ver.210601E
SHOWA DENKO K.K. (https://www.shodex.com/)
4
· If the column was used at an elevated temperature, lower the flow rate to less than half of
the recommended flow rate at the end of analysis. Then, turn off the column oven, and let
the column temperature return to room temperature before stopping the pump. If the pump
was stopped while the eluent inside the column was still hot, as the eluent temperature
decreases, its volume also decreases. This may result in creating an empty space in the
column and deteriorates the column.
Note
· It is recommended to set the pump limiter to avoid exceeding the maximum pressure.
7.4 Solvent Exchange
(1) When replacing the solvent, start the system at less than 0.2 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 highly concentrated buffer solution or salt solution to water/acetonitrile, first run water
and then replace it with the eluent.
(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.
7.5 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.
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
and use the flow rate less than 0.2 mL/min. Introduce 5 to 10 times column volume of the washing solvent.
Method 1: After using eluent not containing buffer salts/additives
Wash the column with solution containing a higher ratio of organic solvent that constitutes the eluent. A
cleaning solution containing THF might be effective when removing highly hydrophobic (lipid-soluble)
substances that are adsorbed onto the gel.
Method 2: After using mobile phase containing buffer salts/additives
First replace with a water/organic solution containing no buffer salts or additives (A ratio of water to organic
solvent should be set at the same proportion as eluent). Then flush the column in accordance with the method
described above.
Attention!
· Flushing with 100% water after using the column around the pH limit might shorten column
life, so flush the column with water/organic solution.
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 7.4 Solvent Exchange for how to replace the eluent.
Attention!
· Never allow inside of the column to dry. It can damage the column.