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P O S I V A O Y
FI -27160 OLKILUOTO, F INLAND
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Jan i Mäke lä
Pent t i Mann inen
May 2007
Work ing Repor t 2007 -23
Humic and Fulvic Acids in Groundwater
May 2007
Working Reports contain information on work in progress
or pending completion.
The conclusions and viewpoints presented in the report
are those of author(s) and do not necessarily
coincide with those of Posiva.
Jan i Mäke lä
Pent t i Mann inen
Rambo l l F in l and Oy
Work ing Report 2007 -23
Humic and Fulvic Acids in Groundwater
HUMIC AND FULVIC ACIDS IN GROUNDWATER
ABSTRACT
A non-ionic polymeric sorbent DAX-8 (XAD-8) was applied to the isolation of humic
and fulvic acids from groundwater. This procedure was able to isolate approximately
40% of the DOC as humic solutes. For the investigation of the structure and molecular
size distribution of the isolated humic solutes, the hyphenated SEC-ESI-MS system
with a quadrupole mass spectrometer and SEC-UV and fluorescence detectors was
utilized. For the higher-molecular-weight humic acids, the ESI-MS loses sensitivity
compared with the parallel UV detection, because of the difficulty in getting the ionized
humic compounds to fly efficiently through the mass spectrometer.
Keywords: Humic and fulvic acids, groundwater, XAD, DAX, SEC, mass
spectrometry.
HUMUS- JA FULVOHAPOT POHJAVEDESSÄ
TIIVISTELMÄ
Humus- ja fulvohappojen eristämiseen pohjavedestä käytettiin adsorptiota polymeeri-
sorbenttiin (DAX-8). Tällä menetelmällä saatiin eristettyä noin 40 % liuenneesta
orgaanisesta aineksesta. Eristettyjen humus- ja fulvohappojen rakenteen ja molekyyli-
kokojakauman tutkimiseen käytettiin SEC-ESI-MS laitteistoa kvadrupolimassa-
spektrometrillä sekä UV ja fluoresenssi detektoreita. Laitteisto osoittautui tehokkaaksi
erityisesti fulvohappojen tutkimuksessa. Raskaamman molekyylipainon omaavilla
humushapoilla ESI-MS laitteiston herkkyys laskee koska ionisoidut humus aineet on
vaikea saada lentämään tehokkaasti massaspektrometrin läpi.
Avainsanat: Humus- ja fulvohapot, pohjavesi, XAD, DAX, SEC, massaspektrometria.
1
TABLE OF CONTENTS
ABSTRACT
TIIVISTELMÄ
1. INTRODUCTION .................................................................................................... 2
2. EXPERIMENTAL .................................................................................................... 3
2.1 Sampling ......................................................................................................... 32.2 Chemicals and reference compounds ............................................................ 32.3 Isolation........................................................................................................... 32.4 TOC analysis .................................................................................................. 42.5 HPLC fluorescence and UV ............................................................................ 42.6 HPLC-MS........................................................................................................ 4
3. RESULTS ............................................................................................................... 6
3.1 TOC results..................................................................................................... 63.2 HPLC with a fluorescence detector................................................................. 63.3 MS results ....................................................................................................... 9
3.3.1 Direct injection......................................................................................... 93.3.2 SEC-MS ................................................................................................ 10
4. DISCUSSION........................................................................................................ 18
5. CONCLUSIONS.................................................................................................... 20
REFERENCES ............................................................................................................. 21
2
1. INTRODUCTION
Aquatic humic substances are polar brown-coloured organic acids, which are derived
from soil humus and terrestrial and aquatic plants. They generally comprise one third to
over half of the dissolved organic carbon (DOC) in natural surface waters.
