7/77 - Institute of Oceanology, Polish Academy of Sciences (IO PAS)

OCEANOLOGY No. 7 (1977)
105
R Y SZ A R D B O JA N O W SK I
JA N U S Z PE M P K O W IA K
P o lish A ca d em y of S c ien ces
D ep a rtm en t of O cean ology — S op ot
G O T FR Y D K U P R Y SZ E W SK I
U n iv e r sity o f G dansk
D ep a rtm en t o f C h em istry
HUMIC SUBSTANCES IN THE BALTIC SEDIMENTS*
I. ISO L A T IO N A N D P R E L IM IN A R Y C H A R A C T E R IZ A T IO N OF H U M IC
SU BSTA NC ES
C o n t e n t s : 1. In tro d u ctio n 105, 2. E x p erim en ts 106, 3. D iscu ssio n 110, 4. C o n c lu ­
sio n s 117; S tr e szc ze n ie 118; R eferen ces 119.
1. INTRODUCTION
Organic m atter plays an im portant role in physico-chemical and bio­
chemical processes taking place in the bottom sedim ents of shallow m a­
rine basins. The energy released upon decomposition of organic compounds
is utilized by a v ariety of m arine organism s to m aintain th eir life activity.
Organic substances contained in bottom deposits influence th eir physical,
m echanical and chemical properties, such as hum idity, consistency, red-ox
potential ec., and determ ine the direction of diagenetical reactions w ithin
the sedim ents [3],
N aturally occurring organic substances contain functional groups
w hich are capable of binding ions of m etals, thereby expressing ionexchange properties [11], It is assum ed th a t hum ic substances, w hich
usually represent a substantial p art of the m arine organic m atter in bot­
tom sedim ents, play a significant role in the turnover of a num ber of
heavy m etals and transition elem ents in the m arine environm ent.
In order to elucidate th e physical and chemical properties of hum ic
substances and characterize th e ir ion-exchange capability, they have
to be isolated from the bulk of m arine sedim ent in reasonably pure state.
A common isolation procedure consists in the m ultiple extraction of the
sedim ents w ith aqueous solutions of sodium or potassium hydroxide of
various strengths (usually 0.1—0.5 M) [2—5, 7, 9], The crude alkaline ex­
*
T h is w o rk w a s fin a n c ia lly su p p orted b y P A N
o f IA E A R esearch A g reem en t N o 598/PO L/R2.
p ro b lem N o 5 and is p art
trac t containing humic and fulvic acids is then acidified to pH 2__3. The
hum ic acids form an insoluble precipitate, which is separated by filtra ­
tion or centrifugation from the m other solution containing fulvic acids.
Purification of hum ic acids can be effected by consecutive dissolution —
precipitation steps, high-speed centrifugation, dialysis and ion-exchange
chrom atography [9, 11].
Humic substances are characterized by th eir elem entary composition
and presence of functional groups [13, 15, 16], complexing properties
[11, 12], isotope composition [9], grow th prom oting properties [8], etc.
The use of strong hydroxide solutions has been reported to alter
the n atu re of isolated compounds significantly [1], The alteration can
even go fu rth er during th e subsequent purification steps, e.g. dialysis
causes loss of substances w ith low m olecular weight, and ion exchange
chrom atography leads to retention of some compounds on the resin bed
due to adsorption effects.
In this w ork an attem p t has been made to modify the commonly
used extraction procedures for humic acids, the aim being to shorten
the extraction tim e and reduce the num ber of extractions w ithout
affecting the overall yield of humic acids. Efforts w ere made to find a
suitable purification m ethod by which substantial losses of the m aterial
could be avoided.
2. EXPERIMENTS
2.1. M A T E R IA L S
All experim ents w ere carried out on bottom m aterial from the Bay
of Gdańsk (southern Baltic). Samples were taken w ith a snap m ud sam ­
pler in the region of V istula riv er discharge, about 1 km off the river
m outh, from a depth of 7 m. Geographical coordinates of the sampling
site w ere as follows: 54° 22.3'Ni. e= 18° 59.5'E.
P a rt of the sedim ent representing the upperm ost 10 cm layer was
transferred to tw ist-off top glass jars and brought to the laboratory.
Analyses w ere commenced on fresh m aterial w ithin 20 hours after col­
lection. The m oisture content of the sedim ent was 78.5% and th e organic
carbon content was 5.45% d ry weight.
P relim inary preparation of the sedim ent sample for the isolation of
humic acids was made using a procedure described by Rashid and King
[11], w ith slight modifications. A sam ple of n atu ral m oisture was centri­
fuged for 20 m inutes at 5,000 g and the supernate, which contained
negligible am ounts of coloured substances, was discarded. The sedim ent
was treated w ith an equal volume of 0.1 M HC1 to decompose carbonates
and the m ixture was adjusted to pH 2 w ith 1 M HC1 while thoroughly
swirling. A fter 16 hours th e m ixture was centrifuged, the supernate was
discarded and the residue washed w ith distilled w ater and carefully neu­
tralized to pH 7.0 w ith 0.05 M KOH. The m ixture was centrifuged again,
and after rem oving the liquid th e sample was homogenized and the
m oisture determ ined. The sample was then divided into th ree sub­
samples on w hich subsequent extraction experim ents w ere perform ed
using different extraction procedures.
