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Gel permeation chromatography of water-soluble organic matter with deionized water as eluent—I. Examination of the method
Org. Geochem. Vol. 15, No. 4, pp. 413-417, 1990
0146-6380/90 $3.00 + 0.00 Copyright ~ 1990PergamonPress plc
Printed in Great Britain.All rights reserved
Gel permeation chromatography of water-soluble organic matter with deionized water as elnentml. Examination of the method LUDWIG HAUMAIER,WOLFGANGZECH and GABRIELEFRANKE Lehrstuhl f/Jr Bodenkunde und Bodengeographie, Universit~it Bayreuth, Postfach l0 12 51, D-8580 Bayreuth, West Germany (Accepted 2 April 1990)
Abstract--The elution behaviour of water-soluble organic substances, from mor organic layers under Norway spruce, in deionized water gel permeation chromatography on Fractogel TSK is examined. Sample or gel pretreatments strongly influence the elution patterns. Various interaction mechanisms control fractionation to different degrees, when cation exchange sites on the gel are occupied by K, Ca or H. Retardation effects, previously attributed to cleavage of macromolecules at low pH, can be explained by alteration of gel properties. Size exclusion plays a minor role. Deionized water GPC provides separation primarily by differences in structural composition. Key words--water-soluble organic matter, forest humus, gel permeation chromatography, Fractogel TSK
INTRODUCTION Water-soluble humic substances play an important role in soil forming processes, as well as in the global carbon cycle. They have been investigated extensively, but there is still considerable lack of kowledge about their structural compositions (Aiken et al., 1985; Thurman, 1985). Modern spectroscopic methods, like ~3CNMR, might give some insight into the chemical nature of this class of natural compounds. For spectroscopic investigations, however, fractionation of the complex polydisperse mixture of polyfunctional molecules, into chemically more homogeneous fractions, is advantageous. Gel permeation chromatography (GPC) is widely used as a fractionation method in research on humic substances from soils and waters (Aiken et al., 1985). Separation into fractions differing in molecular size is intended in most cases. However, as is clearly shown by some authors (e.g. Swift and Posner, 1971; Sfchtig, 1975; Saito and Hayano, 1979), GPC gives no fractionation of humic substances, solely on the basis of molecular size differences, when deionized water is used as eluent. Gel-solute interactions, such as adsorption or coulombic repulsion, influence the fractionation to a higher degree than molecular size differences. On the other hand, the gel-solute interactions in deionized water elution can be exploited to give fractions differing in chemical characteristics (Gardner and Landrum, 1983). Another advantage of deionized water elution is avoidance of electrolyte abundance in the fractions which may be a disruptive factor in spectroscopic investigations. In an attempt to find a suitable fractionation procedure for water-soluble organic matter from
forest humus, we investigated the deionized water GPC elution behaviour of an aqueous extract from mor organic layers under different conditions. The chromatography medium we preferred was Fractogel TSK HW because of its resistance to microbial degradation, as well as its greater chemical and mechanical stability compared to the most widely used Sephadex gels.
MATERIALS AND METHODS
The mor organic layer sample was taken in autumn 1985 from a Cryochrept under Norway spruce developed from phyllite near Oberwarmensteinach, Fichtelgebirge, F.R.G.; no distinction was made between Oi, Oe, and Oa horizons. The field moist sample was stored in a freezer. Thawed material (1 kg) was extracted with deionized water (3 1) for 2 h with occasional shaking at room temperature. Coarse particles were removed by filtration through a polyamide net (mesh size 0.053 mm). The filtrate was centrifuged at 4000 rpm for l h and finally filtered through silver membrane filters (0.45 #m, Selas). To ensure identical sample composition for all chromatographic runs, the filtrate was divided into 100 ml aliquots which were stored in a freezer. Tenfold concentrated samples were obtained by rotary evaporation of thawed aliquots at 30°C just before chromatography. Some characteristics of the extract are given in Table 1. The chromatography equipment consisted of a peristaltic pump LKB 2132, a Rheodyne injection valve 5020 equipped with a 5 ml sample loop, a Pharmacia SR 25/100 glass column, an ISCO UA 5
413
LUDWIGHAUMAIERet al.
