Fractionation and Characterization 

of Natural Organic Matter from 

Certain Rivers and Soils by 

Free-Flow Electrophoresis

GEOLOGICAL SURVEY WATER-SUPPLY PAPER 1817-E
Fractionation and Characterzation 

of Natural Organic Matter from 

Certain Rivers and Soils by 

Free-Flow Electrophoresis

By J. A. LEENHEER and R. L. MALCOLM

ORGANIC SUBSTANCES IN WATER

GEOLOGICAL SURVEY WATER-SUPPLY PAPER 1817-E

UNITED STATES GOVERNMENT PRINTING OFFICE, WASHINGTON : 1973
UNITED STATES DEPARTMENT OF THE INTERIOR 

ROGERS C. B. MORTON, Secretary

GEOLOGICAL SURVEY 

V. E. McKelvey, Director

Library of Congress catalog-card No. 73-600209

For sale by the Superintendent of Documents, U. S. Government Printing Office

Washington, D. C. 20402 - Price 35 cents (paper cov«?r)

Stock No. 2401-02400
CONTENTS

Page

Abstract ___________________________________________ El

Introduction __________________________________ __ 1

Methods and materials __________________________________ 3

Sample preparation ______ _______ _ __ 3

Sample description _________________________________ 3

Separation procedure _______ ___________ __ 4

Assay of fractions ___________________________________ 4

Results and discussion _ _______________ 5

Conclusions ________________________________________ 12

References _____________________________________ __ 14

ILLUSTRATIONS

Page

FIGURES 1-3. Graphs showing:

1. Organic-carbon and optical-density electrophoretic fractionation curves for organic materials from soil and from river 

water ___________________________ E6

2. Organic-carbon and polysaccharide electrophoretic fractionation curves for fulvic acid from soils __________ 7

3. Organic-carbon and polysaccharide electrophoretic fractionation curves for dissolved organic materials from rivers _ 8

4. Infrared spectra of Fairbanks fulvic acid fractions ______ 11

TABLES

Page

TABLE 1. Percentage of polysaccharide carbon at the peak of the polysaccharide

fractionation curve ____________________________ E9 

2. Elemental analysis of Fairbanks fulvic acid fractions _________ ___ 13
ORGANIC SUBSTANCES IN WATER

FRACTIONATION AND CHARACTERIZATION

OF NATURAL ORGANIC MATTER FROM

CERTAIN RIVERS AND SOILS BY FREE-FLOW

ELECTROPHORESIS

By J. A. LEENHEER and R. L. MALCOLM

ABSTRACT

Soluble river organic matter and soil fulvic acids from a variety of environments were compared by examining the free-flow electrophoretic fractionation curves of organic carbon, color, 

and polysaccharides. Significant amounts of virtually colorless organic material were found in 

both the soil and the river preparations. Polysaccharides comprised 20 75 percent of the 

colorless material in the soil fulvic acids but only 3.2-7.0 percent of the colorless material in 

the river preparations. A significant amount of polysaccharides was complexed with organic 

materials having negative charges. Amounts of polysaccharides were greater in the Fairbanks 

soil from Alaska than in the soils from North Carolina or Iowa, and they were greater in the 

Tahquamenon River in Michigan than in the two rivers in Florida; this suggests that 

polysaccharide degradation is slower in cooler environments.

For all of the organic preparations which were fractionated, the intensity of th°- yellow color 

increased as the charge on the organic anion increased. Highly colored, negatively charged 

organic material was found to be present in greater amounts in the subsurface spodic soil 

horizon of the Lakewood and Fairbanks soils than in the surface mollic horizon of the 

Macksburg soil.

Infrared spectroscopy and elemental analysis of four pooled fractions of the Fairbanks fulvic 

acid indicated increasing aromatic character with increasing negative charge. An increase in 

the carboxyl content with negative charge suggests the carboxyl group as the primary source of 

the negative charge.

