Abstract Electrospray ionization mass spectrometry(ESI-MS) was used to evaluate the average molecularmass of terrestrial humic substances, such as humic (HA)and fulvic (FA) acids from a soil, and humic acid from alignite (NDL). Their ESI mass spectra, by direct infusion,gave average molecular masses comparable to those pre-viously obtained for aquatic humic materials. The soil HAand FA were further separated in size-fractions by prepar-ative high performance size exclusion chromatography(HPSEC) and analyzed with ESI-MS by both direct infu-sion and a further on-line analytical HPSEC. Unexpectedly,their average molecular mass was only slightly less thanfor the bulk sample and, despite different nominal molec-ular size, did not substantially vary among size-fractions.The values increased significantly (up to around 1200 Da)after on-line analytical HPSEC for the HA bulk sample, atboth pH 8 and 4, and for the HA size-fractions when pHwas reduced from 8 to 4. It was noticed that HA size-frac-tions at pH 8 were separated by on-line HPSEC in furtherpeaks showing average masses which progressively in-creased with elution volume. Furthermore, when the HAand NDL bulk samples were sequentially ultracentrifugedat increasing rotational speed, their supernatants showedmass values which were larger than bulk samples and in-creased with rotational speed. These variations in mass val-ues indicate that the electrospray ionization is dependenton the composition of the humic molecular mixtures andincreases when their heterogeneity is progressively re-duced. It is suggested that the dominance of hydrophobiccompounds in humic supramolecular associations may in-hibit the electrospray ionization of hydrophilic compo-nents. Our results show that ESI-MS is reasonably applic-

able to humic substances only after an extensive reductionof their chemical complexity.

Keywords Electrospray ionization mass spectrometry ·Humic substances · Soil · Supramolecular associations ·Molecular mixtures · Molecular sizes · Hydrophobiccompounds

Introduction

Humic substances, the end product of decayed organicmatter, play a significant role in environmental and eco-logical processes by controlling transport and transforma-tion of toxic chemicals in soil and water, metal complexa-tion, nutrient availability, maintenance of soil structure,and carbon and nitrogen exchange between soil and atmo-sphere [1, 2, 3, 4]. However, the spacial and temporal het-erogeneity and molecular complexity of humic matter haveso far prevented identification of a specific molecular struc-ture. The long-standing assumption that humic substancesare macromolecular polymers had never been unambigu-ously proved [5]. Within the polymeric concept, the con-formational structure of humic substances has been as-sumed to be similar to either the random coil of proteins[6] or to micellar or membrane-like structures [7, 8]. Thecommonly reported high molecular weights for soil humicsubstances ranging from a few thousands to hundred ofthousands of Da [9] was recently questioned. Experimentswith High Performance Size Exclusion Chromatography(HPSEC) suggested that humic substances are non-cova-lent associations of relatively small molecules [10] andthat less heterogeneous size fractions can be separated byHPSEC after interactions of humic solutions with organicacids [11]. The complex molecular mixture of humic sub-stances, rather than high molecular weight polymers, shouldthen be better described as supramolecular associations ofheterogeneous molecules self-assembling in only appar-ent high molecular size materials by non covalent interac-tions such as dispersive forces (van der Waals, π-π, CH-π)at neutral pH, and classical hydrogen bonds at lower pH

A. Piccolo · M. Spiteller

Electrospray ionization mass spectrometry of terrestrial humic substances and their size fractions

Anal Bioanal Chem (2003) 377 : 1047–1059DOI 10.1007/s00216-003-2186-5

Received: 11 April 2003 / Revised: 28 May 2003 / Accepted: 17 July 2003 / Published online: 4 September 2003

ORIGINAL PAPER

A. Piccolo (✉)Dipartimento di Scienze del Suolo, della Pianta e dell’Ambiente,Università di Napoli Federico II, Via Università 100, 80055 Portici, Italye-mail: alpiccol@unina.it

M. SpitellerInstitute of Environmental Research (INFU), University of Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany

© Springer-Verlag 2003

[11, 12]. This view was confirmed by other experimentsemploying fluorescence spectroscopy [13], NMR spec-troscopy [14, 15, 16] and thermal analysis [17], therebysubstantiating the supramolecular rather than the polymericnature of humic materials.

