HPLC Column Management

1.0 OBJECTIVE :

To lay down the procedure for handling, storage, maintenance, verification and disposal of chromatographic column.

2.0 SCOPE :

This SOP is applicable for handling, storage, maintenance, verification and disposal of chromatographic column in Quality Control Department.

3.0 RESPONSIBILITY :

Quality Control Officer and above

4.0 ACCOUNTABILITY:

Quality Control Head

5.0 PROCEDURE :

5.1 Handling of Column:

Receive the chromatographic column with proper documents i.e. Invoice/ challan, certificate etc., by the person authorized by department head.

Ensure that the documents are proper and as per requirement.

Ensure that the column is properly packed and in well condition.

Make counter sign with date of received in documents.

Take entry in Chromatographic Column Entry Log Book, Annexure No.: 02.

After receiving the column, ID Number for the column is allocated by numbering system as,

A/H-C/XX/YY

Where, A = for Company name initials,

H = for HPLC,

C = for Column,

XX = for last two digit of the year,

YY = for sequential number starting from 01.

Label with ID Number of column is affixed on the column.

Performance of HPLC column is verified as per verification procedure of performance of chromatographic column.

After completion of column performance, enter performance observation of chromatographic column in record of performance of chromatographic column – HPLC (Annexure No. 01).

5.2 Storage and Maintenance :

All chromatographic columns are stored in cabinet, dedicated as per column dimension/type.

Chromatographic column should be handled with care.

Use the column for analysis as required.

Usage of column shall be recorded in Column Usage Log Book as per the Annexure No.: 03

An analyst shall follow manufacturer’s instruction strictly for usage of the column which was provided with the column.

HPLC Column washing procedure :

Column shall be washed properly as per follows;

After completion of analysis:

In case of reverse phase column

Use water as the preliminary solvent and switch over to organic solvent based on the mobile phase used.

Flush the column with the water for at least 45 minutes followed by the respective 80% solvent which is used for analysis, for 45 minutes and again with water for 45 minutes.

In case of normal phase column (Silica column)

The column shall be washed properly using organic solvent such as isopropyl alcohol, n-Hexane and methanol.

Do not use water as solvent for washing the silica column. Water can be used only when any buffer solution is used in preparation of mobile phase.

Flush the column with the isopropyl alcohol for at least 45 minutes followed by n-Hexane for 45 minutes and then with methanol for 45 minutes.

Washing period shall be increased to 1 hour instead of 45 minutes whenever required.

Then the column shall be flushed with the storing solvent for at least 15 minutes.

Before analysis:

In case of reverse phase column

Flush the column with the water for at least 30 minutes followed by the respective solvent which will be used for analysis for 30 minutes.

If the mobile phase contains methanol or acetonitrile as organic solvent, flush with 100 % concentration of the same solvent for washing, for at least 30 minutes.

If the mobile phase contains both acetonitrile and methanol as solvent, flush with 100 % of the solvent whose concentration is maximum in the mobile phase, for at least 30 minutes.

In case of normal phase column (Silica column)

Flush the column with the isopropyl alcohol for at least 30 minutes followed by methanol for 30 minutes.

Then the column shall be flushed with the storing solvent for at least 15 minutes.

5.3 Verification for Performance of HPLC Column :

Checking of New Column

Column efficiency of chromatographic column is evaluated in terms of number of theoretical plates. Column with high theoretical plate numbers are considered to be more efficient than columns with a lower theoretical plates.

Check the column with respect to certificate of analysis for retention time, theoretical plates, resolution, tailing factor and method specified by vendor.

Carry out the column efficacy for theoretical plates, resolution and tailing factor.

Determination of Column Efficacy:

Selection Criteria:

Perform the column performance by one of the procedure (either by procedure A, B or C)

If the column is used for only one product, then follow the procedure A.

If the column is used for more than one product, prefer the procedure B.

In case of unavailability of solvent, Reagent or WS/RS, follow the procedure C.

Column performance by using System Suitability (Procedure A):

Perform the system suitability of material for which the column is procured.

Prepare the solutions used for checking of system suitability of specific material as mentioned in pharmacopoeia.

Acceptance criteria: The system suitability criteria of monograph should complies with the solution.

Column performance by using Benzene and Toluene (Procedure B):

Prepare mobile phase freshly on the day of use with combination of 60:40 v/v Acetonitrile and HPLC water. Filter the mobile phase by using 0.45 µm membrane filter and degas the mobile phase by using sonicator.

Chromatographic Conditions:

Flow Rate : 1.0 ml/min

Wavelength : 254 nm

Injection Volume : 20 µl

Temperature : Ambient

Run the mobile phase to saturate the column.

Prepare the test solution as follow:

Take 1 ml of Benzene and 1 ml of Toluene in 100 ml volumetric flask and dilute to volume with acetonitrile. Dilute 1 ml of this solution to 100 ml with mobile phase. Mix well and filter through 0.45 µm syringe filter.

Inject the filtered test solution and check the number of theoretical plates, tailing factor, resolution between the peak of toluene and benzene.

Acceptance criteria:

Resolution between the peak of toluene and benzene should not be less than 2 or as given by manufacturer / supplier criteria.

Theoretical plate for benzene and toluene should not be less than 2000 or as given by manufacturer / supplier criteria.

Tailing factor of benzene and toluene should not be more than 2.0 or as given by manufacturer / supplier criteria.

Column performance as per procedure of Manufacturer (Procedure C):

Column performance is carried out by using the procedure given on column performance record provided by vendor.

Prepare mobile phase as mentioned on vendor record of column performance.

Chromatographic condition such as flow rate, wavelength, injection volume, temperature is adjusted as mentioned on vendor record of column performance.

Test solution is prepared as per vendor record of column performance.

Inject the filtered test solution and check the test parameter against the specification mentioned in vendor record of column performance.

Acceptance criteria:

The results of defined test parameter should be met with manufacturer / supplier criteria.

Inform to QC Head for replacement of the column if column does not meet with acceptance criteria of column performance.

QC Head informs to the purchase department for procurement of the column as replacement.

Report the observation in Annexure No.: 01 ‘Performance of Chromatographic Column – HPLC’.

Frequency: On receipt of the new column.

5.4 Destruction of HPLC column :

The following HPLC column shall be destructed.

If the column does not meet with verification for HPLC column performance criteria.

If system suitability criteria as per method of analysis is not matched.

Affix the label “REJECTED” on the column and store in containers reserved for rejected column.

All documents regarding last non-compliance of system suitability data (Chromatogram etc.) are recorded and maintained as HPLC column destruction record.

Destroy the rejected columns as per following procedure.

Remove all labels or marking from column with the help of water.

Open the end of fitting of column with the help of Engineering department.

Columns are to be sent to scrap yard for further disposal.

After destruction of column, enter the details in HPLC Column Disposal Record Annexure No.: 04.

6.0 ABBREVIATIONS:

Abbreviation Expanded form

HPLC High Performance Liquid Chromatography

% Percentage

v/v Volume per volume

°C Degree Celsius

ppm Part per million