A Selection Guide for Each Column
Mode Packing materials Samples Mode Packing materials Samples
Reverse
phase
C18、C8、C4、TMS、Ph Uracil, benzene and naphthalene Dedicated PTH PTH amino acids
(20 components)
Normal phase CN Uracil, benzene and naphthalene PTC PTC amino acids
(17 components)
NH2 Benzene and thymidine DNA Oligonucleotide
Hydrophilic
interaction
AS-AQ Toluene and thymidine Cu Oxine copper
Ion
exchange
SAX Uridine and 5’-UMP Agri-9 Oxine copper, MCPP,
isoprodione and TPN
SCX Uridine and cytidine GP-N6、PAHs、AS-Aqua Uracil, benzene and naphthalene
Gel filtration Diol Protein molecular weight marker DNPH-Ⅱ DNPH aldehydes
Adsorption SIL Benzene and nitrobenzene Fluofix、Fluofix-Ⅱ Uracil, toluene and
hexafluorobenzene




1. Do not give shock to the column. Feed a liquid in the direction of arrow shown on the label.
2. Confirm the kind of solvent contained in the column. Examine whether the solvent can be mixed with the mobile phase and whether problems, such as salt precipitation, will not occur in the case of use of a buffer solution.
3. As a mobile phase, use solvents for high-performance liquid chromatography, and deaerate the phase. Particularly, in the case of an aqueous solvent with an organic solvent to be used for reverse-phase or ion exchange system, air bubbles may be generated in the process of analysis even if there are no air bubbles at the initial stage of mixing. Some buffer solutions may generate large quantities of air bubbles depending on the kind of salt used in them.
4. Deaeration is often performed under reduced pressure when a deaeration device is not available. In this case, care must be taken to avoid changes in the composition of the mobile phase. Helium deaeration using ultrasonic wave is effective.
5. Connect the column to HPLC while feeding a liquid at a low flow rate of about 0.1 mL/min. This can prevent ingress of air bubbles into the column. When using a pre-column, connect the pre-column to HPLC as stated above, fit the connecting joint,and connect the analytical column after ensuring that the liquid can flow out.
6. When using the column at a high flow rate, start feeding a liquid at a rate of 1 mL/min or so if possible, and increase the flow rate gradually to the working flow rate. While increasing the flow rate, check for leakage from connections.
7. If possible, filter the sample through a filter as fine as 0.45 μm before pouring it.
(Reference) Solvents for HPLC (page 146 of this catalog)


1. Although the quantity of liquid necessary for equilibration from the solvent contained in the column to the applied mobile phase varies depending on the packing material, column size, detector and mobile phase type, the approximate quantity is regarded as 15 to 20 times the column capacity. Particularly, in the case of ion-pair chromatography, it may take a long time for equilibration.
2. In the case of a silica gel packed column in normal-phase adsorption chromatography, the separation time and elution time depend greatly on the water content of the silica gel and the moisture in the mobile phase. To solve this problem, 0.1 to 0.2% of a polar solvent may be added to a non-polar solvent, or a water-saturated solvent may be used.
3. When replacing the mobile phase, carefully check the inside of the HPLC equipment as well as the inside of the column. Some columns may be deteriorated by ingress of the mobile phase used before. The injector (in the sample loop), damper and mixer shall be checked with particular care.


1. Before removing the column, make sure that the pressure gauge of HPLC indicates 0.
2. The optimum pH of mobile phase is 2.5 to 7 from the viewpoint of the column life. (For Wakosil® AR type, the optimum pH is 2 to 8.)
3. The working pressure and flow rate vary depending on the kind of packing material, particle diameter, column size and mobile phase viscosity. It is favorable to the column life to examine the separation capacity and analysis time and use the column at as low pressure as possible. (See the column manual.)
4. Use the column in a temperature range from 10 to 50℃.
5. If the column is heated (higher than room temperature), do not stop feeding the liquid immediately after the completion of measurement. Stop feeding the liquid after the column reaches room temperature.


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