Water Research 36 (2002) 4552–4562

Characterization, differentiation and classification of aquatichumic matter separated with different sorbents: synchronous

scanning fluorescence spectroscopy

Juhani Peuravuori*, Riitta Koivikko, Kalevi Pihlaja

Laboratory of Physical Chemistry, Department of Chemistry, University of Turku, FIN-20014 Turku, Finland

Received 6 June 2001; received in revised form 8 February 2002; accepted 12 April 2002

Abstract

Aquatic humic solutes were separated by the non-ionic macroporous XAD-8 and DAX-8 resins and a weakly basic

DEAE cellulose anion exchanger from seven different fresh water sources. Synchronous fluorescence spectroscopy was

applied for characterization, differentiation and classification of the different humic-solute aggregates. Fluorescence

properties verified that humic-solute fractions isolated parallelly with the non-ionic XAD-8 and DAX-8 resins

resembled very closely each other speaking strongly for their structural similarities. DAX-8 resin separated ca. 19%

more aquatic humic matter than did the analogous XAD-8 resin. It was possible to tentatively differentiate the

untreated water samples according to their fluorescent materials. Several distinct classes of chromophores were detected

in both DOM and isolated humic fractions by the synchronous technique: lex=lem 280/298, 330/348, 355/373, 400/418,

427/445, 460/478, 492/510 and 516/534 nm. r 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Fluorescence; Dissolved organic matter; DAX/XAD-resin isolation; Chromophores

1. Introduction

Over more than three decades fluorescence spectro-

scopy has been applied extensively to characterization,

differentiation and classification of natural organic

matter (OM) such as humic matter (HM). Likewise,

attempts to identify certain structural and functional

constituents (fluorophores) in natural HM have been

carried out. Many investigations have been performed

solely with the conventional fluorescence methods in the

emission or excitation modes. Fluorescence is sensitive

and many environmental factors (type of solution, pH,

ionic strength, temperature, redox potential of the

medium and interactions with metal ions and organic

substances) affect it (e.g. [1]). Despite the effects of the

structural units of DOM/aquatic HM on the intensity

and wavelength of the molecular fluorescence are

extremely complicated and partly unknown, some

fundamental hypothesis have been stated (e.g. [1–4]):

The intensity will decrease with increasing molecular size

of humic aggregates. Electron-withdrawing groups

decrease and electron-donating groups increase the

intensity of fluorescence in aromatic compounds.

Carbonyl-containing substituents, hydroxyl, alkoxyl

and amino groups tend to shift fluorescence to longer

wavelengths. Structural factors in HM which fluoresce

at long wavelengths with low intensity could be the

linearly condensed aromatic rings and other unsaturated

bond systems capable of a high degree of conjugation.

Fluorescence studies may sometimes be surprising and

contradictory. For example, fluorescence does not show

significant correlation with total organic carbon (TOC)

of certain model materials [3] or seawater TOC.

However, a linear correlation between fluorescence and

TOC has been found in surface waters [5] and in run-off*Corresponding author. Fax: +358-2-333-6700.

E-mail address: juhpeur@utu.fi (J. Peuravuori).

0043-1354/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.

PII: S 0 0 4 3 - 1 3 5 4 ( 0 2 ) 0 0 1 7 2 - 0

waters of peatlands [6]. Also, a nice correlation has been

found between fluorescence and biochemical oxygen

demand (BOD) of wastewater [7].

Fluorescence spectrometers equipped for synchronous

scanning in the constant-wavelength or constant-energy

modes have been used with good results to differentiate

fluorescent contaminants such as oils and polycyclic

hydrocarbons in the environment (e.g. [8–10]). Synchro-

nous fluorescence spectroscopy (SFS) has also the same

limitations inherent to the basic fluorescence technique,

such as spectral distortions caused by intermolecular

interactions and by static and dynamic quenching

processes. SFS offers, however, a potentiality to reduce

overlapping interferences and a possibility for each

fluorescent component to be identified in a specific

spectral range.

The idea of the synchronous technique was first

suggested by Lloyd [11]. In SFS the excitation wave-

length, lex; and the emission wavelength, lem; are

scanned synchronously with a constant-wavelength

interval, Dl ¼ lem � lex: For well-defined absorption

and quantum yield maxima, the optimum value of the

bandwidth Dl is set by the difference in wavelength of

the emission and excitation maxima which is known as

Stoke’s shift. As pointed out by Vo-Dinh [12,13], one of

the most essential limitations in multi-component

analysis by synchronous or any other luminescence

technique is spectral overlap. When more and more

components are present, as is the case with the HM

solution, the chances of spectral overlap increase, which

may lead to problems such as distortion of the

synchronous signal.

