ver.211105E
SHOWA DENKO K.K. (https://www.shodex.com/)
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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 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 to a mixture of buffer solution and acetonitrile, take
a pre-equilibrium step: First run 10 - 20 column volumes of the buffer solution without
acetonitrile, and then replace it with the desired eluent.
*If replacing to a buffer solution alone or a mixture of water and acetonitrile, pre-equilibrium
step is generally not required.
·
When replacing the eluent from a mixture of buffer solution and acetonitrile to
water/acetonitrile, first wash the column with acidic 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.
If a guard column is used with the analytical column, first remove the guard column and check the performance
of the analytical column alone. If the problem is solved, most likely the cause is from the guard column. In this
case, clean the guard column.
If the problem is not solved by removing the guard column, clean both guard and analytical columns. Make sure
to clean the guard and the analytical columns separately. 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.
Washing Solution
Cleaning Time
1 H
2
O
6 min
2
0.1 M HNO
3
aq. solution
75 min
3 H
2
O 12
min
4 Eluent
40
min
Attention!
· Keeping acidic 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.
8. Column Storage
Remove the column from the system after replacing the in-column solvent with the shipping solvent. Securely
tighten the end caps and store the column at a location with stable temperature (a cool and dark space is
recommended). Refer to section 7.3 Solvent Exchange for how to replace the eluent.
Attention!
· Never allow inside of the column to dry. It can damage the column.