2.2. E X T R A C T IO N — M ETH OD A
A 145 g undried sub-sam ple w hich contained 31% m oisture, was
m ixed w ith 300 cm3 0.5 M KOH solution and left for 16 hours w ith
occasional stirring. The m ixture was centrifuged for 20 mins. at 5,000 g,
and the residue washed w ith 100 cm3 0.5 M KOH and separated by
centrifugation. The solutions w ere combined and the residue was m ixed
w ith a second 300 cm3 portion of 0.5 M KOH, then the extraction and
washing w ere repeated as above. The to tal num ber of extractions was
ten. The solutions from each extraction w ere fu rth er processed to obtain
pure preparations of hum ic acids. M inute m ineral particles w ere rem oved
by centrifugation for 30 m inutes at 6,000 g then th e extracts w ere acidi­
fied w ith conc. HC1 to pH 2 and left overnight. The precipitates w ere
separated by centrifugation and washed w ith 50 cm3 distilled w ater and
the liquids saved to retriev e fulvic acids. Purification of hum ic acids
was achieved by consecutive dissolution of the precipitates in 0.5 M
KOH, centrifugation and re-precipitation w ith conc. HC1 to pH 2, this
having been done four to six tim es to achieve a satisfactory degree of
dem ineralization of the preparations. The final precipitates w ere washed
w ith 0.01 M HC1 followed by redistilled w ater, u n til the w ashing solution
was no longer acidic and w eighed after oven-drying at 70°C. The p re­
parations w ere th en analyzed for the to tal ash content and elem entary
composition, and IR, VIS and UV absorption spectra w ere recorded.
The solutions rem aining after the isolation of humic acids contained
fulvic acids and large quantities of inorganic salts. They w ere joined and
neutralized w ith 0.05 M KOH to pH 7 and vacuum -evaporated at
70°C in a rota-vapour device to about 100 cm3. The solution w as acid­
ified w ith a few drops of conc. HC1 to pH 2 and left overnight. A ny
precipitate was rem oved by filtering the solution through a W hatm an
GF/A glass-fibre filter. To remove salts the solution w as applied on th e
top of a glass column 0 4 X 40 cm filled w ith Sephadex G — 10 and the
column bed was washed w ith redistilled w ater. The eluate was collected
in several fractions, and each fraction was checked for the presence of
chloride ions. The salt-free fractions were combined and evaporated in
Final HA product
discard
procedure
Rye. 1. Schemat wyodrębniania
Fig. 1 . Isolation
Fina/ FA product
osadów
sed im en ts
kwasy fu/winowe
z morskich
substances in bottom
humusowe
substancji humusowych
for humic
kwasy
odrzut
a rota-vapour at 70°C to dryness. The residue was then analyzed for
the total ash content, elem entary composition, and IR, VIS and UV
absorption spectra w ere recorded. The schem atic diagram of th e isola­
tion of hum ic and fulvic acids from sedim ents is shown in Fig. 1 and the
course of alkaline extraction in Fig. 2.
Corg
m
extraction number
numer ekstrakcji
F ig. 2. C han ges o f o rg a n ic carb on con ten t in th e sed im e n t sa m p le in th e cou rse
o f e x tra c tio n w ith 0.5 M K O H (m eth od A)
R ye. 2. Z m ian y w za w a rto ści w ę g la o rgan iczn ego w p róbce o sad ów ek stra h o w a n ej
p rzy u ży ciu 0,5 M (m etod a A)
2.3. E X T R A C T IO N — M ETH OD BI
A portion of th e sedim ent, which had been p re-treated as described
in paragraph 2.1. was consecutively extracted w ith eight 300 cm3 portions
of 0.1 solution of EDTA (disodium salt). The extracts w ere dialysed
through a cellophane bag (Kalle A ktiengesellschaft, West G erm any)
against distilled w ater, the dialysates w ere evaporated under reduced
pressure to 1/4 of th e original volume, and acidified w ith conc. HC1 to
pH 2. A fter 24 hrs. the precipitate of humic acids was centrifuged, washed
w ith diluted HC1 and distilled w ater, and dried. The yield of hum ic
acids for each extraction w as determ ined gravim etrically, and th e p rep ­
arations w ere thoroughly m ixed to obtain a homogeneous composite
sample. On this sam ple the determ ination of the total ash content and
elem entary composition was carried out, and absorption spectra in IR,
VIS and UV light w ere taken.
The sedim ent, after the Na2EDTA extractions, was w ashed four tim es
w ith 400 cm3 of distilled w ater and brought in contact w ith 300 cm 3 of
0.2 M KOH for 16 hrs., w ith occasional stirring. The alkaline extraction
was repeated three times and the humic and fulvic acids w ere isolated
using the same procedure as in Method A.