The Stoke’s shift is dependent on the solvent

environment but of most interest, the width of the

synchronous spectrum can be simply compressed or

expanded just by decreasing or increasing the Dltotal(Dl+the spectral overlap range) parameter [12]. The

decrease of the bandwidth of the synchronous signal is

advantageous since the spectral overlap greatly reduces.

Lloyd [14] has suggested that the interval of the

bandwidth should be between 20 and 30 nm for

condensed aromatic compounds. It has been later

proved experimentally (e.g. [4,15–18] that a constant

wavelength difference of 18 nm (Dl ¼ lem � lex) pro-

vides the best optimized resolution of the synchronous

signal for the natural HM in its different solutions.

The natural DOM has been nowadays generally

classified and modelled by different column chromato-

graphic methods into, structurally quite simple, non-

humic solutes and into very complicated heterogeneous

humic-solute aggregates. Sorption of humic solutes onto

wide variety of different sorbents takes place via

different mechanisms at given chemical conditions.

Thus, humic aggregates retained on different sorbents

do not have necessarily the same chemical and physical

properties although they possess somewhat acidic

character. The most popular sorbent has been the

XAD-8 (poly methyl methacrylate) resin at preadjusted

acidity. However, the manufacture of the XAD-8 resin

has been ceased for some years ago. It has been stated

[19] that XAD-8 can be substituted by Supelitet DAX-8

since their technical specifications are quite close

together. It is also possible to isolate with a very good

yield of macromolecular organic acids as an integrated

whole of HM from fresh waters without any pH-

adjustment of the original sample using weakly basic

anion exchange resins (namely Sigmas DEAE cellulose,

diethylaminoethylcellulose: –OC2H4N(C2H5)2).

In the present study the SFS technique was applied for

characterization and differentiation of several lake

aquatic HM samples originating in seven slightly

different environmental sources—also some coarse

structural classification of these, with different sorbents,

isolated HM samples was attempted to carry out. The

sorbents utilized for isolation of the HM samples were:

non-ionic macroporous (1) XAD-8 and (2) DAX-8

copolymers and (3) weakly basic DEAE cellulose anion

exchange resin. The objectives were: (I) study the effect

of the sorption resin on the structural compositions of

the different HMs, (II) examine the environmental

impact on the quality of the HMs, (III) study the effect

of the freeze-drying process on the structural composi-

tions of the HMs, (IV) try to find general similarities-

dissimilarities occurring between the different original

DOMs, (V) try to find similarities occurring between the

isolated HM samples and the original DOM, and (VI)

try to name some structural units as potential con-

tributors to molecular fluorescence.

2. Experimental

2.1. Origin and isolation of samples

Natural fresh water humic samples were collected

from five forest lakes (Kakarlampi, Ka; Mustaj.arvi, Mu;

Kuljuj.arvi, Ku; Pikkuj.arvi, Pi; Pappinen, Pa), from one

reservoir (Maaria, Ma) and from one river (Aura, Au).

All surface waters situate around the city of Turku in

southwestern Finland. The aquatic environments of Au

and Ma are sources of raw waters for the city Turku.

The water samples (ca. 50 l) were collected from 1m

below the surface into glass bottles between October

1999 and January 2000. The samples were prefiltrated

(0.2 mm, Nuclepore 611101 and Gelman Sciences 12117

polycarbonate filter cartridges) directly after sampling

and, thereafter, stored in the hermetic containers in the

dark at 41C during the analysis and isolation proce-

dures. Table 1 shows some characteristic properties for

the studied natural fresh water samples.

Aquatic humic solutes were isolated from the seven

water samples (Ka; Mu; Au; Ma; Ku; Pi and Pa) by the

J. Peuravuori et al. / Water Research 36 (2002) 4552–4562 4553

conventional so-called XAD technique at pH 2 using the

non-ionic Amberlites XAD-8 resin (isolated humic

samples are sublabelled as [XAD.HM]. The humic

solutes were also isolated from the water samples of

Ka, Mu, Au and Ma using the non-ionic Supelitet

DAX-8 resin (isolated humic samples are sublabelled as

[DAX.HM]. The isolation procedure with the DAX-8

resin was analogous to that of XAD-8 resin. Further-

more, in the case of the Mu-water sample the DEAE

cellulose was applied for isolating at the natural acidity

the integrated whole of practically all humic solutes

(isolated humic sample is sublabelled as [DEAE.HM].