414
Table I. Characteristicsof the mot organic layer extract DOC Conductivity (mgl i) pH (,uScm-I) Original sample 63 4.13 110 Tenfold concentratedsample 3.40 854 absorbance monitor with type 6 optical unit set at 280 nm, and an ISCO Foxy fraction collector. Fractogel TSK HW-40 (F) (Merck, Darmstadt, F.R.G.) was suspended in 0.1 M KCI and vacuum degassed. The gel, now in the K form, was packed into the column (gel bed dimensions 70 x 2.5 cm) and equilibrated with deionized water. For the conversion of the gel to the Ca form, 0.1 M CaCI 2 was pumped through the column until the effluent contained Ca 2÷. In a similar manner, the H form was obtained by rinsing with 0.01 M HC1 until the column effluent reached pH 2. In each case, equilibration with deionized water followed. Samples, 5 ml of each, were applied to the column and eluted at a constant flow rate of 6 0 m l h -j with deionized water. Void volume (V0) and total volume (Vt) of the gel column were determined with Blue Dextran and acetone, respectively. The exchange capacity of the gel was determined by eluting K or Ca from the gel in its respective form with 21 of 0.1 M HCI. The amount of K or Ca, respectively, in the eluate was determined by AAS. The exchange capacity was found to be 8 pmol ml -~ wet gel for both, K and Ca. Spectrapor membrane tubing (molecular weight cutoff 3500) was used for dialysis. 100 ml portions of a maleic acid solution (100mgl -~) were dialyzed against 1 1 of water adjusted to pH 3, 7, or 9 with HCI or NaOH. During the first eight hours the water was renewed every hour. Maleic acid concentrations in the dialysis tubings were measured by absorbance at 250 nm. RESULTS AND DISCUSSION GPC of the aqueous extract from mor organic layers, using deionized water as eluent, yielded extremely varying results. Chromatograms, obtained under different conditions of sample or gel treatment, are shown in Fig. 1. The same patterns were obtained consistently, when the original gel properties were readjusted. At the original sample concentration no fractionation occurred on the gel in K form [Fig. l(a)]. All the UV-detectable organic constituents were eluted at the void volume (V0) of the gel column. It is known, from aqueous GPC of simple electrolytes on various gels, that the elution b~haviour of electrolytes strongly depends on sample concentration (Neddermeyer and Rogers, 1968; Rochas et al., 1980; Rinaudo and Desbrieres, 1980; Kadokura et al., 1982). At low ionic strengths, simple salts such as NaCI or NaNO3 are partly excluded from the gel pores and elute at or near the void volume depending on the pore sizes of the gel. This effect is due to
electrostatic repulsion between solute ions and negative charges on the gel matrix. Polyelectrolytes behave similarly (Rochas et al., 1980; Rinaudo and Desbrieres, 1980). The observed elution behaviour at low concentration is quite reasonable, because water-soluble humic substances contain carboxyl groups and therefore are electrolytes. As expected of electrolyte behaviour, a small increase in the elution volume was observed at tenfold sample concentration [Fig. l(b)]. Additionally, a slight splitting of the peak indicated the presence of compounds differing in properties. Concentration effects in GPC of aquatic humic substances have been attributed to an increase in the ionic strength of samples during evaporation (Aho and Lehto, 1984). In order to determine whether ionic strength alone is responsible for stronger splitting, and retardation, of concentrated samples we added a large excess (100#molml -~) of KCI to both the original and the tenfold concentrated sample. The differences in total ionic strength should have been negligible now since the initial ionic strengths of unconcentrated and concentrated samples were low (cf. conductivity data in Table 1). The well known "salt boundary" effect (Posner, 1963; Swift and Posner, 1971), that is separation into an excluded and a retarded fraction, was observed for the unconcentrated sample [Fig. l(c)]. However, the elution volume of the retarded fraction did not exceed the total volume of the column, as it did with humic acids (Swift and Posner, 1971). The chromatogram of the concentrated sample [Fig. I(d)] showed a stronger decrease of the excluded fraction, and a third peak with medium elution volume. We infer from these different elution patterns, that either the concentration of the organic molecules themselves also influences the separation, or some other ions in the sample act by other mechanisms than simply by ionic strength at higher concentrations. Gardner and Landrum (1983) concluded, from high performance size exclusion chromatography of river water samples, with different metal salts having been added, that some organic components interacted more strongly with divalent copper and calcium than with monovalent sodium ions. In our case, addition of a small amount of CaC12 (5 #mol ml-~ ) to the unconcentrated sample, prior to chromatography, also altered the elution behaviour [Fig. I(e)]. Three distinct fractions were obtained, the excluded one being much more diminished than in the corresponding KC1 treatment [Fig. l(c)]. This clearly shows that factors other than ionic strength influence elution patterns, if calcium (or presumably other dior trivalent metal) ions are present in a sample. Only a very small portion of the added calcium was eluted together with the sample: more than 99% of the total of 1 mg was retained by the gel. This amount was high enough to alter the column properties, as evidenced from the chromatogram of an original sample
