Products for Liquid Chromatography 31

Instructions and directions for use of columns (pp.30-31)

Instructions and directions for use of columns 4

1. After using the column, clean the column with a solvent which can elute the sample well and does not affect the column, and
store it. As the solvent for storage, the solvent contained in the supplied column is optimum. When a buffer solution has been
used, remove salts from the inside of the column by flowing distilled water into it.
2. When ion-pair chromatography has been performed, the ion-pair reagent in the column can be removed efficiently by cleaning
it with methanol and water at a ratio of 50/50 (v/v) after washing it with water.
3. Do not use a chlorinated solvent for storing.
4. Fit an airtight stopper to the column to prevent the inside of the column drying.
5. If the inside of the column dries, feed the stored solvent at a low flow rate of 0.1 to 0.2 mL/min by HPLC, and, after making
sure that the solvent flows out of the column outlet, suspend feeding. After leaving the column for about 5 to 10 minutes, feed
the solvent as usual. The column may be reused although its performance is lower than the initial performance.
6. When the column is stored for a long period, feed a liquid into the column every one to two months.
7. To maintain the column performance, avoid cleaning the column frequently or using a large quantity of cleaning liquid as far as
possible. The column life is reduced only by feeding a liquid. Frequent change of the kind of solvent will lead to the same result.

Column cleaning method and solvent for storage

The column cleaning method varies depending on the kind of column, mobile phase and sample. Normally, clean the column in
accordance with the following procedures. For storing, generally, use the liquid contained in the supplied column.
Reverse-phase silica gel
1. Packing material contamination can be removed by flowing Water → Methanol → Chloroform → Methanol
a medium polar solvent, such as THF, chloroform and
dichloromethane. However, do not store the column with such a Pouring DMSO (200 μL) several times
chlorinated solvent contained in it.
(Use each cleaning liquid in a quantity ten
2. When storing the column for a long period, replace the liquid in the
column with methanol or acetonitrile. times as much as the column volume.)
3. After using an inorganic salt buffer as the mobile phase, clean the
inside of the column with water, replace the water successively
with 0.1% phosphoric acid aqueous solution (as much as the column
volume), and store the column with acetonitrile or methanol.

Normal-phase silica gel

1. Deterioration of silica gel is caused mainly by adsorption of polar
●Silica gel column
substances. If the adsorption capacity is reduced by moisture, the IPA → Chloroform → Chloroform → n-hexane
capacity can be restored by the following procedures. The method
2% 2,2-dimethoxypropane
of cleaning chemically bonded columns varies depending on 1% acetic anhydride
whether they are used in a normal-phase or reverse-phase system.
General methods are shown right. However, when replacing the ●Chemically bonded column (used on normal-phase system)
used mobile phase with a cleaning liquid, pay attention to the Methanol → Chloroform → n-hexane
miscibility between them.
2. When storing the column for a long period, store the column with ●Chemically bonded column (used on reverse-phase system)
the following solvent. Water → Methanol → Chloroform → Methanol
・ Silica gel column: n-hexane (Use each cleaning liquid in a quantity ten times as much as
the column volume.)
・ Chemically bonded column: Ethanol

Ion-exchange silica gel

1. Deterioration of columns is caused mainly by adsorption of ionic Water → 0.1M EDTA・2Na → Water → Methanol → Water
substances (metal ions, halide ions, etc.). Deteriorated columns can
be regenerated by the procedures shown right.
2. When storing the column for a long period, clean the column well with water to remove inorganic salts, and replace the water
with methanol or acetonitrile 100%.

Rise of column pressure

While the column is being used, the column pressure may rise abnormally. The abnormal rise of pressure may be caused by
clogging in the column. The clogging may be solved by feeding a solvent having high eluting power. If it cannot be solved by this
method, connect the column in the reverse direction, and feed a liquid. Then, the clogging may be solved. However, the column
performance may be degraded by this operation. Use this method as a last resort.
Wako Product Information − Chromatography − 8th Edition
For other products, please visit the Wako Online Catalog: http ://www.e-reagent.com/