INTRODUCTION

Similarities have been found between fulvic acid extracts from soils and 

the colored natural organic materials in many lakes and streams. Wilson 

(1959) held that colored organic matter in water is fulvic acid derived from 

soil organic matter. Because fulvic acid is the most water soluble of the extractable organic-matter fractions from soils, more fulvic acid than any 

other soil organic-matter fraction is likely to be found in water. Midwood 

and Felbeck (1968) found that over 80 percent of the carbon present in 

colored organic matter dissolved or suspended in pond water is in the fulvic 

acid fraction. They found the pond organic matter to be resistant to both 

chemical and biological degradation, as is characteristic of soil filvic acids. 

Yellow organic acids of lake waters were named "humolimnic" acids by

El
E2 ORGANIC SUBSTANCES IN WATER

Shapiro (1957) to indicate their probable relation with the natural organic 

acids of the type found in soils.

Black and Christman (1963a) applied soil-science techniques developed 

for characterization of soil organic matter to the chemical characterization 

of colored organic materials in water. Through the use of ar?ular-lightscattering data and ultraviolet spectroscopy, they found that the material 

responsible for color in water exists primarily in colloidal suspension. Data 

obtained from solubility relationships, chemical spot tests, and infrared absorption spectra indicated that fulvic acids found in surface waters are 

aromatic polyhydroxy methoxy carboxylic acids. In a study of organic extracts from some colored streams in western Washington, Christman and 

Ghassemi (1966) found substantial variation in the color-producing properties of organic materials in different streams. They assumed that the water 

did not contain substantial quantities of non-color-producing organic 

materials. Studies on the degradation of organic matter provided evidence 

for the presence of phenolic nuclei in the color macromolecuK

In the past, chemical characterizations of natural organic materials found 

in soils and water have often been performed on gross extracts called humic 

and fulvic acids. These acids are not specific compounds; rath°r, they are 

complex mixtures of organic polyelectrolytes. Thus, these humh and fulvic 

extracts frequently defied characterization because the mixtures were not 

fractionated into more homogeneous components prior to analysis. Recent 

advancements in gel-permeation chromatography and preparative electrophoresis now permit fractionation of humic and fulvic acids in aqueous 

media to be routinely performed prior to further analysis.

Until recently, preparatory electrophoretic methods were limited by such 

problems as adsorption interactions upon an electrolyte supporting curtain, 

inadequate cooling at high voltage and current, and small amounts of sample. High-resolution electrophoretic fractionations of organic-matter extracts were difficult to obtain when an electrolyte supporting medium such 

as filter paper was used in continuous electrophoresis. Adsorption of the 

organic material on the supporting media was the major hindrance, particularly when acidic electrolytes were used. Consequently, organic-matter 

extracts were often hydrolyzed prior to electrophoresis to produce constituents of greatly different mobilities which could be resolved (Johnson, 

1959; Waldron and Mortensen, 1961). Clapp (1957) and Mortensen and 

Schwendinger (1963) resolved colorless soil polysaccharides from a more 

highly colored organic fraction of soil-water extracts.

The development of continuous free-flow electrophoresis has circumvented the adsorption problem. In this method, electrophoretic 

separations are carried out in a self-supported film of buffer between glass 

plates, and large amounts of sample can be fractionated in a relatively short 

time. Free-flow electrophoresis was found to be highly suitable for fulvic 

acid fractionations in aqueous media (Leenheer and Malcolm, 1973).
ORGANIC MATTER FROM CERTAIN RIVERS AND SOILS E3

This report compares free-flow electrophoretic fractionations of soil 

fulvic acids and soluble river organic materials from a variety of environments. Limited characterization of the polysaccharide constituents 

along with visible and infrared spectral analysis aid in the interpretations of 

the electrophoretic fractionations.

METHODS AND MATERIALS

SAMPLE PREPARATION

The soil fulvic acids were extracted according to the following procedure. 