Gel permeation chromatograph '--I
a)
~
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~
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)
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~
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, ~
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aufs1.0 100 200 Ve [ml]
300 ,0
100
200
300
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Fig. 1. Fractionation of the mor organic layer extract on Fractogel TSK HW-40 under various conditions (original sample = sample 0, tenfold concentrated sample = sample 10). obtained after chromatography of the calcium containing sample [Fig. l(f)]. Two peaks can now be clearly distinguished instead of a single peak at the void volume [Fig. l(a)]. In our opinion, the observation that free calcium ions are strongly bound to the gel, and alter the gel properties, is of great relevance to GPC of natural water samples such as soil solutions, river waters, or lake waters, and especially their concentrates. These solutions nearly always contain varying amounts of metal ions. Calcium, or other di- and trivalent metal ions, e.g. Fe 2÷, Fe 3+, AI 3+, not strongly bound to natural ligands can accumulate during consecutive runs on the same gel bed and thus alter the column properties drastically. Ghassemi and Christman (1968) noted that "practically all of the iron became irreversibly bound and could not be eluted" (p. 593) when they applied solutions of ferric chloride, or ferrous sulphate, to a column of Sephadex G75. On the other hand, "in some cases, total iron detected in the effluent was higher than that originally present in the sample" (p. 593). The authors explain this by the
low precision of their test. However, strong complexing natural ligands, not already saturated with metal ions in those samples, may have removed accumulated iron from the gel and thus led to enhanced iron contents in the effluent. Dunemann and Schwedt (1984) used GPC for determination of "free" AI and Fe ions in a soil solution. They reported that these ions were retained by Sephadex gel, but they gave no information about the consequences for the fractionation patterns of subsequent samples. Occupation of all the exchange sites on the gel by calcium (gel in Ca form) led to a fractionation of the aqueous mor layer extract, as shown in Fig. l(g). Again three fractions were obtained, but intensity distribution, as well as elution volumes, were different to those of the sample with calcium having been added prior to chromatography [Fig. l(e)]. Obviously, in the two procedures calcium acts in different ways. We explain this as follows. Addition of calcium ions to the sample results in complex formation and thereby a reduction of negative charges on the solute molecules. Electrostatic repulsion is diminished, and
416
LUDWIGHAUMAIERel al.
other fractionation mechanisms become more important. As can be seen from Fig. l(e), exclusion is in fact drastically reduced. Saturation of the gel with calcium converts the originally negative charges on the gel to positive charges surrounded, however, by again negatively charged chloride ions (Fig. 2). This is obvious from the exchange capacity of the gel which is 8 #mol m1-1 wet gel for both, K and Ca ions, and from the fact that chloride is strongly bound to the Ca-loaded gel. Electrostatic exclusion therefore works again for solute ions not able to replace chloride ions, whereas stronger complexing ligands are retarded by interactions with the calcium ions bound to the gel. Since those replacement reactions obey the law of mass action, higher solute concentration should result in reduced exclusion, and enhanced retardation, due to ligand exchange processes. These considerations are corroborated by the result of chromatography of the tenfold concentrated sample on the gel in Ca form [Fig. l(h)]. Aluminium bound to the gel should expose two positive charges to passing solute molecules. We therefore expected the elution pattern to change markedly by chromatography on aluminium loaded gel. Indeed, the effect was drastic. Nearly all UVabsorbing substances were strongly adsorbed and could not be eluted with deionized water. This, on the one hand, supports our conclusion of ligand exchange processes being predominant in GPC on metal loaded gels, and on the other hand again emphasizes the ability of polyvalent metals to alter gel properties (see above). Protonation of the gel should result in reduction of ionic sites because carboxyl groups, thought to be responsible for ion exchangeability of gels (Gelotte, 1960), are only partly dissociated in aqueous systems. Electrostatic repulsion of ionic solutes therefore should be diminished. The result of GPC of our sample on the gel, in its H form [Fig. l(i)], confirmed these considerations. The excluded fraction was strongly reduced. Again, at higher sample concentration, effects were more drastic [Fig. l(k)]. The more acid concentrate might have suppressed dissociation of carboxyl groups on the gel, and thus led to stronger reduction of electrostatic effects. On the whole, our results are similar to those of researchers who studied elution patterns of aquatic humic substances at different pH, by varying the pH of eluents (Ghassemi and Christman, 1968; Lehto et al., 1986), samples (Gjessing, 1971; Stabel and Steinberg, 1976), or both (De Haan et al., 1983). In each case intensities of the peaks at or near the void volume decreased, and intensities of peaks with
gel/ ^70 Q Ke CaCI2 matrix/--U~O -KCl " - C/O"'~Ca ~,0 j (~) CIG Fig. 2. Conversion of the gel from the K form to the Ca form.