2.4. E X T R A C T IO N — M ETH OD B2
An aliquot portion of the p re-treated sedim ent sample was extracted
w ith two 300 cm3 portions of 0.1 M Na2EDTA for 16 hrs. each, the
residues w ere washed th ree tim es w ith distilled w ater and the extrac­
tion was continued using th ree 300 cm3 portions of 0.2 M KOH for each
extraction. Subsequent separations w ere perform ed on the combined
alkaline extracts as described in 2.1.2.
3. DISCUSSION
The data characterizing extraction efficiency of hum ic and fulvic
acids by the three different methods are presented in Table 1. The results
indicate th a t all methods tested yield sim ilar quantities of both th e acids,
and for the sedim ent sample used in this tria l the am ounts of humic
and fulvic acids were 2.4 to 3.1 and 0.49 to 0.54 percent of dry sedim ent,
respectively. The higher values for both acids w ere obtained when
alkaline extractions w ere made on the NaaEDTA p re-treated samples.
T ab le 1
T ab ela 1
R eco v ery o f h u m ic and fu lv ic acid s from sed im e n t sa m p les b y d iffe r en t e x tra ctio n
m eth o d s
W yd ajn ość k w a só w fu lw in o w y c h i h u m u so w y ch u z y sk a n y ch z osad u d en n ego
po za sto so w a n iu ek str a k cji m etod ą A oraz m eto d ą B 1 i B 2
E xtraction
m ethod
S a m p le
size
(dry)
M etoda
e k stra k cji
Ilo ść
osadu
R eco v ery
W yd ajn ość
C
org.
C
org.
FA
KF
HA
KH
E x tra ctio n
co n d ition s
HA
KH
FA
KF
W arunki
e k stra k cji
g
%
A
100
5,45
2,420
0,485
22,4
4,5
0.5M ,K O H 8x
BI
100
5,45
0,255
—
2,3
—
N a 2E D T A 8x
2,715
0,525
24,9
4,8
0.2M K O H 3x
—
—
—
—
N A 2E D T A 2 x
3,070
0,540
28,1
5,0
0.2M K O H 3x
B2
100
5,45
g
%
Assuming the to tal organic m atter in th e sedim ent to be tw ice the
am ount of organic carbon, hum ic acids constitute about 28% of the
organic m atter, w hereas fulvic acids are m uch less abundant and am ount
to some 5% of th e to tal organic m atter.
The to tal ash content in the respective fractions of humic and fulvic
acids is shown in Table 2. The results for humic acids vary w ith in a
narrow range of 2.2 to 2.8% irrespective of the m ethod of extraction.
The only result w hich fell outside this range was for the B1 (OHA)
fraction, w hich had not been subjected to m ultiple purification proce­
dure. The ash content in the fulivc acids is m uch greater and am ounts
to up to 15.3%, w hich can be indicative of g reater affinity of this tow ard
cations.
T a b le 2
T ab ela 2
E lem en ta ry co m p o sitio n o f h u m ic and fu lv ic acid s iso la ted
fro m B a ltic se d im en ts
S k ła d e lem e n ta r n y k w a só w h u m u so w y ch i fu lw in o w y c h
w yo d ręb n io n y ch z o sad ów d en n y ch M orza B a łty ck ieg o
E xtraction
m eth od
(fraction
num ber)
M etoda
ek stra k cji
(nr frak cji)
A
A
A
A
B
B
B
B
B
A
B
B
(1KH**)
(3KH)
(5KH)
(7KH)
1 (OKH)
1 (1KH)
1 (2KH)
2 (1KH)
2 (2KH)
(K F ***)
1 (KF)
2 (KF)
A sh
co n ten t
E lem en ta ry co m p o sitio n *
Z a w a r­
tość
p op iołu
S k ła d elem en ta rn y
%
C%
H%
N%
0 + S%
2,2
2,7
2,3
2,8
5,28
2,8
2,4
2,3
2,2
15,1
13,3
12,9
53,94
55,39
55,96
56,61
53,63
55,73
56,92
55,17
56,81
47,32
46,59
46,68
6,31
6,75
6,26
6,28
6,49
6,41
6,17
6,35
6.14
7,77
7,38
7,75
5,74
6,44
5,98
5,88
5,50
5,83
6,02
5,93
5,96
5,61
5,88
6,13
34,01
31,42
31,80
31,23
33,98
32,33
30,89
32,55
31,09
39,30
40,15
30,84
C /H
C/'N
8,54
8,20
8,93
9,01
8,26
8,60
9,22
8,61
9,26
6,09
6,31
6,35
9,39
8,60
9,35
9,62
9,08
9,52
9,41
9,30
9,53
8,43
7,92
7,61
C, H a n d N co n te n ts a re ex p ressed on ash fre e substance.
Z aw artość C, H, N podano w % p rzeliczonych d la su b sta n c ji pozbaw ionych popiołu.
