Gel permeation chromatography applied to the study of the nitrohumic acids from coal

Gel permeation chromatography applied to the study of the nitrohumic acids from coal Rafael

Moliner,

J. O&car

and Jose Ma Gavilhn

lnstituto de Carboquimica, C.S.I.C., Apartado 509, Zaragoza-5, (Received 13 October 1981; revised 12 January 1982)

Spain

Gel permeation chromatography on Sephadex (G-types) was applied to nitrohumic acids (NHA) from Iignites. Deionized water and TRIS (Tris-hydroxymethylaminomethane) buffer were tested as eluents. Deionized water provided the same number of fractions and better resolution than TRIS. In both cases, interactions between the gel matrix and the NHA occurred. Five NHA fractions were obtained and characterized by elemental and spectral analysis, which showed significant differences between them. Molecular weights of the fractions, calculated from Cameron’s calibration for soil humic acids, are < 9000 or > 100 000 with no significant fractions having molecular weights in the range 9000-I 00 000. Keywords:

lignites;

nitrohumic

acids; separation

methods

The humic acid fraction extracted from nitrolignite obtained by nitration of lignite, is named nitrohumic acids (NHA); the method is described in a previous paper’. NHA, like other humic acids (HA) extracted from peats, lignites or soils, are composed of different molecular-weight fractions. Isolation of these fractions facilitates a better study of NHA. Gel permeation chromatography (g.p.c.) on Sephadex is a recognized technique for separating fractions of HA from soils and coals and, accordingly, this gel was chosen for the fractional separation of NHA from coal. The use of Sephadex gels can lead to difficulties caused by interactions between the gel matrix and the humic acid molecules. There are two kinds of interactions: the first one operates through the electrons of the aromatic? and has non-polar character; the second one has polar character and acts by means of functional groups such as OH, NH, and COOH, bonded to the aromatic rings3. The intensity of the interactions varies with HA nature, ionic strength and pH of the solvent and the eluent used and hence, choice of these factors is very important for a good chromatographic performance. Several workers have described different chromatographic procedures. Ferrari and Dell’Angnola4 used sodium tetraborate as solvent and eluent. Posne? added electrolytes to the HA samples and used deionized water as eluent. Lindquist6 did not agree with this method and proposed to add the electrolyte to the eluent instead of the the sample. Many authors’** have used NaOH (0.1 N) as solvent and deionized water as eluent, but Swift and Posnerg rejected this eluent as they observed the molecular-weight distribution obtained changed significantly with the HA concentration in the sample solution. According to these authors, the best eluent is an alkaline buffer which contains high-molecular-weight amines, such as TRIS (Tris-hydroxymethylaminomethane, dissolved in hydrochloric acid at pH = 9). TRIS is a widely used buffer eluent in the g.p.c. of HA, but several workers”,’ ’ have continued using deionized water as eluent when studying the fractions, as they are 0016-2361/82/050443~4$3.00 0 1982 Butterworth & Co (Publishers)

Ltd.

(chemical

analysis))

collected without foreign substances which are introduced when an eluent different to deionized water is used. In this Paper, deionized water and TRIS are tested as eluents for NHA and the results are compared. However, to determine the molecular weight of HA fractions obtained by g.p.c. the difficulty of finding suitable standards for column calibration has to be overcome. Many workers have used dextrans and proteins as calibration standards or have taken the exclusion limits provided by the manufacturers for the gels ‘,12-15 as standards. Cameron et ~1.‘~ calibrated a Sephadex G-100 column with TRIS using as a standard soil humic fractions for which molecular weights had been determined by ultracentrifuge sedimentation. HA from soil and NHA from coal are similar substances and hence, Cameron’s calibration was considered adequate for the NHA molecular-weight determination.

EXPERIMENTAL Two different NHA samples, obtained from a black lignite from Utrillas, Spain, (UNHA) and a brown lignite from Puentes, Spain, (PNHA) were chromatographed without mineral matter removal so as not to break molecular aggregates stabilized by the mineral matter. Water eluent experiments

G.p.c. was carried out on Sephadex G-25, G-50 and G-75 grades ” . The position of each fraction in the gel chromatogram was characterized by means of its Kav value which was defined by Laurent and Killanderlg as:

I&v=!?!&? t 0

(1)

where; V, elution volume; V,, void volume; and V, total volume of the gel bed. The excluded fraction (Kav =0) obtained from each gel grade was chromatographed on

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1982,

Vol 61, May

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G.p.c. of nitrohumic acids from coal: R. Moliner et al. TRIS eluent experiments