Because of the dilute nature of aquatic humic substances, the material must first be
isolated and concentrated for further studies. Dissolved humic matter contains a large
number of diverse chemical functionalities, and no single analytical method is available
to reveal the actual concentration of these macromolecular organic solutes in the
sample. The main difficulty is that the aquatic humic solutes must be separated from the
other organic and inorganic solutes in natural water. Different methods for
concentrating and isolating the dissolved organic matter such as freeze drying, chemical
precipitation, solvent extraction, reverse osmosis, ultra filtration and adsorption to solids
have been presented. All of these methods have some disadvantages of not isolating the
aquatic humic matter and, at the same time, keeping the chemical and structural
composition unaffected as it occurs in its original state in an aquatic environment
(Aiken et al. 1985).
The classification of dissolved organic matter into humic and non-humic solutes is
based on the isolation method. The isolation method and the chemical conditions
applied determine the degree of separation of humic from non-humic matter. The
fraction of humic substances that are not soluble in water at a pH lower than 2, are
defined as humic acids. The fraction of humic substances that are soluble under all pH
conditions are referred to as fulvic acids (Malcolm 1990).
The most frequently used isolation procedure for humic substances is the column
chromatographic method by non-ionic sorbents such as XAD resins (polymethyl
methacrylate) or analogous materials (Leenheer 1981, Thurman et al. 1981 and
Peuravuori et al. 1997). The XAD resin classifies organic solutes in an acidified water
sample into artificial hydrophobic and hydrophilic fractions according to their random
ability to adsorb onto non-ionic macroporous resin. This operational classification of
aquatic humic substances is clear but, on the other hand, the XAD isolation technique
under strongly acidic conditions might involve certain risks for uncontrolled
fractionation or reactions.
The goal of this study was to determine the amount of humic substances and their
properties such as the structure and molecular size distribution in the groundwater from
a shallow depth (about -85 m b.s.l.) in the bedrock at Olkiluoto.
The isolation procedure applied was the adsorption of humic substances on non-ionic
macroporous resin (polymethyl methacrylate, DAX-8). The hyphenated SEC-ESI-MS
system with a quadrupole mass spectrometer and SEC with fluorescence and UV
detection were applied to the investigation of the structure and molecular size
distribution.
3
2. EXPERIMENTAL
2.1 Sampling
Groundwater samples were collected from groundwater station ONK-PVA3 (depth
about –85 m b.s.l.) in ONKALO, Olkiluoto, Finland. The samples were on-line pressure
filtered (Pall-Gelman) in situ, using Millipore HA 47 mm, 0.45µm filters. The tubing
material used in the sampling was nylon. The glass containers (1-5 L) that were used to
store the samples were washed with 6% HNO3, high-purity MQ water and ethanol. The
samples were stored at 6º C and protected from the light by wrapping the glass
containers in aluminium foil.
2.2 Chemicals and reference compounds
1.0 M HCl and NaOH (Reagecon, Shannon Free Zone, Shannon, Co. Clare, Ireland)
were used by diluting to the desired concentration with MQ water.
n-Hexane (Fluka) and methanol (J. T. Baker) were of the “for residue analysis” or
“Baker Analyzed” quality.
Standard solutions containing humic and fulvic acids were made by dissolving IHSS
(International Humic Substances Society) Nordic Aquatic Humic Acid Reference
(1R105H) and IHSS Nordic Aquatic Fulvic Acid Reference (1R105F) in an 80/20 (v/v)
water/methanol mixture with 10 mM NH4HCO3.
PEO (polyethylene oxide) standards (Polymer Laboratories) were used for the
investigation of the molecular size distribution. PEO standards were dissolved in an
80/20 (v/v) water/methanol mixture with 10 mM NH4HCO3 using sonication at 40° C
during several hours.
QCWW4 reference solution (Eurofins A/S) was used as a quality control sample for the
TOC determinations.
DAX-8 resin (Supelite DAX-8, Supelco, a new substitute for XAD-8 resin) was used
for the isolation of humic substances from groundwater.