C [rag I"1]
211
[~ ½ '~ 6 8 ' 2r4 hours Fig. 3. Decrease of maleic acid concentration with time during dialysis against water adjusted to (a) pH 9, (b) pH 7, and (c) pH 3. higher elution volumes increased as the pH decreased. The authors conclude that molecular sizes are significantly reduced at low pH. However, our results clearly show alteration of gel properties by ion exchange to be responsible for changes in elution patterns. Ghassemi and Christman (1968) observed identical elution profiles for dextrans and glucose at different pH and concluded that there was no change in Sephadex properties. In contrast to charged humic molecules, however, non-ionic solutes such as saccharides and polysaccharides are hardly affected by the presence or absence of charges on the gel and therefore no influence of pH on elution should be expected. De Haan et al. (1983) compared their results from GPC with those of dialysis experiments and found that a rise in pH increased the amounts of their fulvic acids not passing a dialysis membrane. We studied the dialysis of maleic acid, a small molecule with molar mass 116 for which no great changes in molecular size can be expected, against water of pH 3, 7, and 9. We observed that the passage of maleic acid through the dialysis membrane was strongly retarded at high pH (Fig. 3). Presumably, the same electrostatic effects that are important in GPC also have a determining influence on the dialysis of charged molecules. This leads us to conclude that dialysis too is unsuitable for the determination of changes in molecular size dependent on pH. Results from GPC experiments with the "model compound" maleic acid (Table 2) confirm our conclusions from GPC of the aqueous mor layer extract. Maleic acid is excluded from the gel in K form. During chromatography it is converted to its potassium salt by ion exchange. Retardation occurs on the gel in Ca, A1, and H form. The dependence of the elution volume (V~) on the sample concentration, in chromatography on the protonated gel, suggests again mechanisms other than size exclusion to be responsible for retardation. Table 2. Elutionvolumes V~(ml) of maleicacid in chromatography on various gel forms Maleic acid concentration K form Ca form A1 form H form 0.5 mgml- J 130 295 310 350 25.0 mg ml L 430
Gel permeation chromatography--I The diversity of mechanisms controlling fractionation in deionized water GPC may be used for obtaining chemically different fractions. For instance, we know from preliminary investigations, on other samples not used in this study, that fractions excluded from the gel in K form contain an enormously high amount of carboxyl groups, whereas fractions excluded by the gel in H form contain only few. In addition, differences in the composition of deionized water GPC fractions have been evidenced by the IR and NMR spectroscopic investigations of some other authors (e.g. Tan and Giddens, 1972; Hall and Lee, 1974; Ruggiero el al., 1981) who, however, were not aware of the fact that they did not fractionate by molecular size.
CONCLUSIONS
(l) Molecular sizes have little, if any influence on the fractionation of water-soluble organic matter by gel permeation chromatography with deionized water as eluent. In this respect, our findings agree with results from previous GPC studies of humic acids (Swift and Posner, 1971; S6chtig, 1975). (2) Fractionation mechanisms strongly depend on gel pretreatments. (3) Ion exchange reactions can lead to alteration of gel properties during each chromatographic run. Reproducibility of fractionation therefore requires careful readjustment of the original gel properties before each subsequent run. (4) GPC provides useful fractionation into chemically different fractions. But it always has to be kept in mind that fractions obtained from one sample, need not correspond to fractions with similar elution volumes of another sample, because too many factors (sample concentration, pH, electrolyte contents, presence of metal ions) influence separation. Spectroscopic methods should be used to rationalise differences. Acknowledgements--We thank Dr R. Candler for valuable discussions and the Deutsche Forschungsgemeinschaft for financial support (SFB 137).
REFERENCES
Aho J. and Lehto O. (1984) Effect of ionic strength on elution of aquatic humus in gel filtration chromatography. Arch. Hydrobiol. 101, 21-38. Aiken G. R., McKnight D. M., Wershaw R. L. and MacCarthy P. (Eds) (1985) Humic Substances in Soil, Sediment and Water. Geochemistry, Isolation and Characterization. Wiley, New York.
De Haan H., Werlemark G. and De Boer T. (1983) Effect of pH on molecular weight and size of fulvic
417
acids in drainage water from peaty grassland in NW Netherlands. Plant Soil 75, 63-73. Dunemann L. and Schwedt G. (1984) Zur Analytik yon Elementbindungsformen in Bodenl6sungen mit Gelchromatographie und chemischen Reaktionsdetektoren. Fresenius Z. Anal. Chem. 317, 394-399. Gardner W. S. and Landrum P. F. (1983) Characterization of ambient levelsof ultraviolet-absorbing dissolved humic materials in natural waters by aqueous liquid chromatography. In Aquatic and Terrestrial Humic Materials (Edited by Christman R. F. and Gjessing E. T.), pp. 203-217. Ann Arbor Science, Ann Arbor. Gelotte B. (1960) Studies on gel filtration. Sorption properties of the bed material Sephadex. J. Chromatogr. 3, 330-342. Ghassemi M. and Christman R. F. (1968) Properties of the yellow organic acids of natural waters. Limnol. Oceanogr. 