Among the soft ionization methods, the ElectrosprayIonization Mass-Spectrometry (ESI-MS) technique, whenapplied to humic substances in either single (MS) or tan-dem (MS/MS) mode, has recently received great attention[18, 19, 20, 21, 22, 23, 24, 25, 26]. The interest in elec-trospray ionisation is due to its advantage in being an ex-tremely soft ionization process and threfore providing un-fragmented ions for which not only the absolute molecu-lar weight (ESI/MS) but also unequivocal structural infor-mation (MS/MS) can be obtained [27, 28]. These featureswere proved for ESI of humic molecules in combina-tion with high-resolution instrumentation such as FourierTransform Ion Cyclotron Resonance Mass Spectrometry(FT IRC-MS) providing a resolving power up to 120,000for low-mass molecules [29, 30, 31, 32]. A possible draw-back in soft ionization of humic substances was indicatedby Novotny and Rice [33] who, by using laser desorptionionization (LDI), suggested that formation of multiplycharged ions may represent a bias in favor of low averagemolecular weight of humic molecules. Plancque andcoworkers [25], using quadrupole time-of-flight (Q-TOF)ESI-MS, showed that singly charged ions were by far themost abundant ions generated from a fulvic acid. TheirMS/MS experiments revealed that humic ions were frag-mented by losing m/z 44 or 18, corresponding to the neutralmasses of CO2 and H2O from singly charged ions, whereasdoubly charged ions would loose m/z 22 and 9 fragments,respectively. The predominance of singly charged ions inhumic materials was definitively confirmed by high-reso-lution mass spectra [24] and ESI-FTIRC mass spectrome-try in both positive and negative mode [30, 32], therebyeliminating inadequate accounting for multiply chargedions as a cause of any low molecular weight bias. Thesefindings are consistent with the reported formation ofmainly monocharged ions from small (<1000 Da) mole-cules [27, 28, 34] and with the supramolecular concept ofhumic substances that involves self-assembling non-cova-lent association of small molecules [5, 10, 11].

In some works [21, 31] the ESI ionization of humicmolecules was reported as dependent on solvent used andcone voltage. Others observed that the very low intensitythat was still found for humic clusters containing up tothree singly charged molecules did not vary with ESI conevoltage [25]. Hence, with the aim to control the extent ofionization of humic molecules and optimize ESI condi-tions, the use of calibration standards has been attempted[24, 26]. In a most detailed study, it was concluded thathumic materials behave differently in ESI mass spectrafrom well-characterized ionization standards such as mix-tures of polyethylene glycol [32].

The evidence collected on the capacity of ESI-MS toproduce spectra of unfragmented and predominantly singlycharged humic ions supports the application of this tech-nique to attempt the evaluation of average molecular

weights of humic samples. Previous works have constantlyutilized humic and fulvic acids originating from aquaticsources. It is the objective of this work to apply the ESI-MStechnique to obtain average molecular weights of humicsubstances extracted from soil and the relative size-fractionsseparated from the bulk extracts with preparative HPSEC.

Experimental

Humic samples and treatments

A humic (HA) and a fulvic acid (FA) from a Fulvudand soil of thevolcanic caldera of Vico, near Rome, Italy, and a humic acid (NDL)from a North Dakota Lignite were isolated by standard methodsand characterized as reported elsewhere [10, 35]. The humic acidsamples were titrated to pH 7 with a 0.5% NH4OH solution in anautomatic titrator (VIT 90 Videotitrator, Radiometer, Copenhagen)under N2 atmosphere and stirring. After having reached the con-stant pH 7, the solution containing ammonium-humate was left undertitration for two more hours, filtered through a Millipore 0.45 µm,and freeze-dried. Three replicates of 10 mg of both HA and NDLammonium humates were dissolved in 10 mL of Milli-Q water andthe solution ultracentrifuged at 96,000×g in a Heraeus-Omikronultracentrifuge. The supernatant was separated and ultracentrifugedagain at 140,000×g. Both supernatants were stored below 4 °C forESI-MS analysis.