2.3 Isolation
The resin was first cleaned according to Thurman et al. (1981) with slight
modifications. The DAX-8 material was first cleaned by rinsing the resin in a beaker
with 0.1 M NaOH and thereafter with methanol until the pH was neutral. Next, the resin
was Soxhlet-extracted sequentially for 6 h with methanol and n-hexane. DAX-8 was
stored in methanol at 6º C until used.
4
Isolation procedure with DAX-8
The cleaned DAX-8 polymer in methanol was slurry-packed into a 160-mm I.D. class
column, resulting in an 8-cm sorbent bed. The packed column was rinsed with MQ
water until free of methanol. The groundwater sample, acidified to a pH just above 2
with 1.0 M HCl, was placed in a separatory funnel and treated on the polymer. The
humic and fulvic substances adsorbed on the resin were eluted with 0.1 M NaOH.
2.4 TOC analysis
The TOC-V CPH analyzer is able to analyse samples only in liquid state. In TOC
analysis, both inorganic and organic forms of carbon (TC, total carbon) are converted
into carbon dioxide (CO2) using a chemical oxidizing agent (platinum on alumina
pellets) and a high temperature such as 680° C. The CO2 formed is then detected using
non-dispersive infrared adsorption (NDIR). When measuring the amount of inorganic
carbon in the sample, it is first acidified with HCl. The CO2 formed from the inorganic
forms of carbon (IC) is purged from the sample with air and detected with an NDIR
detector. The amount of total organic carbon (TOC) can then be calculated as a
difference between total carbon and inorganic carbon. The TOC analyzer was calibrated
in the range of 0.5 to 100 mg/l for organic and inorganic carbon. Quality control
samples (2.0 and 20.0 mg/L, QC WW4) were analysed at the beginning and at the end
of each run.
The TOC measurements were performed with the TOC-V CPH analyzer connected to
an ASI-V autosampler (Shimadzu Corporation). The samples were neutralized with 1.0
M HCl before the measurements.
2.5 HPLC fluorescence and UV
Size exclusion chromatography (SEC) with fluorescence detection was used for the
investigation of the molecular size distribution of isolated humic substances. The HPLC
instrument was a Shimadzu LC-6A pump coupled to a Shimadzu RF-551 fluorescence
detector and an SPD-6A ultraviolet detector. The excitation and emission wavelengths
of the fluorescence detector were 340 nm and 435 nm, respectively. The wavelength of
the ultraviolet detector was set at 254 nm. A PL Aquagel-OH 30 column from Polymer
Laboratories (Shropshire, U.K) of 250 × 4.6-mm i.d. and a particle size of 8 µm was
used as the SEC column. The separation was performed at a flow rate of 1.0 ml/min
with an eluent consisting of an 80/20 (v/v) water/methanol mixture with 10 mM
NH4HCO3 at ambient temperature. The injection was performed manually and the
injection volume was 100 µl.
2.6 HPLC-MS
Size exclusion chromatography (SEC) was performed on an HP 1100 (Hewlett-
Packard) liquid chromatography system, which was coupled to a Quattro LC triple-
quadrupole mass spectrometer (Micromass, Manchester, U.K) with a Z-spray
5
electrospray source. The drying gas (N2) flow rate was 650 l/h and the nebulizer gas
(N2) flow rate was 65 l/min. The source temperature was 110° C and the desolvation
temperature was 200° C. A capillary voltage of 3.26 kV was used and the cone voltage
was set at 20 kV. The mass spectrometer was operating in the negative ion mode. The
m/z range of 150-750 was scanned in 5 s. The samples were also screened by injecting
the samples straight into the mass spectrometer. The injection was performed through a
T piece with the eluent from the SEC column. For the analysis of the high-molecular-
weight fraction (humic acids), two separate analyses were performed with the ranges
m/z 200-1200 and 1000-2000. The low-molecular-weight fraction (fulvic acids) was
scanned in the range of m/z 100-1100. The SEC column was the same as used in the
investigation of the molecular size distribution. The separation was performed at a flow
rate of 0.3 ml/min with an eluent consisting of an 80/20 (v/v) water/methanol mixture
with 10 mM NH4HCO3 at ambient temperature. The injection volume was 20 µl.
