196 M.V. Moreno-Arribas et al.
the small pore size to favor the passage of wines (Dos Santos et al. 2000; Desportes
et al. 2000, 2001).
After precipitation or ultracentrifugation of proteins, classical preparative chro-
matographic methods are generally used, such as molecular exclusion chromatog-
raphy, ion exchange and, in a less extent, reversed phase chromatography. To
separate wine peptides, low-pressure molecular exclusion chromatography on cross-
like dextran (Sephadex G-10 and Sephadex LH-20) is widely employed. This type
of chromatography allows the separation of peptides according to their molecular
size, which is traditionally very useful as an initial step in the fractionation of pep-
tides from foods. The choice of pore size is determined by the range of molecular
weights of the peptides in the sample. At the same time, it may also be convenient,
the clean up of the samples by removing interfering smaller or higher molecular
components, such as salts, organic acids, carbohydrates, phenolic compounds and
amino acids. Peptides can be eluted from the column with water, solutions of acetic
acid or sodium chloride, or mixtures of water and methanol, or water and ethanol.
This is a suitable strategy for analyzing low molecular weight peptides (<1000 Da).
Since these compounds usually elute together with amino acids, it will be essen-
tial to separate them from the amino acids prior to analysis. Sephadex G-10 with
acetate buffer as eluent has been used to separate peptides from amino acids in the
ethanol-soluble fraction of wines (Moreno-Arribas et al. 1996, 1998a,b; Bartolom ́e
et al. 1997; Mart ́ınez-Rodr ́ıguez et al. 2002) and Sephadex LH-20 has been used by
Acedo et al. (1994), Desportes et al. (2000, 2001) and Pozo-Bay ́on et al. (2005) to
eliminate the amino acids contained in thewine after ultrafiltration. Several succes-
sive chromatographic systems have been used by Takayanagi and Yokotsuka (1999),
Yanai et al. (2003) and Alcaide-Hidalgo et al. (2008).
6B.2.3 Separation by Chromatographic Techniques
HPLC is the most widely used method for peptide analysis. It offers many advan-
tages, such as versatility, short analysis times, high resolution, effective separations,
and it is well suited to automation procedures, There are many mechanisms that
could be employed in the chromatographic separation of peptides, e.g. those based
on molecule size (GPC), charge (IEC), hydrophobicity (reversed-phase and inter-
action chromatography), and even on combinations of them. Nevertheless, only
reversed-phase chromatography has been used to separate mixtures of peptides from
wines (Table 6B.1). The mobile phase most frequently used is a mixture of water
and acetonitrile, with trifluoroacetic acid as an ion pairing reagent, and in gradi-
ent conditions. Less employed are the mixtures of methanol, phosphate buffer, and
tetrahydrofurane, also in gradient conditions.
In the first study appearing in the literature on wine peptides, Acedo et al. (1994)
applied RP-HPLC to separate peptides from wine after the formation of pep-
tide derivatives witho-phthaldialdehyde. Also an RP-HPLC procedure using a
Nova-Pak C 18 column under gradient conditions was applied to analyze peptides