To lay down a standard procedure for purchase, installation, storage, performance evaluation, usage, cleaning and withdrawal of HPLC column.
This procedure is applicable for all HPLC columns used in the Quality Control Laboratory
Officers / Executive – Quality Control shall be responsible for complies of this SOP
Head QC and Head QA shall be accountable for compliance of this SOP.
Label for Column Attachment-I
Index for Column Attachment-II
Column Performance Test Record Attachment-III
Column Washing Record Attachment-IV
Column Usage Record Attachment-V
Column Regeneration Record Attachment-VI
- PURCHASE OF COLUMNS:
- Indent & receive the column from Purchase department and verify whether correct columns are
Received against to specify the column make, brand name, dimensions and particle size.
- Check the column for physical damage which may have occurred during shipping.
- INSTALLATION OF COLUMNS:
- Retain safely the column performance certificate sent by the manufacturer.
- Ensure that the column ends are securely closed.
- Store the column in the designated area.
- Test the column immediately to verify quality and Labeled as per Attachment-I.
- COLUMN PERFORMANCE EVALUATION:
- Prior to column performance, flush the column with suitable solvent (compatible) based on the solvent in which the column is shipped.
- Read and follow the instructions such as temperature, pH, back pressure limits mentioned for one particular column.
- Test the column for performance by the following procedure:
- For reverse phase bonded packings (C18, C8, Cyano, Amino etc.)
Uracil stock solution: Weigh accurately about 50 mg of Uracil in a 50 ml volumetric flask. Dissolve in 25 ml mobile phase and dilute upto the mark with the mobile phase.
- Standard solution: Weigh accurately 40 mg of phenol and 60 mg of anisole transfer in a 50 ml volumetric flask, containing about 20 ml of mobile phase and dissolve.
Add 2 ml of uracil stock solution and dilute up to the mark.
- Mobile phase: Methanol: water (70: 30)
- Flow: 1.0 ml/minute
- Wavelength: 254 nm
- Injection volume: 20 µl
- Procedure: Inject the standard solution 5 times. Calculate the column efficiency (theoretical plates). Symmetry factor and capacity factor for the peaks of phenol and anisole. Use the retention time of uracil as void volume for determination of capacity factor. Relative standard deviation of peak area for 5 replicate injections should not be more than 2.0% for phenol and anisole.
Acceptance criteria :
For phenol : Not less than 0.30
For Anisole : Not less than 1.00
For phenol : Not less than 2000
For Anisole : Not less than 2000
USP tailing : Not more than 2.0 (each for phenol and anisole)
- For Adsorption/Normal Phase columns
Preparation of the standard: Weigh about 200 mg each of acetophenone, 80 mg of 2,4-dinitrotoluene and 200 mg of toluene in a 100 ml volumetric flask. Dissolve in 25 ml of mobile phase and dilute upto the mark with mobile phase. Dilute 3 ml of the above to 100 ml in a volumetric flask with mobile phase.
Mobile phase: Hexane: Ethyl acetate (98:2)
Flow: 1.0 ml/minute
Wavelength: 254 nm
Injection volume: 20 µl
Run time: 20 Minutes for 250mm and above 250mm length columns.
10 Minutes for below 250mm length columns.
Procedure: Inject the standard solution 5 times. Calculate the column efficiency (theoretical plates), symmetry factor and capacity factor for the peaks of acetophenone and 2,6-dinitrotoluene. Use the retention time of toluene as void volume for determination of capacity factor. Relative standard deviation of peak area for 5 replicate injections should not be more than 2.0% for acetophenone and 2,4-dinitrotoluene.
For Below 250 mm length columns
Capacity factor: For acetophenone: Not less than 0.15
For 2,4 dinitrotoluene : Not less than 0.30
Theoretical plates: Not less than 1000 (Each for acetophenone and 2,4 dinitrotoluene)
USP tailing: Not more than 2.0 (Each for acetophenone and 2,4 dinitrotoluene)
For 250 mm and above 250mm length columns Capacity factor: For acetophenone : Not less than 0.40 For 2,4 dinitrotoluene : Not less than 2.0
Theoretical plates: Not less than 1000 (Each for acetophenone and 2,4 dinitrotoluene)
USP tailing: Not more than 2.0 (Each for acetophenone and 2,4 dinitrotoluene).
After completion of the performance test, attach a tag to the column indicating the column number, date of performance check and signature and update column performance test as per Attachment-III.
- Update the index of column to include the new column as per Attachment-II
- If the column fails to meet the acceptance criteria, then return the column to the supplier and arrange for replacement.
- After meeting the requirements of column performance allot the ID no as given below:-
SC/QC/C00, C indicates Column and 00 is the serial number for column. Dedicates the columns individually for finished product and stability testing of each product (where ever possible) and labels the same on the label as per Attachment-I.
- USAGE OF COLUMNS:
- 6.4.1 Handle the column with extreme care. Never allow the column to experience mechanical shocks or vibrations.