Dry Sephadex gels were swollen with TRIS buffer, and NHA were dissolved in 0.1N NaOH or TRIS. Chromatographic conditions were similar to those used in the water eluent experiments. RESULTS AND DISCUSSION Deionized water experiments

98 95 Elution

68 volume

52 (cm3)

Figure 1 NHA fractions obtained through G-25 using deionized water as eluent. Optical density of the effluent at 254 nm is plotted versus the eluted volume (cm3). DL: Bed dimensions 20 cm x 2.5 cm; V,: sample volume; C3: solute concentration in the sample, -, 1 mg ml-l; - - -, 0.5 mg ml-l ; UF : flow eluent, 30 ml h-l; Vt : total volume of the bed; Vo : void volume of the bed. A, Fraction 3U; B, fraction 2U;C. fraction U

160 Elutron

volume

120 92 4 (cm31

NHA adsorption on the matrix gel but with norepeatibility were observed from the first to the fifth runs, but in the following runs repeatibility was obtained and the column was considered ready for use. Figures I,2 and 3 show the UNHA fractions. Table 1 lists the Kav values, elution pH and weight ratios for the UNHA and PNHA fractions. Abrupt pH variations of eluents were not observed when fractions were eluted from the column. Most of NaOH eluted as a peak with Kav=2. A set of experiments was carried out to test the performance of the chromatographic column: (1) Fractions lU,and 2U were chromatographed again on Sephadex G-25. Differences between the Kav values were lower than the accuracy of the measurement (5%). (2) Fraction 7U was chromatographed on Sephadex G25. All the material eluted with Kav = 0. (3) UNHA were chromatographed on Sephadex G-25 and G-50: the fraction excluded (Kav = 0) from G-25 was larger than that excluded from G-50, in agreement with theoretical behaviour. The anomalous behaviour described by Swift and Posner* was not observed. (4) Different concentrations of samples were tested on Sephadex G-50: elution volumes and fraction ratios did not vary appreciably when sample concentrations were lower than 2 mg ml-‘. Changes were observed with higher concentrations, such as those described by Swift and Posner for HA from soil’. Nevertheless, this behaviour is not only due to the eluent, as it has been observed with deionized water and TRIS. (5) The pH of sample solutions influences the results: when NHA solution at pH = 2.5 is chromatographed on

5

Figure 2 Fractions obtained from fractions 1 U plus 2U through G-50 using deionized water as eluent. Optical density of the effluent at 254 nm is plotted versus the eluted volume (cm31. DL : Bed dimensions, 32.5 cm x 2.5 cm; l/s: sample volume, 3 ml; Cs: solute concentration in the sample, 1 mg ml-l; UF : flow eluent, 30 ml h-l; V, : total volume of the bed; Vo : void volume of the bed. A, Fraction 7U; B, fraction 6U;C. fraction 5U; 0, fraction 4U

the next grade. NHA samples were introduced into the column dissolved in O.lN NaOH and eluted with deionized water. Absorbance of the eluents at 254 nm and 258 nm was measured and recorded. The weights of the fractions were determined by weighing the dried precipitate obtained after the fractions had been precipitated with HCl. NHA concentrations in the sample solutions were 1 mg ml-’ or lower, as higher concentrations modify the elution volume of the fractions because of variations in effective molecular size and viscosity effects18. Various eluent flows up to 0.5 ml mini, a sample volume of 1% of bed volume and bed lengths up to 30 cm were used.

444

FUEL, 1982, Vol 61, May

VO I

124 118 100 Elutron volume (cm31

44

Figure 3 Fractions obtained from fraction 4U though G-75 using deionized water as eluent. Optical density of the effluent at 254 nm is plotted versus the eluted volume (ems). DL : Bed dimensions, 23.5 cm x 2.5 cm; Vs : sample volume, 30 ml: Cs: solute concentration in the sample, 1 mg ml-t; U,z: flow eluent, 25 ml hl/r: total volume of the bed; V, : void volume of the bed. A, Fraction 1OU; B, fraction 9U; C, fraction BU

G.p.c. of nitrohumic Table 1 Fractions obtained from the UNHA

and PNHA

using deionized

acids from coal: R. Moliner

et al.

water as eluent UNHA

G-25 Fraction Kav value PH” Weight ratio f% total NHA)

1ub 0 5.7

G-75

G-50

2lJb 0.36 7.1

3u 0.93 8.6 2.4

4u 0 5.7

5Ub 0.38 6.4

6Ub 0.83 7.9 20.1

7U 1.16 9.1 23.7

8U 0 7.3 50.3

9u 0.75 7.5 0.8

1ou 1.08 7.8 1

PNHA G-25 Fraction Kav pHa Weight ratio (% total NHA)

