Procedure For HPLC column washing procedure

1.0 Objective
Objective of this Standard Operating Procedure is to provide guidelines

for HPLC column washing procedure.
2.0 Scope
Standard Operating Procedure is to provide guidelines for HPLC column washing procedure at abc Pvt. Ltd.
3.0 Responsibility
3.1 Executive – Quality Control.
3.2 In charge/Head-Quality Control.
4.0 Abbreviations and Definitions
SOP : Standard Operating Procedure
QC : Quality Control
CC NO : Change Control Number
cGMP : Current Good Manufacturing Practice
G : General
5.0 Procedure
5.1 Unpacking:
5.1.1 Inspect the column and check calibration certificate with respective serial

number/lot number immediately at the time of received.
5.1.2 If there is any damage or missing of certificate, immediately inform to Q.C Head.
5.2 Installation of column :
5.2.1 Remove the plugs of column and install the column from injector to detector carefully.

Start pump with minimum flow and check the leakage from the edges of the column.
5.3 Shipping solvent of columns :
5.3.1 Ship the Column with the solvent used for the final test of the column. When switching

between solvents with vastly different polarities follow the manufacturer instruction for

particular column. It may be necessary to first purge the column with a mutually miscible solvent such as methanol and water.
5.4 Column care:
5.4.1 Shock: Do not drop or bump columns. Subjecting columns to shock may cause peak splitting.
5.4.2 pH: Columns are designed for use within the pH range of 2.0 to 7.5. Higher pH will

dissolve the silic gel and lower pH can strip away some of the bonded phase.
5.4.3 Pressure: To maximize column life operate at pressures up to 4000psi.
5.4.4 Sample solvents: Samples should be dissolved in the eluent or weaker solvent

than the eluent. This will prevent the sample precipitation at the column inlet. Filter

sample solution with 0.45μm membrane filters to remove particulate matter.
5.4.5 Solvent: Use HPLC grade solvent that have been filtered through a 0.45μm

membrane filter. Filter all buffer solution before use.
5.4.6 Column flushing: After completion of analysis clean the column

by the following solvents as mobile phase.
5.5 For reverse phase columns:
5.5.1 Flush with the same mobile phase which was need for analysis for 15 minutes with 1.0 ml flow rate.
5.5.2 Flush with mixture of water: methanol or Acetonitrile (90:10) for 30 minutes.
5.5.3 Then flush with the mixture of methanol or Acetonitrile: Water (90:10) for 30 minutes at a flow rate of 1.0 ml/minute.
5.6 For normal phase columns:
5.6.1 Flush with the same mobile phase which was need for the analysis for 15 minutes at a flow

rate of 1.0 ml /minute. Then flush with n -Hexane at a flow rate of 1.0 ml / minute.
5.7 Storage of columns:
5.7.1 For Reverse phase:
After using the column with eluent containing buffer or ion – pair reagent, wash the column

completely with water for minimum 30 minutes at 1.0 ml/min flow rate followed by a

salt free eluent (Methanol) before Storing. When storing the column for a long period,

store it filled with a 100% organic solvent (Methanol).
5.7.2 For Normal phase:
After using the column, wash the column completely with n-Hexane for minimum 30 minutes

at 1.0 ml/min flow rate before storing. When storing the column for a long period, store it filled with a 100% n-Hexane.
5.8 Precautions:
5.8.1 Avoid the dusty place
5.8.2 Cleanness of the instrument.
5.8.3 Connect the column carefully with instrument.
5.8.4 Ensure the mobile phase without any such type of particles.
6.0 Forms and Records
6.1 None.
7.0 Distribution
7.1 Master copy – Documentation Cell (Quality Assurance)
7.2 Control copy – Quality Assurance, Quality Control
8.0 History