* O + S v alues w ere obtain ed by su b tra c tio n of th e sum of C + H -f N fro m 100%.
W artość O + S w yliczono z różnicy: 100%> — (%>C + %H + %N).
** H um ic acids
K w asy hum usow e
*** F ulvic acids
K w asy fulw inow e
The course of alkaline extraction of the sedim ent in m ethod A is
shown in Fig. 2. Num bers on the ordinate show the to tal organic carbon
left in the sedim ent after consecutive extraction steps. The overall
decrease of C org. in the sedim ent after ten extractions was 43%.
It should be pointed out th a t a little higher recovery of hum ic and
fulvic acids w as obtained when Na2EDTA treatm en t was applied prior to
alkaline extraction, even though few er extractions w ith m ore diluted
KOH solutions w ere carried out. The effectiveness of Na2EDTA alone in
solubilizing hum ic acids in the sedim ent proved to be negligible in
the first th ree extractions and appreciable quantities of hum ic substan­
ces began to appear startin g from the fo u rth extract. The rate of ex­
traction was slow though, the overall yield of eight Na2EDTA extractions
having been but 250 mg per 100 g of d ry sediment.
The enhancing effect of Na2EDTA on th e alkaline extraction of
humic acids can presum ably be attrib u ted to complexing properties of
ethylen diam ine tetraacetic acid molecules tow ard positively charged ions
of metals. Humic m a tter is 'known to form insoluble compounds w ith
m ultivalent cations, and adsorb on m ineral particles [10, 12, 16]. In the
presence of Na2EDTA exchange of ligands takes place and m ultivalent
cations complexed w ith EDTA are no longer capable of rendering the
hum ic m a tter in insoluble form. If adsorption of hum ic acids on clay
particles is effected via cations loosely bound to alum inosilicate stru c­
tures [16] by complexing these cations w ith Na2EDTA th e bonds are
broken and hum ic anions are m ade available for alkaline extraction.
The results of elem entary analysis of hum ic and fulvic acids are given
in Table 2. They are expressed on an ash-free substance basis. The
results indicate th a t th e carbon content of consecutive humic acids
fractions obtained in m ethod A increases gradually from 53.94% to
56.61%. A t th e same tim e th e hydrogen cointent decreases in th e same
order, w hereas the values for nitrogen are w ith in th e range of 5.74 to
6.44%, the variation being irregular. The C/H ratio, w hich is regarded
as indicative of the degree of condensation of hum ic acids [2], increases
w ith the increase of th e carbon content. Such results m ay speak in
favour of the belief th a t during the consecutive alkaline extractions
hum ic acids of increasingly condensed molecules pass into th e solution.
The elem entary composition of hum ic acids extracted using the th ree
different m ethods is m uch th e same, which can be regarded as evidence
th a t no substantial alteration of th e natu re of extracted substances has
occurred. In comparison w ith humic acids fulvic acids contain higher
proportions of hydrogen and a low er carbon content, w hich results in
great differences in the C/H ratio for both acids.
Absorpion spectra, in visual and u ltraviolet light, of selected humic
and fulvic acid fractions are shown in Figs. 3 and 4. Spectra of hum ic
acids bear the same character for all th e fractions, and indicate two def­
lections on the absorption curves, one at 405 nm and th e other at
665 nm, w ith continual increase of absorbance tow ards a shorter
w avelength region. On absortion spectra of fulvic acids no such deflecwove length
dtugość fa li
r„i
I 1
wave number
liczba falo wa
F ig. 3. A b sorp tion sp ectra o f h u m ic (K H ) an d fu lv ic (K F) acid s in v is ib le lig h t
R ye. 3. W idm o a b so rp cy jn e k w a só w h u m u so w y ch (KH) i k w a só w
(K F) w za k resie w id z ia ln y m
wave length
długość fa li
fu lw in o w y c h
r i
1 1
wave number U f '. / t f ']
l/czba falowa L
J
F ig. 4. A b sorp tio n sp ectra o f h u m ic (KH) an d fu lv ic (KF) acid s in u ltr a v io le t lig h t
R ye. 4 W idm o a b so rp cy jn e k w a só w h u m u so w y ch (KH) i k w a só w fu lw in o w y c h (KF)
w za k resie n a d czerw o n y m
8 — O ceanologia n r 7
tions can be seen in visible light, and absorbance values are generally
m uch lower th an for humic acid solutions of th e same concentration.
