Before proceeding with the ion exchange chromatography,
the pH and the initial ionic strength
were selected by preliminary tests with continuous
and simultaneous gradients of both pH and ionic
strength (Bermejo et al., 2001). Results showed
that an ionic strength discontinuous gradient was
enough for separation of the phycoerythrin. The
R-PC was eluted at 0.05 M acetic acid–sodium
acetate buffer (pH 5.5), whereas B-PE was eluted
at 0.25 M acetic acid–sodium acetate buffer (pH
5.5). The chromatography step was optimised on
a small scale by using a column (2.5×15.0 cm)
with different flow rates and different loadings of
phycobiliproteins mixture. The best results were
obtained with a flow rate of 100 ml h−1 (0.34 ml
cm−2 min−1), whereas the optimum loading was
7.3 mg of phycobiliproteins. The recovery of pure
fractions with A545/A2803 remained constant