Organic Acids in 

Naturally Colored 

Surface Waters

GEOLOGICAL SURVEY WATER-SUPPLY PAPER 1817-A
Organic Acids in 

Naturally Colored 

Surface Waters

By WILLIAM L. LAMAR and DONALD F. GOERLITZ

ORGANIC SUBSTANCES IN WATER

GEOLOGICAL SURVEY WATER-SUPPLY PAPER 1817-A

An analytical study of the organic acids 

in naturally colored surface waters

UNITED STATES GOVERNMENT PRINTING OFFICE, WASHINGTON : 1966
UNITED STATES DEPARTMENT OF THE INTERIOR 

STEWART L. UDALL, Secretary

GEOLOGICAL SURVEY 

William T. Pecora, Director

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

Washington, D.C. 20402 - Price 15 cents (paper cover)
CONTENTS

Page

Abstract.________________________________________________________ Al

Introduction. _____________________________________________________ 1

Previous research______________________________________________^_ 2

Laboratory studies._________-___-_-_________._-__-_--__-----_---_ 4

Water samples__-_--___-.---_----_-----__----__------------_--_ 4

Apparatus. ______-_-_-________--_-_--_-__----_----__---_-_--__ 5

Isolation of organic acids_____________-______-______----------__ 5

Gas chromatography_________________________________________ 7

Paper chromatography_________________________________________ 11

Column chromatography___________________--_____-___-__---___ 11

Infrared spectroscopy____________________________________________ 12

Discussion and summary______________..____-___------________-__-__ 14

References cited__________________________________________________ 17

ILLUSTRATIONS

Page 

FIGURE 1. Drawing of gas chromatographic effluent collector.__________ A6

2. Graph of effective chain lengths of carboxylic acid methyl

ester s _ _______________________________________________ 8

3. Chart of infrared spectra of organic acids__________________ 3

TABLE

TABLE 1. Effective chain lengths for methyl esters of carboxylic acids_

in

Page 

A9
ORGANIC SUBSTANCES IN WATER

ORGANIC ACIDS IN NATURALLY COLORED 

SURFACE WATERS

By WIDLIAM L. LAMAR and DOXALD F. GOERLITZ

ABSTRACT

Most of the organic matter in naturally colored surface waters consists of a 

mixture of carboxylic acids or salts of these acids. Many of the acids color the 

water yellow to brown; however, not all of the acids are colored. These acids 

range from simple to complex, but predominantly they are nonvolatile polymeric 

carboxylic acids.

The organic acids were recovered from the water by two techniques: continuous liquid-liquid extraction with n-butanol and vacuum evaporation at 50 °C. 

(centigrade). The isolated acids were studied by techniques of gas, paper, 

and column chromatography and infrared spectroscopy.

About 10 percent of the acids recovered were volatile or could be made volatile 

for gas chromatographic analysis. Approximately 30 of these carboxylic acids 

were isolated, and 13 of them were individually identified. The predominart part 

of the total acids could not be made volatile for gas chromatographic analysis. 

Infrared examination of many column chromatographic fractions indicated that 

these nonvolatile substances are primarily polymeric hydroxy carboxylic acids 

having aromatic and olefinic unsaturation. The evidence suggests that some of 

these acids result from polymerization in aqueous solution. Elemental analysis 

of the sodium fusion products disclosed the absence of nitrogen, sulfur, and 

halogens.

INTRODUCTION

In 1786, Achard extracted a brown substance from soil and peat 

using alkali solutions. Since that time, colored organic substances 

have been extracted from soil, peat, coal, water, and decaying plants 

by many investigators. The early literature abounds with ideologic 

confusion. No less than 45 substances or fractions have been "discovered" and generically named. The literature contains many names, 

such as humus, ulmin, humus acid, humic acid, crenic acid, apocrenic 

acid, ulmic acid, torfic acid, fumic acid, hymatomelanic acid, fulvic 

acid, and liumolimnic acid. Waksman (1938, p. 22-64) provided a significant historical summary of the studies and concepts of these natural 

organic substances.

Al
A2 ORGANIC SUBSTANCES IX WATER

The names most frequently used for the organic acids occurring in 

naturally colored water are humic, hymatomelanic, and fulvic. This 

generic nomenclature is based on solubilities in water, alkali, acid, 

and alcohol. Because of the lack of specific separations and identifications, these generic names have continued in common use.

