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  • No : 4961
  • Displayed Date : 2021/02/08 18:08
  • Updated DTM : 2021/02/17 10:02
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(HPLC: Amino Acid Analysis) Precautions on Handling the Column

(HPLC: Amino Acid Analysis) Precautions on Handling the Column
Category : 

Answer

• Parts Number: Na-type
  228-18837-91: Analysis Column Shim-pack Amino-Na
  228-14206-91: Ammonia Trap Column ISC-30Na
• Parts Number: Li-type
  228-18837-92: Analysis Column Shim-pack Amino-Li
  228-00821-91: Ammonia Trap Column ISC-30Li
*There is no guard column available for either column.However, an in-line filter PEEK (P/N: 228-41748-91) for removing insoluble materiales can be attached in front of the analytical column.
 
• Flushing the Column (When using the separation column (Amino-Na/Li) for the first time or if the column is stained due to previous analyses)
  Rinse the column with mobile phase C (AA-MC).
  1) Set the following parameters.
    Pump A T.Flow (LC-20AB total flow rate): 0.3 mL/min
    Pump A B.Conc (LC-20AB-B ratio (%)): 0 %
    SV (Pump A) (FCV-11ALS port): B (Mobile phase C(AA-MC))
    B.Flow (Flow rate of LC-40D 1st): 0.2 mL/min
    C.Flow (Flow rate of LC-40D 2nd): 0.2 mL/min
  2) Pump mobile phase for about 15 minutes.
    Pump reaction reagents at 0.2 mL/min to prevent backflow.
    For Shim-pack Amino-Li column, change the flow rate to 0.6 mL/min and run for at least 120 minutes after cleaning.
 
• Flushing the Analysis Column
  Irreversible adsorption of sample components on the column packing may result in retention time change and/or poor resolution. In such a case,the following column cleaning procedure may be effective.
  • Shim-pack Amino-Na
    1) Flush the column with 0.2 mol/L sodium hydroxide aqueous solution or mobile phase C (Na type).
      Flush with the cleaning solution at 0.2 mL/min for at least 30 minutes, although these cleaning condition depends on the degree of contamination of the column.
    2) After the cleaning, run with mobile phase A to flow at 0.5 mL/min for 20 minutes.
 
  • Shim-pack Amino-Li
    1) Flush the column with 0.2 mol/L lithium hydroxide or mobile phase C (Li type).
      Flush with the cleaning solution at 0.2 mL/min for at least 30 minutes, although these cleaning condition depends on the degree of contamination of the column.
    2) After the cleaning, allow 0.15 mol/L lithium citrate or mobile phase A (Li type) to flow at 0.6 mL/min for 120 minutes.
      NOTE: Step 2 is necessary to obtain reproducibility of the peak area of sarcosine (Sar)).
 
• Rinsing Ammonia Trap Column (When using ammonia trap column first time)
    rinse the flow line upstream of the ammonia trap column by flushing with purified water. Then rinse the ammonia trap column as follows:
  1) Prepare mobile phase C. Fill one of the LC-20AB (mobile phase delivery pump) flow lines with mobile phase C.
    NOTE: For mobile phase C, use 0.2 mol/L sodium hydroxide aqueous solution and 0.2mol/L lithium hydroxide aqueous solution for Na-type analysis and Li-type analysis, respectively.
  2) Reattach the ammonia trap column. Pump the mobile phase at 1 mL/min for 30minutes.
    CAUTION: NEVER pump lithium salt aqueous solution to Na-type columns. NEVER pump sodium salt aqueous solution to Li-type columns. Otherwise, load pressure may increase and damage columns.
  3) Upon completion, purge the pump with purified water.
 
• Store of Column
  If the column is to be long out of service (for six months or longer), store it in the following procedure:
  • Shim-pack Amino-Na
    1) Clean the column throughly with 0.2 mol/L sodium hydroxide aqueous solution or mobile phase C (Na-type).
    2) Replace the solution in the column with 10% ethanol aqueous solution containing 0.01% caprylic acid.
    3) Store the column in a cool dark place.
      Prior to using the column, run with mobile phase A (Na type) at 0.1 mL/min overnight.
 
  • Shim-pack Amino-Li
    1) Clean the column throughly with 0.2 mol/L lithium hydroxide aqueous solution or mobile phase C (Li-type).
    2) Replace the solution in the column with 10% ethanol aqueous solution containing 0.01% caprylic acid.
    3) Store the column in a cool dark place.
      Prior to using the column, run with mobile phase A (Li type) at 0.1 mL/min overnight.

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