Preparative HPSEC separation

A Biosep SEC-S-2000 (600 mm×21.2 mm i.d.) column precededby a Biosep SEC-S-2000 Guard Column (78.0 mm×21.2 mm i.d.)by Phenomenex was used. A Gilson 305 pump, a Gilson autosam-pler model 231, a Gilson FC205 fraction collector, and a Perkin-Elmer LC-75 UV/Vis detector set at 280 nm were used to automat-ically isolate humic fractions in continuous. A Perkin-Elmer Nel-son 1022S integrator was used to automatically record all chromato-graphic runs. Elution flow rate was set at 1.5 mL min-1. Due to thelack of proper calibration standards for HS [10], protein standardsof known molecular weights (205, 64, 20 and 6.0 kDa) by Pharma-cia (Standard Molecular-Weight Kit) were used to calibrate thecolumn with nominal molecular-weight ranges.

The freeze-dried HA ammonium salt and FA were dissolved ina 0.05 M NaCl solution (0.2 mg mL–1) and subjected to preparativeHPSEC, as described elsewhere [11], under a 0.05 M NaCl elutingsolution. The shapes of the preparative chromatograms for HA andFA (not shown) had the classic bimodal and monomodal size dis-tribution, respectively. Eleven size-fractions were collected for HAover 50 HPSEC chromatographic runs by changing vials in a auto-matic fraction collector every 5 minutes between 60 and 115 minof each elution. Nine size-fractions were collected likewise for FAwithin the 65–105 min interval of the HPSEC elution. Negligiblelosses by adsorption of humic material on HPSEC columns werepreviously verified [11, 36]. Over 5 consecutive runs, the percentOC recovery by a Shimadzu TOC analyzer was 98.7±0.6% of theinitial OC injected in the preparative column. The column calibra-tion was verified by running standards every tenth chromatographicseparation of humic material. The collected fractions were firstfreeze-dried, dialyzed against Milli-Q water until chloride-free,and freeze dried again. Both the bulk HA and FA and their sepa-rated size-fractions were again solubilized in Milli-Q water, adjustedto pH 8 with a 25% ammonia solution, in order to reach a concen-tration of 1 mg mL-1, and subjected to direct infusion injections.

Electrospray Ionization Mass spectrometry

Mass spectra of humic matter were obtained using a TSQ 7000 massspectrometer (Thermo Finnigan, Bremen, Germany) equipped with

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a ESI source operating in both positive and negative mode. ForESI, the best conditions were found to be: ionization voltage at 4 kV;transfer capillary temperature at 220 °C; tube lens and skimmer at60 V. Nitrogen was employed as both the drying and nebulizationgas. The ionization current was fixed at 5 mA and the detectorvoltage at 1.3 kV. The mass spectrometer was used in the singlescan mode and the first quadrupole was scanned in the mass rangeof 200–2500 amu at a scan speed of 2 s in centroid mode, due tocommon lack of signals in higher amu ranges. Number-averaged(Mn) and weight-averaged (Mw) molecular masses were calculatedby weighted summation of averaged and background subtracted MSspectra, assuming single charged ions, over the entire scan range[37].

Direct Infusion

Sample injection (10–50 µL for measurements by direct infusion;100 µL for on-line HPSEC) was performed with a GINA 50 au-tosampler (Gynkotek) and with a Gynkotek P 580 HDG HPLCpump. For direct infusion injection the flow rate was 0.1 mL min-1

with a solution of 0.1 M ammonium acetate-acetonitrile (9:1). In-fusion measurements were made in triplicates for all samples. Therelative intensities of the base peaks in ESI mass spectra were be-tween 1–3×10-5 and 1–5×10-3.