6
3. RESULTS
3.1 TOC results
Three parallel 2.0-L samples of groundwater (ONK-PVA3) and one blank sample (MQ
water) were preconcentrated with the DAX-8 procedure. The adsorbed humic and fulvic
substances were eluted with 60 mL of 0.1 M NaOH. The results from the TOC
measurements and the calculated recovery are shown in Table 1.
Table 1. Recovery percentages of the total organic carbon (mg/isolated sample) using
the XAD procedure.
ONK-PVA3 (1) ONK-PVA3 (2) ONK-PVA3 (3)
TOC, original
sample, mg
12.95 11.06 17.15
TOC, isolated
sample, mg
5.95 4.64 6.37
% of original
TOC
45.9 42.0 37.1
3.2 HPLC with a fluorescence detector
The fluorescence chromatograms of humic and fulvic acid standards are shown in
Figures 1 and 2. The fluorescence chromatogram of the isolated groundwater sample is
shown in Figure 3. In the humic acid standard, there is one peak with the highest
intensity at 6.1 min and two peaks at 6.5 and 7.6 min. Similar peaks can be seen in the
fulvic acid standard. Humic substances in the isolated groundwater sample exhibit one
peak with high intensity at 6.1 min and two peaks with lower intensity at 6.5 and 8.0
min.
7
Minutes
3 4 5 6 7 8 9 10 11 12 13 14
mV
olts
0
50
100
150
200
250
300
mV
olts
0
50
100
150
200
250
300
1
6,0
98
0
,00
0
R F -551H A flu o r
Nam eRetention T im eEST D concen tra tion
Figure 1. SEC-fluorescence chromatogram of the humic acid standard (80 mg/l).
Minutes
4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 10,0
mV
olts
0
100
200
300
400
500
600
700
800
900
1000
mV
olts
0
100
200
300
400
500
600
700
800
900
1000
1
6,0
73
0
,00
0RF-551FA fluor
Nam eRetention T im eEST D concentration
Figure 2. SEC-fluorescence chromatogram of the fulvic acid standard (100 mg/l).
8
Minutes
4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 10,0 10,5 11,0
mV
olt
s
0
25
50
75
100
125
150
175
200
225
250
mV
olt
s
0
25
50
75
100
125
150
175
200
225
250
1
6,0
90
0
,00
0
R F-551XAD 2_10xlaim
Nam eRetention T im eEST D concentra tion
Figure 3. Fluorescence chromatogram of the isolated groundwater sample (ONK-
PVA3) diluted 10 fold.
PEO standards (Polyethylene oxide) with a molecular weight of 520500 amu, 128000
amu, 43580 amu and 20000 amu were used for the investigation of the molecular size
distribution of the isolated humic substances. The concentration of standards was 100
mg/l. The fluorescence intensities of the PEO standards were found to be low and thus
the determination of the exact retention times was not reliable. The fluorescence
chromatogram of the 128000-amu PEO standard is shown in Figure 4 as an example.
Minutes
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
mV
olt
s
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
mV
olt
s
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
0
,10
0
0,0
00
0
,26
7
0,0
00
0
,36
8
0,0
00
0
,52
7
0,0
00
0
,61
7
0,0
00
0
,79
8
0,0
00
0
,90
8
0,0
00
1
,05
2
0,0
00
1
,15
8
0,0
00
1
,20
2
0,0
00
1
,28
2
0,0
00
1
,36
7
0,0
00
1
,44
2
0,0
00
1
,60
7
0,0
00
1
,66
3
0,0
00
1
,77
8
0,0
00
1
,91
7
0,0
00
(1)
0,0
00
BD
L
RF-551uusi MW std Mp128000
NameRetention T imeESTD concentration
Figure 4. Fluorescence chromatogram of the 12800-amu PEO standard.