Organic acids are the most predominant organic material in naturally colored waters. They are produced by biological and oxidative 

processes and perhaps by condensation reactions, and the result is a 

complex mixture of many acids both colored and colorless The mixture of these substances ranges from the well-defined simple acids to 

complex polymeric nonvolatile ones which apparently consist of several or more building units. In this mixture the polymeric nonvolatile 

acids predominate, and these substances, after more than 150 years of 

study, remain the most difficult to separate and identify.

PREVIOUS RESEARCH

In isolating organic acids from naturally colored water, a mixture 

of many acids is obtained. It is difficult to isolate a fraction which 

contains only the nonvolatile colored acids and to separate the complex 

nonvolatile acids into individual components. Usually, rtudies of the 

colored organic acids in water relate to a mixture of many acids in 

which the colored nonvolatile acids predominate. Many values have 

been reported for the molecular weight of the colored organic acids 

because these values usually represent an undetermined mixture of 

many acids colored, colorless, simple, and complex.

Mueller, Larson, and Lennarz (1958) studied the organic acids in 

surface water and sewage. The acids were isolated by continuous extraction with ether, separated by column chromatography, and characterized by measuring the characteristic effluent volumes obtained by 

titration of the fractions. In a later paper, Mueller, Larson, and Ferretti (1960) used both column and paper chromatography for the analysis of the organic acids in water. The fractions separated by column 

chromatography were tentatively identified by their elution volumes, 

and the presence of some of the acids was confirmed by paper 

chromatography.

The colored organic acids exert a holding action for a number of 

metallic ions. This holding action has been referred to as adsorption, bonding, complexion, and chelation. In water chemistry it has 

been particularly observed that many naturally colored waters carry 

up to a part per million of iron or more in apparent solution, and this 

iron is not readily precipitated by aeration. In other aerated waters 

whose pH is above about 5, ferric iron can be in excess of 0.01 ppm only 

as a suspension of oxide or hydroxide (Hem and Cropper, 1959).
ORGANIC ACIDS IN NATURALLY COLORED SURFACE WATERS A3

The fouling of anion-exchange resins by the colored organic substances in water has been discussed by many authors. Friscli and 

Kunin (1960) conducted a number of experiments with the eluted 

foulant recovered from commercial anion-exchangers that were used 

to treat surface water. They called the fouling material humic acids 

and referred to them as large organic acids commonly found in surface 

waters. They eluted the organic foulant from the resin with an 8 

percent sodium chloride solution, whereas Rice, Simon, and Rice 

(1962) used concentrated hydrochloric acid for the same purpose. In 

both papers the fouling material was identified as a carboxylic acid 

by infrared analysis. Rice, Simon, and Rice concluded that a part of 

the organic material eluted from fouled anion-exchange resins must 

be unsaturated aliphatic polycarboxylic acids.

Ruchti (1962) reported on the fractionation of low-molecularweight substances extracted from the B horizon of podzol soils. He 

noted that the ether-soluble part condensed in aqueous solution and 

was oxidized on exposure to air. The observed condensation in aqueous solution could be significant to the complex structure of th°, colored organic acids occurring in water.

Lamar and Goerlitz (1963) conducted studies of the organic acids 

in natural waters and developed procedures for the isolation and identification of a number of organic acids that are volatile or could be 

made volatile for gas chromatographic analysis. The present paper 

includes studies of the volatile and nonvolatile organic acids in naturally colored surface waters.

Shapiro (1957, 1964) used vacuum evaporation, freeze-out, ard extraction to concentrate the organic acids from large volumes of colored 

water (up to 900 liters) collected primarily from Linsley Pond near 

New Haven, Conn. He used a number of techniques in the study of 

the iron-holding characteristics and other properties of these acids. 

Black and Christman (1963a) studied the organic matter as it occurs 

in colored waters. On the basis of angular-light-scattering data and 

ultraviolet spectroscopy they concluded that the organic substances 

responsible for color in water exist primarily in colloidal suspension. 

This observation on the raw water is in contrast to Shapiro's (1957, 

1964) findings of molecular solution for the colored organic matter 

after it was concentrated and extracted. The different conditions in 

these experiments may account for this disagreement. In ar other 

paper Black and Christman (1963b) described isolation techniques for 

fractions of organic matter which they called fulvic acid, hymatomelanic acid, and humic acid. The fulvic acid fraction comprised over 

80 percent of the acids so isolated. They concluded from solubility
A4 ORGANIC SUBSTANCES IN WATER

relationships, chemical spot tests, and infrared spectra that the fulvic 

acids are aromatic polyhydroxy methoxy carboxylic acids.

