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 Location: Home > TechToids > Cleaning Procedures > Cleaning Superdex 75 & 200 HR 10/30
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Frequently Asked Questions

The suggested flow rates and volumes in the following procedure are appropriate in most cases, but may need to be adjusted, depending on the condition of the column. Always keep the backpressure below 1.5MPa for Superdex 200 and 1.8MPa for Superdex 75.

Two cleaning procedures are recommended:

A. A simple procedure for regular cleaning to prevent
contamination.

B. A rigourous procedure, when the column is
already contaminated.


A. Regular cleaning

Clean the column at least every 10 - 20
separation cycles.

1. Wash with 25 ml 0.5 M NaOH at 0.5 ml/min.

2. Immediately equilibrate with at least 2 bed
volumes of buffer until the UV baseline is
stable.

B. Rigourous cleaning to remove contamination
may be required if you notice an increased back-pressure

¤ Before cleaning, make sure that the high
back-pressure in the system is in fact
caused by the column. Disconnect one piece
of equipment at a time
(starting at the fraction collector) with
the pumps working. Check the pressure
reading after each piece is disconnected,
to determine the source of the back-pressure.
(You will often find that a dirty pre-filter
causes the increased back-pressure.)

¤ Check the back-pressure at the same stage
during each run, since the back-pressure
can vary within one run, e.g. injecting a
sample and mixing different eluents may
cause increased back-pressure.

Also clean the column if you notice any of the following:

- A space that has become visible between the
adaptor and the gel bed

- A color change at the top of the column
and/or
- A loss of resolution

The following steps should be performed:

1. Change the filter at the top.
(Instructions for changing the filter are
supplied with the Filter kit.)
¤ Since the contaminants are introduced
with the liquid flow, many of them are
caught by the filter.

2. Perform cleaning with 1 M NaOH (30 min to 2 hr cycle)
Rinse with water or aqueous buffer.
Clean with 0.1 M HCl (30 min to 2 h cycle).
Equilibrate with buffer until the baseline
is stable.

To remove proteins and peptides:
Fill the column with 1 mg/ml pepsin in 0.1 M acetic acid and 0.5 M NaCl.
Incubate overnight at room temperature, or 1 h at 37°C.
Note: After enzymatic digestion, careful rinsing is
required.


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