9
3.3 MS results
3.3.1 Direct injection
Humic and fulvic acid standards (80 mg/l and 100mg/l) were first screened by injecting
them directly into a mass spectrometer. This resulted in unrepeatable results most likely
due to pulsed flow of the direct injection pump. Because of this, the direct injection of
the standards was performed straight into the eluent flow of the SEC column using a T
piece.
Directly injected fulvic acid (Figure 5) and humic acid (Figures 6 and 7) standards show
the most intensive mass spectra in the mass range of m/z 150-750. This mass range was
selected for further studies.
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050m/z0
100
%
FA CONE20 ELUENTILLA 1 (0.085) Scan ES- 4.24e5466.07
112.80
419.08
364.65112.98309.22
231.38215.25
191.01
169.22
258.08
283.21
353.19
401.82468.15
483.96
554.14488.62
523.27 570.90
655.19631.19
665.20
672.83
823.51
Figure 5. A mass spectrum of the directly injected fulvic acid standard in the mass
range of 100-1100 m/z.
10
200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150m/z0
100
%
HA CONE20 200-1200 ELUENTILLA 1 (0.085) Scan ES- 2.24e5486.16
479.23
470.66469.71
415.35
248.05
220.09
385.43365.77
251.33
273.37 287.10
344.04
432.99
629.35585.63
553.56
486.91
764.54651.77
718.74665.38
692.78753.83
914.21
834.27775.37
805.36839.31
860.29
1076.46920.45
976.25
1070.29
990.43
1060.91
1100.14
1118.41
1150.72
Figure 6. A mass spectrum of the directly injected humic acid standard in the mass
range of 200-1200 m/z.
1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950m/z0
100
%
HA CONE20 1000-2000ELUENTILLA 1 (0.085) Scan ES- 1.98e51366.49
1082.09
1019.42
1036.30
1316.241201.631106.53
1148.79
1233.05
1262.97
1320.40
1702.46
1546.261430.09
1410.51 1519.621468.941567.16
1622.69
1652.79
1700.95
1839.031797.91
1710.96
1910.431839.72
1891.41
1841.991991.591939.33
1986.43
Figure 7. A mass spectrum of the directly injected humic acid standard in the mass
range of 1000-2000 m/z.
3.3.2 SEC-MS
Figures 8 to 19 show the results of the size exclusion chromatography (SEC) with mass
spectrometry detection of the blanks, standards and samples. Chromatographic data is
presented as the total ion chromatogram of all masses together. A background-
11
subtracted mass spectrum, which was summed across the detected mass peak, is also
shown.
Figures 8 and 9 show the total ion chromatogram and the mass spectrum of a blank
sample. The peak in the total ion chromatogram consists probably of some compounds
originating from the isolation procedure. The mass spectrum does not resemble the mass
spectrum of humic substances. The same peak can also be found in the isolated
groundwater samples but not in the humic and fulvic acid standards and in the elution
solvent, which have not gone through the isolation procedure.
nollanayte
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00Time0
100
%
040107_18 Scan ES- TIC
2.56e623.13
12.765.19
23.81
30.5343.62
32.65 39.80 47.1153.40
Figure 8. Total ion chromatogram of the blank sample.
12
nollanayte
150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750m/z0
100
%
040107_18 272 (23.129) Cm (266:295-24:226) Scan ES- 1.33e5168.80
170.83
228.00
172.84226.87
210.89
228.89
230.90
286.16248.94 357.01
301.99410.04383.85 463.03436.50 516.08489.62 623.78570.92596.60 677.06
695.01
Figure 9. A mass spectrum of the peak in the blank sample.
The total ion chromatogram of the fulvic acid standard (100 mg/L of fulvic acid in
20/80 (v/v) MeOH/MQ water) is shown in Figure 10. The main peaks in the total ion
chromatogram are at about 20.5, 21.4 and 23.6 min. Mass spectra of these peaks are
shown in Figures 11, 12 and 13. It can be seen that earlier eluting peaks contain higher
mass compounds.