The specific humic solutes (referred also as hydrophobic

humic substances) eluted with 0.1M NaOH from the

XAD-8 and DAX-8 resins were not further divided at

pH 1 into functional humic- (HA) and fulvic- (FA) acid

fractions. Similarly, the macromolecular organic acids

of the [DEAE.HM] sample were not divided at pH 1

into fictional HA and FA subfractions. The organic

constituents not eluted from the XAD-8 and DAX-8

resins with base (so-called hydrophobic neutrals being

relatively too hydrophobic to be eluted with base) were

eluted with methanol and labelled as [XAD.MeOH] and

[DAX.MeOH], respectively. The different analytical

flow procedures have been reported in detail previously

(e.g. [20,21]). The amounts of the humic samples

obtained by the different sorbents and their relation-

ships to the original DOCs are shown in Table 2. It is

noteworthy that the most hydrophobic constituents

eluted only with organic solvents from non-ionic sorbing

solids are not, according to an adopted definition,

usually counted to aquatic HM/substances. In this

study, however, the [-.MeOH] fractions are also

terminologically considered to form a part of the HM

samples.

2.2. Chemical analysis

Colour, pH, CODMn, Fetot and conductivity of the

original water samples were measured, for the sake of

general comparison, in the laboratory of Turku Water

Works using the standard methods. The methods and

equipments used for the determination of dissolved

organic carbon (DOC) in the original water samples, the

moisture and ash contents of the isolated HMs as well

their elemental analyses (carbon, hydrogen, nitrogen

Table 1

Some characteristic properties of studied fresh water samplesa

Water source pH (201C) Colour

(mg Pt l�1)

CODMn

(mg l�1)

DOC

(mg C l�1)

Fetot(mg l�1)

Conductivity

(251C, mSm�1)

Lake Kakarlampi, Ka 5.8 100 77 17.2 0.4 4

Lake Mustaj.arvi, Mu 5.6 150 83 18.9 1.1 3

Aura river, Au 6.8 175 72 16.4 0.7 13

Maaria reservoir, Ma 6.6 175 70 15.2 0.9 14

Lake Kuljuj.arvi, Ku 5.2 175 94 21.2 1.7 4

Lake Pikkuj.arvi, Pi 5.5 70 55 11.6 0.7 2

Lake Pappinen, Pa 6.3 175 83 18.7 2.1 10

aCODMn=chemical oxygen demand as mg KMnO4 l�1. Colour values were measured in the laboratory of Turku Water Works with

a Lovibond 1000 comparator.

Table 2

Amounts (mg l�1) and carbon distributions (% of original DOC) of different humic isolates obtained by different sorbents (moisture-

and ash-free basis)a

Water source [XAD.HM]

(mg l�1, %)

[DAX.HM]

(mg l�1, %)

[DEAE.HM]

(mg l�1, %)

[XAD.MeOH]

(mg l�1, %)

[DAX.MeOH]

(mg l�1, %)

Lake Kakarlampi, Ka 18.7 59 20.6 65 1.7 6 0.3 1

Lake Mustaj.arvi, Mu 20.8 57 22.2 63 30.1 82 2.4 8 0.8 2

Aura river, Au 15.5 51 20.7 68 1.3 4 0.4 1

Maaria reservoir, Ma 14.9 54 18.5 67 0.7 3 0.3 1

Lake Kuljuj.arvi, Ku 19.9 53 1.9 5

Lake Pikkuj.arvi, Pi 9.3 48 1.2 7

Lake Pappinen, Pa 18.6 53 1.9 6

aDifferent [-.HM] fractions were not divided into so-called humic- (HA) and fulvic- (FA) acid subfractions thus representing their

combined mixtures.

J. Peuravuori et al. / Water Research 36 (2002) 4552–45624554

and sulphur; the content of oxygen was taken as a

difference from 100%) have been thoroughly discussed

previously [22]. Table 3 shows the major elemental

composition on ash- and moisture-free basis for the

different humic isolates.

2.3. Fluorescence measurements

Fluorometric analyses at 201C were performed on a

Model LS-50B computer controlled spectrofluorimeter

(Perkin-Elmer, Norwalk, CT, USA). SF spectra

were collected in the 250–600 nm excitation wavelength

range using the bandwidth of Dl ¼ 18 nm between the

excitation and emission monochromators. All spectra

were recorded with a 5-nm slit width on both mono-

chromators. The filter response was 1 and the scan speed

of spectra 120 nmmin�1 and resolution 0.5 nm. The

light-path of the cuvette (FUV, Spectrocell, volume

3.5ml, with screw cap) applied for SFS was 10� 10mm.

Although no further corrections for fluctuation of

instrumental factors and for scattering effects (e.g.

primary and secondary inner filter effects) were applied

to experimental spectra, a comparative discussion on the

spectra is acceptable, at least on a qualitative basis, since

all of them were recorded on the same instrument using

the same experimental conditions.