13, 583-597. Gjessing E. T. (1971) Effects of pH on the filtration of aquatic humus using gels and membranes. Schweiz. Z. Hydrol. 33, 592~00. Hall K. J. and Lee G. F. (1974) Molecular size and spectral characterization of organic matter in a meromictic lake. Water Res. 8, 239-251. Kadokura S., Miyamoto T. and Inagaki H. (1982) Aqueous gel-permeation chromatography of electrolytes and polyelectrolytes I. Effect of electrolytic nature of gel. Polym. J. 14, 993-998. Lehto O., Aho J. and V/ih/isarja E. (1986) Elution patterns of aquatic humus in size exclusion chromatography based on different eluants. Aqua Fenn. 16, 47-55. Nedderrneyer P. A. and Rogers L. B. (1986) Gel filtration behavior of inorganic salts. Anal. Chem. 40, 755-762. Posner A. M. (1963) Importance of electrolyte in the determination of molecular weights by "Sephadex" gel filtration, with especial reference to humic acid. Nature 198, 1161-1163. Rinaudo M. and Desbrieres J. (1980) Aqueous gel permeation chromatography of polyelectrolytes and salt exclusion effect. Eur. Polym. J. 16, 849-854. Rochas C., Domard A. and Rinaudo M. (1980) Aqueous GPC of electrolytes and polyelectrolytes. Eur. Polym. J. 16, 135-140. Ruggiero P., Interesse F. S., Cassidei L. and SciacovelliO. (1981) ~H NMR and i.r. spectroscopic investigations on soil organic fractions obtained by gel chromatography. Soil Biol. Biochem. 13, 361-366. Saito Y. and Hayano S. (1979) Application of high-performance aqueous gel permeation chromatography to humic substances from marine sediment. J. Chromatogr. 177, 390-392. S6chtig H. (1975) Gel chromatography as a method for characterization of humic systems. In Humic Substances. Their Structure and Function in the Biosphere (Edited by Povoledo D. and Golterman H. L.), pp. 321-335. Centre for Agricultural Publishing and Documentation, Wageningen. Stabel H. H. and Steinberg C. (1976) Cleavage of macromolecular allochthonous soluble organic matter. Naturwissenschaften 63, 533. Swift R. S. and Posner A. M. (1971) Gel chromatography of humic acid. J. Soil Sci. 22, 237-249. Tan K. H. and Giddens J. E. (1972) Molecular weights and spectral characteristics of humic and fulvic acids. Geoderma g, 221-229. Thurman E. M. (1985) Organic Geochemistry of Natural Waters. Nijhoff/Junk, Dordrecht.
0146-6380/90 $3.00 + 0.00 Copyright ~ 1990PergamonPress plc
Printed in Great Britain.All rights reserved
Gel permeation chromatography of water-soluble organic matter with deionized water as elnentml. Examination of the method LUDWIG HAUMAIER,WOLFGANGZECH and GABRIELEFRANKE Lehrstuhl f/Jr Bodenkunde und Bodengeographie, Universit~it Bayreuth, Postfach l0 12 51, D-8580 Bayreuth, West Germany (Accepted 2 April 1990)
Abstract--The elution behaviour of water-soluble organic substances, from mor organic layers under Norway spruce, in deionized water gel permeation chromatography on Fractogel TSK is examined. Sample or gel pretreatments strongly influence the elution patterns. Various interaction mechanisms control fractionation to different degrees, when cation exchange sites on the gel are occupied by K, Ca or H. Retardation effects, previously attributed to cleavage of macromolecules at low pH, can be explained by alteration of gel properties. Size exclusion plays a minor role. Deionized water GPC provides separation primarily by differences in structural composition. Key words--water-soluble organic matter, forest humus, gel permeation chromatography, Fractogel TSK
INTRODUCTION Water-soluble humic substances play an important role in soil forming processes, as well as in the global carbon cycle. They have been investigated extensively, but there is still considerable lack of kowledge about their structural compositions (Aiken et al., 1985; Thurman, 1985). Modern spectroscopic methods, like ~3CNMR, might give some insight into the chemical nature of this class of natural compounds. For spectroscopic investigations, however, fractionation of the complex polydisperse mixture of polyfunctional molecules, into chemically more homogeneous fractions, is advantageous. Gel permeation chromatography (GPC) is widely used as a fractionation method in research on humic substances from soils and waters (Aiken et al., 1985). Separation into fractions differing in molecular size is intended in most cases. However, as is clearly shown by some authors (e.g. Swift and Posner, 1971; Sfchtig, 1975; Saito and Hayano, 1979), GPC gives no fractionation of humic substances, solely on the basis of molecular size differences, when deionized water is used as eluent. Gel-solute interactions, such as adsorption or coulombic repulsion, influence the fractionation to a higher degree than molecular size differences. On the other hand, the gel-solute interactions in deionized water elution can be exploited to give fractions differing in chemical characteristics (Gardner and Landrum, 1983). Another advantage of deionized water elution is avoidance of electrolyte abundance in the fractions which may be a disruptive factor in spectroscopic investigations. In an attempt to find a suitable fractionation procedure for water-soluble organic matter from
forest humus, we investigated the deionized water GPC elution behaviour of an aqueous extract from mor organic layers under different conditions. The chromatography medium we preferred was Fractogel TSK HW because of its resistance to microbial degradation, as well as its greater chemical and mechanical stability compared to the most widely used Sephadex gels.