Spectra in visible light provide some inform ation about th e m olecular
structure of hum ic acids. Skopintsev [17] has shown th a t the absorbance
ratio: K = —■465 nm depends on the degree of condensation of humic
-^•665 nm
acid molecules, this being higher for stru ctu res w ith a low er degree of
condensation, and vice versa. The validity of this finding was confirm ed
by Nissenbaum and K aplan [9],
T a b le 3
T ab ela 3
K = A 4e5nm : A 665nm v a lu e s fo r d iffe r e n t fu lv ic and h u m ic
acid fra ctio n s
W artości K = E465nm : E665nm d la p o szczeg ó ln y ch fra k cji
su b sta n c ji h u m u sow y ch
E xtra ctio n
m eth od
(fractio n
num ber)
K
M etoda
ek str a k c ji
A (I K H )
Ą (3KH)
A (5KH)
A (7KH)
E x tra ctio n
m eth od
(fra ctio n
num ber)
K
M etoda
e k stra k cji
4.8
4.65
4.55
4.25
B
B
B
B
B
1 (1KH)
1 (2KH)
2 (1KH)
2 (2KH)
1 (OKH)
E x tra ctio n
m eth od
(fraction
num ber)
K
M etoda
e k stra k cji
4.7
4.3
4.6
4.35
4.75
A (K F)
B 1 (KF)
B 2 (KF)
5.8
6.1
5.95
The K values for hum ic acids obtained by consecutive alkaline ex­
tractions of the bottom sedim ent are shown in Table 3. It can be seen
from these data th a t the K values decrease w ith the increasing num ber
of extractions, which indicate the growing contribution of more conden­
sed molecules in each subsequent alkaline extract. The same general
conclusion can be draw n from the results of elem entary analyses
perform ed on the respective fractions of hum ic acids (Table 2).
Fulvic acids show higher K values th an hum ic acids which m ay be
associated w ith lower m olecular w eight of the form er [9],
Both hum ic and fulvic acids comprise a v ariety of chem ical com­
pounds w ith different m olecular sizes and elem entary composition [10, 15]
which fact makes them a difficult object of study. In frared spectrom etry
provides useful m eans of elucidating th e functional composition of such
complex substances. In exam ining IR spectra of hum ic and fulvic acids
attention is given to th e following w ave num ber regions: 3200 cm-1 —
stretching vibrations of — OH and — NH2 bonds, 1720 cm-1 stretching
vibrations of > C = 0 in esters and aliphatic aoids, 1650 cm-1 — s tre t­
ching vibrations of peptide bonds in proteins and > C = C < in arom atic
rings 1530 cm-1 —• amide II bonds in proteins, 1240 cm 1 — usually
accompanied w ith 1530 cm-1 absorption band, 1200 cm-1 — usually
wave number r -n
liczba ptowa
J
F ig. 5. In frared tr a n sm issio n sp ectra o f sep a ra te fra ctio n s of h u m ic (KH) and fu lv ic
(KF) acid s
R ye. 5. W idm o a b so rp cy jn e p o szczeg ó ln y ch fra k c ji k w a só w h u m u so w y ch
i k w a só w fu lw in o w y c h (KF) w za k resie p od czerw o n y m
(KH)
accompanied w ith a carbonyl band at 1720 c m '1, 1200 — 1100 cm -1
cyclic ethers, 1050 cm -1 — a band characteristic fo r > C = 0 in polysa­
ccharides, 1000
700 cm-1 — a band characteristic for silicoorganic
compounds.
Specimen exam ples of IR spectra taken for selected fractions of humic
and fulvic acids are shown in Fig. 5, and the m ore im portant absorption
bands are tab u lated in Table 4. Interp retatio n of the spectra is compliT ab le 4
T ab ela 4
M ore im p o rta n t in fra red a b sorp tion b an d s fo r h u m ic su b sta n ces
iso la ted from B a ltic sed im en ts
W ażn iejsze m a k sim a a b sorp cji w p o d czerw o n y m za k resie w id m a
dla su b sta n cji h u m u so w y ch z o sa d ó w d en n y ch M orza B a łty ck ieg o
E xtraction
m ethod
(fraction
num ber)
IR
M etoda
ek strak cji
3200
1720
n e w o il
m ax
(cm -1 )
IR
n u jol
m ax
(cm -1)
1650
1530
1240
1260 1200-1100
1030
A (1KH)
S ( v e r y w id e )
S
M
M
W
M
A (3KH)
S
S
S
M
M
A (5KH)
S
s
s
M (w ide)
szer.
M
M
W
M
A (7KH)
S
s
s
M
M
W
M
S
M
B 1 (1KH) S
B 1 (2KH) S
B 2 (1KH) S
B 2 (2KH) S
A (KF)
S
B 1 (KF)
S
B 2 (KF)
S
S
b. sz e r .
S
(v e r y w id e )
b. sz e r .
( v e r y w id e )
S
b. sz e r .
S
( v e r y w id e )
b. sz e r .
S
(v e r y w id e )
b . sz e r .
s
( v e r y w id e )
b. sz e r .
s
( v e r y w id e )
b . sz e r .
s
(v e r y w id e )
b. sz e r .
(very w id e)
M
b. szer.
(v ery w id e)
M
b. szer.
(v ery w id e)
M
b. szer.
S tro n g
silne
S (w ide)
szer.
S (w ide)
szer.
S
M
s
M
M
w
M (w ide)
szer.
M
M
S (w ide)
szer.
