Interaction of the analyte and its matrix with the stationary phase of the column can result in contamination of the column and change in the column characteristics. The chromatographic performance of a contaminated column can be different from an uncontaminated column.
Column contamination can result in failure to meet system suitability requirements, poor peak shape, baseline disturbances, baseline drift, high column back pressure, etc.
In such cases, regeneration procedures can often restore the column to its original working condition.
The regeneration procedure for the column depends upon the nature of the column and contaminants.
If the column exhibits high back pressure, first determine the cause of the high back pressure - whether it is due to the column or the HPLC/UPLC system. This can be done by checking the back pressure when the column is connected to the system and disconnected from the system. Always disconnect the column from the detector during this check.
If the high back pressure is due to the column, reverse the column and flush the column at 0.5ml/min (0.1 ml/min for UHPLC column) for 10-15 minutes with buffer free mobile phase, then increase the flow rate slowly upto a maximum of 2.0ml/min (provided system back pressure is below 4000psi in case of HPLC or below 15000 psi in case of UHPLC).
Wash the column with 10-20 column volumes of a strong eluent (high organic solvent like methanol or acetonitrile for reverse phase and high salt for ion exchange columns) and run 2 to 3 blank gradients to remove contaminants.
When regeneration of the column is necessary, disconnect the column from the detector and arrange the setup to collect the eluent directly in a waste beaker.
Carry out the regeneration procedure at typical flow rates of 1.0 to 2.0 ml/min. However, the flow rate can be chosen over a range of 0.2 to 2.0 ml/min, based on the column dimensions to avoid higher back pressures.
Calculate the column volume using the following formula:
V= pi *r 2*L
Where,
V is the column volume in ml.
r is the column inner radius in mm.
L is the column length in mm.
pi value is 22/7
REGENERATION OF BONDED - SILICA REVERSE PHASE COLUMNS:
Connect the column in reverse direction and flush the column using the mobile phase in the sequence mentioned below.
10 column volumes of water.
10 column volumes of 1 % v/v acetic acid in water.
10 column volumes of water.
10 column volumes of methanol.
10 column volumes of 5 % v/v tetrahydrofuran in methanol
10 column volumes of isopropyl alcohol.
10 column volumes of hexane.
10 column volumes of isopropyl alcohol.
10 column volumes of methanol.
10 column volumes of water.
If the column is to be stored after regeneration, flush the column with 10 column volumes of acetonitrile : water in the ratio of 90:10 v/v or with appropriate column storage solvent.
At each stage of the regeneration procedure, ensure miscibility of solvents being used in the series.
REGENERATION OF POLAR BONDED - SILICA USED IN REVERSE PHASE MODE (AMINO, CYANO AND DIOL):
For polar bonded silica columns used in reverse phase mode, follow the same procedure as for bonded silica reversed phase columns
REGENERATION OF POLAR BONDED - SILICA COLUMNS USED IN NORMAL PHASE MODE (AMINO, CYANO AND DIOL):
Connect the column in reverse direction and flush the column using mobile phase in the sequence mentioned below. At each stage ensure the miscibility of solvents being used in the series. If immiscible, flush the column with a miscible intermediate solvent such as iso propyl alcohol before proceeding to the next solvent.
10 column volumes of chloroform.
10 column volumes of methanol.
10 column volumes of Acetonitrile.
10 column volumes of methylene chloride.
10 column volumes of methanol.
If the column is to be stored after regeneration, flush the column with an intermediate solvent such as isopropyl alcohol to prevent solvent immiscibility, followed by 10 column volumes of the storage solvent.
REGENERATION OF SILICA COLUMNS:
Before proceeding with the regeneration of the column, check the miscibility of the column storage solvent with n-hexane; if immiscible, flush the column with a miscible intermediate solvent such as isopropyl alcohol, followed by the solvents in the sequence mentioned below.
10 column volumes of n-hexane.
10 column volumes of methylene chloride.
10 column volumes of isopropyl alcohol.
10 column volumes of methanol.
10 column volumes of methylene chloride.
10 column volumes of n-hexane.
If the column is to be stored after regeneration, flush the column with 10 column volumes of the storage solvent. Check the miscibility of the storage solvent with n-hexane; if immiscible, flush the column with a suitable intermediate miscible solvent.
REGENERATION OF SILICA BASED ION-EXCHANGE COLUMNS:
Connect the column in reverse direction and flush the column by the solvents in the sequence mentioned below.
10 column volumes of hot water (40°C - 60°C).
10 column volumes of methanol.
10 column volumes of acetonitrile.
10 Column volumes of methylene chloride.
10 column volumes of methanol.
If the column is to be stored after regeneration, flush the column with 10 column volumes of the storage solvent.
At each stage of the regeneration procedure, ensure miscibility of solvents being used in the series.
REGENERATION OF POLYMER BASED COLUMNS AND OTHER COLUMNS:
Due to variations in the pressure stability and solvent compatibility of polymer based columns as a result of the different nature of polymers used by different manufacturers, always follow the instructions of the manufacturer for regeneration of these columns. In case of doubt, contact the technical support group of the manufacturer for clarity.
POST-REGENERATION PROCEDURE:
If a column continues to show the performance characteristics that prompted the regeneration procedure, such as, failure to meet system suitability requirements, poor peak shape, baseline disturbances, baseline drift, high column back pressure, etc., the column is no longer suitable for use for that particular analysis.
Do revert at connect@bhaskarnapte.com in case if you have any query about column regeneration procedure.
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