The solid HM isolates were dissolved (50mg l�1) into

sodium acetate buffer (pH 7.0, conserved under N2).

Likewise, small aliquots of concentrated stock solutions

of HMs (before their freeze-drying) were diluted with

sodium acetate buffer (pH 7.0) to the HM concentration

of 50mg l�1. The SF spectra of original water samples

were recorded directly from their filtrated (0.2 mm)solutions without any dilution or concentration. The

precision (P0.95) of the spectra was ca. 10% (including

the sampling and pretreatment errors). The row spectra

(701 data points) were smoothed by a 12-point

averaging adjacent method before further interpreta-

tions. Relative emission intensities, based on a milligram

of organic carbon per unit volume of solvent, were

calculated in order to normalize the spectra.

3. Results and discussion

3.1. Effect of the sorption resin. Differentiation of humic

isolates according to their origin

Table 2 indicates that the quality of a sorbent has a

significant effect on the quantities of the different humic

isolates obtained. The DAX-8 resin isolated ca. 1976%

more organic, so-called humic solutes (defined as HM or

Table 3

Major elemental composition of different humic isolatesa

Humic fraction,

isolation methodbAsh-% C (%) H (%) N (%) S (%) O (%)

Ka.[XAD.HM] 2.5 54.7 4.7 2.1 1.2 37.4

Ka.[DAX.HM] 2.5 54.0 4.9 1.9 1.3 37.9

Mu.[XAD.HM] 1.1 51.4 4.6 2.8 0.9 40.3

Mu.[DAX.HM] 2.3 53.3 4.6 1.9 1.0 39.2

Mu.[DEAE.HM] 3.8 51.6 4.2 1.8 1.3 41.1

Au.[XAD.HM] 1.9 53.5 4.5 1.5 0.9 39.6

Au.[DAX.HM] 2.3 53.8 5.0 2.0 1.1 38.1

Ma.[XAD.HM] 2.4 55.4 4.9 1.6 0.9 37.1

Ma.[DAX.HM] 2.1 55.2 5.0 1.7 0.8 37.3

Ku.[XAD.HM] 1.1 56.3 6.1 2.2 1.7 33.8

Pi.[XAD.HM] 2.8 60.1 5.7 2.5 1.1 30.6

Pa.[XAD.HM] 2.7 53.1 4.7 2.1 0.8 39.3

Ka.[XAD.MeOH] 3.8 59.6 6.6 2.3 2.5 29.0

Ka.[DAX.MeOH] 3.7 57.2 6.5 3.6 1.3 31.4

Mu.[XAD.MeOH] 1.6 59.5 5.9 2.3 2.1 30.2

Mu.[DAX.MeOH] 3.3 56.9 6.2 3.5 1.6 31.9

Au.[XAD.MeOH] 2.7 57.8 6.3 3.8 3.8 28.3

Au.[DAX.MeOH] 3.1 54.9 5.9 4.9 1.7 32.6

Ma.[XAD.MeOH] 2.7 59.0 6.6 2.5 1.7 30.2

Ma.[DAX.MeOH] 3.2 58.9 6.3 3.2 1.7 29.9

Ku.[XAD.MeOH] 3.6 58.9 6.1 1.9 2.9 30.2

Pi.[XAD.MeOH] 3.7 68.0 7.9 3.6 1.6 19.0

Pa.[XAD.MeOH] 1.9 55.1 4.8 2.1 0.9 37.1

aElemental composition is given on ash- and moisture-free basis.bKa=Lake Kakarlampi; Mu=Lake Mustaj.arvi; Au=Aura river; Ma=Maaria reservoir; Ku=Lake Kuljuj.arvi; Pi=Lake

Pikkuj.arvi; Pa=Lake Pappinen.

J. Peuravuori et al. / Water Research 36 (2002) 4552–4562 4555

humic substances) than did XAD-8 resin. This is very

well in agreement with the report of Farnworth [19]. The

high binding capacity of the DEAE cellulose (ca. 45%

more HM was obtained than with the XAD technique)

was analogous to that in a previous study [21]. The

amount of the most hydrophobic organic constituents

([-.MeOH]) back eluted from the DAX-8 resin with

methanol was considerably smaller (ca. 6975%) than

that found by the XAD-8 resin. The major elemental

compositions in Table 3 were, in turn, practically similar

for the different [-.HM] fractions and also for different

[-.MeOH] fractions (except that the carbon contents of

[XAD.MeOH] fractions were slightly, ca. 471%, great-

er than those of [DAX.MeOH]. Table 1 shows that the

characteristic properties of the original water samples

were fairly similar, except those for Lake Pikkuj.arvi.