Christman (1964) studied the colored solids extracted from garden 

soil, Douglas-fir bark, and water. He compared the degradation 

products and reported evidence of the aromatic and phenolic nature 

of the color molecules by identification of 3-methoxy-4-hydroxybenzaldehyde in oxidation mixtures of the color solids.

Hoak (1962) described problems in the recovery and identification 

of organic substances in water. Several extraction techniques were 

used, and it was reported that continuous liquid-liquid extraction in 

a Scheibel 18-stage countercurrent column appeared to offer advantages over adsorption on carbon. Examinations were made by colorimetric tests, ultraviolet and infrared spectroscopy, and paper and gas 

chromatography. Phenol, guaicol, m-cresol, and 3,5-rylenol were 

identified by paper chromatography in extracts from river waters 

and oak-leaf fermentations; several other phenolic compounds and a 

few steam-volatile fatty-acids were tentatively identified.

Packham (1964) provided a discussion of the nature of organic 

color. Macroporous resins were utilized to isolate the complex organic 

acids. The fractions, which he called humic, hymatomelanic, and 

fulvic acids were obtained by solubility techniques. It appears that 

the differences in these fractions may be more related to the process 

used and to the physicochemical state of the solution because solubility 

techniques have not provided separations than can be relied upon.

LABORATORY STUDIES

The organic acids were removed from the water by two techniques: 

Continuous liquid-liquid extraction and vacuum evaporation. A 23.5 

liter sample of water was used in both of these processes. The organic 

acids were isolated by selective extraction and examined by techniques 

of gas, paper, and column chromatography and infared analysis.

WATER SAMPLES

The water samples were collected from unpolluted streams in California, Hawaii, and Washington. If the sample contained particulate 

matter or turbidity that did not settle overnight, the water was filtered 

by suction through a Biichner funnel having a sintered-glass disc of 

medium porosity. The temperature of the waters when collected 

ranged from 46° to 72°F. The values for certain characteristics of 

these waters ranged as follows: Bicarbonate 2-270 ppn, carbonate 

none, specific conductance 27-509 /*mho (micromhos) at 25°C (centigrade), pH 5.2-7.7, and color 7-250 units (platinum-cobalt scale). 

The iron measured in selected samples ranged from 0.23 to 0.83 ppm.
ORGANIC ACIDS IN NATURALLY COLORED SURFACE WATERS A5

APPARATUS

Extractor. A specially designed cylindrical extractor of 26-liter 

capacity was used to separate the organic matter from the water sample. 

The solvent is dispersed continuously in fine bubbles from four matched 

dispersion discs at the bottom of the extractor and travels through the 

water sample into a 1-liter boiling flask, where the extracted organic 

compounds are concentrated. The solvent is distilled from the concentrate to complete the cycle of continuous operation.

Evaporator. A Buchler rotating vacuum evaporator was used for 

concentration of large volumes, and a Rinco rotating vacuum evaporator was used for the concentration of small volumes.

Gas chromatograph. Gas chromatographic analyses were performed with the Perkin-Elmer, model 154-D, isothermal gas chromatograph having a thermistor thermal-conductivity detector. The analytical columns were stainless steel, 4 mm ID, 1.5- and 2-meters long.

Recorder. A Leeds and Northrup Speedomax O strip chart recorder having 1- and 5-millivolt nominal spans.

Gas chromatographic effluent collector. Because difficulty occurred 

in the collection of pure gas chromatographic samples for infrared 

analysis, a suitable collector as shown in figure 1 was designed for this 

purpose (Goerlitz and Lamar, 1964). The 18-gage stainless-steel admission tube (hypodermic tubing) is shown in the illustration in the 

vertical position, but it may be placed in the horizontal arm of the 

collector, dependng upon the access to the detector vent. Each collector is easily handled, individually cooled, and conveniently sealed 

from atmospheric moisture and coolant-bath vapor. Importantly, the 

apparatus allows collection of each component very near the end of the 

analytical column in an individually complete assembly.

Infrared spectropJiotometer. Infrared analyses were made on a 

Perkin-Elmer model 21 spectrophotometer equipped with sodium chloride optics. A 4X beam condenser with microcells and micropellets 

was used where necessary.

