neutral throughout the experiments. (24) Zhou, Q.; Cabaniss, S. E.; Maurice, P. A. Wat. Res. 2000, 34, 3505-3514.

Molecular weight calibration curve data were analyzed using a linear regression curve with a 95% confidence using SigmaPlot software (version 4) from SPSS, Inc., (Chicago, IL). The molecular weight distribution in each humic acid sample and fraction was determined using the following equation,

MX )

∑[H M ∑[H M i

n i

]

n-1

i

i

]

(1)

where n is equal to 1, 2, and 3 for the number-average molecular weight (Mn), weight-average molecular weight (Mw), and Z-average molecular weight (Mz), respectively; the polydispersivity, P, is Mw/ Mn; and Hi is the intensity of the chromatographic band for molecular weight, Mi. Mass Spectral Characterization. Preliminary mass spectral analysis of fractions of peat, compost, and soil were also undertaken. All experiments were performed on a Micromass (Q-TOF) II (Micromass, Wythenshawe, U.K.) mass spectrometer equipped with an orthogonal electrospray source (Z-spray) operated in positive ion mode. Sodium iodide was used for mass calibration for a calibration range of m/z ) 100-2000. Humic samples were prepared in a solution containing distilled water at pH 7.0 and were infused into the electrospray source at a rate of 5-10 mL/ min. Optimal ESI conditions were capillary voltage 3000 V, source temperature 100 °C, and a cone voltage of 60 V. The ESI nebulizer gas was nitrogen. Q1 was set to optimally pass ions from m/z 100-2000, and all ions transmitted into the pusher region of the TOF analyzer were scanned over an m/z range from 200 to 2000 with a 1-s integration time. Data were acquired in continuum mode until acceptable averaged data were obtained. RESULTS AND DISCUSSION The molecular weight moments and the polydispersivity for the three bulk humic acid samples of varying origin were determined using size-exclusion chromatography (Table 2). Figure 1A shows the calibration curves of both poly(acrylic acid) and sodium polystyrene sulfonate operating in the SEC mode utilizing a mobile phase of 99 vol % 5 mM phosphate buffered H2O/1 vol % acetonitrile. At these conditions, the calibration curves for both sets of standards overlap, demonstrating that characteristics other than hydrodynamic volume of the samples were not influencing the SEC separation. The average molecular weights decreased in the order of peat > compost > soil, as shown in Figure 1B. Similarly, Kudryavtsev et al. compared the number-average and weight-average molecular weights of humic acid from a soil and peat sample and also determined that the molecular weight of the peat HA was substantially greater than that of the soil.25 Although this experiment established the average molecular weight distributions for the bulk HA sample, more information was gained with the application of water-based LCCC. Sodium Polystyrene Sulfonate-LCCC of Humic Acids. The critical condition for each model polymer was determined by starting with the SEC conditions and carefully changing the salt concentration and organic component of the mobile phase until (25) Kudryavtsev, A. V.; Perminova, I. V.; Petrosyan, V. S. Anal. Chim. Acta 2000, 407, 193-202.

Figure 1. (A) SEC calibration curves of (b) polystyrene sulfonate and (9) poly(acrylic acid) with 99 vol % 5 mM phosphate buffered H2O/1 vol % ACN. (B) Size-exclusion chromatographs of humic substances at the conditions described in 1A. (1) Peat humic acid, (2) compost humic acid, and (3) soil humic acid.