Fig. 1 shows the fluorescent behaviours of the humic

isolates separated with different methods from different

sources. The spectra of all the [XAD.-], [DAX.-] and

[DEAE.HM] type fractions in Fig. 1(a) were fairly

similar with each other as was also the case for different

[XAD.-] and [DAX.MeOH] type fractions in Fig. 1(b).

The characterization of the spectra can at the first sight

look somewhat confusing because of the poorly resolved

‘‘peaks’’ between wavy valleys. The first thing which

can be clearly deduced from Figs. 1(a) and (b) is that

the distribution of the fluorescent components is

fully different for the [-.HM] and the corresponding

275 300 325 350 375 400 425 450 475 500 525 550 575 600

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

(a)

Ka.[XAD.HM]

Ka.[DAX.HM]

Mu.[XAD.HM]

Mu.DAX.HM]

Mu.[DEAE.HM]

Au.[XAD.HM]

Au.[DAX.HM]

Ma.[XAD.HM]

Ma.[DAX.HM]

Ku.[XAD.HM]

Pi.[XAD.HM]

Pa.[XAD.HM]Rel

ativ

e em

issi

on in

tens

ity

250 275 300 325 350 375 400 425 450 475 500 525 550 575 600

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

(b)

Ka.[XAD.MeOH]

Ka.[DAX.MeOH]

Mu.[XAD.MeOH]

Mu.[DAX.MeOH]

Au.[XAD.MeOH]

Au.[DAX.MeOH]

Ma.[XAD.MeOH]

Ma.[DAX.MeOH]

Ku.[XAD.MeOH]

Pi.[XAD.MeOH]

Pa.[XAD.MeOH]Rel

ativ

e em

issi

on in

tens

ity

Excitation wavelength (nm), λem = λex+ 18 nm

Excitation wavelength (nm), λem = λex+ 18 nm

Fig. 1. SF spectra recorded for different humic isolates. In (a) the [-.HM] samples were back eluted from the sorbents with 0.1M

NaOH. In (b) the [-.MeOH] samples were back eluted from the sorbents with methanol. The freeze-dried solid samples were dissolved

in sodium acetate buffer (pH 7.0) before SF measurements. Abbreviations of different water-sample sources are given in Table 3.

J. Peuravuori et al. / Water Research 36 (2002) 4552–45624556

[-.MeOH] fractions. A closer inspection of the spectra

indicates that the valleys between the ‘‘tops’’ of the

different waveforms locate fairly precisely at the

same positions for both the [-.HM] and [-.MeOH]

type fractions. Therefore, the whole spectral range

was divided into eight sections: A ¼ 2602302 nm;B ¼ 3022340 nm;C ¼ 3402370 nm; D ¼ 3702420 nm;E ¼ 4202438 nm; F ¼ 4382487 nm;G ¼ 4872510 nm;H ¼ 5102550 nm:For better extracting the information from the spectra

they were mathematically manipulated to fix the set of

‘‘primary’’ peaks between the different valleys. The

baseline drifts of the spectra were removed (e.g. [23]), i.e.

the peaks and adjacent valleys were defined by a

modified multi-point method originally described by

Marshall [24] and Marshall et al. [25]. The ‘‘real’’ peak

heights were then detected and their relative proportions

calculated for every eight spectral regions of A–H. In

this way the very large multi-dimensional spectral data

sets of Fig. 1 (12� 701 in Fig. 1(a) and 11� 701 matrixes

in Fig. 1(b)) were reduced into smaller size matrixes

(12� 8 in Fig. 1(a) and 11� 8 in Fig. 1(b)) without

significant loss of the original information. These new

data sets were computationally [26] examined closer with

a statistical-graphical principal components analysis,

PCA (e.g. [27]). The basic goal of the PCA is to reduce

the numerous variables and to seek linear combinations

(e.g. so-called scores) of those variables explaining most

of the variability.

Fig. 2 shows the biplot of the eight variables for the

different humic isolates (12 [-.HM] cases in Fig. 2(a) and

11 [-.MeOH] cases in Fig. 2(b)). The level of statements

were 72% and 15% for PC1 and PC2 in Fig. 2(a) and

52% and 36% for PC1 and PC2 in Fig. 2(b), respec-

tively. The eight arrowhead lines intersecting at (0,0)

represent the weights of the variables. The direction and

length of each vector (variable) are proportional to

its contribution to the principal components, and the

angle between any two is inversely proportional to

the correlation between them. It is clearly seen that the

fluorescent units of A, B, C, D and F have the most

powerful differentiating abilities.