Organic matter was extracted from the soils by using 0.17V sodium hydroxide under nitrogen to prevent auto-oxidation of the organic extract. The 

suspended clay was removed by centrifugation and filtration, and the humic 

acid was precipitated from the fulvic acid by acidifying the filtrate to pH 1 

with hydrochloric acid. The fulvic acids remaining in solution were dialyzed 

against distilled water for removal of the hydrochloric acid. Final purification of the fulvic acid was done by hydroxyl saturation in a hydroxylsaturated anion-exchange resin, followed by hydrogen saturation in a 

hydrogen-saturated cation-exchange resin. The fulvic acid solutions were 

freeze-dried for preservation.

The dissolved organic materials in surface waters were sep" rated and 

purified in the following manner. The sediment material was filtrated out by 

a partly clogged vinyl metricell membrane filter, the metallic cations were 

removed by using a hydrogen-saturated cation-exchange resin, and the 

water was eliminated by freeze-drying. Because the predominant anion in 

the waters selected for this study was the organic anion, removal of the inorganic anions was not necessary to obtain organic preparations having an 

ash content of 5 percent or less.

The freeze-dried fulvic acids and the organic materials extracted from 

river waters were usually dissolved simply by adding disti'led water. 

However, if some of the organic material refused to go into solution, it was 

dissolved in a minimum amount of 0.17V sodium hydroxide solution and 

then was hydrogen saturated by passage through a hydroge^-saturated 

cation-exchange resin. The organic-matter preparations used for electrophoretic fractionation were initially dissolved in water at concentrations 

of 10 20 milligrams per milliliter. The solutions were diluted 1:1 with the 

separation buffer to give final concentrations of 5 10 mnT'grams per

milliliter.

SAMPLE DESCRIPTION

Fulvic acids were extracted from the spodic horizon of soils of the Fairbanks series, collected near Fairbanks, Alaska; from the spodic horizon of 

soils of the Lakewood series, collected near Wilmington, N.C.; and from 

the mollic horizon of soils of the Macksburg series, collected near Des 

Moines, Iowa. The spodic horizon is characterized by illuvial accMmulations 

of free sesquioxides and by metallo-organic salts and complexes of humic 

and fulvic acids which have been leached from the upper sections of the soil
E4 ORGANIC SUBSTANCES IN WATER

profile. The mollic horizon is the deep, darkly colored surface horizon found 

under grass vegetation, which is the source of the organic matter.

The river waters selected for this study were also in a variety of environments. A water sample was taken from Jane Green Creek near 

Melbourne, Fla., to examine the organic material in waters draining a 

hardwood Cyprus swamp. The Sopchoppy River, sampled near Tallahassee, 

Fla., drains an upland area containing coniferous vegetatior; however, 

small areas of Cyprus swamps also drain into the Sopchoppy River. Lastly, 

the Tahquamenon River was sampled at Newberry, Mich., to examine the 

organic material from a northern cedar and tamarack swamp.

SEPARATION PROCEDURE

In this study, the Brinkman Free-Flow Electrophoretic Separator (Model 

FF4) 1 was used. The instrument and its method of operation have been 

described elsewhere (Brinkman Instrument Co., 1969). The methodology 

and the applications of free-flow electrophoresis to the fractionation of 

natural organic materials were discussed in a paper by Lecnheer and 

Malcolm (1973).

The separation buffer selected for this study was 0.1 6M phosphoric acid, 

which gave a pH of 2.2. The buffering capacity of this solution was not overcome even after 15 hours of continuous electrophoresis, and the pH was sufficiently low to give good fractionations based on isoelectric pH differences. 

The electrode rinse buffer was the same concentration as the separation 

buffer. The separation-buffer flow rate was about 100 milliliters per hour. 

The electric field strength was held at 450 volts (30v/cm). Lastly, the 

temperature of each run was set at 10°C, and the sample was injected into 

the separation chamber at the rate of about 6 milliliters per hour. A onehour electrophoresis run at the specified separation-buffer and sampleinjection flow rates produced 106 milliliters of sample diluted by the separation buffer. After the 106 milliliters was equally divided into 90 fractions, 

each fraction contained about 1.2 milliliters. A four-hour electrophoresis 

run produced sufficient volume in each fraction for determinations of 

organic carbon, polysaccharides, and color.