ISOLATION OF ORGANIC ACIDS

In the liquid-liquid extraction process, the water sample was adjusted 

to pH 2 and continuously extracted for 40 hours with n-butanol. The 

extract was made slightly basic with sodium hydroxide and concentrated to about 100 ml (milliliters) at 50°C on a rotary vacuum evaporator. The organic acids were removed from this concentrate by 

batch extraction with one 100-ml and two 50-ml amounts of aqueous 5 

percent sodium bicarbonate. After acidification to pH 2, the acids 

were extracted back into n-butanol, titrated to pH 7.0 with sodium

210 395 66 2
A6 ORGANIC SUBSTANCES IN WATER

hydroxide, and evaporated to dryness at 30°C on a small rotary vacuum 

evaporator.

In the evaporation process, the water sample was adjusted to pH 9 

and then evaporated at 50 °C to about 100 ml on a rotary vacuum evaporator. Batch extraction of this concentrate was performed with one 

100-ml and two 50-ml amounts of 1:1 ethyl ether-ethanol to remove the 

neutral and basic components. The aqueous layer containing the organic acids was acidified to pH 2 with sulfuric acid and steam distilled. The steam distillate, containing steam volatile acids, was 

flushed with helium to remove carbon dioxide and titrated with sodium 

hydroxide to the phenolphthalein end-point. This neutralized disFIGURE 1. Gas chromatographic effluent collector.
ORGANIC ACIDS IN NATURALLY COLORED SURFACE WATERS A7

tillate was evaporated to dryness on a steam bath to remove water and 

nonacidic volatile substances. The acids were released from their corresponding salts by the method of Gehrke and Lamkin (1961). The 

organic acids remaining in the steam distillation residue were extracted 

with n-butanol and evaporated to dryness at 30°C on a small rotary 

vacuum evaporator.

GAS CHBOMATOGBAPHY

The organic acids, isolated by the evaporation technique, were 

separated into steam-volatile acids and higher boiling acids, whereas 

the acids from the extraction process were not. Because the evg.poration technique was performed at pH 9, all the organic acids were retained in the concentrate. The most volatile acids were not expected 

in the extraction process, owing to the relatively high boiling point of 

the solvent. The acids were analyzed by gas chromatography; those 

that were not volatile or sufficiently stable for the gas chromatographic 

analysis were converted to their methyl ester derivatives by the procedure of Radin, Hajra, and Akahori (1960).

The steam-volatile acids in ether solution were chromatographed on 

a 2-meter column packed with 80-100 mesh Chromosorb W which had 

previously been coated with a mixture of 9:1 Dow Corning 550 silicone 

oil and stearic acid. This coating (liquid phase) was 15 percent of 

the weight of the uncoated material (solid phase). The chromatographic analysis was made at a temperature of 140°C with a carrier gasflow rate of 30 ml per minute.

The steam distillates yielded three steam-volatile acids. These components were identified as acetic, propionic, and butyric acids by direct 

comparison of retention volumes of the samples with standard retention volumes obtained by chromatographing solutions of known g teamvolatile acids. There was indication of minute quantities of other 

volatile acids, such as formic and higher boiling homologues.

The methyl ester derivatives of the higher boiling acids, in methylene 

chloride solution, were gas chromatographed on a 2-meter polar column 

at 140°C with a carrier gas-flow rate of 69 ml per minute, and on a 1.5- 

meter nonpolar column at 160° C with a carrier gas-flow rate of 32.5 

ml per minute. The polar column (3:1 solid-liquid by weight) was 

prepared by coating 80-100 mesh silanized Chromosorb W (solid 

support) with diethylene glycol succinate polyester, purified by the 

method of Corse and Teranishi (1960). The nonpolar column (4:1 

solid-liquid by weight) was prepared by coating the same solid support 

with Apiezon L grease.

To characterize the methyl ester derivatives, the samples and standards were run on the polar and nonpolar columns. Retention volumes
A8 ORGANIC SUBSTANCES IN WATER

were recorded for each component in the samples and stmdards. A 

graph, as shown in the figure 2, was prepared by plotting log retention 

volume against the carbon number of the esterified fatty-acid homologues used as standards. The curves are used to obtain reproducible 

values for the carboxylic acids which were chromatographed 011 both 

polar and nonpolar columns. These values have been designated as 

effective chain lengths by Miwa, Mikolajczak, Earle, and Wolff (1960), 

and they are independent of column temperature, column dimensions, 

and carrier gas-flow rate.