The statistical graphical analysis of Fig. 2(a) discloses

the important finding that the spectral features are

practically similar to the parallelly isolated [XAD.HM]

and [DAX.HM] fractions, i.e. they were positioned in

pairs quite close to each other. In other words, the

XAD-8 and DAX-8 resins seem to isolate humic-solute

bulks equally, producing mixtures with similar structur-

al compositions. This essential outcome was absolutely

analogous to that obtained previously [22] for the same

HM fractions by means of solid-state carbon-13

measurements confirming the of the SFS utilization.

Furthermore, the distant position of the Mu.[-

DEAE.HM] fraction from that of the corresponding

Mu.[XAD.HM] fraction confirms the perception (e.g.

[21]) that the structural composition of the humic-solute

mixture isolable with the DEAE procedure differs

partially from any special HM fraction obtained

by the multi-stage XAD technique. Fig. 2(b) shows

interestingly that the organic (humic) fractions

[DAX.MeOH], independent of the water sources,

formed their own compact cluster far from their

[XAD.MeOH] homologues. This may mean that the

hydrophobic–hydrophilic sorption-desorption proper-

ties of the DAX-8 resin are more precise than those of

the XAD-8 resin, as also the obtained quantities in

Table 2 indicate.

The environmental impact of the various fresh water

sources on the quality of the different humic-solute

isolates can be seen also from Fig. 2 by inspecting the

distribution of the different fractions. In Fig. 2(a) every

sampling site differs more or less from each other, and

Lake Pikkuj.arvi (Pi) is clearly individual. According to

the distinct distributions it is evident that the water

sources in question play their own essential roles in the

structural fine-chemistry of the HM isolates. Fig. 2(b)

demonstrates, on the contrary, that the water source has

no strong effect on the distribution of the [-.MeOH] type

fractions but they are rather random.

The bands in the SF spectra of Fig. 3 obtained for all

seven original filtrated water samples resemble widely

speaking those recorded for the different [-.HM] isolates

in Fig. 1(a). This is a quite natural consequence because

the [-.HM] isolates accounted up to ca. 80% for the

original DOC. The water-sample sources can be divided

into two subgroups based on the average fluorescence

unit-contents of their original DOM as demonstrated in

Figs. 3(a) and (b). It is, however, noteworthy that the

differentiation of the water sources in Figs. 3(a) and (b)

is totally different from that obtained especially in

Fig. 2(a) and also in Fig. 2(b) for their humic isolates.

This implies strongly that the isolation procedure (e.g.

drastic changes of acidities between ca. pH 2–11) has a

given effect on the structural composition of the isolates

verifying the difficulty to isolate humic solutes as they

occur in their natural conditions. On the other hand,

Fig. 3 supports the statement [15,28,29] that the type of

raw water can be tentatively defined with the aid of

certain so-called fingerprints of the SF spectra.

3.2. Effect of freeze-drying on the composition of humic

isolates

An uncertainty connected to the isolation procedure

of humic solutes is the freeze-drying process for

obtaining solid matter for further analyses. It is essential

that the composition and also the conformation of

the freeze-dried and back dissolved solid HM remain

as equal as possible with those prevailing in their

original solution straight after isolation and purification

procedures, i.e. structural changes during freeze-drying

J. Peuravuori et al. / Water Research 36 (2002) 4552–4562 4557

should be minimized. SFS permits for this purpose an

applicable control technique. Fig. 4 demonstrates the

effect of freeze-drying on the average fluorescence

properties (structural changes) of the different humic

isolates. The average SF spectra (both after and before

freeze-drying) accounted for ca. 96% of the total

variance of the individual [-.HM] fractions and for ca.

83% in the case of [-.MeOH] fractions. Fig. 4(a)

indicates that structural changes happened during the

freeze-drying process are quite minor for the [-.HM]

isolates back eluted from the sorbing solids with base,

only the quantities of different fluorescence units

fluctuated slightly on both sides of ca. 417 nm. This

finding will strengthen the general significance of

different [-.HM] type fractions as an applicable model

for natural aquatic humic solutes.

However, the situation was not so straightforward in

the case of [-.MeOH] fractions as shown in Fig. 4(b).

The strong spectral intensities lying in the spectral range

of 420–438 nm, centred at lex=lem around 427/445 nm,

obtained before freeze-drying for [-.MeOH] fractions

almost vanished during the freeze-drying and dissolving

procedures. This speaks strongly for significant structur-

al changes taking place in the freeze-drying process of

this minor and the most hydrophobic humic-solute

fraction.