MATERIALS AND METHODS
The mor organic layer sample was taken in autumn 1985 from a Cryochrept under Norway spruce developed from phyllite near Oberwarmensteinach, Fichtelgebirge, F.R.G.; no distinction was made between Oi, Oe, and Oa horizons. The field moist sample was stored in a freezer. Thawed material (1 kg) was extracted with deionized water (3 1) for 2 h with occasional shaking at room temperature. Coarse particles were removed by filtration through a polyamide net (mesh size 0.053 mm). The filtrate was centrifuged at 4000 rpm for l h and finally filtered through silver membrane filters (0.45 #m, Selas). To ensure identical sample composition for all chromatographic runs, the filtrate was divided into 100 ml aliquots which were stored in a freezer. Tenfold concentrated samples were obtained by rotary evaporation of thawed aliquots at 30°C just before chromatography. Some characteristics of the extract are given in Table 1. The chromatography equipment consisted of a peristaltic pump LKB 2132, a Rheodyne injection valve 5020 equipped with a 5 ml sample loop, a Pharmacia SR 25/100 glass column, an ISCO UA 5
413
LUDWIGHAUMAIERet al.
414
Table I. Characteristicsof the mot organic layer extract DOC Conductivity (mgl i) pH (,uScm-I) Original sample 63 4.13 110 Tenfold concentratedsample 3.40 854 absorbance monitor with type 6 optical unit set at 280 nm, and an ISCO Foxy fraction collector. Fractogel TSK HW-40 (F) (Merck, Darmstadt, F.R.G.) was suspended in 0.1 M KCI and vacuum degassed. The gel, now in the K form, was packed into the column (gel bed dimensions 70 x 2.5 cm) and equilibrated with deionized water. For the conversion of the gel to the Ca form, 0.1 M CaCI 2 was pumped through the column until the effluent contained Ca 2÷. In a similar manner, the H form was obtained by rinsing with 0.01 M HC1 until the column effluent reached pH 2. In each case, equilibration with deionized water followed. Samples, 5 ml of each, were applied to the column and eluted at a constant flow rate of 6 0 m l h -j with deionized water. Void volume (V0) and total volume (Vt) of the gel column were determined with Blue Dextran and acetone, respectively. The exchange capacity of the gel was determined by eluting K or Ca from the gel in its respective form with 21 of 0.1 M HCI. The amount of K or Ca, respectively, in the eluate was determined by AAS. The exchange capacity was found to be 8 pmol ml -~ wet gel for both, K and Ca. Spectrapor membrane tubing (molecular weight cutoff 3500) was used for dialysis. 100 ml portions of a maleic acid solution (100mgl -~) were dialyzed against 1 1 of water adjusted to pH 3, 7, or 9 with HCI or NaOH. During the first eight hours the water was renewed every hour. Maleic acid concentrations in the dialysis tubings were measured by absorbance at 250 nm. RESULTS AND DISCUSSION GPC of the aqueous extract from mor organic layers, using deionized water as eluent, yielded extremely varying results. Chromatograms, obtained under different conditions of sample or gel treatment, are shown in Fig. 1. The same patterns were obtained consistently, when the original gel properties were readjusted. At the original sample concentration no fractionation occurred on the gel in K form [Fig. l(a)]. All the UV-detectable organic constituents were eluted at the void volume (V0) of the gel column. It is known, from aqueous GPC of simple electrolytes on various gels, that the elution b~haviour of electrolytes strongly depends on sample concentration (Neddermeyer and Rogers, 1968; Rochas et al., 1980; Rinaudo and Desbrieres, 1980; Kadokura et al., 1982). At low ionic strengths, simple salts such as NaCI or NaNO3 are partly excluded from the gel pores and elute at or near the void volume depending on the pore sizes of the gel. This effect is due to
electrostatic repulsion between solute ions and negative charges on the gel matrix. Polyelectrolytes behave similarly (Rochas et al., 1980; Rinaudo and Desbrieres, 1980). The observed elution behaviour at low concentration is quite reasonable, because water-soluble humic substances contain carboxyl groups and therefore are electrolytes. As expected of electrolyte behaviour, a small increase in the elution volume was observed at tenfold sample concentration [Fig. l(b)]. Additionally, a slight splitting of the peak indicated the presence of compounds differing in properties. Concentration effects in GPC of aquatic humic substances have been attributed to an increase in the ionic strength of samples during evaporation (Aho and Lehto, 1984). In order to determine whether ionic strength alone is responsible for stronger splitting, and retardation, of concentrated samples we added a large excess (100#molml -~) of KCI to both the original and the tenfold concentrated sample. The differences in total ionic strength should have been negligible now since the initial ionic strengths of unconcentrated and concentrated samples were low (cf. conductivity data in Table 1). The well known "salt boundary" effect (Posner, 1963; Swift and Posner, 1971), that is separation into an excluded and a retarded fraction, was observed for the unconcentrated sample [Fig. l(c)]. However, the elution volume of the retarded fraction did not exceed the total volume of the column, as it did with humic acids (Swift and Posner, 1971). The chromatogram of the concentrated sample [Fig. I(d)] showed a stronger decrease of the excluded fraction, and a third peak with medium elution volume. We infer from these different elution patterns, that either the concentration of the organic molecules themselves also influences the separation, or some other ions in the sample act by other mechanisms than simply by ionic strength at higher concentrations. Gardner and Landrum (1983) concluded, from high performance size exclusion chromatography of river water samples, with different metal salts having been added, that some organic components interacted more strongly with divalent copper and calcium than with monovalent sodium ions. In our case, addition of a small amount of CaC12 (5 #mol ml-~ ) to the unconcentrated sample, prior to chromatography, also altered the elution behaviour [Fig. I(e)]. Three distinct fractions were obtained, the excluded one being much more diminished than in the corresponding KC1 treatment [Fig. l(c)]. This clearly shows that factors other than ionic strength influence elution patterns, if calcium (or presumably other dior trivalent metal) ions are present in a sample. Only a very small portion of the added calcium was eluted together with the sample: more than 99% of the total of 1 mg was retained by the gel. This amount was high enough to alter the column properties, as evidenced from the chromatogram of an original sample