2000

1000

500

100

50

Nonpolar Column

Z

7

7

7

 Polar Column

7

1 9 

EFFECTIVE CHAIN LENGTH

11 13

FIGURE 2. Effective chain lengths of carboxylic acid methyl esters.
ORGANIC ACIDS IN NATURALLY COLORED SURFACE WATERS A9 

TABLE 1. Effective chain lengths for methyl esters of carboxylic acids

Reference acid

Effective chain length

Reference

Water sample

1 2 3 4

Polar Column

Valeric.

Unknown

Do

Unknown. _

Do

Oxalic _ _ _ _

Malonic. ___ - ___

FumaricSuccinic

Lauric

Unknown _

Unknown

4.8

5.4

5.9

6.2

7.8

8.0

8.3

9. 8

10. 0

10.9

11.6

11. 8

12.0

12.8

4.8

5.5

6.2

7.5

7. 9

8.3

8.6

9.2

9.8

10.9

11.2

11.8

12. 3

12. 8

13.0

5.5

6.3

7.9

9. 2

9. 8

10. 9

11.8

5. 5

6. 3

7.9

8.3

9.2

9. 8

10.9

11.8

13.0

5.5

6.4

7.9

9.8

10.9

11.8

12.3

Nonpolar Column

Pyruvic

Crotonic

Valeric

Caproic

Maleic _

Do

Unknown

Do_ _ __

4.0

4.0

4. 3

4. G

4.7

5.0

5.5

6.0

6.6

6.7

6.9

8.0

8.9

3.7

4.3

5.0

6.6

6.6

6.7

7.3

7.9

8.3

8.7

4.3

5.0

6.0

7.3

8.3

5.6

6.0

6. 6

7.3

8. 3

8.9

5.0

5.5

6.0

6.7

6.9

7. 3

8.3

8.9

Table 1 gives the effective chain lengths of the esterified carbixylic 

acids reference acids and acids isolated from the water samples. 

These substances are reported in the order of their emergence from the 

polar column and from the nonpolar column. Identifications of the
A10 ORGANIC SUBSTANCES IN WATER

carboxylic acids in the extracts were made by comparison of their 

effective chain lengths with those of reference acids.

Primarily, the effective chain lengths are nearly the same for the 

less polar aliphatic saturated methyl esters on both types: of columns 

used in this study but are usually quite different for more polar methyl 

esters. If there is little difference in the peak areas of a complex mixture, the emergence of a particular component is difficult to follow on 

both columns. Greater confidence can be placed in the identification 

of the components of a mixture if each component is collected individually and then rechromatographed on another type of column.

The gas chromatographic analysis of the methyl esters revealed a 

number of carboxylic acids of which 10 were identified; namely, adipic, 

butyric, caproic, fumaric, lactic, maleic, malonic, oxalic, succinic, and 

valeric. Butyric acid, identified in the steam-volatile fraction from 

the evaporative process, was also found in the esterified fraction from 

the extractive process. Butanol was used as a solvent ir the liquidliquid extraction, but it is significant that it was not used in the evaporative process. Thus, the butyric acid did not result from oxidation 

of the butanol solvent.

During the course of this investigation it was found that the yield 

of the methyl ester derivatives decreased in the presence of increasing 

amounts of inorganic salts. The results of an experiment to improve 

the yield indicated that a good yield of the ester derivatives, in the 

presence of appreciable inorganic salts, can be obtained by esterifying 

the organic acids with sulfuric acid catalyzed n-butanol. In addition, 

gas chromatographic analysis of the butyl ester derivatives revealed 

phthalic acid which had not been previously identified by gas chromatography of the methyl esters.

In all, the gas chromatographic analyses revealed approximately 

30 carboxylic acids which were volatile or could be made volatile for 

this purpose. Thirteen of these acids were identified. Confirmatory 

results were obtained by several methods of analysis that included 

isolation of the acids by both liquid-liquid extraction and evaporative 

processes and by gas chromatographic analysis on both polar and nonpolar columns. The presence of phthalic and succinic acids was additionally confirmed by infrared analysis. All the gas chromatographically volatile carboxylic acids were present in small amounts. The 

highest individual concentration measured was about 600 parts per 

billion for valeric acid.