ASSAY OF FRACTIONS

The organic-carbon determination of the electrophoretic fractionation of 

the organic-matter preparations was made by monitoring tl ? organiccarbon parameter on an organic-carbon analyzer. Because the phosphoric 

acid buffer caused the pH of the fractions to be below 4.5, inorganic carbon 

was absent from the fractions, and organic carbon could be directly determined. Each individual organic-carbon determination was made on only 40 

microliters of sample. Aliquots were taken from every other fraction of the 

90 fractions except near the peaks of the organic-carbon curves, where

'Mention of a specific product is for identification only and does not constitute endorsement by the U.S. Geological Survey.
ORGANIC MATTER FROM CERTAIN RIVERS AND SOILS E5

determinations were made on every fraction for high resolution. A complete 

determination of the organic-carbon electrophoretic fractionation required 

2 hours or less.

Polysaccharide concentrations were monitored by a procedure used by 

Clapp (1957). In this procedure, the polysaccharides were hydrolized to the 

constituent sugars by sulfuric acid, and the sugars reacted with a-naphthol 

to form a colored complex, which was measured by absorbance at 570 nanometers. Sucrose was used to determine the standard curve, and watersoluble starch was found to give equivalent results as the polysaccharide 

standard. The absorbance sample blanks of the colored fractions were subtracted from the sample absorbance to obtain the true absorbance values for 

sugars in the colored fractions.

The color of the electrophoretic fractions was determined by absorbance 

at 400 nanometers. Although natural organic materials do not exhibit maximums in the visible and ultraviolet absorbance spectra, 400 nanometers is 

the wavelength commonly selected for the measurement of color of organicmatter solutions.

More extensive characterization of the separates required removal of the 

buffer and drying of the samples. On the basis of the organic-carbon fractionation curve, the 90 fractions were pooled into four fractions for 

characterization. The phosphoric acid buffer was removed by dialysis 

against deionized water in 8-centimeter dialysis tubing for a period of 2 

days. After desalting, the samples were freeze-dried to give fluff}' watersoluble preparations suitable for analysis.

Infrared spectra of the four pooled fractions were run from 2 to 25 

micrometers. Elemental analysis for carbon, hydrogen, oxygen, nitrogen, 

sulfur, and phosphorus and an ash determination were performed b^ Huffman Laboratories, Inc., Wheatridge, Colo.

RESULTS AND DISCUSSION

The initial electrophoretic fractionations indicated that a large proportion of the organic matter extracted from both soils and rivers had little 

color. Figure 1 presents the optical-density (color) curves and the organiccarbon fractionation curves for the Fairbanks fulvic acid and the Jane 

Green Creek organic matter. Although the organic-carbon fractionation 

curves of the two preparations were greatly different, the color curves were 

very similar. For both preparations, the slightly charged fraction which 

peaks near the point of application only gives a slight absorbance for the 

color curve, and the color maximum at tube 52 does not correspond with 

any peak of the organic-carbon fractionation curve. Thus, color serves as a 

very poor index of organic-carbon concentration. Figure 1 indicates that 

color increases as the electrophoretic mobility and the ionic charge increase. 

The highly charged, colored fractions may be more aromatic than the fractions of lesser charge and color because light absorbance occurs in the pi
E6 ORGANIC SUBSTANCES IN WATER

electron system of organic compounds having conjugated double-bond 

systems.

