1. 
   

Materials:

Equipments :

1. 1. A UV/Visible spectrophotometer (if elution is not monitored by an HPLC instrument)

2. Peristaltic pump

3. A gradient mixer

Reagents and chemicals :

1. 1. Sepharose ion exchanger (Carboxymethyl-cellulose)

2. 20 mM phosphate buffer, pH 7.0

3. 0.1 N NaOH

4. 3 M NaCl solution

Glassware, pasticware, and other materials :

1. 1. Glass column (30 cm × 2.5 cm )

2. Laboratory stand

Procedure:

1. 1. Follow the manufacturer's instructions for swelling the ion exchanger matrix. Alternatively follow the following steps:

   a.  Weigh 10 g of CM-cellulose and suspend it in 200 ml of binding buffer (20 mM phosphate buffer, pH 7.0 in this case) containing 1 M NaCl.

   b.  Allow swelling of the matrix for at least 24 hours.

   c.  Remove the fines by decanting the buffered saline.

   d.  Wash the swollen matrix with the buffered saline 2-3 times.

   e.  Wash the matrix with distilled water to remove the salts (if the binding buffer does not have a neutral pH, achieving a neutral pH is an indicator of removal of salts).

   f.  Add 200 ml of 0.1 N NaOH in the matrix and leave it for 30 minutes. This ensures that the matrix functional groups, carboxylate groups in this case, are completely ionized.

   g.  Wash the matrix with the binding buffer 3-4 times. This is to remove the excess NaOH present in the matrix gel.

2. Mount the glass column on a laboratory stand.

3. Place a cotton plug or glass wool plug at the bottom (Figure 24.1A). This is done to prevent the loss of the matrix gel from the column. Alternatively, fritted columns (Figure 24.1B) can be used.

Figure 24.1: A column with cotton/glass wool plug (A); a fritted column (B)