M
S
s
S
s
s
s
M — m edium
śre d n ie
S (w ide)
szer.
M (w ide) M (w ide)
szer.
szer.
M
S
M
W
S(ujide) S (w ide)
S (w ide)
szer.
S (w ide)
szer.
szer.
szer.
M
M
M
M
S (w ide)
b. szer.
S (w ide)
szer.
w _w eak
słabe
■
cated by the presence of strong and wide absorption bands. It can be
seen, however, th a t the spectra of fulvic acid fractions are all alike,
w ith all the aforem entioned bands being w ell visualized. The more
pronounced absorption peaks occur at 1050-1 and w ithin a range of 1200
— 1100 cm-1 , w hich w ere not observed in th e hum ic acid spectra.
T here are also some additional peaks in hum ic acid spectra, at 1240 cm -1
and 1200 cm“ 1 w hich are not seen in the spectra of fulvic acids. Humic
acids do not seem to undergo alteration in the course of alkaline ex tra ct­
ion significant enough to be revealed by the IR spectra.
4. CONCLUSIONS
A comparison of different extraction m ethods for isolating hum ic
substances from m arine sedim ents indicates th a t treatm en t of th e sedi­
m ents w ith 0.1 M solution of Na2EDTA prior to alkaline extraction w ith
0.2 M KOH solution results in faster dissolution rate and greater yield
of hum ic substances com pared w ith m ethods th a t m ake use of hydrox­
ide solutions alone. The resu lts of elem entary analysis and IR, VIS
and UV spectra show no substantial differences in the physical and
chem ical properties of the hum ic substances isolated by both the m eth ­
ods. The effectiveness of N a2EDTA in enhacing solubilization of organic
m a tter contained in sedim ents m ay be due to com plexation of heavy
m etal ions th a t form insoluble compounds w ith hum ic substances. A nother
factor th a t m ay contribute is th e action of Na2EDTA on organo-m ineral
aggregates, w hich brings th e organic com ponent to solution.
An advontage of the p re-tre atm e n t of sedim ent sam ples w ith
Na2EDTA solution is th a t by using more dilute hydroxide extractants
and shortening th e extraction procedure organic compounds are less
exposed to the adverse environm ent, thereb y yielding products which
resem ble m ore closely th eir original structure.
OCEANOLOGIA N r 7 (1977)
118
R Y SZ A R D B O JA N O W SK I
J A N U S Z P E M P K O W IA K
P o lsk a A k a d em ia N a u k
Z akład O cea n o lo g ii — S op ot
G O T FR Y D K U P R Y SZ E W SK I
U n iw e r sy te t G dański
In sty tu t C h em ii — G d ań sk
S U B S T A N C J E H U M U SO W E
W O S A D A C H D E N N Y C H M O RZA B A Ł T Y C K IEG O
I. W Y O D R Ę B N IE N IE I W S T Ę P N A C H A R A K T E R Y ST Y K A
S U B S T A N C J I H U M U SO W Y C H
S tresz cz e n ie
S u b sta n c je org a n iczn e za w a rte w o sad ach d en n y ch m órz o d g ry w a ją w a ż n ą rolę
w fizy k o ch em ic zn y ch i b io ch em iczn y ch p rocesach zach od zących w ty ch osadach.
N a jw ię k sz ą fra k cję su b sta n cji o rg a n iczn y ch o sa d ó w d en n y ch sta n o w ią su b sta n cje
h u m u sow e. W ła sn o ści su b sta n cji h u m u so w y ch p ozn an e zo sta ły w m a ły m stop n iu ;
w iad om o jed n ak , że w sk ła d fr a k c ji h u m u so w ej za w a rtej w m orsk ich osa d a ch d en ­
n y ch w ch od zą zw ią z k i zn a czn ie ró żn ią ce się ciężarem czą steczk o w y m , p osia d a ją ce
szereg gru p fu n k c y jn y c h , ta k ich jak h y d ro k sy lo w e, k a rb o n y lo w e, k a rb o k sy lo w e,
a m in o w e, u g ru p o w a n ia a ro m a ty czn e i h etero cy k liczn e. P o ró w n a n ie w y n ik ó w badań
nad su b sta n cja m i h u m u so w y m i, k tó re z o sta ły u z y sk a n e przez różn ych badaczy,
sp ra w ia tru d n o ści ze w z g lęd u n a sto so w a n ie różn ych m etod izo lo w a n ia ty ch
zw ią zk ó w z o sa d ó w oraz m o żliw o ści zm ia n w n a tu rze w y o d ręb n io n y ch su b sta n cji.
W p racy p rzed sta w io n o m eto d ę szy b k ieg o w y o d r ęb n ia n ia su b sta n cji h u m u so ­
w y c h z osad ó w i o czy szcza n ia u z y sk a n y ch su b sta n c ji w w a ru n k a ch za ch o w a w czy ch .