The preponderant part of the isolated organic acids was nonvolatile 

and could not be made volatile for gas chromatographic analysis. 

These nonvolatile acids, commonly referred to as humic acids, or by 

other generic terminology, were examined by paper and column 

chromatography and infrared spectroscopy.
ORGANIC ACIDS IN NATURALLY COLORED SURFACE WATERS All 

PAPER CHROMATOGRAPHY

Paper chromatograms were prepared by spotting 1-3 pi (microliter) 

amounts of 5 percent solutions of the isolated organic acids on Whatman No. 1 chromatographic paper. Reference compounds, phenol, 

pyrogallol, and m-digallic acid were spotted alongside and the paper 

was allowed to dry. The chromatograms were developed usirg the 

ascending technique with four parts n-butanol, one part glacial acetic 

acid, and five parts water. These chromatograms were air dried, 

sprayed with 2 percent ferric chloride, and then dried in an oven at 

100°C. Observations before and after spraying indicated that although individual spots were vaguely discernible, there was an overall 

smear from the starting line to the solvent front. The lack of distinct 

separations was characteristic of the total acids from all water samples 

examined, whereas well-defined rounded spots were obtained from the 

reference compounds. Although no real separations were observed, 

the paper chromatograms augmented the conclusion that the nonvolatile acids were polymeric.

COLUMN CHROMATOGRAPHY

Repeated column chromatographic analyses indicated that the nonvolatile colored acids in natural water are polymeric, condensed 

molecules. The majority of the column chromatographic separations 

were made on 1-centimeter-diameter columns containing 7 grams of 

100 mesh silicic acid coated with 4.9 ml of 0.5N sulfuric acid. A 50- 

100-mg (milligram) amount of the organic acids was chromatographed each time. The elution was performed with chloroform and 

increasing amounts of n-butanol in a graded series of solvent mixtures, 

from 0 to 45 percent n-butanol. Each solvent mixture was equilibrated 

with 0.5N sulfuric acid prior to addition to the column. The solvent 

flow rate through the column was 3-5 ml per minute, and each f ruction 

was collected with a 5-ml volumetric siphon. Additional separations 

of the components of the organic acids were attempted on powdered 

cellulose using a graded series of eluents, namely, hexane, carbon 

tetrachloride, benzene, ether, acetone, chloroform, methanol, g-lacial 

acetic acid, and water.

From time to time during the elution, the column was observed in 

long-wave ultraviolet light. A true chromatographic separation of 

the colored acids was not visually observed to pass through the column 

but rather with each new solvent front a surge of yellow-browr solution was seen. Selected fractions from each solvent mixture were 

washed with a small amount of distilled water and evaporated to 

dryness, either on a steam bath or with the aid of a rotary evapc rator. 

Some of the chromatographic fractions of the acids were taken into
A12 ORGANIC SUBSTANCES IN WATER

solution with, carbon tetrachloride or carbon disulfide for infrared 

analysis, whereas others were pressed into potassium bromide pellets 

for this analysis.

IETBABED SPECTBOSCOPY

A number of infrared spectroscopic examinations were made on the 

total acids but the most extensive examinations were performed on the 

chromatographic fractions. Figure 34 shows the infrared spectrum 

of the total organic acid fraction isolated from an unpolluted stream 

in California. Spectra of the total acids isolated from the other 

natural water samples were always nearly identical. Similar spectra 

were obtained by Shapiro (1957) and Black and Christman (1963b) 

for the colored acids they had isolated from water and also by Frisch 

and Kunin (1960) for the deionized foulant that was eluted from an 

ion-exchange resin, previously used in the treatment of surface water. 

In figure 34, the band at 3.I/A (microns) represents a bonded hydroxyl 

and carboxyl absorption. Normally, the free OH absorbs at 2.75/x 

and the bonded OH maximum absorption is shifted to longer wavelengths over a broader range (2.7-3.2/A). The carboxyl OH absorbs 

over the broad range from 3.0-3.5/t. Absorption due to NH is ruled 

out because nitrogen was absent in the elemental analysis.

In figure 34 the carbon-hydrogen stretch absorption at 3.36^ is 

probably due to asymmetric methyl and asymmetric methylene groups. 