The assumption by Christman and Ghassemi (1966) that river water does 

not contain substantial quantities of non-color-producing organic material 

does not appear to be valid for the Florida rivers of this study. This electrophoretic study indicates that the relationship between color and organic 

matter concentration is dependent on the ratios of the concentration to 

color of the individual organic constituents in solution. In data not 

presented here, the color was found to intensify slightly wh^n the pH was

240

180-

120

60

240

180

120

60

T

T T I T

Sample injected

1.2

0.9

0.6

0.3

1.2

0.9

0.6

0.3

20 40 

TUBE NUMBER

60 90

FIGURE 1. Organic-carbon and optical-density electrophoretic fract; onation curves 

for organic materials from soil and from river water. A, Fairbanks soil fulvic acid, 

Alaska; B, Jane Green Creek, Fla.
ORGANIC MATTER FROM CERTAIN RIVERS AND SOILS E7

raised from 2.2 to 7.0 by addition of sodium hydroxide to the phosphoric 

acid buffer. The absorbance of the entire color curve increased by about 8 

percent for the organic matter from Jane Green Creek. The effect of pH on 

color was also noted by Black and Christman (1963b), who attributed the 

increase in color to the greater amount of dispersion of the organic matter 

at higher pH levels.

The main comparisons of this study are graphically illustrated in figures 2 

and 3. Figure 2 presents the organic-carbon and polysaccharide elec40 

TUBE NUMBER

80 90

FIGURE 2. Organic-carbon and polysaccharide electrophoretic fractionation curves 

for fulvic acid from soils. A, Lakewood series, North Carolina; B, MacksHurg series, 

Iowa; C, Fairbanks series, Alaska.
E8 ORGANIC SUBSTANCES IN WATER

trophoretic fractionations of the soil fulvic acids, and figure 3 presents the 

same parameters for the river preparations. The polysaccharide concentration is based on milligrams per liter of sucrose equivalents.

The organic-carbon curves of the fulvic acid fractionations 1 ave a few 

features in common, but in the main, they differ greatly. All three have a 

slightly charged, slightly colored fraction whose peak at tube 25 corresponds to the main peak of the polysaccharide fractionation, and they all 

have a highly charged, highly colored fraction which peaks at tuHs 46-50.

40 

TUBE NUMBER

FIGURE 3. Organic-carbon and polysaccharide electrophoretic fractionat; on curves 

for dissolved organic materials from rivers. A, Jane Green Creek, Fla.; fl, Fopchoppy 

River, Fla.; C, Tahquamenon River, Mich.
ORGANIC MATTER FROM CERTAIN RIVERS AND SOILS £9

The relative distribution of organic carbon, however, differs greatly for the 

three fulvic acid preparations.

The Lakewood fulvic acid (fig. 2A) is characterized by its large ariount of 

colored, charged material, which peaks at tube 49. Table 1 indicates that 

nearly three-quarters of the organic carbon at the polysaccharide maximum 

is bound in polysaccharides at tube 24. The percentage carbon in 

polysaccharide was estimated to be 40 percent, the same as in glucise. The 

slightly charged peak of the Lakewood fulvic acid has the highest 

polysaccharide percentage in comparison with the other fulvh acids. 

However, inspection of the curves in figure 2 shows that the total amount of 

polysaccharides in the Lakewood preparation is the lowest of the three 

preparations because of the small ampunt of organic carbon in the slightly 

charged fraction. Comparisons of peak areas under the polysaccharide fractionation curve show that more than half of the polysaccharide material 

appears to be complexed with charged organic materials. Secondary 

polysaccharide peaks appear at tubes 40 and 46. In an infrared study of the 

polysaccharide constituents in fulvic and humic acids, Tan and Clark (1969) 

also found chemically bonded polysaccharides in humic materials.

TABLE 1. Percentage of polysaccharide carbon at the peak of the polysaccharide

fractionation curve

Polysaccharde carbon 

Preparation (percent of total carbon)

Soil fulvic acids:

Lakewood soil ___________________________ 74.7 

Macksburg soil ____________________________ 20.0 

Fairbanks soil ___________________________ 42.6

Soluble river organic matter: 

Jane Green Creek _________________________ 3.2

Sopchoppy River __________________________ 5.6

Tahquamenon River ________________________ 7.0

Organic material with greatly different charge, color, and polysaccharide 

characteristics is seen in the Macksburg fulvic acid (fig. IB). Mo^t of the 

material possesses a slight negative charge and has little color, but a significant amount of material is positively charged, as indicated to the left of the 

point of application. The polysaccharide percentage is only 20 percent at 

tube 24 (table 1). The total polysaccharide content is greater than in the 

Lakewood fulvic acid because of the large quantity of slightly charged 

material where most of the polysaccharides occur. The characteristic peak 

at tube 49 shows up only as a slight shoulder in this preparation.