the critical condition was found.19 A mobile phase of 46% H2O/ 54% acetonitrile with an acetate buffer concentration of 23 mM at room temperature and 61.2 atm column head pressure was established as the critical condition for sodium polystyrene sulfonates using a 150-mm-long × 2.0-mm-i.d. column packed with the same packing as used in the SEC studies. The exclusion limits of this column corresponded to 0.18-0.26 ( 0.02 mL. When operating at the critical condition for sodium polystyrene sulfonate, the poly(acrylic acid)s were found to elute through sorptive interactions with retention volumes greater than the critical retention volume of 0.26 mL.19 The aliquots of peat, compost and soil humic acid samples were next separated at the critical condition of polystyrene sulfonate sodium salt. The resultant chromatograms of all three humic acid samples at the LCCC conditions for sodium polystyrene sulfonate are shown in Figures 2-4. The chromatogram of the peat humic acid (Figure 2A) shows four unresolved chromatographic bands. The first band eluted at the critical retention volume of the sodium polystyrene sulfonate, and the remainder of the sample eluted at retention volumes greater than the critical elution point (i.e., sorptive interactions were predominating for those components of the peat humic acid), which was similar to the behavior of the poly(acrylic acid) standards at the CC for polystyrene sulfonate. The concentration of the injected sample was varied to determine if the low chromatographic resolution was due to overwhelming the column with too high of a concentration of humic acid and to test the possibility that the humic acids could self-associate or dissociate. This possibility was recently proposed Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

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Figure 4. LCCC of concentrated soil humic acid sample at the critical condition for PSS. Vertical lines delineate where the three fractions were collected.

Figure 2. (A) LCCC of peat humic acid at the critical condition for polystyrene sulfonate sodium salt (PSS). Vertical line shows the critical retention volume of PSS. Chromatogram 1 corresponds to a concentration of 1 mg/mL, number 2 corresponds to 0.3 mg/mL, and 3 is 0.1 mg/mL. (B) LCCC of concentrated peat humic acid sample at the critical condition for PSS. Vertical lines delineate where the four fractions were collected.

Figure 3. LCCC of concentrated compost humic acid sample at the critical condition for PSS. Vertical lines delineate where the four fractions were collected.

by Conte and Piccolo.11 Figure 2A shows that as the sample concentration decreased, the resolution remained approximately the same, which indicates self-association is not likely, and saturation of the stationary phase did not occur. Similarly, concentration variation studies on the peat and compost humic acids showed no evidence of self-association. The humic acid originating from compost had the bulk of its elution profile centered at the critical elution volume (0.26 mL) of the sodium salt of the polystyrene sulfonates. The soil humic acids were completely eluted by sorptive interactions; i.e., no signal was observed at retention volumes before or at the critical condition of the sodium polystyrene sulfonate. Utilizing the same chromatographic conditions, the sample concentration was raised so that an appreciable amount of sample 5548 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

could be collected from the chromatographic system for further characterization. The chromatograms of the concentrated samples and the fraction collection regions are indicated in Figures 2B, 3, and 4. The overall shape of the chromatographic bands was similar under dilute concentration conditions or concentrated conditions. For all humic acids studied, the chromatograms for the more concentrated conditions are included here, and the chromatograms under dilute sample conditions are included in the Supporting Information for this work. Four fractions were collected for the peat and compost humics and three fractions were collected for the soil humic. The fractions collected from each sample varied in color, as indicated in Table 3. Further characterization of the fractions will be described later. Next, the LCCC conditions for poly(acrylic acid) were applied to these humic acid samples. Poly(acrylic acid)-LCCC of Humic Acids. A mobile phase system of 53 vol % H2O/47 vol % acetonitrile with a phosphate buffer concentration of 17 mM was established as the critical mobile phase composition for standards of poly(acrylic acid) using a 250 × 2.0-mm-i.d. column which had exclusion limits of 0.310.42 ( 0.02 mL.19 As expected, the retention volume at the critical condition was 0.42 mL, which is also the retention volume at the exclusion limit. At these conditions, the sodium polystyrene sulfonates were found to elute in an exclusion mode. Next, the humic acids were injected into the column under these conditions. Species eluting between 0.31 and 0.42 mL were undergoing a separation based on exclusion interactions where the sodium polystyrene sulfonates were eluted. Those species with a retention volume of 0.42 mL were eluting with the poly(acrylic acid)s at the critical elution volume, and those species with an elution volume greater than 0.42 mL were undergoing sorption-based retention. Figures 5-7 show the resultant chromatograms of the peat, compost, and soil humic acids separated at the critical condition of the poly(acrylic acid)s. Figure 5 shows that like the polystyrene sulfonate sodium salts, a significant fraction of the peat humic acid eluted at a retention volume less than the critical condition of the poly(acrylic acid), with a tail portion of the band experiencing primarily sorptive interactions. Figure 6 shows the chromatogram of the compost humic acid. Elution volumes also covered the entire exclusion region of the chromatographic system, with the tail-end of the peak eluting at the critical retention volume. Figure 7 shows the chromatogram of the soil humic acid at the critical condition of the poly(acrylic acid)s. The soil humic acid

