M. De Nobili et Y. Chen, Size exclusion chromatography of humic substances: Limits, perspectives and prospectives, SOIL SCI, 164(11), 1999, pp. 825-833
The fractionation range of chromatographic media used in size exclusion chr
omatography (SEC) depends strongly on the kind of substances eluted, Althou
gh the fractionation range reported by the manufacturer for globular protei
ns on Sephadex G-200 is 5000 to 250,000 daltons and that for dextrans 1000
to 150,000 daltons, phenolcarboxylic acids (pH = 8, 0.1 mmol/L NaOH) are el
uted much earlier than would be predicted based on their molecular weight (
MW). The actual MWs for this group of compounds ranges from 200 to 500 dalt
ons, whereas their MWs determined by SEC calibrated with proteins is much h
igher (7200-180,000 daltons). Ion exclusion, which is the basis for this be
havior, originates from residual ionized groups and limits the possibility
of applying SEC to the study of conformational changes of humic substances
(HS). A correct selection of experimental conditions enables the use of SEC
for various applications such as the study of metal-binding equilibria and
fractionation according to molecular size. To determine the MW distributio
n of HS, columns should be calibrated with standard humic and fulvic acids
with known MWs determined by colligative properties such as vapor pressure
osmometry. It should be noted, however, that SEC studies on fractions of HS
originating from various soils, result in a series of significantly differ
ing calibration curves. The Linearity of the relationship between the distr
ibution coefficient (K-av) values and the logarithm of the MWs implies the
constancy of partial specific volumes. It also suggests that HS extracted f
rom the same soil constitute a homologous series in which the structure of
the larger components is formed by the repetition of structurally similar u
nits and is, therefore, more regular than previously believed. The experime
ntal results obtained by SEC also suggest that in regard to the sensitivity
of the technique, charge density differences are small within a certain se
ries compared with size differences.