The dispersion step is one of the most delicate operations. On the one hand, a weak dispersion will leave aggregates intact and may lead to an over-estimate of heavy fraction C; on the other hand, too vigorous a dispersion step could cause re-distribution of C across fractions by partial dispersion of organo-mineral complexes. Weak organic-sand associations may be particularly vulnerable to this process. Since occlusion within aggregates and surface sorption are processes occurring along a continuum (Kögel-Knabner et al. 2008), no perfect solution exists.

The application of ultrasonic energy is one of the most commonly used dispersion method. Reported sonication energies range from 60 to 5000 J mL-1 but the consensus seems to be that 100 J mL-1 is sufficient to destroy macroaggregates and effectively disperse sandy soils, while 500 J mL-1 will destroy microaggregates and disperse reactive soils (Spielvogel et al., 2007; Heckman et al., 2013; Schrumpf et al., 2013a; Asano and Wagai, 2014).

In physical fractionation schemes, complete dispersion of silt- and clay-sized aggregates may not be necessary, since the protection mechanism is more likely to become apparented to sorptive stabilization in these size ranges. A reasonable objective of dispersion prior to size or density fractionation may be to disrupt macro- (> 250 μm) and large micro- (> 53 μm) aggregates. Energies of 100 J mL-1 (sandy soils) to 200J mL-1 (loamy soils) may be appropriate choices. An energy of 200 J mL-1 may already extract a portion of microbial metabolites (supposedly mineral-associated) (Höfle et al., 2012), thus the use of higher sonication energies should be subject to caution. However, reactive soils with cemented aggregates could require up to 500 J mL-1 to disperse.

There are certain fractionation methods, in which incomplete dispersion shall be achieved to isolate a "stable" aggregates (Zimmermann et al., 2007). For incomplete dispersion, Poeplau and Don (2014) highlighted the importance of standardizing the ultrasonic output power (J s-1; W), instead of standardizing the output energy (J) alone. This is because power is what influences cavitation, the mechanism which breaks aggregates during ultrasonication. In a fractionation ring trial, Poeplau et al. (2013) showed that differing power settings across laboratories led to a significantly different distribution of carbon in fractions. This underlines the importance of dispersion, as the very first step of many fractionation methods. Calibration of the ultrasonic device is usually performed using warming of a defined amount of water in a dewar vessel (Schmidt et al., 1999; Poeplau and Don, 2014). This procedure should be repeated in regular intervals or before every larger set of samples to be fractionated, since the output power of the ultrasonic tip decreases over time. For the same reason, the tip needs to be changed after a certain operation time.

Besides ultrasonication, another mechanical dispersion method is the use of glass beads. A general problem of mechanical methods is, that certain fragile fragments of e.g. particulate organic matter might also disintegrate and redistribute into other, smaller fractions. Therefore, chemical dispersion with agents like hexametaphosphate (HMP) might also be an option. In the comprehensive method comparison (Poeplau et al., in review), this method tended to yield the highest reproducibility. However, HMP should only be used, if a more or less complete dispersion of particles is desired.


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