SOP Title-Management of HPLC/GC Column
- OBJECTIVE
To lay down the procedure for Management of chromatographic column to be followed in QC Department.
- SCOPE
This SOP is applicable to Management of chromatographic column used for HPLC/GC in Quality Control laboratory.
- RESPONSIBILITY
- QC personnel shall be responsible for implementation of this procedure.
- QC Head /designee shall be responsible for compliance of this procedure.
- PROCEDURE :
- Precaution
- Ensure that mobile phase is properly filtered using 0.45µ filters and suitably degas before its use.
- Do not cross the maximum temperature (in case of GC columns) and minimum temperature (in case of HPLC column) of column specification of manufacture.
- Always close inlet and outlet of columns by using end cap, when not in use.
- Handle column carefully to prevent it from any mechanical shock.
- Do not store HPLC column in buffer.
- Mobile phase pH should not cross its recommended values.
- Read the instruction regarding column packing before handling the column.
- Qualify every column prior to use.
- Always labeled the column immediately after receiving.
- Procurement
- Based on the requirement of QC Department shall raise the Indents for purchase of chromatographic column from the authorized sources.
- Receipt and Issuance
- On the receipt of new column QC Executive, Sr.Executive/designee is responsible for checking the physical condition of column such as dimension, end caps, labels, and damage.
- On the receipt of new column verify the column with purchase indent and tally the serial number, make etc of the column with the respective Certificate Of Analysis.
- Unique identification numbering of new Liquid chromatographic column shall be done as per below mentioned procedure.
QC/LC/YY/ZZZ
Where;
QC – Quality Control
LC – Liquid Chromatography
YY – Last two digit of the current calendar year
ZZZ – Serial Number (Starts from 001)
- Unique identification numbering of new Gas chromatographic column shall be done as per below mentioned procedure.
QC/GC/YY/ZZZ
Where;
QC – Quality Control
GC – Gas Chromatography
YY – Last two digit of the current calendar year
ZZZ – Serial Number (Starts from 001)
- After receiving of column maintain record in column receiving log as per format .
- If the column performance passes the column performance criteria then only column can be used for routine analysis.
- If column fails the system suitability criteria then immediately reject the column and sent to purchase department.
- Handling and Usage :
- Always store the column as per recommended solvent by its manufacturer. Unless otherwise specified follow In-House column washing instructions.
- Routinely monitor the column performance by its theoretical plate count or System Suitability parameter.
- Reverse phase columns shall be kept in mixture of Acetonitrile: water. (90:10).
- If the eluent is having buffer then always wash the column with water before its storage.
- Silica Cyno, Amino columns are shipped in n-Hexane or n-Heptane by its manufacturer. when such column are to be used for slightly polar eluent than column should be washed with ethanol or Isopropanol before equilibration with mobile phase.
- Always increased or decreased the flow rate gradually to avoid pressure shock to the stationary phase in column.
- Pre conditioning and post conditioning shall be provided to GC column.
- Before starting any analysis pre condition the GC column above 10˚C of product .
- Column shall be flushed properly after the analysis is complete. The solvent used for flushing should be miscible.
- Performance of Column :
- Qualify the column as per the party COA or with the product for which column is dedicated.
- COA of column shall be attached with the column performance record.
- Maintain HPLC/GC Column Usage logbook.
- Maintain list of HPLC/GC column.
- Maintain HPLC/GC column performance record.
- Storage of Column :
- Short Term Storage:
- For overnight storage, keep the mobile phase flowing at 0.2 to 0.4 ml/min. this is very helpful when using complex mobile phase (buffers and ion-pairing reagents), as it reduces equilibration time to few minutes rather than an hour or more.
- Long Term Storage :
- If intended mobile phase has buffer, then first wash the column with same mobile phase composition without buffer. Then flush it with water and store the column in 90% Acetonitrile or Methanol.
- Column Maintenance: The column shall be flushed properly after the analysis is complete. The solvent used for flushing shall be miscible. The Column flushing procedure for HPLC column is as follows:
- Reverse phase HPLC columns:
- Before starting the analysis flush the column with water, then switch over to mobile phase and saturate the column with mobile phase and perform the analysis.
- After completion of analysis, run the mobile phase for about 10 minutes. Switch over to water and wash the column with water for about 30 minutes at the flow rate 1.0 ml per minutes/as per specified STP.
- Switch over to Methanol/Acetonitrile (used in mobile phase as per specified STP) and wash the column with methanol/Acetonitrile (used in mobile phase as per specified STP) for about 30 minutes.
- Set the flow to 0ml/minute after completion of washing and remove the column from the system. Put the end plug and keep the column at respective place.
- Normal phase HPLC columns:
- After completion of analysis, run the mobile phase for about 10 minutes.
- If Hexane is used in the mobile phase , switch over to 100% Dichloromethane.
- Wash the column for about 60 minutes at flow rate 1.0 ml/minute.
- If hexane is not used in the mobile phase, switch over to 100 % Methanol.
- Wash the column with Methanol for about 30 minutes at the flow rate of 1.0 ml/minute.
- Switch over to 100 % Dichloromethane and wash the column for about 30 minutes.
- Set the flow to 0 ml /minutes after completion of washing and remove the column from the system. Put the end plug and keep the column at the respective place.
- Re-Generation Procedure for HPLC Column:
- Re-Generation procedure for Reversed phase (C18, C8, & Phenyl) :
- Wash the column with Hot water (40ᵒC), inject 50 µl x 4 DMSO (Dimethyl sulfoxide) injections for a flow rate 0ml/min. up to 30 minutes.
- Then wash with 1.0ml/min up to 30 minutes of solvents mentioned in the following order-
- Methanol (100%)
- Isopropanol (100 %)
- Dichloromethane (100%)
- Hexane (100%)
- Dichloromethane (100%)
- Isopropanol (100 %)
- Chloroform (100%)
- Methanol (100%)
- Re-Generation procedure for Normal phase (Silica & CN) :
- Wash the column with flow 1.0ml/min up to 30 minutes of solvents mentioned in the following order:
- Heptane (100%)
- Chloroform (100%)
- Acetone (100%)
- Ethanol (100%)
- Water (100%)
- Re-Generation procedure for ION Exchange Column:
- Wash column with flow 1.0ml/minutes up to 30 minutes of solvents mentioned in the following order-
- Buffer wash – 5X as concentrated as that is used previously.
- Distilled water
- Acid wash – 0.5 M H3PO4, 01 M H2SO4 or 1.01 M HNO3
- Distilled water
- Chelating wash: 0.1 M Disodium EDTA
- Distilled water
- Methanol wash: To remove absorbed organics from the bonded organic phase.
- Distilled water
- DISCONTINUATION AND DISPOSAL :-
- Maintain the column regeneration record as per define format.
- If System suitability parameters are within limits than reuse the column.
- If system suitability parameters are out of limit than discard the column and mention that the column is discarded in column usage logbook.
- Dedicated columns can be used till it meets the requirements of system suitability of respective method.
- Columns with high back pressure and not complying system suitability parameter should be discarded with the approval of Head quality.
- Column should be labeled as “Not to use” and stored separately from other columns.
- While discarding, column performance shall be evaluated for system suitability parameter.
- ABBREVIATION(s)
| Abbreviation | Full Description |
| QC | Quality Control |
| QA | Quality Assurance |
| SOP | Standard Operating Procedure |
| No. | Number |
*Note – Ready to use SOP available in “DOWNLOAD” Section.