The shoulder at 3.45^ is due to symmetric methyl and methylene 

absorption. The band at 6.2^ is indicative of unsaturatioii. The 

most intense band, shown at 5.78^, is in the region for carbonyl absorption. Ester carbonyl occurs at 5.76/i whereas carboxylic acid carbonyl 

absorption is shifted up in wavelength and is usually fonnd at 5.86/x. 

For carboxylic acid salts, the absorption wavelength moves even 

higher to 6.1-6.4ju.. An electron withdrawing group, influencing the 

acid carbonyl would shift the absorption band down in wavelength 

as may be the situation here. The rather broad and strong band at 

8.3-8.4j«. is possibly due to carbon-oxygen stretch such as vith esters or 

carboxyl groups.

Spectra of column chromatographic fractions of the acids isolated 

from highly colored water indicated very little difference from fraction to fraction except to reveal some increased band intensities in 

the 11.5-14.5/i range. Spectra of the acids of chromatographic fractions from lightly colored water, however, were varied in the initial 

fractions; a typical example is shown in figure 35. The spectra of 

later fractions returned to the characteristic spectrum shewn in figure 

34.

The spectrum shown in figure 35 confirmed the presence of phthalic 

acid which was also identified in the gas chromatographic analysis.
FIGURE 3. Infrared spectra oforganic acids.A, Total organic acids; R chro 

matographic fraction ; C, esterified acids D, acetylated acids.

23 456789101 

WAVELENGTH, IN MICRONS

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A14 ORGANIC SUBSTANCES IN WATER

Because the acids were eluted from, a sulfuric acid-coated silicic acid 

column with chloroform-butanol, the resulting spectrum, figure 32?, is 

the spectrum of dibutyl phthalate. The hydroxyl band in this spectrum may be due to the possible elution of silicic acid with this fraction. 

Black and Christman (1963b, p. 912) in their paper on fulvic acids 

showed a spectrum similar to figure SB which they called fulvic acid 

oxidation products. The rechromatographing of a number of fractions (combined eluates from a solvent system) yielded a large number 

of partially separated polymers or other compounds.

Esterification, using sulfuric acid catalyzed butanol, decreased the 

OH absorption band and made it sharper. A spectrum of the esterified organic acids is shown in figure 3(7. The OH absorption observed 

after esterification indicates that in addition to carboxylic acid groups 

there are alcoholic and probably phenolic hydroxy groups present. 

Because the esterification had no noticeable effect on the b^nd at 6.2/*T 

unsaturation is strongly suspected.

Reaction of acetic anhydride with the total organic acid fraction 

almost completely eliminated the hydroxy absorption. T^is elimination can be observed in figure 3Z>, which is a spectrum of the acetylation reaction products. This spectrum indicates that most of the hydroxy groups are free to react. Again, the band at 6.£/x remained 

unchanged. Bands characteristic of anhydrides are evider t.

DISCUSSION AND SUMMARY

The organic acids in naturally colored waters result from biologic 

and oxidative processes. Most of the organic matter in these waters 

consists of a complex mixture of carboxylic acids or the salts of the 

acids which impart a yellow to brown color to the water. The colored 

acids have an indicator effect the intensity and shade of color increases with pH. Although some of the acids isolated are not colored, 

the mixture of the acids is dark brown to black. The mixture of acids 

in solution in various solvents fluoresces under ultraviolet light, 

whereas the acids in the solid state were not observed to do so.

The colored organic acids generally appear to be stable and remain 

in apparent solution for considerable periods of time. Four samples 

retested for color after prolonged storage (approximately 1-2 yr) 

indicated that considerable reduction in color had occurred. This loss 

in color was accompanied by the formation of a flocculent brown 

organic precipitate. Active samples may show considerable variation 

in short periods of time. For example, the color of the vater of one 

sample was 7,17, and 26 units on successive days after collection. At 

6 and 28 days after collection, the color of this water had decreased to 

17 and 4 units, and this decrease was accompanied by the formation of 

a flocculent brown organic precipitate.
ORGANIC ACIDS IN NATURALLY COLORED SURFACE WATERS A15

The observed increase in color after collection was confined to some 

of the lightly colored waters. Processing these waters before the 

color increased made the elution of more distinct column chromatographic fractions most apparent. The spectrum, shown in figure 3B, 

was obtained from a lightly colored water that noticeably darkened 

after collection but was processed promptly before the color increased. 

This sample was collected after a period of surface runoff following 

an extended period of drought. As these observations were not evident 

with the more mature and darkly colored waters, it seems probable 

that some of the complex nonvolatile acids result from polymerization 

in aqueous solution.