Table 3. SEC of Sodium Polystyrene Sulfonate-LCCC Fractions fractiona

retention modeb

appearancec

Mn (KDa)

Mw (KDa)

Mz (KDa)

Pd

peat 1 peat 2 peat 3 peat 4 compost 1 compost 2 compost 3 compost 4 soil 1 soil 2 soil 3

critical adsorption adsorption adsorption SEC critical adsorption adsorption adsorption adsorption adsorption

dark brown brown dark yellow pale yellow dark yellow/orange dark amber/brown pale yellow yellow dark brown brown pale yellow

13.2 7.5 6.8 7.9 16.0 12.9 8.3 9.8 8.6 9.7 5.2

21.4 14.9 12.5 13.5 21.3 20.2 14.8 17.0 14.5 15.2 8.4

27.1 21.5 17.3 17.3 24.9 24.9 19.6 22.2 19.2 19.8 11.4

1.6 2.0 1.8 1.7 1.3 1.6 1.8 1.7 1.7 1.6 1.6

a Peat, compost, and soil fractions as shown in Figures 2B, 3B, and 4B, respectively. b Retention mode where each fraction was collected at the LCCC mode for sodium polystyrene sulfonate. c Each fraction was clear and colored. d P ) Mw/Mn

Figure 5. LCCC of concentrated peat humic acid sample at the critical condition for poly(acrylic acid). Vertical lines delineate where the four fractions were collected.

Figure 7. LCCC of concentrated soil humic acid sample at the critical condition for poly(acrylic acid). Vertical lines delineate where the four fractions were collected. Table 4. SEC of Poly(acrylic acid)-LCCC Fractions

Figure 6. LCCC of concentrated compost humic acid sample at the critical condition for poly(acrylic acid). Vertical lines delineate where the three fractions were collected.

chromatographic band has most of its peak area at retention volumes equal to or greater than the critical condition for the poly(acrylic acid)s. Note: the shape of the chromatographic bands for all three humic acids separated at the critical condition for the poly(acrylic acid)s were significantly different from those separated at the critical condition of the sodium polystyrene sulfonate-LCCC conditions. Greater resolution of the soil humic acid was observed at the critical condition for the poly(acrylic acid) than at the critical condition of the sodium polystyrene sulfonates. Sample concentrations were increased, and fractions were collected from the chromatographic system operating at the LCCC

fractiona

retention modeb

appearancec

Mn (KDa)

Mw (KDa)

Mz (KDa)

Pd

peat 1 peat 2 peat 3 peat 4 compost 1 compost 2 compost 3 soil 1 soil 2 soil 3 soil 4

SEC critical adsorption adsorption SEC SEC critical SEC critical adsorption adsorption

brown dark brown dark yellow pale yellow dark amber light brown pale yellow brown dark brown dark yellow pale yellow

