The column is packed with porous particles and it’s made of polymer and is bounded by a thin layer of silica and polystyrene. In the early years, the Chromatography work carried out by the glass column with the diameter 01 to 05 cm and length is 50 to 500 cm, now a day HPLC columns are made of stainless steel. Normally the columns are in length from 30 to 250 mm, diameter 01 to 05 mm and pore size is 03, 05 and 10 microns.
System dead volume:
Minimize the dead volume in the system by using connection tubing of small internal diameter, for the analytical columns that lengths are 0.25 mm or less. Keep the length of tubing short as much as possible between the injector, column and the detector.
Equilibration or saturation of the column:
A new column has a storage solvent mobile phase that is used to evaluate the column unless or as specified on the chromatogram. Primarily be concerned about to do pass any material through the analytical column, which can be passed in storage solvent. Make sure the column is completely equilibrated or saturated in the mobile phase before starting the HPLC analysis. A normal phase silica column generally needs extra conditioning than the reverse phase column. The flow direction is always marked on the analytical column.
Performance testing of the column:
It is suggested that every column performance should be tested during the arrival and periodic intervals
HPLC solvents:
Make use of only HPLC grade solvents and the freshly prepared buffer solution to reduce any bacterial growth in the mobile phase. To ensure the column's life maximum, keep the pH of the mobile phase between 2 to 9.5 and always filter with a 0.45-micron filter.
Pressure on the column and system:
Exposure of analytical columns to fast change in backpressure or pressure is more than 400 bars it may reduce the column performance and life.
Fittings - torque:
Extreme tightening of end fittings of the column will damage the column fittings or tubing. To prolong column life and performance, it does not remove an end fitting of the column.
Mechanical damage:
Keep columns safe from mechanical shock, banging or Dropping of columns can spoil its performance.
Safety:
Highest working pressure = 400 bars
Highest working temperature = 60°C
Storage:
Wash out all the water and buffer from the bonded analytical columns and stores in the organic solvent, for Unbonded silica columns can be stored in the hexane or compatible organic solvent. Place the column in a cool area. To prevent drying replace the end caps.
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