Installation Tips and Column Handling
1. Purge the system with mobile phase first. Pump solvent through the system to remove air and to prevent that air is entering the colums. Dont’ forget to flush the injection loop.
2. Use the column connections supplied from PSS to connect columns in series.
Make sure that the fitting tubing protrudes a maximum of 1 mm inside the column head
3. Be aware of column flow direction. Operating the flow rate in the reverse direction is only part of troubleshooting or operating after a long storage time.
4. Thread the column fittings finger tight into the system. Do not over tighten the fitting. Overtightening may damage the column and the column head.
5. Flush the column with 10 times of the column volume at 1/5 of the recommended flow rate.
Recommended flow rates
|Flow||I.D. 8 mm||I.D. 4.6 mm||I.D. 20 mm|
|Operating||1 ml/min||0.33 ml/min||6.25 ml/min|
|Reduced||0.25 ml/min||0.1 ml/min||1.5 ml/min|
|Idle||0.1 ml/min||0.03 ml/min||0.6 ml/min|
6. Eluent should come out after 2-3 minutes at 0.2 ml/min flow rate. If not, see recovering partially dry columns, below.
7. When the solvent exchange is completed, connect to the detector(s).
8. Slowly increase the flow rate to the typical operating flow rate which depends on the column I.D. (inner diameter) (see table above).
9. Check the column pressure. Maximum pressure should never exceed twice the pressure specified in the column certificate.
10.Give the columns time to equilibrate.
11.Check the plate count of the whole chromatographic system incl. columns (see column user documentation). If this test fails, first check each column separately when using a column combination, then contact PSS or a PSS representative.
Tip: For Multiple Columns Use (column combinations of single porosity columns)
• Install the middle porosity first and use the largest porosity connected into the detector.
• Use only column combinations recommended by PSS.
• Do not combine linear and single porosity columns.
• Do not combine different particle sizes.
Recovering partially dry columns
Occasionally solvent is lost during long-term storage or due to high storage temperatures. When after (re-) installing the column no solvent overflows or the expected system pressure does not build up, this can be an indication for a partially dry column.
To re-wet a partially dry column:
• Install the column in the reverse direction.
• Fill the column with solvent, using a flow rate of 0.1 ml/min, until no more bubbles appear at the column outlet.
• Change the column to the correct flow direction and use 0.1 ml/min for 2h.
• Increase the flow rate slowly to the operating flow rate with an increment of 0.2 ml/min per 5 minutes.
When columns loose efficiency (Rsp, Asymmetry) or you suspect the presence of foreign material adsorbed on the column, PSS recommends the following procedures:
• Remove the column from the detector.
• Install the column in reverse direction for clean-up.
• Flush the column at 0.1 ml/min flow rate with a solvent that dissolves the suspect impurities and is fully compatible with your system, until the impurities are removed.
To clean aqueous columns, use variation of pH, buffer concentration, or mixtures of organic and aqueous eluents. Organic columns are cleaned best by variation of eluent polarity. Use mixtures of appropriate solvents (e.g. THF in toluene) resp. chloroform) or solvent additives (TFAc in THF).