On-line Size Exclusion Chromatography

Solutions of HA and its previously separated size-fractions at bothpH 8 (adjusted with a 25% ammonia solution) and 4 (adjusted withglacial acetic acid) were also analyzed by ESI mass spectrometryafter a further on-line separation by size-exclusion chromatography.This second HPSEC separation was performed on a PhenomenexGPC3000 (3600 mm×21.2 mm i.d.) analytical HPSEC columnpreceded by a Phenomenex Biosep SEC-S-2000 Guard Column(78.0 mm×21.2 mm i.d.). The eluting solution for the on-lineHPSEC separation was a 9:1, Milli-Q water/acetonitrile, at 0.5 mLmin-1. These HPSEC runs were obtained from triplicate samples. Adiode array spectrophotometer Dionex UVD 340S was used tomonitor the interaction of the eluted fractions with electromagneticradiations. The relative intensities of the HPSEC peaks in ESImass spectra were between 1–5×10-4 and 1–9×10-3. Lower intensi-ties (2–4×10-2) were recorded for some of the peaks from the on-lineHPSEC.

Results and discussion

Mass spectra by direct infusion

The direct infusion into the ion source by negative modeproduced electrospray ionization mass spectra of HA andFA from soil which are similar to the ones previously re-ported by other authors for humic substances from aque-ous sources (Fig. 1). The difference in mass distributionbetween soil humic and fulvic acid is more evident thanfor aquatic samples [19, 21] in that the molecular ions arevisibly spread towards higher masses in soil HA. Also theNDL sample from lignite produced an ESI mass spectrumwith a similar shape to that of soil HA (Fig. 1). The valuesfor Mn and Mw calculated from negative ionization of thesehumic substances were, respectively, 791 and 966 Da forHA, 589 and 834 Da for FA (Table 1), and 761 and 911 Dafor NDL. No significant change in mass values was ob-served when varying tube lens and skimmer from 30 to 60 Vduring ESI ionization.

Positive ion spectra were less intense and had morespurious noise ions than for the negative ion spectra, ashad been already reported for aquatic humic matter [21].Spurious ions in positive mode were attributed to multiplesodium adducts preformed in solution or during ionization[26]. In fact, a progressive decrease in intensity with in-creasing NaCl concentration was experimentally observedin positive ion spectra for fulvic acid from a river [31]. Inthe case of soil humic acids, interference from sodiumions is possibly larger than aqueous humic samples due tothe increased difficulty in purifying a notably less soluble

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Fig. 1 ESI mass spectra by direct infusion of bulk samples: a HA;b FA; c NDL

material from the sodium introduced during extraction withNaOH solution. Since negative ion electrospray spectrawere simpler and more intense, the negative mode waspreferred in the present study.

Results on the bulk extract from terrestrial sources in-dicate not only that the number- and weight-averaged mo-lecular weight of a humic and a fulvic acid from soil arecomparable to those found for aquatic humic material [19,31], but also that the average molecular mass of soil humicmatter is far lower than the values that are commonly re-ported for molecular mass of humic “polymers” in soil [9].

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Table 1 Mn and Mw values (Da) of HA and FA bulk samples and their size-fractions by electrospray negative ionization. Standard de-viation in parentheses

HA FA

Fraction Nominal Mw Mn Mw Fraction Nominal Mw Mn Mw(kDa)a (kDa)a

Bulk 791 (±15) 966 (±33) Bulk 589 (±12) 834 (±22)1 205–186 648 (±29) 911 (±16) 1 167–148 476 (±8) 756 (±33)2 186–167 686 (±13) 943 (±32) 2 148–129 538 (±9) 790 (±33)3 167–148 676 (±25) 921 (±19) 3 129–110 477 (±8) 771 (±44)4 148–129 659 (±28) 888 (±23) 4 100–91 584 (±15) 792 (±36)5 129–110 652 (±27) 868 (±16) 5 91–72 570 (±10) 787 (±31)6 100–91 651 (±14) 871 (±22) 6 72–54 603 (±12) 822 (±45)7 91–72 603 (±18) 831 (±24) 7 54–36 594 (±13) 808 (±35)8 72–54 586 (±15) 821 (±20) 8 18–7 538 (±8) 759 (±34)9 54–36 586 (±21) 823 (±17) 9 7–1 550 (±8) 753 (±57)

10 18–7 598 (±27) 839 (±20)11 7–1 576 (±21) 816 (±31)

a Nominal MW were based on a column calibration obtained with standard proteins.