16.6 7.4 7.5 9.6 13.6 20.0 15.7 6.9 3.2 7.8 10.2

27.5 16.8 17.6 11.2 21.1 50.5 24.4 12.4 9.05 16.1 18.3

34.8 24.6 25.2 12.6 25.9 72.9 32.3 17.5 15.1 21.9 24.0

1.7 2.3 2.3 1.2 1.6 2.5 1.6 1.8 2.8 2.1 1.8

a Peat, compost, and soil fractions as shown in Figures 5B, 6B and 7B, respectively. b Retention mode where each fraction was collected at the LCCC mode for poly(acrylic acid). c Each fraction was clear and colored. d P ) Mw/Mn

conditions for poly(acrylic acid). Four fractions were collected from the peat and soil humics, and three fractions were collected from the compost sample. Table 4 describes the appearance of each fraction collected from the LCCC system. The characterization of these fractions is described in the next section. The results obtained for these humic substances at the LCCC conditions established for both sodium polystyrene sulfonate and poly(acrylic acid) indicate that the sodium polystyrene sulfonate is a good model for a significant portion of the “lignin-rich” compost humic acid. The poly(acrylic acid) provides a good model for a significant portion of the soil humic acid and a small portion Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

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of the peat humic acid. The sodium polystyrene sulfonate was not a good model for the soil humic acids, which also points to the fact that it would not be a suitable size-exclusion standard for this soil humic acid. The specific attributes of the humics that cause the observed similarities and differences will require further investigation. SEC of Humic Fractions from LCCC Analysis. In an effort to further characterize the humic acid samples that were separated at the critical conditions for the sodium polystyrene sulfonates and the poly(acrylic acid)s, SEC analysis of each collected fraction was undertaken. Tables 3 and 4 list the molecular weight moments calculated from the SEC analysis of each fraction collected from the LCCC analyses of the humic samples. In addition, Tables 3 and 4 list the color variation of the collected fractions. As indicated in the tables, the color variation among the fractions is substantial. Recall that at the critical condition of a subunit, all molecular weight variation is removed for that subunit. Therefore, the shape and molecular weight moments for the collected fractions should not be the same as that of the original bulk media. For the purpose of comparison, we will focus primarily on the Mw values. Figures 8A-C and 9A-C show distributions that are very different from those observed in Figure 1B. In addition, the variation in calculated Mw values among the fractions [note: the variation among soil fraction (12.4, 9.05, 16.1, 18.3 KDa) and peat fractions (27.5, 16.8, 17.6, 11.2 KDa) in Table 4] is not continually decreasing, as expected if the LCCC separation were merely based on hydrodynamic differences, but is randomly varying. This is a strong indication that the separation is based on unique chemical differences among the fractions. The results described in Table 4 were established by SEC analysis of the fractions collected at the LCCC conditions for poly(acrylic acid). At these conditions, the fractions collected at the critical elution volume for PAA (0.42 mL/min) behave chromatographically similarly to the PAA and are anticipated to have components that are structurally similar to PAA. As anticipated, the compost sample showed the bulk of the chromatographic band eluting at the critical retention volume for PSS (Figure 3) and well before the critical retention volume for PAA (Figure 6), indicating that PSS and the compost fraction behave chromatographically similarly. Figure 8B shows discreetly different molecular weight distributions from the fraction of the compost humic acid sample at the PSS critical condition (Figure 3). The distributions demonstrated for the compost sample in Figure 9B, generated through fractionation using LCCC of PAA, are not markedly different from each other, unlike those observed for the peat and soil samples generated through the same experimental conditions. This supports the conclusion that two discreet separation mechanisms were observed when analyzing these samples at the critical condition for the two different polymer models. In addition, note that in the SEC of the compost fractions, a much higher molecular weight species (Mw ) 50.5 KDa) was observed in the collected fraction number 2 than what was observed in the bulk sample (Figure 1B). This observation will be discussed further later. Mass Spectral Analysis of the Fractions. Preliminary mass spectral analysis of some of the fractions collected after the LCCC separation was undertaken. The humic acid fractions were collected and mass-analyzed using offline electrospray mass 5550 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

Figure 8. (A) SEC of the peat fractions described illustrated in Figure 2. (B) SEC of the compost fractions described in Figure 3. (C) Size-exclusion chromatography of the soil fractions described in Figure 4.