M etoda p o leg a n a w stę p n e j ob rób ce osad u 0,1 M ro ztw o rem w erse n ia n u d w u so d o w eg o (N a2E DTA ), a n a stę p n ie ek str a k c ji osad u 0,2 M ro ztw o rem w o d o ro tlen k u
p o ta so w eg o (KOH). T a k i sp osób iz o la cji p o zw a la z w ię k sz y ć w y d a jn o ść e k stra k cji
oraz ogran iczy ć ilo ść d estru k c y jn y ch e k stra k cji a lk a liczn y ch
W w y p a d k u sto so w a n ia w y łą c z n ie ek stra k cji a lk a liczn y ch k o n ieczn a je st d z ie się ­
ciok rotn a ek stra k cja 0,5 M ro ztw o rem KO H, co sp raw d zon o ozn aczając za w a rto ść
w ę g la organ iczn eg o (corg) w o sa d zie po k o lejn y ch ek stra k cja ch . U z y sk a n e su b sta n ­
c je h u m u so w e rozd ziela n o n a k w a sy h u m u so w e i k w a sy fu lw in o w e w y k o rzy stu ją c
różn icę rozp u szcza ln o ści ty ch zw ią zk ó w w śro d o w isk u k w a śn y m (pH 2). K w a sy
h u m u so w e oczyszczan o drogą w ir o w a n ia , a k w a sy fu lw in o w e — na k o lu m n ie w y ­
p ełn io n ej S ep h a d e x e m G -10.
W celu sch a ra k tery zo w a n ia u z y sk a n y ch w tra k cie k o le jn y c h e k stra k cji fra k cji
k w a só w h u m u so w y ch i k w a só w fu lw in o w y c h w y k o n a n o w id m a ab so rp cy jn e w o d ­
n y ch roztw orów ty ch k w a só w w ś w ie tle w id z ia ln y m i n a d fio le c ie oraz w id m a w
p od czerw ien i. W yk on an o r ó w n ież a n a lizę sk ła d u elem en ta r n e g o p o szczeg ó ln y ch
frak cji, ozn aczając za w a rto ść w ę g la , w od oru i azotu.
W idm o a b so rp cy jn e w ś w ie tle w id z ia ln y m o trzy m a n e d la ro ztw o ró w k w a só w
h u m u so w y ch w y k a z u je d w a p u n k ty p rzegięcia: p rzy d łu g o ści fa li 405 n m i 665 nm .
N a to m ia st w id m o k w a só w fu lw in o w y c h m a ch a ra k ter m o n o to n iczn y w ty m z a k re­
sie . W idm o a b so rp cy jn e w n a d fio le c ie d la k w a só w h u m u so w y ch i fu lw in o w y c h
p osiada p u n k t p rzeg ię cia p rzy ok oło 280 nm . W idm a ab sorp cyjn e w ś w ie tle w id z ia l­
nym i n a d fio lecie w y k a z u ją w z ro st absorp cji w ra z ze z m n iejsza n iem d łu g o ści fa li.
Z n aczn ie bardziej in te r e su ją c e są w id m a a b so rp cy jn e w p o d czerw ien i. P om im o
u tru d n ion ej, ze w z g lęd u n a bardzo siln e i bardzo szero k ie p asm a, in te r p r e ta c ji w e
w sz y stk ic h a n a lizo w a n y ch fra k cja ch w y k a za n o is tn ie n ie pasm ab sorp cji w y w o ła ­
n ych o b ecn ością gru p h y d ro k sy lo w y ch , k a rb o n y lo w y ch , k a rb o k sy lo w y ch , a m in o ­
w y ch , a tak że w ią z a ń p ep ty d o w y c h u gru p ow ań a ro m atyczn ych i p olicu k rów .
A n a liza elem en ta rn a w y k a za ła , że k w a sy h u m u so w e za w iera ją 53,94— 56,61% w ę ­
gla, 6,14— 6,75% w o d o ru oraz 5,74— 6,44% azotu. S k ła d elem e n ta r n y k w a só w fu lw in o w y ch je st n ieco in n y . Z a w a rto ść w ę g la w y n o si 46,68—47,32%, za w a rto ść azotu
5,61— 6,13%, a zaw a rto ść w o d o ru 7,35— 7,77%. N ie stw ierd zo n o z a leż n o ści p o m ięd zy
ch a rak terem w id m w p o d czerw ien i a sk ła d em elem en ta rn y m b a d a n y ch zw ią zk ó w .
W zrost w y d a jn o śc i e k stra k cji po w stę p n y m u ży ciu w e r sen ia n u d w u so d o w eg o
w y tłu m a czo n o k o m p lek so w a n iem jo n ó w m e ta li przez siln y lig a n d , ja k im je s t w e r sen ia n d w u sod ow y . N ie b ez zn a czen ia m oże b y ć ró w n ież w p ły w w e r se n ia n u na
stru k tu rę m in era łó w ila sty c h , k tó re są g łó w n y m c zy n n ik iem a d sorb u jącym su b sta n ­
cje h u m u so w e z w o d y m orsk iej.