Fig. 2 ESI mass spectra by direct infusion of some HA size-fractionsseparated by preparative HPSEC: a HA1; b HA4; c HA7; d HA11

Moreover, the molecular mass values by ESI-MS are in thesame order of magnitude as those found by DOSY-NMRspectroscopy [16].

The ESI mass spectra of both the HA and FA size-frac-tions previously separated by preparative HPSEC are shownin Figs. 2 and 3. The mass spectra appearance is very sim-ilar for the different size-fractions regardless of their elu-tion volume and associated nominal molecular-size range(Table 1). Likewise, the Mn and Mw values calculated fromthese ESI mass spectra did not differ substantially amongsize-fractions (Table 1). This result was unexpected and sowas the small difference in average molecular mass ob-served between the bulk HA and FA samples and theirfractions separated by size-exclusion at very different elu-tion volumes. A possible explanation is that, despite sim-ilar shapes in ESI mass spectra, the molecular composi-tion of each fraction was somewhat different and thisshould have had an effect on the formation of ions duringelectrospray ionization. It is known that when mixtures ofcompounds, such as humic substances, are analyzed byESI, those present at the surface of the droplets generatedby the electrospray source can completely mask the pres-ence of compounds which are more soluble in the droplet

bulk [34]. Since electrospray desorption of charged mole-cules occurs from the surface of an aqueous droplet, com-pounds in larger concentration at the droplet surface arethe most hydrophobic molecules due to thermodynamicfactors such as the hydrophobic effect [38, 39], and mayhave a preferential ESI sensitivity in comparison to morehydrophilic molecules maintained well dissolved by waterin the inner droplet.

Size-fractions of the NDL humic acid, which were sep-arated by preparative HPSEC, were found by Pyrolysis-GC-MS and 1H-NMR spectroscopy to contain mainly hy-drophobic compounds (saturated and unsaturated long alkylchains and fatty acids) in the apparently large molecular-size fractions, whereas substituted aromatic compoundsand shorter unsaturated alkyl chains as well as more hy-drophilic compounds were progressively present in lowermolecular-size fractions [11]. It is then plausible that thehighly hydrophobic compounds present in the HA and FAsize-associations of larger nominal molecular-size mayconcentrate at the surface of the droplet and be preferen-tially ionized by ESI in respect to the hydrophilic compo-nents which remain confined in the inner droplet. In thiscase, the average molecular mass would be preferentiallydominated by the hydrophobic components. On the otherhand, a progressive decrease of hydrophobic compositionin molecular associations of lower nominal molecular-

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Fig. 3 ESI mass spectra by direct infusion of some FA size-frac-tions separated by preparative HPSEC: a FA1; b FA4; c FA6; d FA8

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Table 2 Mn and Mw values(Da) by electrospray negativeionization of peaks of HA size-fractions eluted (first peak ifnot otherwise indicated) byHPSEC. Standard deviations inparentheses

Fraction pH 8 pH 4

Mn Mw Mn Mw

Bulk 985 (±13) 1214 (±20) 961 (±15) 1206 (±41)

HA1 538 (±16) 690 (±18) 571 (±8) 881 (±32)

HA2 568 (±14) 692 (±16) 602 (±7) 973 (±36)

HA3 541 (±15) 650 (±19) 600 (±10) 987 (±31)

HA4 509 (±15) 606 (±19) 1002 (±12) 1213 (±39)

HA5 466 (±31) 564 (±20) 939 (±11) 1164 (±36)

HA6 456 (±22) 553 (±18) 600 (±14) 941 (±34)

HA7 1st peak 506 (±17) 1st peak 608 (±21) 980 (±13) 1254 (±38)2nd peak 458 (±13) 2nd peak 692 (±19)3rd peak 420 (±15) 3rd peak 680 (±20)

HA8 1st peak 458 (±18) 1st peak 563 (±18) 943 (±15) 1237 (±49)2nd peak 449 (±22) 2nd peak 700 (±14)