The organic acids in naturally colored waters reflect the characteristics of a composite in which polymeric nonvolatile carboxylic acids 

predominate. The polymeric acids in water behave like negatively 

charged colloids. In the raw water they can be coagulated with alum 

in the acid range. The optimum coagulating pH may be as low as 5 

for alum and even lower for ferric coagulants. These substances 

extracted, dried, and taken into solution with dilute NaOH were 

similarly coagulated with alum.

The usual mineral analysis of naturally colored waters frequently 

shows an excess of cations over anions. This imbalance is small in 

terms of equivalents, usually being less than 0.2 equivalent per million 

even for the highly colored waters. The nature of this imbalance can 

be interpreted as indirect evidence that these colored polymeric substances have a high average molecular weight or exist in water as a 

colloidal sol that has complexed or adsorbed cations.

The various techniques for separating the colored acids in water 

by differences in solubilities are not reliably reproducible. If the 

organic acids in a water sample are concentrated by extraction, evaporation, or freezing, the concentrate will contain variable amounts of 

inorganic material. These inorganic substances tend to provide an 

adsorbing media for the organic acids apparently altering their 

solubilities. For example, a water sample concentrated twentyfold 

by vacuum evaporation contained filterable solids consisting of 30 

percent organic and 70 percent inorganic compounds. Acidification 

of the soluble part of the concentrate to pH 2 with hydrochloric acid 

precipitated material which consisted of 60 percent organic s.nd 40 

percent inorganic compounds. The colored organic matter in both 

precipitates redissolved in dilute sodium hydroxide but would not 

again precipitate by adjustment to pH 2.

Although the concentration of the individual acids recovered and 

then identified by chromatography can be determined readily, it is 

difficult to determine accurately the total quantity of the organic acids 

present in the water. Loss occurs in extraction, freeze concentration,
A16 ORGANIC SUBSTANCES IN WATER

and where the acids are sorbed on a carbon filter or ion-exchange 

materials. The contamination with inorganic salts is particularly 

extensive in the evaporative and freezing processes. Experiments 

with commercial electro dialyzers for desalting the concentrates were 

not successful. With the nonionic membrane circulating electrolyte 

desalter, uncontrollable foaming occurred whereas with ion-exchange 

membrane type, loss due to precipitation at the anode occurred. Extraction of the organic acids from the concentrate with n-butanol was 

found to be an acceptable method for removing the inorganic material 

but some loss occurred. In the gas chromatographic studies, it was 

found that esterifying with sulfuric acid catalyzed n-butanol provided 

salt-free esters.

The isolated organic acids were found to be soluble in polar solvents such as acetone and the alcohols, slightly soluble in ether, and 

insoluble in hexane, methylene chloride, and benzene. Th°s solid acids 

were determined to be noncrystalline by X-ray diffraction. Even 

after careful preparation of the sodium salts and analysis by X-ray 

camera technique, no crystallinity was observed. The solid acids 

softened at 110°C and decomposed over a broad temperature range. 

Elemental analysis of the sodium fusion products showed the absence 

of nitrogen, sulfur, and the halogens.

Most of the gas chromatographic analyses were made with an isothermal gas chromatograph having a thermistor thermal-conductivity 

detector. Some of the later work was performed with a dual-column 

temperature-programmed gas chromatograph having a f ame-ionization detector. Generally 23.5 liters of water were used to provide a 

quantity of the organic acids sufficient for repeated examinations. 

Smaller samples, however, may be used, particularly for gas chromatographic analysis with the more sensitive temperature-programmed 

flame-ionization instrument.

About 10 percent of the mixed acids isolated from the water yielded 

carboxylic acids which were volatile or could be made volatile for 

gas chromatographic purposes. The gas chromatographic analysis 

indicated about 30 carboxylic acids of which 13 were identified. The 

individual concentrations of the gas chromatographicaJly volatile 

ucids ranged from trace quantities to about 600 parts per billion.

The predominant part of the organic acids are nonvolatile and could 

not be made volatile for gas chromatographic purpose?, Infrared 

analysis of many column chromatographic fractions indicated that 

these nonvolatile substances are primarily polymeric hydroxy carboxylic acids with aromatic and olefinic unsattiration.
ORGANIC ACIDS IN NATURALLY COLORED SURFACE WATERS A17

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