PC1PC1

-3.8-3.8 -2.4-2.4 -1-1 0.40.4 1.81.8 3.23.2 4.64.6

-2.2-2.2

-1.4-1.4

-0.6-0.6

0.20.2

1

1.81.8

2.62.6

-3.4-3.4 -2-2 -0.6-0.6 0.80.8 2.22.2 3.63.6

-1.6-1.6

-0.6-0.6

0.40.4

1.41.4

2.42.4

3.43.4

PC2

PC2

PC2

PC2

PC1PC1

Ma.[XAD.HM]

Ma.[DAX.HM]

DB

Pa.[XAD.HM]

Mu.[DAX.HM]

Mu.[XAD.HM]u.[XAD.HM

Mu.[DEAE.HM]

(a)

Ka.[DAX.HM]

Ka.[XAD.HM] GG

H

C

E

Pi.[XAD.HM]A

Ku.[XAD.HM]

F

Au.[DAX.HM]

Au.[XAD.HM]

B

Ku.[XAD.MeOH]

Ma.[XAD.MeOH]

Au.[XAD.MeOH]

E

HG

C

A

D

FMa.[DAX.MeOH]

Au.[DAX.MeOH]Mu.[DAX.MeOH]

Ka.[DAX.MeOH]

Mu.[XAD.MeOH]

Pi.[XAD.MeOH]

Ka.[XAD.MeOH]

Pa.[XAD.MeOH]

(b)

Fig. 2. Graphical perspective on the distribution (biplot) of the different humic isolates for first-two principal components. The eight

variables (A–H) indicate different fluoresence units. For abbreviations of cases (samples) see Table 3.

J. Peuravuori et al. / Water Research 36 (2002) 4552–45624558

3.3. Characteristic features of different humic isolates in

relation to DOM

Fig. 5 shows the relation between the fluorescence

properties prevailing for the original DOM and for the

isolated [-HM.] and [-.MeOH] fractions. The average SF

spectrum of DOM accounted for ca. 95% of the total

variance of the seven individual spectra shown in Fig. 3.

The average SF spectra of the [-HM] and [-.MeOH]

fractions are the same as shown in Fig. 4 (after f-d). The

three average SF spectra displayed seven different

‘‘maxima’’ at lex=lem around 280/298, 330/348, 355/

373, 400/418, 427/445, 460/478, 492/510 and 516/534.

The whole spectral range was also divided into eight

sections (A–H) for better illustrating the eight variables

shown in Fig. 2. The fluorescence properties of [-.HM]

isolates are, on the average, reasonably close to those

recorded for the original DOM, only the relative

quantities of different fluorescence units fluctuated to a

certain degree on both sides of ca. 420 nm. This finding

is in principle a corollary, though not necessary self-

evident.

However, in the case of [-.MeOH] fractions a very

strong emission intensity within the spectral range B,

centred at lex=lem around 330/348 nm, was found. The

intensity in the range B was greatly dependent on the [-

.MeOH] sample in question, as shown in Fig. 1(b). The

peculiar behaviour of the range B was hardly visible in

the spectra of original water samples (cf. Fig. 3).

Likewise, the [-.HM] fractions did not show much signs

of this abnormality although according to Fig. 1(a) the

effect varied somewhat with the sample in question. The

250 275 300 325 350 375 400 425 450 475 500 525 550 575 600

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35 Lake Kakarlampi, Ka

Lake Mustajärvi, Mu

Aura river, Au

Maaria reservoir, Ma

Lake Kuljujärvi, Ku

Lake Pikkujärvi, Pi

Lake Pappinen, Pa

Rel

ativ

e em

issi

on in

tens

ity

(b)

Mu

Ku

Pa

250 400 600

0.00

0.35

Rel

ativ

e em

issi

on in

tens

ity

250 400 600

0.00

0.35

(a)

Ka

AuMa

Pi

Rel

ativ

e em

issi

on in

tens

ity

Excitation wavelength (nm), λem

= λex

+ 18 nm Excitation wavelength (nm), λem

= λex

+ 18 nm

Excitation wavelength (nm), λem

= λex

+ 18 nm

Fig. 3. SF spectra of filtered (0.2 mm) water samples at their original conditions.

J. Peuravuori et al. / Water Research 36 (2002) 4552–4562 4559

importance of the range B as a discriminating factor

between different samples is clearly seen from Fig. 2.

Despite the above-mentioned peculiarity of the

spectral range B, the waveforms of all the [-.MeOH]

fractions resemble, in the broad sense, also those

recorded for the original DOM. It has been previously

verified [30,31] that the different [-.HM] and [-.MeOH]

type fractions contained qualitatively the same principal

building blocks and only their relative content was

the most powerful discriminating factor. The outline

of the recorded SF spectra in Fig. 5 supports this

interpretation, irrespective of the fact that certain

structural changes will take place during the isolation

procedures thus affecting the reactivity of fluorescence

units.