FA 80mg/l
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00Time0
100
%
040107_07 Scan ES- TIC
2.11e620.58
20.07
0.77
9.952.98
7.06
21.43
24.23
23.55
Figure 10. Fulvic acid standard (100 gm/l).
13
FA 80mg/l
150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750m/z0
100
%
040107_07 242 (20.578) Cm (229:243-138:209) Scan ES- 6.51e3506.90
479.10437.06
425.23381.13
353.05
243.04
198.99
154.95
187.13
224.98
350.87
341.06
309.09297.12
463.15
590.82
566.83
549.13
613.08643.26
739.18657.32
699.05
723.50
Figure 11. A mass spectrum of the first peak at 20.5 min in the fulvic acid standard.
FA 80mg/l
150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750m/z0
100
%
040107_07 252 (21.428) Cm (248:254-148:205) Scan ES- 9.95e3379.12
365.08325.12
313.09
225.03
164.98
181.06
198.98
283.11
269.02
241.02
339.09
381.16
411.06 423.16
451.20
493.13
477.20495.20
533.15563.09
537.15583.08598.99
689.58625.22697.87
Figure 12. A mass spectrum of the second peak at 21.4 min in the fulvic acid standard.
14
FA 80mg/l
150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750m/z0
100
%
040107_07 285 (24.235) Cm (272:288-144:207) Scan ES- 2.38e3199.15
154.96
197.06
281.19
255.05209.07
223.05
321.20
295.16
353.05 393.27
405.18
409.13
423.10 729.44624.56445.27517.19
493.05477.12
593.28
590.55554.79
702.50
637.59651.67 743.52
Figure 13. A mass spectrum of the third peak at 23.6 min in the fulvic acid standard.
The total ion chromatogram of the humic acid standard (84 mg/L of humic acid in 20/80
(v/v) MeOH/MQ water) is shown in Figure 14. The main peaks in the total ion
chromatogram are at about 20.6 and 21.4 min. Mass spectra of these peaks are shown in
Figures 15 and 16. The mass spectrum is slightly broader and has fewer patterns than
that of the fulvic acid standard.
HA 80mg/l
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00Time0
100
%
040107_14 Scan ES- TIC
1.08e620.49
20.15
18.961.53 6.63
5.70
2.045.02
14.9713.698.16 11.99
15.39
21.34
28.06
23.04
24.9125.42 32.99
32.57
28.9152.8133.93
47.1940.2235.71 42.94
45.3249.15 49.91
53.4057.48
Figure 14. Total ion chromatogram of the humic acid standard (84 mg/l).
15
HA 80mg/l
150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750m/z0
100
%
040107_14 241 (20.493) Cm (232:246-173:201) Scan ES- 5.24e3225.01
198.96
154.93
157.04
180.91
243.01
451.19409.03377.04337.03
281.05309.12
429.13649.24559.10529.05499.05 612.83599.12
659.39
709.19737.32
Figure 15. Total ion chromatogram of the first peak at 20.6 min in the humic acid
standard.
HA 80mg/l
150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750m/z0
100
%
040107_14 251 (21.343) Cm (249:254-159:202) Scan ES- 2.32e4165.00
180.98
211.04
281.00225.02 237.05 419.13363.09335.07320.99 403.05437.10
539.05479.40487.93 683.58629.60555.18583.66 696.93
735.98
Figure 16. Total ion chromatogram of the second peak at 21.4 min in the humic acid
standard.
The total ion chromatogram of the isolated groundwater sample is shown in Figure 17.