HA9 1st peak 460 (±14) 1st peak 567 (±22) 1195 (±8) 1470 (±43)2nd peak 445 (±20) 2nd peak 712 (±18)

HA10 1st peak 450 (±17) 1st peak 562 (±19) 1177 (±15) 1450 (±33)2nd peak 443 (±15) 2nd peak 723 (±26)

HA11 1st peak 470 (±21) 1st peak 587 (±18) 1114 (±9) 1398 (±47)2nd peak 445 (±29) 2nd peak 714 (±22)

Fig. 4 ESI total ion chromatogram and mass spectrum of HA bulk sample at pH 8

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Fig. 5 ESI total ion chro-matogram of HA7 size fractionat pH 8 and relative mass spec-tra of: a first, b second, andc third peak

size may produce a more efficient ionization of the polarcompounds present in the droplet. The ESI spectra ofthese size-fractions may then indicate average molecularmasses which are not smaller than for the largest molecu-lar size-fractions, being given by another set of ionizedmolecules. This phenomenon also explains why the Mn

and Mw values for the bulk Ha and FA samples were sodramatically lower than those for the sum of their respec-tive size-fractions (Table 1). In fact, the large concentrationof hydrophobic compounds in the bulk samples (35% ofalkyl carbon was estimated by CPMAS-NMR [35] in HA)may have prevented the full electrospray ionization ofother more water-soluble components.

On-line Size Exclusion Chromatography/ESI-MS

The HA bulk and its size-fractions were again subjectedto ESI-MS after a further chromatography through an an-alytical HPSEC column. Values for Mn and Mw were alsocalculated from mass spectra of eluted peaks when the in-jected HA solutions had a pH of either 8 or 4 (Table 2).The HA bulk sample at pH 8 produced a single peak byHPSEC separation at an elution volume slightly higherthan the column void volume (V0=9.7 mL) and the relativeESI spectrum had a mass distribution similar to that ob-tained by direct infusion (Fig. 4). Likewise, the HA1 size-fraction and the following size-fractions of progressivelylower molecular-size up to HA6, gave a major single peakeluting at the V0, whose ESI spectrum had similar mass

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Fig. 6 ESI total ion chro-matogram of HA11 size frac-tion at pH 8 and relative massspectra of: a first, and,b second peak

distribution as those by direct infusion, though with somenotable variation in the intensity of specific masses. Fromfraction HA7 to HA11, a second and third smaller peakbecame visible in the HPSEC elution profile detected bythe mass spectrometer. For example, Figs. 5 and 6 showthe elution chromatograms and the mass spectra of elutedpeaks for fractions HA7 and HA11, respectively. The massspectra of the first major peak of both these fractionsshowed a mass distribution again typical of humic matter,though centered at lower m/z values than for the bulk sam-ple and size-fractions of larger nominal molecular size.Less complex mass spectra were produced by the secondand third elution peaks with fewer and more resolved m/zsignals (Figs. 5 and 6). This suggests that the analyticalHPSEC elution was capable of further separating distincthumic molecules from the size-associations previouslyisolated by preparative HPSEC and that they are efficientlyionized by ESI. Moreover, it is notable that such an in-creased separation through the on-line analytical HPSECoccurred to a detectable extent only for the lower nominalmolecular-size fractions in which the content of hydropho-bic components is presumed to be progressively lower [5].

The Mn and Mw values calculated from the mass spec-tra (Table 2) of the bulk sample eluted through the on-lineHPSEC column were higher than for direct infusion(Table 1), whereas those of size-fractions passed throughthe on-line column were significantly lower than the val-ues found for both the eluted bulk sample and the size-fractions analyzed by direct infusion. This finding appearsto be consistent with the method of isolation of the differentsamples by preparative HPSEC, whereby the bulk sampleshould have a larger average molecular mass than itssmaller size fractions. However, the appraisal of an aver-age molecular mass of a size-fraction after an on-linechromatography seems still linked to the effective electro-spray ionization of all the components present in the elutedpeaks. In fact, while the Mn and Mw values, for the elutionat pH 8, somewhat decreased with fractions of lowernominal molecular weight, the sum of fraction values wasstill larger than the value of the bulk sample (Table 2), asalready observed for direct infusion. Moreover, for theHA7-HA11 fractions, the average molecular mass (Mw) ofthe first eluted peak was significantly lower than the sec-ond peak, due to the larger relative importance of highermass peaks in the spectrum of the latter. Again, a differ-ence in fraction composition, namely a selective concen-tration of hydrophobic compounds in the first peak con-

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Fig. 7 ESI total ion chromatogram and mass spectrum of HA bulksample at pH 4

taining material associated in larger size, may be respon-sible for the different ionization sensitivity by ESI-MS forthe two peaks.