spectrometry. Figure 10 shows the resultant mass spectra of compost humic acid fractions near the critical condition of PSS, while Figures 11 and 12 show the mass spectra of the peat humic acid fractions and soil humic acid fractions near the critical conditions of PAA. Like previously observed mass spectra of humic or fulvic acids,26,27 signal was observed at almost every m/z value. For the compost fractions collected at LCCC of PSS (Figure 10), the mass spectra of compost fractions 2 and 3, C2 and C3, have similar general structure, and fractions 1 and 4 are substantially different structure. Fraction C2 is the fraction nearest the critical condition (26) Kujawinski, E. B. Environ. Forensics 2002, 3, 207-216. (27) Kujawinski, E. B.; Freitas, M. A.; Zang, X.; Hatcher, P. G.; Green-Church, K. B.; Jones, R. B. Org. Geochem. 2002, 33, 171-180.

Figure 10. Electrospray mass spectrum of compost humic acid fractions (Figure 3) collected near the critical condition of polystyrene sulfonate sodium salt. (Note: C1 corresponds to fraction 1.)

Figure 9. (A) SEC chromatograms of the peat humic fractions shown in Figure 5. (B) SEC of compost fractions described in Figure 6. (C) SEC of soil fractions described in Figure 7B.

of PSS. For compost fraction C2-PSS, 15 clusters of ions (or fragments ions) were observed, with average spacing between the groups and average ∆m/z ) 60; for fraction C3-PSS, 18 groups of clusters with similar spacing was observed. This was measured by a peak-to-peak m/z value between the most intense bands of the clusters. The mass spectra obtained on fractions on fractions collected near the PSS critical condition contain repeat units rather than what is typically observed for the mass spectra of humic acids, which is signal at almost every nominal mass. Figure 11 shows that fraction P2-PAA of the peat humic acid was the fraction nearest to the critical condition of poly(acrylic acid), and it had five observed repeating units with average peakto-peak spacing between the clusters of m/z ) 120. Fraction number P3-PAA showed mass spectra that may be oligomer repeat units, but fractions P1-PAA and P4-PAA show no such structure. In addition, the soil humic acid sample was well-centered around the critical condition of the poly(acrylic acid). Fractions S1-PAA, S2-PAA, and S3-PAA all show oligomeric-like repeat units that are spaced ∼100 m/z units apart. When these data are generally compared to that published previously, these data show markedly more clustering or grouping at m/z values than others that are typically one big grouping of m/z values.7 This is a strong indication that the critical chromato-

Figure 11. Electrospray mass spectrum of peat humic acid fractions (Figure 5) collected near the critical condition of poly(acrylic acid). (P1 corresponds to fraction 1, etc.)

graphic separation using well-chosen model compounds is able to fractionate the humic acids in a way that has not been accomplished previously. Comparison of Mass Spectral Data and SEC Data on Humics. The order of magnitudes of the SEC data presented in this work is very similar to that published by others for humic acids from similar terrestrial sources. Similarly, the range of m/z values for the humic acids in this work are also similar to that Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

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Figure 12. Electrospray mass spectra of soil humic acid fractions (Figure 7) collected near the critical condition of poly(acrylic acid). (Note: S1 corresponds to fraction 1.)

