3.3 Gel Filtration
1. Suspend Sephadex G-25 in Na-phosphate buffer and allow
swelling (see Note 7).
2. Pour the swollen Sephadex G-25 slurry into the column (see
Notes 8 and 9).
3. After the slurry has settled, rinse the column with 3 column
volumes of Na-phosphate buffer.
4. Load the dialyzed phycocyanin solution on the column using
Pasteur pipette.
5. Elute with the running buffer and collect the phycocyanin
fractions.
6. Combine the collected fractions of high purity and yield.
3.4 Ion-Exchange
Chromatography
1. Suspend DEAE-sephadex in Na-phosphate buffer, and allow it
to stand for 2–4 h.
2. Pour the DEAE-sephadex slurry into the column and allow
settling down.
3. After the slurry has settled, rinse the column with 3 column
volumes of Na-phosphate buffer in order to stabilize and
equilibrate the bed.
4. Load the phycocyanin fractions obtained from gel filtration
chromatography on the DEAE-sephadex column.
5. Rinse the column with Na-phosphate buffer.
6. Elute the phycocyanin with a linearly increasing ionic concentration gradient of NaCl from 0.1 to 0.25 M (C-phycocyanin
(C-PC) usually elute between 0.15 and 0.25 M NaCl).
7. Collect the fractions.
8. Check the phycocyanin concentration and purity of the fractions collected after each step.
3.5
Spectrophotometric
Estimation of
Phycocyanin
1. The PC concentration can be calculated by using Eq. 1 [15]
PC mg mL
À1
À
Á ¼
A 615 À 0:474 A 652
5:34
ð1Þ
where PC is the C-PC concentration (mg mL
À1 ), A 615 is the
absorbance of the sample at 615 nm, and A 652 is the absorbance of the sample at 652 nm.
2. PC purity can be calculated by using Eq. 2 [15]
Purity ¼
A 620
A 280
ð2Þ
Phycocyanin Purification
177
1. Suspend Sephadex G-25 in Na-phosphate buffer and allow
swelling (see Note 7).
2. Pour the swollen Sephadex G-25 slurry into the column (see
Notes 8 and 9).
3. After the slurry has settled, rinse the column with 3 column
volumes of Na-phosphate buffer.
4. Load the dialyzed phycocyanin solution on the column using
Pasteur pipette.
5. Elute with the running buffer and collect the phycocyanin
fractions.
6. Combine the collected fractions of high purity and yield.
3.4 Ion-Exchange
Chromatography
1. Suspend DEAE-sephadex in Na-phosphate buffer, and allow it
to stand for 2–4 h.
2. Pour the DEAE-sephadex slurry into the column and allow
settling down.
3. After the slurry has settled, rinse the column with 3 column
volumes of Na-phosphate buffer in order to stabilize and
equilibrate the bed.
4. Load the phycocyanin fractions obtained from gel filtration
chromatography on the DEAE-sephadex column.
5. Rinse the column with Na-phosphate buffer.
6. Elute the phycocyanin with a linearly increasing ionic concentration gradient of NaCl from 0.1 to 0.25 M (C-phycocyanin
(C-PC) usually elute between 0.15 and 0.25 M NaCl).
7. Collect the fractions.
8. Check the phycocyanin concentration and purity of the fractions collected after each step.
3.5
Spectrophotometric
Estimation of
Phycocyanin
1. The PC concentration can be calculated by using Eq. 1 [15]
PC mg mL
À1
À
Á ¼
A 615 À 0:474 A 652
5:34
ð1Þ
where PC is the C-PC concentration (mg mL
À1 ), A 615 is the
absorbance of the sample at 615 nm, and A 652 is the absorbance of the sample at 652 nm.
2. PC purity can be calculated by using Eq. 2 [15]
Purity ¼
A 620
A 280
ð2Þ
Phycocyanin Purification
177