Similar results were found when the same size-frac-tions were brought to pH 4 by acetic acid addition beforethe on-line HPSEC/ESI-MS. However, the sensitivity ofthese mass spectra was considerably lower (2.2–8.1×102)than for the mass spectra of pH 8 samples. Reduced in-tensity for an aquatic humic acid brought to pH 3 was also

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Fig. 8 ESI total ion chromatogram of HA4 size fraction at pH 4 andrelative mass spectra of: a first, b second, c third, and, d fourth peak

observed by Persson and coworkers [21] when analyzedby on-line size exclusion chromatography/ESI-MS. Thetotal mass chromatograms of the humic samples at pH 4(Figs. 7 and 8) showed that the elution began at highervolumes (15 mL) and was spread over a larger elutionrange than for the samples at pH 8. The capacity of aceticacid to disrupt the loosely-bound association of humic sub-stances, thereby resulting in varied size-exclusion chro-matograms, has been well described [5, 10, 11, 16, 23].The bulk sample eluted a single peak whose mass-spec-trum shape was not different from that at pH 8 (Fig. 7).Conversely, all size-fractions showed from 2 to 4 detectablepeaks. For example, 4 peaks were separated by the on-lineHPSEC of HA4 (Fig. 8). However, while the first peak ofHA4 had a mass spectrum reconducible to the appearanceof other humic samples, the subsequent three peaks gaveinstead a less complex spectrum pattern of four well re-solved signals at 469, 387, 305, and 223 m/z, which showeda constant difference of 82 D. An ESI-MS/MS analysis ofeach of these masses revealed that, instead of molecularions, they are clusters of the 41 D mass, that can be attrib-uted to acetonitrile present in the elution solution. It ispossible that these clusters were formed in the presence ofresidual Na+ ions eluting at, and even after, the total ex-

clusion volume of the HPSEC column. The diode-arraychromatogram did not reveal distinct signals at the sameelution volumes, thereby confirming the assignment toclusters of the peaks visible in the ESI chromatogram andthat these peaks contained ionization artifacts rather thanhumic molecules.

The Mn and Mw values calculated from the on-lineHPSEC/ESI-MS spectra of size-fractions at pH 4 (Table 2)were significantly higher than for direct infusion (Table 1)and they appeared to increase with decrease of nominalmolecular size of fractions (Table 2). An increased effi-ciency in electrospray ionization may be advocated to ex-plain the higher average molecular masses of size-fractionsat pH 4, whereby humic associations may have been dis-rupted into less complex molecular arrangement due tothe dispersing action of acetic acid [5, 10, 11, 16, 23].However, the lower intensity achieved in the mass spectraof the fractions at pH 4 and the consequent higher noise atlarger molecular mass may have biased the Mn and Mw

values towards a higher molecular mass.

Ultracentrifuged humic samples

To verify that the efficiency of electrospray ionization mayvary in humic samples according to their composition andconsequent distribution of their heterogenous moleculesin the ESI droplet, an ultracentrifugation experiment wasconducted. The bulk HA and NDL ammonium-humateswere completely dissolved in water and subjected to ul-