By far the most diversity is found in the electrophoretic fractionation of 

the Fairbanks fulvic acid (fig. 2C). Four distinct peaks and a positively 

charged shoulder are found in the fractionation. A polysaccharide content 

of 42.6 percent (table 1) at tube 25 and substantial quantities of negatively
E10 ORGANIC SUBSTANCES IN WATER

charged complexed polysaccharides give this preparation by far the highest 

total polysaccharide content. The twin peaks at tubes 47 and 50 of the 

organic-carbon curve were also found in two subsequent runs of this fulvic 

acid.

The electrophoretic fractionations of the organic materals extracted 

from river waters exhibited much less diversity. The organic-carbon curves 

in figure 3 show two maximums for each preparation: organic material of 

slight color and charge which peaks near the point of application, and 

colored organic material of high negative charge which p^aks at tubes 

46 58. These characteristic peaks appear near the same positions as the 

peaks in the fulvic acid preparations.

The organic-carbon fractionations from Jane Green Creek and the 

Sopchoppy River are very similar in appearance (fig. 3/4 and E). The 

geographical proximity and climatic similarities of these two streams may 

give rise to closely related types and distributions of organic materials. The 

biggest apparent difference from fulvic acid preparations is tH low level of 

polysaccharides in the river preparations. Table 1 lists only 3.2 7.0 percent 

polysaccharide carbon at the maximums of the polysaccharide fractionations for the river preparations, whereas the polysaccharide carbon 

ranges from 20 to 75 percent for the fulvic acids.

The fractionation of the dissolved organic material from the Tahquamenon River (fig. 3C) differed from the Florida streams ir that the peak 

representing the colored material of high negative charge wa? resolved into 

a more distinct peak. This could be an indication of less diversity in type of 

organic materials in the Michigan river. Also, the polysaccharide content 

was the highest of the three streams.

The Fairbanks fulvic acid was selected for additional characterization by 

infrared and elemental analysis because its fractionation cur^e showed the 

major characteristic peaks common to the other preparations plus peaks at 

tubes 31 and 50, which were not common to the other preparations. A largescale fractionation was performed on more than 400 milligrams of material. 

After fractionation, tubes 22-28, 29-35, 36-48, and 49-65 were respectively pooled and numbered 1 through 4; the four pooled f-actions were 

desalted by dialysis, and the organic material in solution was freeze-dried. 

Each pooled fraction yielded abundant material for both infrared and 

elemental analysis. The infrared spectra of the four fractions are given in 

figure 4.

Various functionalities were ascribed to the different absorption bands. 

The strong absorption band at 2.90/*m (micrometers) is du? to hydroxyl 

stretching, the shoulder band at 3.40/mi is due to aliphatic carbon-hydrogen 

stretching, and the b'road band at 3.7/mi 4.0/im is due to hydrogen-bonded 

hydroxyl indicative of dimeric carboxyl groups. The carbony1 of esters and 

carboxylic acids is at 5.80/mi, the carboxylate ion symmetrical stretch is at 

6.10/im, the nitrogen-hydrogen-bond deformation of mcnosubstituted
ORGANIC MATTER FROM CERTAIN RIVERS AND SOILS Ell
E12 ORGANIC SUBSTANCES IN WATER

amides is at 6.4(Vm 6.5(Vm, and the carboxylate ion asymrretrical stretch 

is at l.\ij,m 1.2iJim.