1P 0 5.6

2Pb 0.92 8.5

5Pb 0.37 7.1

6Pb 0.63 8.8 73.2

“0

I 19 volume

4P 0 5.5

7P 1.08 9.5 4.2

8P 0 6.5 12.6

9Pb 1.25 8.4

1OPb 1.35 9.5 1

was not possible

“t El&on

3Pb 1.03 9.1 9.3

a Taken at the maximum of the peak b Collected together because separation

G-75

G-50

I 6.6 (cm3)

Figure 4 Fractions obtained from fraction 1 U solved in 0.1 N NaOH, through G-50 using TRlS as eluent. Optical density of the effluent at 254 nm is plotted versus the eluted volume (cm3). DL : Bed dimensions, 30 cm x 0.9 cm; Vs: sample volume, 0.6 ml; CS: solute concentration in the sample, 1 mg ml-*; UF: flow eluent, 8 ml h-l; V,: total volume of the bed; V,: void volume of the bed. A, Fraction 4T, Kav = 1.2; 6, fraction 3T, Kav = 0.95; C, fraction 2T, Kav = 0.45; D, fraction lT, Kav = 0

Sephadex G-25, only a fraction (Kav = 0) is obtained. This effect is due to the increase of hydrogen bonds, which increase the molecular size. TRIS buffer experiments

The same number of fractions were obtained from NHA using either TRIS or water, but TRIS provided poorer resolution than water. pH of the sample solution also influenced the number of fractions, so that when the UNHA were dissolved in O.lN NaOH more lowmolecular-weight fractions were obtained than when dissolved in TRIS. Figure 4 shows the chromatograph of fraction 1U dissolved in O.lN NaOH on Sephadex G-50. Fraction 4T eluted with Kav > 1, indicating that there are interactions with the gel matrix. To test this hypothesis the UNHA

fractions listed in Table I were dissolved in TRIS and chromatographed on Sephadex G-100 with TRIS as eluent. All the fractions behaved as individuals and their Kav values kept the same order in relation to that shown when water was used as eluent. Fractions 2U, 6U and 7U exhibited Kav > 1. A low-molecular-weight substance containing aromatic rings and phenolic groups, such as alizarin sulphonic acid, was chromatographed on Sephadex G-100; the Kav value of 1.4 indicated that reversible interactions had occurred. Irreversible interactions were observed when HA from a black lignite with a high aromatic condensation was chromatographed on Sephadex G-100. The majority of the HA were fixed by the gel matrix and only a small fraction was taken off the column when TRIS was passed through. Molecular weight of NHA fractions

Molecular weights of the fractions were determined by means of Cameron’s calibrationi6, using the Kav values listed in Table 2. Table 2 also shows the results of the determinations. Molecular weights of some fractions were not determined because their Kav values were > 1. Molecular-weights of fractions 7U and 7P should be between 4000 and 9000 as these are the exclusion limits given by the manufacturers for Sephadex G-25 and G-50 and it is probable that any particular grade of Sephadex would exclude lower-molecular-weight humic acids than the nominal value indicates5. As shown in Tables 1 and 2, 46.27; of UNHA have molecular weights ~9000 and 50.3% molecular weights > 100000. In the PNHA, 86.7% have molecular weights < 9000 and 12.6% have molecular weights > 100000. From the results provided by g.p.c. it can be concluded that neither UNHA nor PNHA have significant fractions with molecular weights in the range 900&100000. Physico-chemical characterization of NHA fractions UNHA fractions listed in Table 1 were characterized by

means of elemental analysis and i.r. and U.V.spectroscopy to establish the more significant differences. In general, the C content of the fractions increased and the 0 content decreased (Table 3) in increase in molecular weight. 1.r. spectra of the fractions have the same characteristic

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G.p.c. of nitrohumic acids from coal: R. Moliner et al. Table 2 Molecular weighrs of the nitrohumic

acids fractions

UNHA Fraction

Kav value

3u 5U. 6U 7u 8U 9u

>l

Molecular

0.74 >I

4000 0 0.63

a Nominal

Table3

PNHA

exclusion

limits of G-25

Physical-chemical

and G-50

characterization

weight

<400oa 5500 < MWa <9000 >100000 7500

Fraction

Kav value

2P, 3P BP, 6P 7P

>l

Molecular

0.83 >l

8P 9P

4000 0 0.65

weight

<4000 4200 < MWa <9OCQ >100000 7750

gels using water as eluent15

of the nitrohumic

acids fractions

Fraction

C (wt %, daf)