Gel permeation chromatograph '--I
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300
Ve [ml]
Fig. 1. Fractionation of the mor organic layer extract on Fractogel TSK HW-40 under various conditions (original sample = sample 0, tenfold concentrated sample = sample 10). obtained after chromatography of the calcium containing sample [Fig. l(f)]. Two peaks can now be clearly distinguished instead of a single peak at the void volume [Fig. l(a)]. In our opinion, the observation that free calcium ions are strongly bound to the gel, and alter the gel properties, is of great relevance to GPC of natural water samples such as soil solutions, river waters, or lake waters, and especially their concentrates. These solutions nearly always contain varying amounts of metal ions. Calcium, or other di- and trivalent metal ions, e.g. Fe 2÷, Fe 3+, AI 3+, not strongly bound to natural ligands can accumulate during consecutive runs on the same gel bed and thus alter the column properties drastically. Ghassemi and Christman (1968) noted that "practically all of the iron became irreversibly bound and could not be eluted" (p. 593) when they applied solutions of ferric chloride, or ferrous sulphate, to a column of Sephadex G75. On the other hand, "in some cases, total iron detected in the effluent was higher than that originally present in the sample" (p. 593). The authors explain this by the
low precision of their test. However, strong complexing natural ligands, not already saturated with metal ions in those samples, may have removed accumulated iron from the gel and thus led to enhanced iron contents in the effluent. Dunemann and Schwedt (1984) used GPC for determination of "free" AI and Fe ions in a soil solution. They reported that these ions were retained by Sephadex gel, but they gave no information about the consequences for the fractionation patterns of subsequent samples. Occupation of all the exchange sites on the gel by calcium (gel in Ca form) led to a fractionation of the aqueous mor layer extract, as shown in Fig. l(g). Again three fractions were obtained, but intensity distribution, as well as elution volumes, were different to those of the sample with calcium having been added prior to chromatography [Fig. l(e)]. Obviously, in the two procedures calcium acts in different ways. We explain this as follows. Addition of calcium ions to the sample results in complex formation and thereby a reduction of negative charges on the solute molecules. Electrostatic repulsion is diminished, and
416
LUDWIGHAUMAIERel al.
other fractionation mechanisms become more important. As can be seen from Fig. l(e), exclusion is in fact drastically reduced. Saturation of the gel with calcium converts the originally negative charges on the gel to positive charges surrounded, however, by again negatively charged chloride ions (Fig. 2). This is obvious from the exchange capacity of the gel which is 8 #mol m1-1 wet gel for both, K and Ca ions, and from the fact that chloride is strongly bound to the Ca-loaded gel. Electrostatic exclusion therefore works again for solute ions not able to replace chloride ions, whereas stronger complexing ligands are retarded by interactions with the calcium ions bound to the gel. Since those replacement reactions obey the law of mass action, higher solute concentration should result in reduced exclusion, and enhanced retardation, due to ligand exchange processes. These considerations are corroborated by the result of chromatography of the tenfold concentrated sample on the gel in Ca form [Fig. l(h)]. Aluminium bound to the gel should expose two positive charges to passing solute molecules. We therefore expected the elution pattern to change markedly by chromatography on aluminium loaded gel. Indeed, the effect was drastic. Nearly all UVabsorbing substances were strongly adsorbed and could not be eluted with deionized water. This, on the one hand, supports our conclusion of ligand exchange processes being predominant in GPC on metal loaded gels, and on the other hand again emphasizes the ability of polyvalent metals to alter gel properties (see above). Protonation of the gel should result in reduction of ionic sites because carboxyl groups, thought to be responsible for ion exchangeability of gels (Gelotte, 1960), are only partly dissociated in aqueous systems. Electrostatic repulsion of ionic solutes therefore should be diminished. The result of GPC of our sample on the gel, in its H form [Fig. l(i)], confirmed these considerations. The excluded fraction was strongly reduced. Again, at higher sample concentration, effects were more drastic [Fig. l(k)]. The more acid concentrate might have suppressed dissociation of carboxyl groups on the gel, and thus led to stronger reduction of electrostatic effects. On the whole, our results are similar to those of researchers who studied elution patterns of aquatic humic substances at different pH, by varying the pH of eluents (Ghassemi and Christman, 1968; Lehto et al., 1986), samples (Gjessing, 1971; Stabel and Steinberg, 1976), or both (De Haan et al., 1983). In each case intensities of the peaks at or near the void volume decreased, and intensities of peaks with
gel/ ^70 Q Ke CaCI2 matrix/--U~O -KCl " - C/O"'~Ca ~,0 j (~) CIG Fig. 2. Conversion of the gel from the K form to the Ca form.