- 6.4.2 Select the column by referring to the HPLC column list and as indicated in the specification.
- 6.4.3 Allow the column to stabilize on the system under the set parameters of the instrument and the mobile phase.
- 6.4.4 Perform the analysis on the column as recommended in the ‘Tests and Methods’ given under specification.
- 6.4.5 On completion of analysis, flush and clean the column with the relevant solvents as recommended in the section on washing and care of HPLC columns and maintain column usage record as per Attachment-V.
- 6.4.6 Replace back the column to the designated place.
6.5 WASHING OF COLUMNS:
- 6.5.1 Always use filtered and degassed mobile phase/ solvents.
- 6.5.2 Before changeover of a mobile phase, make sure that solvents used for column flushing are miscible with the mobile phase.
- 6.5.3 Equilibrate the column for about 30 minutes at the flow rate as specified in the individual specification. If ion-pairing agents or additives are used, flush the column for about 1 hour at the specified flow rate for complete equilibration.
- 6.5.4 After completion of analysis, flush the column with the appropriate solvents. If the mobile phase contains buffer, flush the column first with sufficient quantity of water and then with an organic solvent, used for mobile phase preparation.
- 6.5.5 For longer storage, store the column in 100% methanol and be sure that the end plugs are firmly in place. Never let the column dry out.
- 6.5.6 Storage conditions for HPLC columns:
Reverse Phase 100% Methanol
(C18, C8, C4, C2, C1 Or Phenyl)
Normal Phase Isopropanol or n-hexane
(Silica, CN, NH2, Diol, Alumina)
Ion Exchange Methanol (flush the column with 50ml water prior to
Size exclusion 0.05% sodium azide in water or in 10% methanol
- 6.5.7 While changing the mobile phase, gradually increase the flow rate of the new mobile phase in increments from 0.1 ml/minute upwards.
- 6.5.8 A shift in retention or resolution is an indication of insufficient flushing of the column. Flush with a organic solvent which may be a part of the mobile phase of the product for which the column is dedicated.
- 6.5.9If buffers like citrate or ammonium carbonate are used in the mobile phase, ensure that the HPLC columns which can withstand the conditions of analysis or the specified HPLC columns as given in the specification are used. Other HPLC columns may get deactivated in such conditions (condition this point while selecting a column for particular applications).
- 6.5.10Maintain column washing record as per Attachment-IV.
6.6 WITH DRAWAL OF COLUMNS:
- 6.6.1 Withdraw the column from usage in case of any of the following instances:
- 6.6.2 Physical damage.
- 6.6.3 Failure to comply system suitability requirements as per specification. (Note: When failure to the system suitability requirements as per specification, evaluate once again the column performance and take the decision of withdrawal).
- 6.6.4 Failure to comply with performance checks requirements.
- 6.6.5 Indicate the withdrawal on the column inwards register and update the column index.
6.7 COLUMN REGENERATION FOR REVERSE PHASE COLUMN:
- 6.7.1 Flush the column using warm water (55 °C) at flow rate of 0.7ml/min. for 10 minutes. Maintain the column at (55°C) in column oven during flushing.
- 6.7.2 Inject 100µl of Dimethylsulphoxide four times at interval of 2 minutes.
- 6.7.3 Flush it with methanol at flow rate of 0.7 ml/min in reverse direction for 70 minutes.
- 6.7.4 After flushing with methanol, flush the column with chloroform for 70 minutes at flow rate of 0.7ml/min in reverse direction.
- 6.7.5 At the end, wash the column with methanol for 70 minutes at flow rate of 0.7ml/min. in reverse direction.
- 6.7.6 After regeneration of column, perform the column performance test and maintain record as per Attachment-VI.
- 6.7.7 Store the column in methanol, after flushing with methanol for 20 minutes at flow rate of 1.0ml/min. in normal direction.
6.8 COLUMN REGENERATION FOR NORMAL PHASE COLUMN:
- 6.8.1 Flush the column with hexane for 60 minutes at flow rate of 2.0ml/min.
- 6.8.2 After flushing with hexane, wash the column with methylene chloride for 60 min. at flow rate of 2.0ml/min.
- 6.8.3 After flushing with methylene chloride, wash the column with isopropyl alcohol for 60 minutes at flow rate of 2.0ml/min.
- 6.8.4 At the end, flush the column with methanol for 60 minutes at flow rate 2.0ml/min.
- 6.8.5 After regeneration of column, perform the column performance test and maintain record as per Attachment-VI.
- 6.8.6 Store the column in Hexane, after flushing with hexane for 20 minutes at floe rate 2.0 ml/min in normal direction.
SOP: Standard Operating Procedure
QC: Quality Control
QA: Quality Assurance
HPLC: High Performance Liquid Chromatography
- DISTRIBUTION LIST:
- HISTORY OF REVISION:
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