Several changes in functional-group content were noted from fraction 1 

to fraction 4. Firstly, aliphatic hydrogen at 3.4(Vm is greater in fractions 1 

and 2 than in fraction 3 and especially in fraction 4, where tH band is present only as a weak shoulder. This suggests that fractions 1 and 2, which 

possess a slight charge, have a much greater aliphatic nature than fractions 

3 and 4, which have a higher charge. Secondly, the broad hydrogen-bonded 

hydroxyl band at 3.7//m 4.(Vm becomes progressively stronger from fraction 1 to fraction 4, suggesting that the negative charge in tl °. higher fractions may originate from the greater carboxyl content.

Thirdly, although changes occurred in the carbonyl, carboxyl, and carboxylate bands at 5.80//m, 6.10//m, and 7.lAtm-7.2//m, no special 

significance can be ascribed to them because the preparations were not 

found to be hydrogen saturated after dialysis. This indicates that separates 

should be run through a hydrogen-saturated cation-exchange column after 

dialysis. The ratio of the 5.80//m band to the 6.10//m band is mainly due to 

the ratio of carboxylic acid to carboxylate salt in the sample.

Lastly, the band at 6.4(Vm-6.5(Vm, which indicates monosubstituted 

amides, is strongest in fraction 1 and progressively decreases until it is completely absent in fraction 4. Mortensen and Schwendinger (1963) also noted 

this band in an electrophoretic separate of soil polysaccharides. They 

ascribed this nitrogen functionality to amino sugars and protein contaminants of soil polysaccharides. Such nitrogenous groups are protonated 

at low pH values and neutralize the negatively charged groups this process 

gives rise to Zwitter ions, which have low electrophoretic mobility, as is 

found in fraction 4.

The elemental analysis of fractions 1 through 4 is given in table 2. The 

analyses for carbon, hydrogen, oxygen, and nitrogen are adjusted to an ashfree basis. The total elemental analysis is slightly greater than 100 percent 

because the oxygen values are high. Inorganic oxygen in the ash was determined along with organic oxygen. Therefore, the oxygen analyses have 

limited value because of ash interference. However, the carbon and 

hydrogen analyses clearly indicate increasing aromatic character from fraction 1 to fraction 4 as the carbon-hydrogen ratio increases from 7.8 in fraction 1 to 13.3 in fraction 4.

CONCLUSIONS

Both soil fulvic acids and organic materials dissolved in river waters commonly contain substantial quantities of organic material which possess no 

color and charge. A large percentage of the colorless material was determined to be polysaccharides for the soil fulvic acids, but only a small 

percentage of the colorless material in the river extracts w?s found to be 

polysaccharides. This difference in relative polysaccharide abundance may
ORGANIC MATTER FROM CERTAIN RIVERS AND SOILS El3

TABLE 2. Elemental analysis (in percent) of Fairbanks fulvic acid fractions

[Percentages of elements are adjusted to an ash-free basis]

Element

Fraction

1

2

3

4

Carbon

46.15 

46.90 

51.59 

51.92

Hydrogen

5.92 

5.66 

4.46 

3.95

Ratio of 

carbon to 

hydrogen

7.8 

8.3 

11.6 

13.3

Oxygen

46.06 

47.21 

43.08 

48.22

Nitrogen

2.63 

2.56 

2.67 

2.09

Total

100.8 

102.3 

101.8 

106.1

Ash

6.97 

7.05 

7.19 

14.52

be due to microbial rates of polysaccharide degradation being slower in soils 

than in rivers, or it may simply point to different organic-extraction efficiencies. However, studies of biochemical oxygen demand have shown that 

polysaccharides in solution in rivers are immediately available fc r microbial 

degradation.

Climate and soil type may have significant effects on the polysaccharide 

content and the type of organic matter. In the frigid environment of the 

Fairbanks soil in Alaska, where microbial degradation of organic material 

in the spodic soil horizon is slow on an average yearly basis, the 

polysaccharide content was high. In the spodic horizon of the Lakewood 

soil in North Carolina, the polysaccharide content was much lower. The 

rate of microbial polysaccharide degradation may also be a factor in the 

river preparations, because the Tahquamenon River in northen Michigan 

had a greater polysaccharide content than did the Florida streams.