C [rag I"1]
211
[~ ½ '~ 6 8 ' 2r4 hours Fig. 3. Decrease of maleic acid concentration with time during dialysis against water adjusted to (a) pH 9, (b) pH 7, and (c) pH 3. higher elution volumes increased as the pH decreased. The authors conclude that molecular sizes are significantly reduced at low pH. However, our results clearly show alteration of gel properties by ion exchange to be responsible for changes in elution patterns. Ghassemi and Christman (1968) observed identical elution profiles for dextrans and glucose at different pH and concluded that there was no change in Sephadex properties. In contrast to charged humic molecules, however, non-ionic solutes such as saccharides and polysaccharides are hardly affected by the presence or absence of charges on the gel and therefore no influence of pH on elution should be expected. De Haan et al. (1983) compared their results from GPC with those of dialysis experiments and found that a rise in pH increased the amounts of their fulvic acids not passing a dialysis membrane. We studied the dialysis of maleic acid, a small molecule with molar mass 116 for which no great changes in molecular size can be expected, against water of pH 3, 7, and 9. We observed that the passage of maleic acid through the dialysis membrane was strongly retarded at high pH (Fig. 3). Presumably, the same electrostatic effects that are important in GPC also have a determining influence on the dialysis of charged molecules. This leads us to conclude that dialysis too is unsuitable for the determination of changes in molecular size dependent on pH. Results from GPC experiments with the "model compound" maleic acid (Table 2) confirm our conclusions from GPC of the aqueous mor layer extract. Maleic acid is excluded from the gel in K form. During chromatography it is converted to its potassium salt by ion exchange. Retardation occurs on the gel in Ca, A1, and H form. The dependence of the elution volume (V~) on the sample concentration, in chromatography on the protonated gel, suggests again mechanisms other than size exclusion to be responsible for retardation. Table 2. Elutionvolumes V~(ml) of maleicacid in chromatography on various gel forms Maleic acid concentration K form Ca form A1 form H form 0.5 mgml- J 130 295 310 350 25.0 mg ml L 430
Gel permeation chromatography--I The diversity of mechanisms controlling fractionation in deionized water GPC may be used for obtaining chemically different fractions. For instance, we know from preliminary investigations, on other samples not used in this study, that fractions excluded from the gel in K form contain an enormously high amount of carboxyl groups, whereas fractions excluded by the gel in H form contain only few. In addition, differences in the composition of deionized water GPC fractions have been evidenced by the IR and NMR spectroscopic investigations of some other authors (e.g. Tan and Giddens, 1972; Hall and Lee, 1974; Ruggiero el al., 1981) who, however, were not aware of the fact that they did not fractionate by molecular size.
CONCLUSIONS
(l) Molecular sizes have little, if any influence on the fractionation of water-soluble organic matter by gel permeation chromatography with deionized water as eluent. In this respect, our findings agree with results from previous GPC studies of humic acids (Swift and Posner, 1971; S6chtig, 1975). (2) Fractionation mechanisms strongly depend on gel pretreatments. (3) Ion exchange reactions can lead to alteration of gel properties during each chromatographic run. Reproducibility of fractionation therefore requires careful readjustment of the original gel properties before each subsequent run. (4) GPC provides useful fractionation into chemically different fractions. But it always has to be kept in mind that fractions obtained from one sample, need not correspond to fractions with similar elution volumes of another sample, because too many factors (sample concentration, pH, electrolyte contents, presence of metal ions) influence separation. Spectroscopic methods should be used to rationalise differences. Acknowledgements--We thank Dr R. Candler for valuable discussions and the Deutsche Forschungsgemeinschaft for financial support (SFB 137).
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