Analysis of Chlorinated Suanee River Fulvic Acid Using Liquid
Chromatography Mass Spectrometry
Bradley D. Harris1, Taylor A. Brown1, Jimmie L. McGehee1, Dominika Houserova1, Benjamin A.
Jackson1, Brandon C. Buchel1, Alexandra C. Stenson1*
1Department of Chemistry, University of South Alabama, Mobile, AL
Abstract
Natural organic matter (NOM) is found in soil and water. When chlorine is
used to disinfect water it reacts with dissolved NOM to form potentially
harmful disinfection byproducts (DBPs).1 Because NOM is so varied the
ways that DBPs form are poorly understood. To better understand the
reactions Suanee River fulvic acid (SRFA), a portion of NOM, was
separated by polarity into 100 fractions (FRCs) using high performance
liquid chromatography (HPLC). Selected fractions were then analyzed
using liquid chromatography mass spectrometry (LCMS) before and after
chlorination to observe changes in polarity and mass distribution. Fifteen
acids with similar polarity to the fractions (Standards) were also analyzed
to compare the results. The analysis showed that early eluting, high
polarity samples appeared to fragment and lose material as volatile
DBPs, whereas non polar material reacted by incorporating chlorine. Loss
of material resulted in a weaker signal for early eluting samples, whereas
later samples increased in signal strength regardless, indicating a higher
ionization potential. Evidence for different reactions occuring according to
polarity agrees with previous literature indicating NOM follows multiple
reaction pathways simultaneously.2,3,4 The HPLC column is currently
being exchanged for a preparative column to collect less abundant FRCs
for analysis.
Objectives
This experiment aims to use LCMS to reveal the dominant reaction
pathways in different portions of SRFA. Because different methods of
water flocculation remove different portions of NOM,5 understanding how
the polarity of FA affects the DBPs produced can be used to inform the
method selected.
Methods
Fractionation:
SRFA was dissolved in water and fractionated using HPLC with a
Water’s Corporation X-Bridge phenyl column (3.5mm, 4.6mm x 150mm).
The mobile phase consisted of deionized water (18.2 MΩ) and 0.1%
formic acid (Optima LC/MS) with a step gradient of increasing
acetonitrile (Optima LC/MS) and 0.1% formic acid concentration. FRCs
were dried using a Labconco Centrivap Cold Trap at 30 oC .
Chlorination:
FRCs were dissolved in deionized water and total organic carbon (TOC)
concentration was determined using a Shimadzu TOC-L CPH/CPN.
FRCs and Standards were diluted to equivalent concentrations of TOC
and chlorinated with 5.65-6.00% NaOCl stock (Laboratory Grade) to a
ratio of 1.6 mg Cl to 1.0 mg TOC. Samples were allowed to sit
undisturbed in a dark cabinet at room temp (~22 oC) for three days and
then frozen at -80 oC until analysis.
LCMS:
Because samples could not be de-chlorinated prior to LCMS analysis
without drowning the parent signal, the samples were thawed and
analyzed in small batches. By spending less time thawed the
discrepancy in how long each sample could chlorinate was minimized.
LCMS analysis was carried out using an LTQ Velos ion-trap mass
spectrometer (Thermo Scientific, USA) in negative ion mode using the
same liquid chromatography method and material as fractionation.
Results
Conclusions
All samples showed a drastic change in mass spectrum after chlorination.
Highly polar FRCs experienced a large loss of higher mass signals, with
remaining signals being concentrated toward a lower mass range. Less polar
FRCs showed a much stronger signal at high mass ranges than before
chlorination. Standards confirmed this trend, with polar Standards losing
parent mass signal and non-polar Standards gaining peaks strongly
indicative of incorporating one or more chlorine atoms.
Citric Acid
Highly-Polar Standard
Before Chlorination
• Different portions of SRFA react with chlorine along different reaction
pathways, resulting in a large variety of DBPs.
• Polarity can be used as an effective indicator of SRFA’s most likely
reaction path.
• Highly polar FRCs experienced the most mass loss, indicating the
production of more volatile DBPs.
