Análisis del proceso de despacho de la Exportadora de uva de la zona de Jayanca –

Various techniques have been used to extract DOM from aqueous samples prior to chemical analysis:

(i) Solid phase extraction (SPE)

Various SPE techniques have been used to extract DOM from aqueous solutions. SPE is used to clean up or purify an analyte, concentrate up an analyte, or to solvent switch. It works by providing a solid surface that the analyte of interest can interact with by adsorption to the surface or penetrating an outer layer of molecules on that surface. An

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equilibrium is established between analyte adsorbed to the solid phase and that remaining in the liquid phase. The analyte is subsequently eluted using a solvent, whereby the solvent provides a more desirable environment for the analyte than the solid phase does. An advantage of SPE versus liquid-liquid extraction is the ability to elute the analyte with a solvent that is miscible with the original sample matrix solvent since in SPE the elution solvent does not come into direct contact with the sample matrix solvent. Therefore a polar solvent can be used to elute an analyte originally in aqueous solution (Simpson, 2000). Hydrophobic macroreticular resins (XAD) have been used to extract fulvic acid from aqueous solutions (Aiken et al., 1979), to

distinguish radiolabelled ‘new’ from ‘old’ DOC in seawater (Lara and Thomas, 1994) and lignin phenols from ocean water (Meyersschulte and Hedges, 1986, Moran et al., 1991). A study comparing different solid phase sorbents (C18, C18EWP, C18OH, C8, PPL,

and ENV) in SPE cartridges (table 3.1) found that whilst the PPL sorbent achieved the highest extraction efficiency of DOC, C18 was more selective for terrigenous

compounds, and was the most efficient silica-based sorbent (Dittmar et al., 2008). Due to the non-specificity of the C18 sorbent, as a cartridge or a disc, molecules with a

hydrophobic nature will sorb to it (Liska, 2000). C18 SPE in disc form was used to

extract riverine DOM and NMR analysis indicated that the C18 extract and original

DOM had a similar distribution of functional groups (Kim et al., 2003). Another extraction study compared hydrophobic SPE (C2, C8, C18, cyclohexyl, or phenyl

sorbents), XAD-2, and ultrafiltration to extract marine humic substances. C18 achieved

the greatest extraction efficiency (Amador et al., 1990).

Table 3.1. Structure and retention properties of polar to highly polar pre-packed polymer and silica-based solid phase sorbents (Dittmar et al., 2008).

Sorbent Structure Pore size (Å) Retention properties

C18 Octadecyl bonded phase, silica-based 60 Retention of nonpolar compounds

C8 Octa bonded phase, silica-based 60 Not as retentive for nonpolar compounds as C18

ENV Styrene divinyl benzene polymer 450 Similar to PPL, larger pore size Retention of highly polar to nonpolar substances from large volumes of water 150

Styrene divinyl benzene polymer PPL

More efficient retention of large molecules, compared with C18

500 Octadecyl bonded phase, silica-based

C18EWP

Enhanced retention of basic compounds, compared with C18

150 Non-endcapped octadecyl bonded

phase, silica-based, with active silanol groups

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Although the PPL sorbent has been used to extract lignin from oilfield product water DOM (Wang et al., 2012c), many studies investigating dissolved lignin degradation products have favoured C18 SPE. These include extracts from freshwaters (Louchouarn

et al., 2000) estuarine waters (Dittmar et al., 2007, Bianchi et al., 2009), oceans (Hernes and Benner, 2006), and from stalagmites (Blyth and Watson, 2009). Using C18 SPE,

Louchouarn et al (2000) achieved 101 ± 4% recoveries of lignin-derived phenols from riverwaters compared with a direct dry down approach using rotary evaporation followed by drying under vacuum in a Savant SpeedVac concentrator. There has even been experimentation with solvents for the C18 extraction protocol where CH3OH or

CH3CN was used to activate and subsequently elute sorbed fresh, estuarine, and marine

DOM from SPE cartridges (Spencer et al., 2010). For freshwater samples they achieved 76.2 – 91.1% recovery (mean = 86.0%) for Ʃ8 lignin phenols using CH3OH versus 48.3

to 77.4% recovery (mean = 67.4%) with CH3CN, compared to the rotary evaporated

sample, and found little fractionation of C:V, S:V, and acid : aldehyde ratios between techniques.

(ii) Freeze-drying (FD), or lyophilisation, is the process whereby a solvent, usually water, is removed from a frozen sample by sublimation (Bruttini et al., 1991). Its main use is preservation of moisture-laden materials, particularly labile biological materials, by preventing microorganism growth and any damaging chemical reactions during sample distribution and storage. Freeze-drying requires the following steps: (i) Sample preparation, whereby samples should have large surfaces and thin uniform cross-

sections, or small spherical shapes; (ii) prefreezing the sample in a freezer; (iii) primary drying which allows the frozen free and weakly bound H2O to sublime slowly under

partial vacuum which is subsequently trapped in a refrigerated condenser; (iii)

secondary drying which removes moisture that was trapped under the ice crystal; (iv) Rehydration, if the dried sample has to be restored to its original hydrated condition. (Mellor, 1978). For the purposes of this thesis, sample rehydration was not required. FD has been used to extract dissolved organic material exuded from freshwater microalgae cultures (Lombardi et al., 2005). However FD in combination with other techniques has successfully been used to extract water borne lignin in other studies e.g. using reverse osmosis followed by FD for lake water (O'Driscoll et al., 2006), or cation exchange column followed by FD (Huang et al., 1998), although FD alone has not been used to extract lignin from aqueous samples to the best of our knowledge.

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(iii) Ultrafiltration has been used to isolate different molecular size fractions of DOM in fresh and marine waters (Amon and Benner, 1996, Opsahl and Benner, 1998), and ocean water at various depths (McCarthy et al., 1996, Benner et al., 1997, Opsahl and Benner, 1997, Opsahl et al., 1999).

(iv) Combined electrodialysis and reverse osmosis has been used to remove inorganic ions and concentrate DOM from freshwater, wastewater (Drewes et al., 2002) and seawater (Vetter et al., 2007).