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SOP for HPLC Column Receipt, Checking and Regeneration

Standard operating procedure to check the performance of the HPLC columns against the certificate received from vendor.


       To lay down the procedure for checking the performance of column with respect to certificate received from vendor and to carry out performance test as per in house method. Also to describe the procedure for regeneration of column.

2.0  SCOPE

       This procedure is applicable to all Reverse Phase (C8, C18, cyno, phenyl) column and Normal Phase (Si) Column regeneration.


3.1  Doing      :   Technical Assistant ,Executive
3.2  Checking :   Executive/Manager


4.1  Head of the Department


5.1  Checking of new HPLC column

5.1.1  On receipt of new column from vendor, condition the column using methanol at 0.5 ml/min.
5.1.2  Check the column with respect to certificate of analysis for back pressure, retention time, theoretical plates using the material provided with column and method specified by 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 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.

Where   XXX indicates the short form of the column, it may be  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 column in Annexure-I.

5.3  Column Regeneration for reverse phase column

5.3.1  Flush the column using warm water ( 55oC) at flow rate of 0.7 ml/min. for 10 minutes. Maintain the column at (55oC) in column oven during flushing.
5.3.2  Inject 100 µl of DMSO four times at interval of 2 minutes.
5.3.4  Flush it with methanol at flow rate of 0.7 ml/min in reverse direction for 70 minutes.
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.


6.1  ml = millilitre

6.2  min. = minute

6.3  oC = Degree centigrade

6.4  IPA = Isopropyl alcohol

6.5  µl = Microlitter

6.6  DMSO = Dimethylsulfoxide

Columns make                             :
Column receipt date                     :
Column per supplier)    :
Column Id .No.                            :
Column inhouse no.                      :
Date of issue                                :
Column dimension                        :        
Check of new column                  :

Data as per COA
Observed Data





Back pressure bar

In-house check

No. of  Theoretical  Plates
Date of
Back pressure

Diethyl phthalate
Diethyl phthalate

Performed By:                                                                                                     Checked By:

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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1 comment:

Unknown said... on 8/6/17 23:35

Dear sir
I want to ask about the above method is it have a validation method for it as it a in house method ?????? or if there is it according any guidelines ????

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