3-1. Mobile Phase 1. The capsule-type strong cation exchange column (CAPCELL PAK SCX) can be used in the pH range from 2 to 7. 2. After degassing completely, 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 "0.2M NH 4H 2PO 4 (pH 3.5)" is sealed in a new column at the time of inspection. To change to a mobile phase containing a large amount of organic solvent, use intermediate liquid (e.g. 50mM buffer/methanol) if necessary, to avoid precipitation in the column. 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 . Flowing 100% water 5. In ion exchange mode, the following factors will change elution behavior significantly: . pH (pH should ideally be 2.0 or more away from pKa for full ion separation from the sample.) . Salt concentration . Amount of organic solvent . Salt type

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 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.

3-3. Notes on Analysis Columns of CAPCELL PAK series generally show similar separation profiles to those of corresponding conventional silica-based columns, although slight selectivity difference may be observed depending on the analyte. When optimizing the conditions for compounds already done with conventional columns, use the same condition as a starting one.

1. Store the column with a water-type mobile phase containing salt. 2. Seal the column with the accessory plug and store it in a cold place where there is little temperature fluctuation to prevent salt precipitation.

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

Table 3 Replacement parts and repair items Part No. Part Name Description EF2042 End fitting (4.6 mm) 2 pieces EF2061 End fitting (6 mm) 2 pieces EF2160 Piping kit (2pcs) Male nuts (1/6) and ferrules (1/16) 2pieces each EF2161 For ferrule (1/16) Ferrules (1/16) 10pieces

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. CAPCELL PAK SCX 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.