The main peak in the total ion chromatogram is at about 21.0. The mass spectrum of
this peak is shown in Figure 18. Most of the second peak in the sample consists of the
impurities originating from the isolation procedure. The mass spectrum of this second
peak is shown in Figure 19 and it shows similar patterns as the mass spectrum of the
16
peak in the blank sample. All the groundwater samples showed equal chromatograms
and spectra.
nayte1
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00Time0
100
%
040107_24 Scan ES- TIC
7.28e620.49
21.34
24.66
23.21
27.64
Figure 17. Total ion chromatogram of the isolated groundwater sample.
nayte1
150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750m/z0
100
%
040107_24 241 (20.493) Cm (226:260-39:196) Scan ES- 5.82e4194.94
181.03
381.14367.13339.11
325.12311.13
295.11283.09196.95281.11
267.13239.10
397.15
413.17 451.17
479.19507.14521.16
579.20591.03
Figure 18. A mass spectrum of the peak at 21.0 min in the isolated groundwater sample
(ONK-PVA3).
17
nayte1
150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750m/z0
100
%
040107_24 290 (24.660) Cm (267:293-59:170) Scan ES- 4.29e4150.83
168.83
170.85
357.04195.93355.06228.02
197.87
210.92
301.99286.16
230.00250.99
253.05
328.63
463.14382.95
409.99 461.06
411.10
465.08
515.99488.49 543.15570.97
623.15595.37676.39 693.44 731.15
Figure 19. A mass spectrum of the peak at 24.0 min (impurities) in the isolated
groundwater sample (ONK-PVA3).
18
4. DISCUSSION
Table 1 shows the results of the TOC measurements of the groundwater samples
isolated with the DAX-8 procedure. The average recovery as total organic carbon is
41.7%. These results are in good agreement with the results by Peuravuori et al. (1997),
where 53% of the DOM (dissolved organic matter) was recovered as hydrophobic acids
and with our previous results, where the average recovery was 39.3% (Manninen et al.
2006). The blank sample (MQ water) showed no significant amount of total organic
carbon. The amount of humic substances in the groundwater at this depth in ONKALO
seems to be the same as in the previous investigation (ONK-PVA1, depth about –20m
b.s.l.).
In the size exclusion chromatography with fluorescence detection, the chromatograms
of humic and fulvic acid standards resemble each other. However, the signal intensity of
the peak in the fulvic acid standard is about 2.5 times higher than the intensity of the
peak in the humic acid standard. When assuming that all of the measured TOC (mg/l) in
the isolated groundwater sample consists of humic substances, the following
calculations can be performed: the fluorescence intensity of the highest peak in the
groundwater sample is about 8 times that of the humic acid standard and about 3 times
that of the fulvic acid standard. This difference in fluorescence is probably due to the
different functional groups and the organic compounds that make up the humic
substances. According to this assumption, the humic substances isolated from the
groundwater differ in structure from the IHSS Nordic aquatic fulvic and humic acid
reference standards.
The determination of the molecular size of humic acids was difficult because of the low
fluorescence intensity of the polyethylene oxide (PEO) standards. With small peaks it
was difficult to determine the exact retention time for the standards, which is needed for
the calibration curve. The PEO standards showed multiple peaks with UV detection and
they were therefore found unsuitable for the molecular weight determination. The humic
substances isolated from the groundwater can be estimated to have about the same
molecular weight as the IHSS (International Humic Substances society) humic and
fulvic acid standards. For the fulvic acid standard, the molecular weight has been
estimated at around 7500 Da (Persson et al. 2000).
The repeatability of the SEC-MS runs was moderate and some instability of the system
was observed. The sensitivity of the instrument seemed to decrease when running
several samples, probably owing to the clogging of the multi-port valve system between
the column and the ion source. The hyphenated SEC-ESI-MS system with the
quadrupole mass spectrometer is a powerful instrument especially for lower-molecular-
weight fulvic acid research, whereas for the higher-molecular-weight humic acids there
seems to be problems to get ionized humic compounds to fly efficiently into and
through the mass spectrometer.
In the fulvic acid total ion chromatogram, two distinctive peaks can be found.