0 (wt %, daf)

I1 l/116a

IrIb

Molecular

3u 5U. 6U 7u 9u 8U

42.25 45.9 56.9 59.6

40.5 48.0 37.6 30.4

1 .I38 1.428 1.377 1.228

-0.00454 -0.00454 -0.00387 -0.00379 -0.00230

<4000 5600 4000 < MW <9000 7500 >100000

a Intensity of band at 1720 cm-l I intensity of band at 1600 cm-* b Slope obtained from log E =6 f mh where E, optical extinction; b, constant;

bands, but there are differences in the ratio of the intensity of the band at 1720 cm- ’ to the intensity of the band at 1600 cm-’ (Il,/Il,). Th is ratio is very significant for humic substances characterization” and is higher when the ratio ofcarboxylic-CO groups to quinonic-CO groups is higher. All u-v.-visible spectra have a unique absorption band at 20&210 cm-‘. Significant differences are observed in the slope of the curve from the visible spectra for the various fractions (Table 3). When the logarithm of the optical extinction, E, is plotted versus 1, a straight line is obtained with slope, m, which increases with the hydrolysable-N and carbonyl group contents2 ‘. This parameter decreases as molecular weight increases. These results agree with those reported by Chen et ~1.~’ for the E,/E, ratio (ratio between the extinction at 465 and 665 cm- ‘) of the HA from soil. CONCLUSIONS Results obtained from water and TRIS experiments indicate that water is a more efficient eluent than TRIS for g.p.c. of NHA from coal on Sephadex G-types, as the same number of fractions and better resolution can be reached. However, in both cases reversible and irreversible interactions with the gel matrix can occur. NHA fractions obtained with deionized water as eluent have different physico-chemical properties and this is a suitable method to separate the NHA in more homogeneous fractions which are more easy to study. G.p.c. also provides the molecular weights of the fractions although for some fractions molecular weights cannot be determined because the fractions interact with

446

FUEL, 1982, Vol 61, May

weight

h. wavelength

the matrix gel and exhibit Kav> 1, both with deionized water and TRIS as eluent. Hence, a more suitable eluent or gel-eluent system should be sought to determine the molecular weights of NHA by g.p.c.

REFERENCES 1 2 3

11 12 13 14 15 16 17 18 19 20 21 22

Moliner, R. and Gavilhn, J. M. Fuel 1981, 60, 64 Eaker, D. and Porath, J. Separation Science 1967,2(4), 507 Brook, A. J. W. and Mounday, K. C. J. Chromatogr. 1970,47(l), 1 Ferrari, G. and Dell’Agnola, G. Soil Science 1963, 96, 418 Posner, A. M. Nature 1963, 198, 1161 Lindquist, Acta Chem. &and. 1967, 21(9), 2564 Bailly, J. R. and Marguliis, H. Pkznr and Soif 1968,29(3), 343 Butler, J. H. A. and Ladd, J. N. Aust. J. Soil Res. 1969,7,229 Swift, P. S. and Posner, A. M. J. Soil Sci. 1971, 22, 237 Dorado, E., Polo, A. and Del Rio, J. Anales de Edafolgia y Agrobiologia 1972, 31, 693 Bailly, J. R. and Tittonel, E. Plant and Soil 1972, 37, 57 Martin, F. and Saiz Jimenez, C. ZeitschrftftFr Pflanzenerniihrung und Bodenkunde 1973, 135, 58 Butler, J. H. A. and Ladd, J. N. Aust. J. Soil Res. 1969,7,229 Dorado, E., Polo, A., Ar6valo, P. and Villalba, L. Boletin Geolbgico y Miner0 1977,88, 244 Gjessing, E. T. Nature 1965, 208, 1091 Cameron, R. S., Swift, R. S., Thornton, B. K. and Posner, A. M. J. Soil Sci. 1972, 23, 343 ‘Sephadex. Gel Filtration in Theory and Practice’, Pharmacie Fine Chemicals Upsala Sweden Yanca, J. Anal. Chem. 1979, 637 Laurent, T. C. and Killander, J. J. Chromatogr. 1964, 14, 317 Stevenson, F. J. and Goh, K. M. Geochim. Cosmochim. 1971,35, 471 Kleist, H. and Mucke, K. Experimenra 1966, 22, 136 Chen, Y., Senesi, N. and Schnitzer, M. Soil Sci. Sot. Am. J. 1977, 41, 352