There are many conceivable possibilities for chromo-

phores which might contribute to the fluorescence of

HM. Likewise, it has been discussed for years (e.g.

[4,32,33]) which are the most suitable candidates for the

molecular components responsible for fluorescence of

HM. By inspecting Fig. 5 it is possible, however, to draw

some rough interpretations in relation to the applied

isolation procedures (i.e. [-.HM] vs. [-.MeOH] vs. DOM):

* The intensities of the spectral region A, centred at

lex=lem around 280/298 nm, can justifiably be as-

signed (e.g. [15,29] mainly to aromatic amino acids

and some other volatile acids containing highly

conjugated aliphatic structures being too hydropho-

bic for back elution from the sorbents with base.

Fig. 4. Average SF spectra for demonstrating the effect of freeze-drying on structural changes of different isolated. f-d=freeze-drying.

For other abbreviations see Fig. 1 and Table 3.

250 275 300 325 350 375 400 425 450 475 500 525 550 575 600

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

HGFEDCBA

516

492

460

427

400

355

330

280

Original DOM

[-.HM] fraction

[-.MeOH] fraction

Average spectra

Original DOM

[-.HM] fraction

[-.MeOH] fraction

Rel

ativ

e em

issi

on in

tens

ity

Excitation wavelength (nm), λem

= λex + 18 nm

Fig. 5. Average SF spectra for interaction prevailing between original DOM and isolated humic fractions. The spectral sections are:

A ¼ 2602302 nm;B ¼ 3022340 nm;C ¼ 3402370 nm;D ¼ 3702420 nm;E ¼ 4202438 nm;F ¼ 4382487 nm;G ¼ 4872510 nm and

H ¼ 5102550 nm:

J. Peuravuori et al. / Water Research 36 (2002) 4552–45624560

* Potential structural descriptor for the abnormal

intensities within the spectral region B, centred at

lex=lem around 330/348 nm, can be naphthalene [9]

with its derivatives. In a recent study [30] it was

found that naphthalene was in the first place enriched

in the [-.MeOH] type fractions according to the

hydrophobic–hydrophilic interactions being in force

during the isolation conditions.* A plain classification of fluorescence units of the

longer wavelengths based solely on the condensation

degree of aromatic compounds, disregarding the

influence of the different substituents (e.g. [9,28]), is

in accordance with the hydrophobic–hydrophilic

qualities of the isolation technique and the recent

study of principal building blocks [30,31].* Potential contributors to the fluorescence behaviour

within the spectral range C, centred at lex=lemaround 355/373 nm, are polycyclic aromatics with

three to four fused benzene rings.* Accordingly, the significant range D, centred at

lex=lem around 400/418 nm, corresponds to polycyc-

lic aromatic with approximately five fused benzene

rings.* The last significant range F, with a maximum at

lex=lem around 460/478 nm, is probably reflecting

the influence of polycyclic aromatics consisting of

about seven fused benzene rings. Also, the most

common lignin descriptors fluorescent within this

region.

4. Conclusions

This study has revealed a number of essential

outcomes for the study of humus chemistry: (I) The

utilization of the synchronous scanning fluorescence

technique seems to be quite reliable also in the study of

multi-component humic solutes. (II) Fluorescence prop-

erties of different humic fractions verified that the

hydrophobic–hydrophilic sorption-desorption proper-

ties of the DAX-8 resin seem to be more precise than

those of the XAD-8 resin. (III) The freeze-drying

process in the isolation procedure had no significant

effect on the fluorescence properties of the bulk of humic

solutes speaking for the stability of the structural

composition. (IV) The environmental source of fresh

water samples had its own effect on the fluorescence

properties of isolated humic-solute fractions. (V) The

fluorescence properties of the bulk of the conventional

humic solutes are quite close to those recorded for the

original DOM except in the case of the minor and

most hydrophobic humic solutes. (VI) Interpretations

about some molecular fluorescence units, based only

on aromatic moieties, were reasonable with the hydro-

phobic–hydrophilic qualities of the applied isolation

techniques.

Acknowledgements

The authors wish to thank Dr. Ronald .Osterbacka

(Department of Physics, (Abo Akademi University) for

his assistance in obtaining the synchronous fluorescence

spectra. Special thanks are expressed to Prof. Jorma

H .ols.a (Laboratory of Inorganic Chemistry, University

of Turku) for his favourable position on the thermal

analyses and to Dr. Ensio Laine (Laboratory of

Industrial Physics, University of Turku) for the practical

efforts. The authors are also thankful to Dr. Christer

Eckerman (Laboratory of Paper Chemistry, (Abo

Akademi) for his assistance in DOC analyses.

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