According to the separation mechanism of SEC, the first eluting peaks are compounds
with a higher molecular weight. This can be clearly seen from the mass spectra from the
different peaks; every peak has a different mass spectrum, and with the increasing
retention times the maximum m/z ratios decrease. These figures demonstrate the
benefits of the SEC separation when comparing them with the direct injection mass
19
spectra. Less complex spectra are obtained with the SEC separation preceding the MS
detection. The most typical differences in mass patterns were -2 and -14 amu. The latter
can be attributed to homologous series differing in their number of methylene groups,
and the former originates probably from the double bonds of the carbon skeleton. With
the high-molecular-weight humic acids, the mass spectrum is not so ordered and these
mass patterns are not so obvious. In the isolated groundwater samples, the -2 and -14
amu spacings are the most distinctive in mass spectra. The groundwater sample mass
spectrum clearly resembles the mass spectrum of fulvic acids rather than that of humic
acids. The obtained results are comparable to those by Reemtsma et al. (2003). They
indicate that fulvic acids are more “symmetrical” in structure than has been thought
before.
20
5. CONCLUSIONS
The isolation method based on a non-ionic polymeric sorbent (DAX-8) has turned out to
be applicable and reliable with groundwater samples. The developed method is shown
to isolate approximately 40% of the total organic carbon as humic substances. With the
DAX technique, adsorption is based on hydrophobic interactions and the salt
concentration of the groundwater does not interfere with the isolation process. In
contrast, the adsorption will increase with an increasing ionic strength.
The SEC-ESI-MS seems to be a powerful technique especially to investigate low-
molecular-weight fulvic acids. With high-molecular-weight humic acids, there is the
well-known problem of sensitivity loss with ESI-MS. When increasing the cone
voltage, increasing signal intensity is obtained, but the average m/z values shift to lower
values, indicating that mostly fragment ions are obtained. Thus, the range of the
compounds suitable for ESI-MS is limited by the stability of higher-molecular-weight
molecules of humic acids in the electrospray process. However, with lower-molecular-
weight fulvic acids, SEC-ESI-MS can provide valuable information on the investigated
compounds.
21
REFERENCES
G. R. Aiken, Isolation and concentration techniques for aquatic humic substances, in: G.
R. Aiken, D. M. McKnight, R. L. Wershaw, P. MacCarthy (Eds.), Humic substances in
soil, sediment and water: Geochemistry, Isolation and Characterization, Wiley, NY,
1985, pp. 363-385.
Ronald L. Malcolm; Analytica Chimica Acta, 232, 1990, 19-30 “The uniqueness of
humic substances in each of soil, stream and marine environments”
Jerry A. Leenheer; Environmental Science & Technology, 1981, 15, 578-587
“Comprehensive approach to preparative isolation and fractionation of dissolved
organic carbon from natural waters and wastewaters”
Earl M. Thurman and Ronald L. Malcolm; Environmental Science & Technology, 1981,
15, 463-466 “Preparative isolation of aquatic humic substances”
Juhani Peuravuori, Kalevi Pihlaja, and Niina Välimäki; Environment International, vol
23, No. 4, pp. 453-464, 1997 “Isolation and characterization of natural organic matter
from lake water: Two different adsorption chromatographic methods”
Pentti Manninen and Jani Mäkelä; Posiva working report 2006-42, 1-24 ”Humic and
Fulvic Acids in Groundwater –A Preliminary Research”
Linn Persson, Thomas Alsberg, Gyula Kiss, Göran Odham; Rapid Communications in
Mass Spectrometry, 14, 286-292, 2000 “On-line size-exclusion
chromatography/electrospray mass spectrometry of aquatic humic substances”
Reemstsma Thorsten, These Anja, Analytical Chemistry, 2003, vol 75, 6, 1500-1507
“On-line coupling of size exclusion chromatography with electrospray ionization-
tandem mass spectrometry for the analysis of aquatic fulvic and humic acids”