•Less polar FRCs showed a much higher tendency to result in high mass
DBPs by incorporating chlorine into existing compounds, perhaps by
adding ClOH to double bonds.
Highly-Polar Standard
After Chlorination
Citric Acid
Future Directions
Figure 1. Mass spectrums of citric acid (highly polar Standard) before and after
chlorination. Mass to charge ratio is indicated along the horizontal axis, with relative
abundance shown on vertical axis. Chlorination has resulted in a significant loss of parent
material.
2-(4-(2,2-Dicarboxy-ethyl)-2,5dimethoxy-benzyl)-malonic acid
Non-Polar Standard
Before Chlorination
Non-Polar Standard
After Chlorination
2-(4-(2,2-Dicarboxy-ethyl)-2,5dimethoxy-benzyl)-malonic acid
• The HPLC column is currently being exchanged for a preparative column
to aid in collecting less abundant FRCs for analysis.
• A method of fractionating Suwannee River humic acid, another portion of
Suwannee River NOM, is being explored.
• Samples of chlorinated FRCs are being shared with collaborators for
bioassay using cancer cells and potentially shrimp, to discover which type
of DBP is most hazardous to living organisms.
Chlorine Peaks
Figure 2. Mass spectrums of 2-(4-(2,2-Dicarboxy-ethyl)-2,5-dimethoxy-benzyl)malonic acid (non-polar Standard) before and after chlorination. Mass to charge ratio
is shown along the horizontal axis, with relative abundance shown on the vertical axis.
Chlorination has resulted in the loss of parent material and the appearance of chlorine
peaks. The appearance of chlorine peaks thirty-six Daltons ahead of the parent peaks
indicate the incorporation of chlorine into the parent material.
The chromatograph of samples was also affected, as elution times shifted
slightly after chlorination and new peaks appeared. Highly polar samples
showed slightly reduced signal strength, whereas slightly-polar and nonpolar samples showed an increase in signal strength despite experiencing
a loss in overall material as volatile DBPs. Signal boosting indicates that
the incorporation of chlorine ions is making non-polar samples more
amenable to ionization and therefore easier to detect by the LCMS. Higher
ionization potential may suggest the addition of ClOH to a double bond.2
The relative boost in signal increased as polarity decreased.
Acknowledgements
We thank the National Science Foundation and the University of South
Alabama for funding. We also thank the Dauphin Island Sea Laboratory
and Mrs. Laura Linn for TOC analysis.
Highly Polar FRC Before
Chlorination
Highly Polar FRC After
Chlorination
Literature Cited
Slightly-Polar FRC
Before Chlorination
(1)
Hrudey, S. E. Chlorination disinfection by-products, public health risk tradeoffs and me. Water Res.
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Slightly-Polar FRC
After Chlorination
(2)
Lavonen, E. E.; Gonsior, M.; Tranvik, L. J.; Schmitt-Kopplin, P.; Köhler, S. J. Selective Chlorinationof
Natural Organic Matter: Identification of Previously Unknown Disinfection Byproducts. Environ. Sci.
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Non-Polar FRC Before
Chlorination
(3)
Zhang, H.; Zhang, Y.; Shi, Q.; Hu, J.; Chu, M.; Yu, J.; Yang, M. Study on Transformation of Natural
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Resolution Mass Spectrometry. Environ. Sci. Technol. 2012, 46 (8), 4396–4402.
Non-Polar FRC After
Chlorination
(4)
Rook, J. J. Chlorination reactions of fulvic acids in natural waters. Environ. Sci. Technol. 1977, 11 (5),
478–482.
(5)
Matilainen, A.; Vepsäläinen, M.; Sillanpää, M. Natural organic matter removal by coagulation during
drinking water treatment: A review. Adv. Colloid Interface Sci. 2010, 159 (2), 189–197.
Figure 3. Chromatograph of FRCs before and after chlorination. Elution time is shown
along the horizontal axis, with signal intensity shown along the vertical axis. Less polar
FRCs experience a stronger relative boost in intensity.