ver.211215E
SHOWA DENKO K.K. (https://www.shodex.com/)
4
7.3 Solvent Exchange
(1) When replacing the solvent, start the system at less than 0.1 mL/min. Recommended solvent volume to
introduce at each step is 3 to 5 times of the column volume.
(2) Check miscibility/solubility of the desired new solvent and the solvent currently filled in the column.
(3)
When replacing the current solvent with a solvent with low miscibility/solubility to the current solvent, first
use a solvent that is miscible/soluble to both eluents, and then replace it with the new solvent.
e.g. When replacing water/acetonitrile to a highly concentrated buffer solution, first run water and then
replace it with the buffer solution.
(4) When using a gradient method, changes in the eluent compositions may increase the column backpressure.
Adjust the flow rate and column temperature so that the column backpressure remains below the usable
maximum pressure.
Attention!
· When replacing the eluent from water/acetonitrile to a mixture of buffer solution and organic
solvent, take a pre-equilibrium step: First run 10 - 20 column volumes of the buffer without
organic solvent, and then replace it with the desired eluent. If replacing to a buffer solution
alone or a mixture of water and ethanol, pre-equilibrium step is generally not required.
·
When replacing the eluent from a mixture of buffer solution and organic solvent to
water/organic solvent, first wash the column with alkaline solution. Please refer to “7.4
Column Cleaning Method 1”.
7.4 Column Cleaning
Problems in peak shapes and elution time changes or elevated column pressure etc. are often caused by
insoluble or adsorbing components present in the eluent and reagents being deposited inside the column. These
problems may be resolved by cleaning the column. When washing the column, let the washing solution flow
from the column outlet go directly into the waste container and not let the solution go through the detector.
<Cleaning Method>
(1) Insoluble components that block the column inlet may be removed by reversing the flow direction, i.e.,
introducing the eluent from the column outlet, with flow rate at less than half of the recommended flow rate.
(2) Follow below cleaning steps for adsorbing components. For an efficient cleaning, reverse the flow direction.
Use the flow rate at 0.1 mL/min.
Method 1: General cleaning
Washing Solution
Cleaning Time
1 H
2
O
10 min
2
0.1 M NaOH aq. solution
120 min
3 H
2
O
20 min
4 Eluent
60 min
Method 2: Adsorption of metal ions or in case Method 1 did not work
Washing Solution
Cleaning Time
1 H
2
O
10 min
2
0.1 M HNO
3
aq. solution
120 min
3 H
2
O
10 min
4
0.1 M NaOH aq. solution
120 min
5 H
2
O
20 min
6 Eluent
60 min
Attention!
· Keeping acidic or alkaline solution in the column for a long time will lead to the column
deterioration. Complete the suggested column cleaning steps all the way up to the eluent
replacement at once and do not hold the procedure in middle way.
· Strong alkaline solvents such as 0.1-M aqueous sodium hydroxide solution can damage
the detector cell. Do not connect the detector while regenerating the column and collect
elute directly from the column outlet to the waste container.