**HPLC Fingerprints of Porewater Organic Compounds as Markers for Environmental Conditions**

Viia Lepane *Tallinn University of Technology Estonia* 

#### **1. Introduction**

310 International Perspectives on Global Environmental Change

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Lake sediments are considered invaluable natural archives that provide long-term records of past changes in climate and environment related to catchment processes as well as in-lake changes in biological communities. Moreover, lake sediments also register anthropogenic activities and man-made environmental problems. Lake sediments are known to accumulate different compounds during their formation and adsorption processes, and thus sediment investigations can be used as an important tool to assess the contamination of aquatic ecosystems. The organic matter in sediments is distributed between the particulate and dissolved phases, and usually the aquatic phase is named porewater (*pw*).

The dissolved organic matter (DOM) is an important component of aquatic ecosystems and of the global carbon cycle. It is known that changes in DOM quality and quantity have effects on the whole ecosystem. Quantitative and qualitative changes in DOM are related to precipitation, runoff, and seasons. DOM consists of a mixture of macromolecular compounds with a wide range of chemical properties and diverse origins. The DOM in lakes can serve as a molecular proxy for identification of previous inputs of organic matter. Moreover, detailed knowledge about DOM is greatly needed in order to reconstruct palaeoclimate or land-use. The biogeochemical transformation of DOM helps to elucidate past and present environmental conditions. For all those reasons, detailed DOM characterization at the molecular level is of utmost importance.

Since DOM is naturally a very complex mixture of molecules, the determination of its exact chemical composition is a complicated task. Only detailed chemical characterization using various analytical methods could be carried out. A part of DOM is optically active, enabling spectroscopic methods based on UV absorption to be used for the characterization. Another possible method of DOM analysis is chromatographic size fractionation using high-performance liquid chromatography (HPLC) with a size exclusion option (high-performance size exclusion chromatography – HPSEC). HPSEC has been widely used in studies of DOM together with spectroscopic methods. The reliability and sensitivity of this method have been reported and discussed previously (Chin et al., 1994; Hoque et al., 2003; Minor et al., 2002; Nissinen et al., 2001; Pelekani et al., 1999; Perminova et al., 1998, 2003; Specht & Frimmel, 2000; Zhou et al., 2000). Although HPSEC characterization of lake sediment *pw*DOM has demonstrated great potential for

HPLC Fingerprints of Porewater Organic Compounds as Markers for Environmental Conditions 313

The second lake investigated is situated in South Estonia, where the anthropogenic pressure is not too high and is expressed mainly through the agricultural activity. L. Rõuge Tõugjärv (57°44'30"N; 26°54'20"E) is a small-size stratified hard-water mesotrophic lake with a surface area of 4.2 ha and a maximum depth of 17 m. The main source of pollutants in L. Tõugjärv sediments is the catchments area. The studied sediment core was visibly laminated, reflecting the annual changes in the lake. Annual laminations, or varves, typically consist of two visible layers (a clastic inorganic layer and a darker organic humic

The topmost 13 cm of the sediment was loose unconsolidated dark gyttja (dated until 1986 AD), while the rest of the sediment sample was laminated gyttja with well-developed varves (dated until the year 1852 AD). The L. Rõuge Tõugjärv sediment core was taken in May 2006 with a Willner-type sampler. The core was transported in a tightly closed Plexiglas tube to the laboratory, immediately sliced into 1-cm thick sub-samples, and packed

The age-scale for the sediment sequence was obtained by correlating marker varve horizons and LOI values with another sediment core sampled in 2001, which had been carefully dated by several parallel dating methods (varve counting, 210Pb, 137Cs, 241Am, and spheroid fly-ash particles) (Alliksaar et al., 2005; Veski et al., 2005; Poska et al., 2008). According to

*Pw* samples for analysis were obtained by extraction of unfrozen sediments by centrifugation at 3,500 rpm for 30 minutes and filtration through 0.45 μm filters (Millex,

Absorbance spectra of the *pw* samples were collected using a Jasco V-530 UV/VIS Spectrophotometer (Japan), with 1-cm-pathlength fused silica cells and ultrapure water as the blank. Spectra were measured over the range of 200–500 nm with a 2.0-nm bandwidth. The dissolved organic carbon (DOC) concentration in *pw* samples was calculated from absorption spectra using the equation given by Højerslev (1988). The absorbance ratio at 250 and 360 nm (A250/A360), which reflects the aromaticity of dissolved molecules (Peuravuori

The molecular characteristics of DOM in sediments were determined using an HPLC system. The HPLC system comprised a Dionex P680 HPLC Pump, Agilent 1200 Series (Agilent Technologies, UK) diode array absorbance detector (DAD), and a Rheodyne injector valve with a 20-μL sample loop. A BioSep-SEC-S 2000 PEEK size exclusion analytical column (length 300 mm, diameter 7.50 mm, Phenomenex, USA) preceded by a suitable guard column (length 75 mm, diameter 7.50 mm, Phenomenex, USA) was used for separation. The applied flow rates were 0.5 mL min-1 (L. Peipsi samples) and 1.0 mL min-1 (L. Rõuge Tõugjärv samples). The column packing material was silica bonded with a hydrophilic diol coating, with a particle size of 5 µm and a pore size of 145 Å. The mobile phase consisted of 0.10 M NH4H2PO4 - (NH4)2HPO4 buffer at pH 6.8. The HPLC system was calibrated using five different molecular mass protein standards (Aqueous SEC 1 Std, Phenomenex, USA) (see Fig. 1). All solutions for HPLC measurements were prepared using ultrapure water passed through a MilliQ water system, filtered with 0.45-µm pore size filters

layer), and each varve can be considered as representing one year's deposition.

this correlation the obtained sediment core covered about 150 years (1850–2005).

into plastic bags to maximally avoid oxygen exposure.

Millipore). Samples were stored at 4 oC in the dark.

& Pihlaja, 1997), was calculated from the spectra.

**2.2 Chemical analyses** 

**2.3 HPLC analyses** 

palaeolimnological research (Leeben et al., 2008a; Lepane et al., 2004, 2010a; Makarõtševa et al., 2010) it is not widely used for evaluating the long-term changes in aquatic ecosystems. At present, no comparative investigations of *pw*DOM from lake sediments are available. Coupling of HPSEC as a separation method with diode-array detection (DAD) allows DOM fingerprints and spectra of DOM molecular fractions to be obtained for qualitative and semi-quantitative analysis. The non-destructive analysis, small sample volume, and minimal sample pretreatment are great advantages of the HPSEC-DAD approach, making the method suitable for environmental studies. HPSEC-DAD has been adapted and optimized for analysis of *pw* samples under various conditions (Lepane et al., 2004; 2010a; O'Loughlin & Chin, 2004). The advantage of the usage of this chromatographic system is a better understanding of the qualitative and quantitative *pw*DOM properties by detecting aromatic fractions (chromophoric compounds). This method has recently been applied for monitoring and detection of organic matter from surface waters after oxidation treatment (Liu et al., 2010).

 This study aims: (1) to investigate temporal changes in *pw*DOM components' qualitative and quantitative characteristics by exploring different sediment core records; (2) to find the similarities and differences in HPSEC-DAD fingerprints of *pw*DOM after applying the statistical data treatment methods; (3) to explore the potential impact of environmental change on *pw*DOM records in investigated sediment cores.