The differences in the electrophoretic fractionation curves for fulvic acid 

preparations from different soil horizons indicate that the highly colored 

organic material of high negative charge is leached from the surface horizon 

and accumulates in the spodic horizon. Some of this material is probably 

carried into lakes and streams, where it comprises the primary organic 

colorative agent. Very little of the colored, charged material w?s found in 

the fulvic acid from the surface mollic horizon of the Macksl nrg soil in 

Iowa.

Infrared and elemental analyses show increasing aromatic character with 

increasing negative charge for the Fairbanks fulvic acid. An inc-ease in the 

carboxyl content with negative charge indicates the carboxyl g-oup as the 

primary source of the negative charge.

Soil fulvic acid and organic materials dissolved in river waters are 

dynamic mixtures whose composition at any certain time depends on 

variables such as the type and quantity of source materials, the rates of 

microbial degradation, the stability of intermediate degradation products, 

and the solubility properties of all the components taken together. Free-flow 

electrophoresis is one technique for fractionating complex natural organic 

mixtures on the basis of electrical charge so the various fractions can be 

studied individually for a more complete characterization.
E14 ORGANIC SUBSTANCES IN WATER

REFERENCES

Black, A. P., and Christman, R. F., 1963a, Characteristics of colored surface waters: Am. 

Water Works Assoc. Jour., v. 55, no. 6, p. 753-769.

____1963b, Chemical characteristics of fulvic acids: Am. Water Works Assoc. Jour., v. 55, 

no. 7, p. 897-912.

Brinkman Instrument Co., 1959, Electrophoretic Separator Model FF4 instrument manual: 

Westbury, New York, 17 p.

Christman, R. F., and Ghassemi, M., 1966, Chemical nature of organic color in water: Am. 

Water Works Assoc. Jour., v. 58, no. 6, p. 723-741.

Clapp, C. E., Jr., 1957, High molecular weight water-soluble muck; isolation and determination of constituent sugars of a borate complex-forming polysaccharide en-'oloying electrophoretic techniques; Cornell University Ph. D. dissertation, Dissert. Abs., v. 17, p. 

963-964.

Johnson, H. H., 1959, Electrophoretic separation of acid-resistant components, part 1 of Soil 

organic matter: Soil Sci. Soc. America Proc., v. 23, p. 293 295.

Leenheer, J. A., and Malcolm, R. L., 1973, Preparative free-flow electrophoresh as a method 

of fractionation, purification, and characterization of natural organic materials: U.S. 

Geol. Survey Water-Supply Paper 1817-D, 14 p.

Midwood, R. B., and Felbeck, G. T., Jr., 1968, Analysis of yellow organic matfer from fresh 

water: Am. Water Works Assoc. Jour., v. 60, no. 3, p. 357 366.

Mortensen, J. L., and Schwendinger, R. B., 1963, Electrophoretic and spectroscopic 

characterization of high molecular weight components of soil organic mat^r: Geochim. 

et Cosmochim. Acta, v. 27, p. 201-208.

Shapiro, J., 1957, Chemical and biological studies on the yellow organic acids of lake water: 

Limnology and Oceanography, v. 2, p. 161-169.

Tan, K. H., and Clark, F. E., 1969, Polysaccharide constituents in fulvic and humic acids extracted from soil: Geoderma, v. 2, p. 245-255.

Waldron, A. C., and Mortensen, J. L., 1961, Electrophoretic separation of organic components, part 2 of Soil nitrogen complexes: Soil Sci. Soc. America Proc., v. 25, p. 29-32.

Wilson, A. L., 1959, Determination of fulvic acids in water: Jour. Appl. Chemistry, v. 9, p. 

501-512.

* U.S. GOVERNMENT PRINTING OFFICE: 1973 O- 543 576/2