Saiga et al. [83] described an example of the complexity of a peptide purification
process. In the first stage, these authors used two sequential chromatography columns, one with cationic exchange resin (AG 50W-X2) followed by a second
anionic exchange resin (AG 1-X4). In the second stage, eluted fractions were
concentrated in a UF membrane (MW < 500 Da) and finally subjected to purification in a reverse-phase HPLC column.
Fig. 5 Photograph of a polyacrylamide gel after isoelectric focusing of samples of HTST
skimmed milk (diluted 20Â, Lane 2), sweet whey (diluted 20Â, Lane 3), sweet whey hydrolysate
supernatant (without dilution, Lane 4), and sweet hydrolysate supernatant with citrate buffer
20 mM pH 4.5 come out from cationic exchanger column during adsorption and washing steps
(diluted 24Â, Lane 5). Lane 1 contains the Broad pI Kit pH 3–10 (GE Healthcare) containing:
amyloglucosidase (pI = 3.50); methyl red (pI = 3.75); trypsin inhibitor (pI = 4.55);
b-lactoglobulin A (pI = 5.20); bovine carbonic anhydrase B (pI = 5.85); human carbonic
anhydrase B (pI = 6.55); myoglobin, acidic band (pI = 6.85); myoglobin, basic band (pI = 7.35);
lentil lectin, acidic (pI = 8.15); lentil lectin, middle (pI = 8.45); lentil lectin, basic (pI = 8.65) and
trypsinogen (pI = 9.30)
490
A. F. A. Salvado et al.