3-1. Mobile Phase 1. Make sure that the concentration of organic solvent will not exceed 20vol%. Avoid using methanol because it is a powerful protein denaturant. Acetonitrile, THF, and IPA are recommended. Example: Phosphate buffer/acetonitrile = 80/20. 2. CAPCELL PAK MF can be used in the pH range from 2.0 to 7.5. To prevent early deterioration of the column, make sure that the pH of the mobile phase will not exceed this range. Never flow a basic mobile phase into the column. 3. 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. 4. Note the replacement procedure to avoid salting out. The shipment solvent is described in the column report enclosed with the column. 5. 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 Compared with conventional C18 columns, this column can greatly simplify analytical procedures of biological samples, such as serum. CAPCELL PAK MF Conventional C 18 Column 1. Serum 1. Serum 2. Precipitating protein by adding denaturant 3. Centrifugation 4. Evaporating the supernatant 5. Re-dissolving in a specific volume 2. Filtration of the above sample using a 0.2-µm filter 7. Filtration of the above sample using a 0.2-µm filter 3. Introduction to HPLC 8. Introduction to HPLC 1. Filtrate biological samples (serum, plasma, urine, etc.) beforehand, using a filter of about 0.2 µm. 2. Dissolve the sample in the mobile phase wherever possible.

1. The column can run approximately 10mL (in accumulative volume) of serum samples, although the value changes depending on the conditions.

1. 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

Table 4 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 (1/16) and ferrules (1/16)2pieces each EF2161 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 MF 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.