REFEREN CES
LITERATURA
1. A k i r a O tsu k i, T a k a n i s h a H an ya, S o m e P re c u rso rs o f H u m ic A c id in
R e c e n t L a k e S e d im e n t, S u g g e ste d b y In fra -R e d S p e c tr a , G eoch im . C osm ochim .
A cta, 1967, 31, 1505.
2. B o r d o w s k i O.K., A c c u m u la tio n o f O rga n ic M a tte r in B o tto m S e d im e n ts ,
Mar. G eol., 1965, 3, 33.
3. B o r d o w s k i O.K., O rg a n ic ze sk o je w ie s z c z e s tw o so w rie m ie n ń y c h o sa d k o w
B e rin g o w o M oria, T ru d y Inst. O k iean oł., 1960, 42, 89.
4. D e g e n s E.T., R e u t e r J.H., S h a w N.T., B io ch e m ic a l C o m p o u n d s in O ffsh o re
C a lifo rn ia S e d im e n ts a n d S ea W a te r, G eoch im . C osm ochim . A cta, 1964, 28, 45.
5. E a rly d ia g e n e sis in S p a n ish In le t, a red u cin g fjo r d , B r o w n E.S., B o e d e c k e r
M.J., N i s s e n b a u m A. , K a p l a n I.R., G eoch im . C osm ochim . A cta, 1972, 36,
1185.
6. G o l d b e r g E.D., A r r h e n i u s G.O.S., C h e m is tr y o f th e P a c ific P e la g ic S e d ­
im en ts, G eochim . C osm och in . A cta, 1958, 13, 153.
7. G o r s z k o w a T.I., O rg a n ic ze sk o je w ie s z c z e s tw o w d o n n ych o tlo że n ija c h B a łtijs k o g o m o ria . C h im ic ze s k ije p ro c e s sy w m o ria c h i o k iea n a ch , N a u k a , Mo'sk w a 1966, s. 55— 59.
8. In flu en ce o f H u m ic S u b sta n c e s on th e G ro w th o f M a rin e P h y to p la n k to n D ia ­
to m s, P r a k a s h A. , R a s h i d M .A., J a n s e n A rn e, S u b b a R a c D .V.,
L im n ol. O ceanogr., 1973, 18, 516.
9. N i s s e n b a u m A. , K a p l a n IR., C h em ica l a n d Is o to p ic E v id e n c e fo r th e
In S itu O rig in o f M a rin e H u m ic S u b sta n c e s, L im n ol. O ceanogr., 1972, 17, 570.
10. P e m p k o w i a k J., S u b sta n c je h u m u so w e w m o rsk ic h o sa d a ch d e n n y c h , S tu ­
dia i M ateria ły K o m itetu B ad ań M orza P A N , C hem ia M orza, 1975, 8, 121.
11. R a s h i d M .A., C o n trib u tio n o f H u m ic S u b sta n c e s to th e C a tio n E x ch a n g e
C a p a c ity o f D iffe re n t M a rin e S e d im e n ts , M ar. S edim ., 1969, 5, 44.
12. R a s h i d M .A., R o le o f H u m ic A c id s o f M a rin e O rig in a n d th e ir D iffe re n t
M o lecu la r W e ig h t F ra ctio n s in C o m p le x in g D i-a n d T r iv a le n t M eta ls, S o il Sci.,
1971, 111, 298.
13. R a s h i d M .A., Q u in o n e C o n te n t o f H u m ic C o m p o u n d s Is o la te d fr o m th e M a­
rin e E n v iro n m e n t, S o il Sci., 1972, 113, 181.
14. R a s h i d M .A., B a c 1 a y D.E., R o b e r t s o n K.R., W z a im o d ie js tw ije h u m in o w y c h k is lo t s r o z lic z n y m i g lin is ty m i m in ie r a la m i i p r ir o d n y m i o sa d k a m i w ilow y c h w o d a c h b lizk ic h k w o d a m e stu a rie w , I M iezdun arodn yj G ieoch im iczesk ij
K on griess. O sadocznoje p rocessy. Tom . IV , K n iga 2, 125. M o sk w a 1973.
15. R a s h i d M .A., K i n g L .H ., C h em ica l C h a ra c te r istic s o f F ra c tio n a te d H u m ic
A c id s A sso c ia te d w ith M a rin e S e d im e n ts , C hem . G eol., 1971, 7, 37.
16. R a s h i d M .A ., K i n g L .H ., M o lecu la r W e ig h t D is tr ib u tio n M e a su re m e n ts of
H u m ic a n d F u lv ic A c id F ra ctio n s fr o m M a rin e C la y s o n th e S c o tia n S h elf,
G eoch im . C osm ochim . A cta, 1969, 33, 147.
17. S k o p i n c e w B .A ., O rg a n ic ze sk o je w ie s z c z e s tw o w p riro d n y c h w o d a c h (w o d n y j g u m u s), T ru d y gos. O k iean ogr. In -ta , 1950, 19, 3.