SUPERIOREXII User‘s Manual

• 1. Handling the Column
• 2. Attaching the Column
• 3. Analysis
• 4. Storing the Column
• 5. End Fittings
• 6. Troubleshooting

Thank you very much for purchasing the SUPERIOREX II ODS column for HPLC.
SUPERIOREXII ODS is a HPLC column filled with modified ODS silicagel particle based on precision-classed, high-quality spherical silicagel.
In order to use SUPERIOREXII ODS for a long period of time and stably, please read this instruction manual carefully, and use it correctly.

1. Handling the Column

1. Handle the column with great care. A strong shock may cause damage.
2. Attach or detach the column when the pressure gage indicates zero.
3. The maximum column operating pressure
   * Please refer to the column index (Osaka Soda HPLC Column Lineup) for the maximum operating pressure of each column.

2. Attaching the Column

1. The column joint is of the male nut type for piping of 1/16 inch OD. Check that the tubing joints of the system fit correctly and that the ferrule tips are deeply inserted into the joints. (See Fig. 1.)
2. Before attaching the column, replace the liquid in the system with the mobile phase to be used.
3. Attach the column according to the direction of the arrow.

3. Analysis

3-1. Mobile phase

1. All solvents acceptable for conventional chemically bonded silica columns can be used.
   * The available pH range is based on the evaluation that the retention time, and the number of theoretical plates that does not decrease at the earlier time of analysis.
2. To prevent premature deterioration of the column, make sure that the pH of a mobile phase will not exceed this specific pH range.
   In addition, a major change of the pH range leads to poor reproducibility, and premature deterioration of the column.
3. Under the conditions with low content of organic solvent, and high column temperature, acid, and alkali resistance are lower.
   *It depends on the measurement of temperature, and the content of organic solvent in the mobile phase.

1. Mobile phase should be degassed, after filtering the mobile phase (A membrane filter 0.45 μm or less) to remove insoluble, and dust.
2. To prevent foreign matter from clogging the column inlet filter, we recommend using a line filter.
3. The new column is sealed with the solvent shown in the included column report. When using a mobile phase containing salt, be careful of the replacement procedure so that the inorganic salt is not deposited.
4. Adding a surfactant such as an ion pairing agent to the mobile phase, the column lifetime will be shortened slightly.
5. Continuous use of 100% water mobile phases leads to poor reproducibility.
6. In general, to prevent column deterioration, avoid the following usage.
   • Frequent change of the mobile phase composition, such as significantly changing the pH range.
   • Continuous column use for a long time over the maximum back pressure, or rapid change in column back pressure exceeding a few MPa.

3-2. Preparing a Sample Solution

1. Dissolve the sample in a solvent of the same composition as the mobile phase wherever possible.
2. Using a solvent with strong dissolving power may lower separation efficiency and cause the peak shapes may become broad.
3. In case the sample is difficult to dissolve in the mobile phase, when the sample dissolved in the sample solvent is injected, the sample may be deposited in the column, and the column pressure may rise rapidly.
4. Sample solution pH should be set appropriate value without exceeding the usable pH range of the column.

4. Storing the Column

1. Seal the column with the accessory plug, and store it in a cold place where there is little temperature fluctuation.
2. In case after using a solvent containing with organic strong acids such as TFA or alkali (pH>8) as a mobile phase, replace it with the same rate composition solvent with the same organic solvent and water (avoid replacing with only water). For long-term storage of more than a week, replace it with acetonitrile.
3. When storing the column for a long time more than one month, after flushing a solvent that does not deposit salt, replace it with a shipping solvent, and store the column in a cool dark place with a plug tightly.
4. Do not use 100% water to wash the column as much as possible (see 3-1-7).

5. End Fittings

See Fig. 1 for the column connection. If the tubing is inappropriate, especially if a tube for a different type of column is used, the length after the ferrule tip (V in Fig. 1) is often different from the end fitting length L, and a problem may occur.
* For the end fitting of each column, check the column index (Specification list).
(L>V) If L is greater than V, dead volume may be generated and cause peak broadening or tailing or deterioration of separation performance.
(V>L) If L is smaller than V, liquid leak may occur because of inadequate ferrule adhesion.
Therefore, we recommend replacing the ferrule together with the column.
* If the column is replaced frequently, the male nut may crush the ferrule and liquid may leak. Since tightening the nut too much may cause its head to come off, replace the ferrule at an early stage.

6. Troubleshooting

Problems in high performance liquid chromatography are attributable to various causes that cannot all be enumerated. The table below describes some comparatively common problems related to the column.

Symptom	Cause	Measures
1. Column pressure rise.	Blocking with foreign matter 1. Dust or insoluble matter in the mobile phase or sample solution. 2. Dirt in the tubing. 3. Plunger seal fragment. 4. Precipitation of sample components.	• Sonicate the filter or replace it. • Filtrate the mobile phase and sample solution in advance using a membrane filter. • Attach a line filter. • Clean the tubing and replace the plunger seal. • Prepare a sample solution with the mobile phase.
2. Peak splitting, tailing, and broadening.	1. Void in the column head. 2. Dead volume due to inappropriate connections. 3. Inappropriate mobile phase conditions. • Ion suppression method: Inadequate suppression (Too much sample). • Ion-pair method: Inadequate concentration of the ion-pair agent (Too much sample). 4. Column deterioration. * Not repairable in the case of column deterioration or damage to the packing condition.	• Replenish the packing material. • Reconnect the tubing. • Review the pH, salt concentration, sample amount, and other conditions. • Review the ion pair agent concentration, pH, sample amount, and other conditions. • Check the column performance using standard inspection solution.
3. Retention time too long or unstable.	1. Liquid leak (Indicated on the pressure gage of the pump). 2. Inappropriate mobile phase conditions. 3. Inadequate column equilibration time.	• Check the pump and tubing for any leaks. See 2-3. • Secure adequate equilibration time.
4. Retention time too short.	1. Hydrolysis (deterioration) of a bonded groups by strong acid or base. 2. Inappropriate mobile phase conditions. 3. Inadequate column equilibration time.	See 2-3. • Secure adequate equilibration time.

SUPERIOREXII ODS is shipped after a strict performance check. However, if you should find any defect, please contact your dealer or Osaka Soda for replacement. Note that Osaka Soda does not warrant the product against column life or deterioration caused by the failure to follow the above handling instructions.
Ten or more days after reception by the customer, Osaka Soda will assume that the product was delivered in good condition, and will not accept a later replacement request.

Update June 1, 2022