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Fig. 9 ESI mass spectra by direct infusion of supernatants of bulkhumic acids after sequential ultracentrifugation: a HA ultracentri-fuged at 96,000×g; b sample from “a” after a second ultracentrifu-gation at 140,000×g; c NDL ultracentrifuged at 96,000×g; d samplefrom “c” after a second ultracentrifugation at 140,000×g

tracentrifugation at 96,000×g. The supernatant was thenseparated from the pellet and further ultracentrifuged at140,000×g. The supernatants from both ultracentrifuga-tions were analyzed by ESI-MS direct infusion in eithernegative or positive mode. The mass spectra obtained bynegative ESI had a baseline sensitivity that varied from of6–8×103 to 2×104 and are shown in Fig. 9. The mass spec-tra of both the HA and NDL supernatants from either96,000 and 140,000×g ultracentrifugation showed a bi-modal mass consisting in a first mass distribution centeredaround 500 m/z and a second one centered at about 1500 m/z.This spectrum shape was different than the monomodaldistribution obtained for the corresponding bulk samplesbefore ultracentrifugation (Fig. 1) which lacked the signalsat higher mass range. In the case of soil HA, quite few dis-tinct masses had large relative abundance at m/z higherthan 1000 Da. The Mn and Mw values calculated from themass spectra (Table 3) were larger than those obtainedwithout ultracentrifugation (Table 1). Moreover, the Mn andMw values in negative mode increased significantly in pass-ing from the 96,000 to 140,000×g for both the HA andNDL samples. These findings suggest that for humic mo-lecular mixtures, the spectrum appearance and the relativeaverage molecular mass is dependent on the efficiency ofelectrospray ionization which seems to be larger in a lesscomplex mixture such as that in the supernatant fromhigher rotational speed. This ultracentrifuge experimentsupports the explanation given above to account for thedifference in average molecular mass found between di-rect infusion and on-line chromatography for both the bulksamples and the size-fractions. This implies that the ab-solute average molecular mass of a humic solution cannotbe accurately measured by ESI-MS if the bulk material isnot made less complex through separation methods.

Conclusions

In conclusion, our results indicate that the average molec-ular masses calculated from ESI-MS spectra of terrestrialhumic substances appear to be in accordance with thosepreviously measured for aquatic humic matter. The valuesfound here for terrestrial humic samples (600–1500 Da)are from one to three orders of magnitude lower thanthose which are commonly reported for molecular weightof soil humic substances. Moreover, these values are inline with the supramolecular model of humic substances

by which relatively small and heterogeneous humic mole-cules self-assemble by multiple non-covalent interactionsin only apparently large molecular sizes. We found incon-sistencies between the average molecular mass of humicsize-fractions when measured by direct infusion ESI-MSand that obtained after an on-line analytical HPSEC.These inconsistencies did not depend on instrumental con-ditions but were attributed to a different relative content ofhydrophobic and hydrophilic compounds in humic size-as-sociations. Their distribution in the humic sample may in-fluence the capacity of electrospray ionization to form ionsfrom all single humic molecules present in ESI droplets.Hydrophobic compounds concentrated at the droplet sur-face may preferentially be ionized, thereby completely in-hibiting ionization of more hydrophilic molecules in theinner droplet. The results of the ultracentrifugation exper-iment confirmed the observed relation between the effi-ciency of electrospray ionization and the composition ofhumic molecular mixtures. This work indicates that a sim-plification of the heterogeneous complex mixture of ter-restrial humic material, through separation methods or se-lective chemical reactions or interactions, is then a pre-requisite to reach viable mass spectra by ESI-MS.

Acknowledgements The first author is grateful for the 1999 awardreceived from the Alexander von Humboldt Foundation that sup-ported his research in Germany. The collaboration of Dr. T. Pfeifer,Dr. K. Sielex, and Mrs. S. Richter in ESI analyses is gratefully ap-preciated.

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Table 3 Mn and Mw values(Da) of supernantants of HAand NDL samples ultracen-trifuged at two rotationalspeeds. Standard deviations inparentheses

Rotational speed, g ESI (+) ESI (–)

Mn Mw Mn Mw

HA96,000 1063 (±20) 1350 (±8) 1194 (±6) 1480 (±9)

140,000 971 (±57) 1285 (±36) 1287 (±43) 1567 (±35)

NDL96,000 1107 (±10) 1376 (±5) 1095 (±2) 1374 (±4)

140,000 1020 (±4) 1330 (±15) 1124 (±14) 1413 (±13)

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