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SOP For HPLC Column Receipt, Checking and Regeneration - Pharmaguideline
SOP For HPLC Column Receipt, Checking and Regeneration - Pharmaguideline
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4.0 ACCOUNTABILITY
4.1 Head of the Department
5.0 PROCEDURE
5.1 Checking of new HPLC column
5.1.1 On receipt of a new column from the vendor, condition the column using methanol at 0.5 ml/min.
5.1.2 Check the column with respect to the certificate of analysis for back pressure, retention time, theoretical plates using the material provided with
column and method specified by the vendor. Record the results in Annexure-I
5.1.3 Carry out the column performance (Theoretical plates, Tailing, resolution and back pressure) using following chromatographic condition. Record
the results in Annexure-I.
5.1.4 Performance of Reversed phase column.
Mobile phase: Methanol: water (70:30) v/v
Flow rate: 1.0ml/min.
Wavelength: 254nm
Inj.Volume: 20µl
Sensitivity: 0.005 AUFS
Test solution for C8 and C18: Mixture of Benzene and Toluene (0.1%) in the mobile phase.
Test solution for Cyano and Phenyl: Mixture of Benzene (0.1% ) and Naphthalene (4mg.)in 100ml mobile phase.
5.1.5 After checking, flush the column with methanol for 20 minutes at flow rate of 1.0ml/min. in normal direction and store the column in methanol.
5.1.6 Performance of Normal Phase (Si) column
Mobile phase: IPA: Hexane (40:60) v/v
Flow rate: 1.0ml/min.
Wavelength: 254nm
Inj.Volume: 20µl
Sensitivity: 0.005 AUFS
Test solution: Mixture of Toluene (0.5 µl/ml) and Diethyl phthalate (2µl/ml) in mobile phase.
5.1.7 After checking, flush the column with Hexane for 20 min. at flow rate of 2.0ml/min and store the column in Hexane.
5.2 Assign column number as follow.
XXX YYY
Where XXX indicates the short form of the column, it may be 2 to 4 digit
e.g. ODS Column = ODS
BDS Column = BDS
Amine Column = NH
Silica Column = SI
Protein pack = PP
High mol. weight protein = HMWP
YYY indicates serial no. in series starting from 001.
5.2.1 Enter the details of the column in Annexure-I.
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2/1/23, 2:33 PM SOP for HPLC Column Receipt, Checking and Regeneration : Pharmaguideline
5.3.5 After flushing with methanol, flush the column with chloroform for 70 minutes at flow rate of 0.7 ml/min in reverse direction.
5.3.6 At the end, wash the column in methanol for 70 minutes at flow rate of 0.7 ml/min. in reverse direction.
5.3.7 After regeneration of column, perform the column performance test as per Step 5.1.4 to 5.1.5
5.3.8 Store the column in methanol, after flushing with methanol for 20 minutes at flow rate of 1.0ml/min. in normal direction.
5.3.9 Maintain the column History card on use of column during analysis as per Annexure-II.
5.4 Column Regeneration for Normal Phase Column
5.4.1 Flush the column with Hexane for 60 minutes at flow rate of 2.0 ml/min..
5.4.2 After flushing with Hexane, wash the column with Methylene chloride for 60 min at flow rate of 2.0ml/min.
5.4.3 After flushing with methylene chloride, wash the column with Isopropyl alcohol for 60 minutes at flow rate of 2.0 ml/min.
5.4.4 At the end, flush the column with methanol for 60 minutes at flow rate of 2.0 ml/min.
5.4.5 After regeneration of column, perform the column performance test as per Step 5.1.6 to 5.1.7.
5.4.6 Store the column in Hexane, after flushing with Hexane for 20 minutes at flow rate of 2.0 ml/min in normal direction.
5.5 If the system suitability fails than only column regeneration required.
6.0 ABBREVIATIONS
6.1 ml = millilitre
6.2 min. = minute
6.3 °C = Degree centigrade
6.4 IPA = Isopropyl alcohol
6.5 µl = Microlitter
6.6 DMSO = Dimethylsulfoxide
ANNEXURE -I
HPLC COLUMN CHECK AND REGENERATION RECORD
Columns make :
Column receipt date :
Column Sr.no.(As per supplier) :
Column Id .No. :
Column inhouse no. :
Date of issue :
Column dimension :
Check of new column :
In-house check
No. of Theoretical Plates Tailing Resolution
Date of Back pressure Benzene Toluene/Naphthalene Benzene Toluene/Naphthalene
Performance Toluene Diethyl phthalate Toluene Diethyl phthalate
check
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of pharmaguideline.com, a widely-read pharmaceutical
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