Column Cleaning for Ion Exchange Chromatography and Chromatofocusing

Correct preparation of samples and buffers and application of a high salt wash (1 M NaCl) at the end of each separation should keep most columns in good condition. However, reduced performance, a slow flow rate, increasing back pressure or complete blockage are all indications that the medium needs to be cleaned using more stringent procedures in order to remove contaminants.

It is recommended to reverse the direction of flow during column cleaning so that contaminants do not need to pass through the entire length of the column. The number of column volumes and time required for each cleaning step may vary according to the degree of contamination. If the cleaning procedure to remove common contaminants does not restore column performance, change the top filter (when possible) before trying alternative cleaning methods. Care should be taken when changing a filter as this may affect the column packing and interfere with performance.

Removal of Common Contaminants

The following procedure should be satisfactory to remove common contaminants:

1. Wash with at least 2 column volumes of 2 M NaCl (Table 21 for recommended flow).
2. Wash with at least 4 column volumes of 1 M NaOH (same flow as step 1).
3. Wash with at least 2 column volumes of 2 M NaCl (same flow as step 1).
4. Rinse with at least 2 column volumes of distilled water (same flow as step 1) until the UV-baseline and eluent pH are stable.
5. Wash with at least 4 column volumes of start buffer or storage buffer (same flow as step 1) until pH and conductivity values have reached the required values.

Removal of Precipitated Proteins, Lipids, Hydrophobically-bound Proteins, or Lipoproteins

To remove precipitated proteins:

1. Inject 1 column volume of pepsin (1 mg/mL in 0.5 M NaCl, 0.1 M acetic acid). Leave overnight at room temperature or for one hour at 37 °C.
2. Rinse with at least 2 column volumes of distilled water (Table 21 for recommended flow) until the UV-baseline and the eluent pH are stable.
3. Wash with at least 4 column volumes of start buffer or storage buffer, same flow as step 2, until eluent pH and conductivity have reached the required values.

Alternatively,

1. Wash with 2 column volumes of 6 M guanidine hydrochloride (Table 21 for recommended flow).
2. Wash immediately with at least 5 column volumes of buffer at pH 7–8 (Table 21 for recommended flow).
3. Rinse with at least 2 column volumes of distilled water (same flow as step 2) until the UV-baseline and eluent pH are stable.
4. Wash with at least 4 column volumes of start buffer or storage buffer (same flow as step 2) until pH and conductivity values have reached the required values.

Table 21 Recommended flow according to medium, column dimensions and eluent.

Column (volume) or medium	Water, buffer, 2 M NaCl or 1 M NaOH+	6 M guanidine hydrochloride	70% ethanol or 30% isopropanol
MiniBeads (0.24 mL)	0.2 mL/min	0.1 mL/min	0.1 mL/min
MiniBeads (0.8 mL)	0.2 mL/min	0.1 mL/min	0.1 mL/min
MonoBeads (1.7 mL)	0.2 mL/min	0.1 mL/min	0.1 mL/min
MonoBeads (1 mL)	0.5 mL/min	0.25 mL/min	0.25 mL/min
MonoBeads (8 mL)	2 mL/min	1 mL/min	1 mL/min
MonoBeads (20 mL)	5 mL/min	2.5 mL/min	2.5 mL/min
SOURCE 15 4.6/100 PE	0.2 mL/min	0.1 mL/min	0.1 mL/min
RESOURCE 1 mL	1 mL/min	0.5 mL/min	0.5 mL/min
RESOURCE 6 mL	6 mL/min	3 mL/min	3 mL/min
SOURCE in larger columns**	40 cm/h	20 cm/h	20 cm/h
HiTrap (1 mL)	1 mL/min	0.5 mL/min	0.5 mL/min
HiTrap (5 mL)	5 mL/min	2.5 mL/min	2.5 mL/min
HiPrep (20 mL)	5 mL/min	2.5 mL/min	2.5 mL/min
HiLoad (20 mL)	3 mL/min	2.5 mL/min	2.5 mL/min
HiLoad (53 mL)	8 mL/min	5 mL/min	5 mL/min
Sepharose High Performance in larger columns**	40 cm/h	20 cm/h	20 cm/h
Sepharose Fast Flow in larger columns**	40 cm/h	20 cm/h	40 cm/h
Sepharose XL in larger columns**	40 cm/h	20 cm/h	40 cm/h
Sepharose Big Beads**	40 cm/h	40 cm/h	40 cm/h

* If contamination is thought to be significant, use a lower flow rate to increase the contact time when using 1 M NaOH.
** When cleaning larger columns, allow a contact time of 1–2 hours for any solution that is used as an initial cleaning step.

To Remove Lipids, Hydrophobically-bound Proteins, or Lipoproteins

Organic solvents or detergents may be required to completely remove contaminants of this type.

Before using organic solvents, wash the medium with at least 4 column volumes of distilled water to avoid any salts precipitating on the column.

When applying organic solvents or solutions it may be necessary to reduce the flow rate to avoid over-pressuring the column.

Use cleaning solutions such as up to 100% isopropanol, up to 100% methanol, up to 100% acetonitrile, up to 2 M NaOH, up to 75% acetic acid, up to 100% ethanol, ionic or non-ionic detergents.

Always check for solvent compatibility in the instructions supplied with the medium or column.

Avoid anionic detergents with Q, DEAE and ANX charged groups. Avoid cationic detergents with S, SP and CM charged groups.

Examples of cleaning procedures:

1. Wash with 4 column volumes of up to 70% ethanol or 30% isopropanol (see Table 21 for recommended flow).
2. Rinse with at least 2 column volumes of distilled water (see Table 21 for recommended flow) until the UV-baseline and eluent pH are stable.
3. Wash immediately with 3 column volumes of start buffer (same flow as step 2).

Alternatively,

1. Wash with 2 column volumes of detergent in a basic or acidic solution, e.g. 0.1–0.5% non-ionic detergent in 0.1 M acetic acid (see Table 21 for recommended flow).
2. Rinse with 5 column volumes 70% ethanol to remove residual detergent (see Table 21 for recommended flow).
3. Rinse with at least 2 column volumes of distilled water (same flow as step 1) until the UV-baseline and the eluent pH are stable.
4. Wash with 3 column volumes of start buffer (same flow as step 1).