HPLC Column Performance Evaluation and Column Care

New column

For new HPLC column, column performance test will be carried out as per the procedure for existing columns in operation and as per manufacturer’s instruction. (If respective chemicals as per manufacturer's certificate are available)

Existing columns

For existing column operation, column performance test will be carried as per following procedure.

For all C18 column and C8 column:

Chromatographic conditions:

Mobile phase: Acetonitrile: Water (60:40)
Wavelength: 254nm
Injection volume: 20 ml
Column oven temperature:25°C +1°C.
For remaining parameters follow column performance method parameters.
Prepare test solution of 0.1% of toluene in the mobile phase.
Check chromatogram for theoretical plates and asymmetry (10%) of the peak obtained.

Acceptance criteria for C8 & C18 column :

RSD of 5 replicate injections: NMT 2.0%
Theoretical Plate: For new column note down the theoretical plates. For existing column theoretical plates more than the initial value or –10% of initial value is acceptable.
Asymmetry (10%): NMT 2.0
Enter the test results in the column performance evaluation report.

For Silica column (Existing columns in routine operation):

Mobile phase: N-Hexane and Ethanol in the ratio of 95:5
Wavelength: 254 nm
Column temperature: 40°C
For remaining parameters follow column performance method parameters.
Prepare test solution of benzene of 0.1% in n-Hexane.
Check chromatogram for theoretical plates and asymmetry (10%) of the peak obtained.

Acceptance criteria for silica column:

RSD of 5 replicate injections: NMT 2.0%
Theoretical Plate: For new column note down the theoretical plates. For existing columns in operation more than initial value or –10% of initial value is acceptable.
Asymmetry (10%): NMT 2.0
Enter the test results in the column performance evaluation report.
Phenyl column, Cyano column, Chiral column etc will be tested as per manufacturer’s instructions or as per column performance method parameters.

An alternative method for Column Qualification or Performance :

For all column including C8 and C18 inject five replicates of respective system suitability or standard as per specified method for which column has to be used.
Calculate the respective system suitability results. The result should meet system suitability parameters.
Theoretical plates (N) = 16 (t/W)2

Asymmetry (10%) = w/2f
Where,
t - Retention time
W - Width of the peak measured by extrapolating the relatively straight sides to the baseline
w - Width of the peak at 10% of peak height
f - Distance between perpendicular dropped from the apex of the peak and leading edge of the peak at 10% of peak height
Frequency: After completion of 1500 injections or earlier if required.

Flushing of columns:

Before commencement and after the completion of day's work on HPLC, the particular column must be flushed with suitable HPLC grade solvents.
Increase or decrease the flow rate of the solvent gradually to avoid the sudden shock to the column bed.
Flushing timings given below are only for guidance and they will be as per requirements. Flushing time may be changed by considering No. of samples injected or if the column is more contaminated.
Record all washing details in HPLC column logbook.
Related: Calculation of Relative Response Factor (RRF)

For C18 & C8 Columns:

Before analysis flushes the column with the same solvent in which column is stored for 30 minutes with a flow rate of 1 ml/min.
Flush the column with acetonitrile or methanol whichever is present in mobile phase with a flow rate of 1 ml/min for about 30 minutes.

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