3-1. Mobile Phase 1. All solvents acceptable for the conventional chemically bonded silica columns can be used. 2. After full degassing, filtrate the mobile phase using a membrane filter 0.45 µm or smaller to remove dust. A 2-µm filter is used at the column inlet. To prevent foreign matter from clogging the column inlet filter, we recommend using a line filter.     3. The mobile phase stated in the report is sealed in a new column. To change to a mobile phase containing inorganic salt, note the replacement procedure. 4. To prevent column deterioration, avoid the following: •Frequent change of the mobile phase composition or direct change to a mobile phase of low compatibility •Rapid change in column inlet pressure •High column pressure due to the use of a high-viscosity mobile phase

3-2. Preparing a Sample Solution 1. Dissolve the sample in a solvent of the same composition as the eluate wherever possible. 2. Using a solvent with strong dissolving power may lower the separation efficiency or cause the sample to precipitate at the column head. 3. If there is insoluble matter remaining in the sample solution, filtrate the solution using a filter 0.45 µm or smaller. 4. The pH of the sample solution should be set in acceptable pH range for packing material.

3-3. Notes on Analysis If the efficiency of catalytic reduction goes down, the reduction column can be activated by the following procedure. (1) Dismount the separation column, and joint the reduction column with the pump. (2) Wash the column with distilled water (500µL/min, 10min) (3) Further wash with 0.1M aqueous ascorbate at the same flow rate for 30min. (4) Do same as (2). (5) Change to mobile phase for aging, activation is finished. (6) Re-mount the separation column. (7) Confirm the sensitivity of the column by standard solution.

1. After usage, wash the column and replace with methanol without electrolyte. 2. Seal the column with the accessory plug and store it in a cold place where there is little temperature fluctuation.

1. An analytical column of up to 6-mm ID uses a filter-embedded end fitting as shown in Fig. 1. The filter cannot be changed alone. If the filter is clogged or the column pressure is high, replace the end fitting. See Table 3 for the replacement parts and repair items. 2. 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. If L is greater than V, dead volume may be generated and cause peak broadening or tailing or deterioration of separation performance. If L is smaller than V, liquid may leak 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. 【Fig. 1】 Column connection