published by others. For all of the electrospray mass spectra, the observed m/z values were significantly lower than the molecular weights determined by size-exclusion analysis of the same fractions (Tables 3 and Tables 4). The mass spectra indicated m/z values in the low 1000s, while the SEC indicated some portions of the humic acids had molecular weights in the 10 000-50 000 range. This is a very common phenomenon for humic materials; however, this is the first publication that has published both in the same paper. Piccolo11 hypothesizes that humic acids may be small oligomers that are held together through multiple hydrogen bonds. The electrospray ionization process may also break the hydrogen bonds before mass analysis is possible or metal-humic acid association proposed by Wrobel et al.3 Variation in the mass distribution shape is different between the fractions for a given terrestrial source as well. For example, C1-PSS and C2-PSS have similar average molecular weight values as measured by SEC but very different m/z distributions as measured by ESI-MS. In addition, by SEC, the average molecular weight for fraction C3-PSS is significantly lower than that of fraction C2-PSS. The observed m/z distributions of the fractions C2-PSS and C3-PSS are actually very similar. The peat mass spectra (Figure 11) show the same discrepancies in that the highest average m/z is observed for the fraction P2-PAA, whereas the SEC shows the high mass should be fraction P1-PAA. For the soil humic ESI-MS, the average m/z value for all fractions is similar, which is approximately observed using SEC. One other possible reason for the observed discrepancies is that the charge on the observed ions is different from +1. Minimal signal is observed in m/z ranges between the nominal masses, which indicates that high-resolution mass spectra do not contain substantial contributions from multiply charged ions. However, differences in ionization efficiencies of the measured ions could 5552

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cause the different portions of the humic acids to be more represented in the mass spectrum. As mentioned earlier, the bulk SEC distributions have order of magnitude mass moments similar to that previously observed by others. In addition, we closely followed the optimum SEC conditions proposed by Cabaniss to avoid non-SEC interactions for the SEC analysis. However, Wrobel et al. recently showed with a combined size-exclusion inductively coupled plasma mass spectrometry study that the presence of trace levels of metal ions can have a significant effect on the measured apparent molecular weight of the humic acid. This may be the cause of the higher value of Mw calculated for the compost fraction 2 near the PAA critical condition that was mentioned earlier. It is also possible that the increase in this compost fraction is merely due to the inherent heterogeneity of the humic matter. Both of these possibilities would cause the ESI-MS data to have lower m/z values than the mass distributions observed by SEC. On the basis of the care taken in doing the SEC, LCCC and ES-MS data, and the similarity between the published general SEC distributions and m/z distributions, we believe that the mass spectral analysis and the SEC analysis are providing different information. The cause of these differences will be discerned in later studies. CONCLUSIONS This work describes the initiation of studies of humic acids using LCCC. Two of the three humic acids studied are standards that were obtained from the International Humic Substances Society. In doing so, we have provided standard LCCC conditions that should allow others to characterize the same fractions that we have produced. Although actual structure elucidation was not attempted in this study, a qualitative assessment of three different humic species was shown. Furthermore, this study suggests that humic acid species derived from differing environmental origins may not be adequately modeled with one universal polymer standard. The LCCC data indicate that PSS is a good model for a significant portion of the compost humic acids and PAA is a good model for portions of the peat and soil humic acids; however, PSS was not a good model for any portion of the soil humic acid and, therefore, is not recommended as a SEC standard for it. The chromatograms of the humics acid samples at the CC of the two different polymer standards were markedly different from each other. In addition, the size-exclusion and mass spectra data on the fractions collected near the critical condition of the two polymer standards clearly indicate that unique selectivity between and within the three humic samples was accomplished using LCCC. Future studies that involve improvements in the LCCC and addition of other spectroscopies (FTIR and ESCA) to better characterize the humic acids are planned. Future applications of LCCC to the analysis of other complicated heterogenious systems are expected. We have previously illustrated the orthogonal nature of the LCCC separation and mass spectrometry.16 LCCC is capable of providing functionality distribution information, and the mass spectrometer provides detailed mass distribution information.

ACKNOWLEDGMENT We would like to thank the staff of the Campus Chemical Instrument Center for the ESI mass spectral analysis. We would also like to thank the Ohio Board of Regents (Hayes Investment Fund), which allowed the purchase of the mass spectrometer. We thank Professor Patrick Hatcher’s research group for providing the humic acid samples. This work was supported by the National Science Foundation as part of the efforts of The Ohio State University Environmental Moleular Science Institute.

SUPPORTING INFORMATION AVAILABLE Chromatograms under dilute sample conditions. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review May 8, 2003. Accepted August 6, 2003. AC0344891

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