CALIBRATION OF ALLIANCE HPLC WITH FLUORESCENCE DETECTOR

• OBJECTIVE:

• To lay down the procedure for Calibration of waters Alliance HPLC with Fluorescence detector.

• SCOPE:

This SOP is applicable to the procedure for Calibration of waters Alliance HPLC with Fluorescence detector at {Company Name} {Location}.

• RESPONSIBILITY:
• Executive/Designee Quality Control is responsible to calibrate the instrument as per SOP.
• Head QC: Ensure the compliance of SOP.
• ACCOUNTABILITY:

             QA Head shall be Accountable for implementation of SOP.

• PROCEDURE:
  • Procedure for noise:
    • Prepare a filtered and degassed mixture of water and Acetonitrile in the ratio of 30:70 (v/v).
    • Keep filtered and degassed mixture of water and Acetonitrile (Ratio 30:70), purge the channel by following operating instructions mentioned in the individual SOPS.
    • Fix Hypersil BDS, C18, 150 × 4.6 mm, 5 µ column to the system.
    • Keep the column temperature at 25°C and sample temperature 15°C.
    • Purge the detector for NLT 30 minutes at a flow rate of 1.0 ml/min.
    • Stabilize for 30 min. at a flow rate of 1.0 ml/min.
    • Fix the Excitation wavelength at 360nm and Emission wavelength at 450nm.
    • Start run by following operating instructions mentioned in the individual SOP.
    • Inject the mobile phase (Run time: 12 min. Injection volume: 10µl) 6 times by following the operating instructions mentioned in the individual SOP and record the Results.
    • Take the report by zooming the chromatogram. For ‘Noise’ calculation. Record Maximum absorbance (x) and minimum absorbance (y) in µV. Calculate the ‘NOISE’ By using the formula as given in Format-III and record the details in same Format.
    • Acceptance criteria: Noise £ 100µV
  • Procedure for Detector reproducibility:
    • Prepare a filtered and degassed mixture of water and Acetonitrile in the ratio of 30:70 (v/v).
    • Keep filtered and degassed mixture of water and Acetonitrile (Ratio 30:70), purge the channel by following operating instructions mentioned in the individual SOP.
    • Fix Hypersil BDS, C18, and 150 × 4.6 mm, 5µ column to the system.
    • Keep the column temperature at 25°C and sample temperature 15°C.
    • Purge the detector for NLT 30 minutes at a flow rate of 1.0 ml/min.
    • Stabilize for 30 min. at a flow rate of 1.0 ml/min.
    • Fix the Excitation wavelength at 360 nm and Emission wavelength at 450 nm.
    • Weigh accurately 20 mg of Anthracene and dissolve in 175 ml of Acetonitrile, sonicate for 5 min to dissolve and make up to 250 ml with water. (Concentration: 0.08 mg / ml). Stock Solution.
    • Further dilute 10 ml of stock solution to 100 ml with Water: Acetonitrile (30:70 v/v) (Concentration: 0.008 mg / ml) Solution A.
    • Further dilute 10 ml of solution A to 100 ml with Water: Acetonitrile (30:70 v/v) (Concentration: 0.0008 mg / ml) Solution B.
    • Inject the solution of 0.0008 mg/ml concentration (Run time: 12 min. Injection volume: 10µl) 6 times by following the operating instruction mentioned in the individual SOP and record the results in Format-I.
    • Acceptance criteria: The % RSD for area of anthracene peak from 6 replicate injections should be NMT 1.0.
    • Calibration schedule: Once in 6 months or after any major maintenance job.
  • Procedure for Detector response Linearity:
    • Prepare a filtered and degassed mixture of water and Acetonitrile in the ratio of 30:70 (v/v).
    • Keep filtered and degassed mixture of water and Acetonitrile (Ratio 30:70), purge the channel by following operating instructions mentioned in the individual SOP.
    • Fix Hypersil BDS, C18, and 150 × 4.6 mm, 5µ column to the system.
    • Keep the column temperature at 25°C and sample temperature 15°C.
    • Purge the detector for NLT 30 minutes at a flow rate of 1.0 ml/min.
    • Stabilize for 30 min. at a flow rate of 1.0 ml/min.
    • Fix the Excitation wavelength at 360 nm and Emission wavelength at 450 nm.
    • Weigh accurately 20 mg of Anthracene and dissolve in 175 ml of Acetonitrile, (Concentration: 0.08 mg / ml) Stock Solution.
    • Further dilute 10 ml of stock solution to 100 ml with Water: Acetonitrile (30:70 v/v) (Concentration: 0.008 mg / ml) Solution A.
    • Further dilute 10 ml of solution A to 100 ml with Water: Acetonitrile (30:70 v/v) (Concentration: 0.0008 mg / ml) Solution B.
    • Further dilute 10 ml of solution B to 100 ml with Water: Acetonitrile (30:70 v/v) (Concentration: 0.00008 mg / ml) Solution C.
    • Inject the solution of 0.008 mg/ml, 0.0008 mg/ml and 0.00008 mg/ml Concentration (Run time: 12 min. injection volume: 10µl) 2 times by Following the operating instructions mentioned in the individual SOP and Record the results in Format-II.
    • After completion take reports by following the operating instructions mentioned in individual SOP.
    • Acceptance criteria:
• The co-relation coefficient from Anthracene peak areas obtained at 3 levels (0.008 mg/ml, 0.0008 mg/ml and 0.00008 mg/ml) should be NLT 0.9990 by regression analysis.
• Calibration schedule: Once in 6 months and after any major maintenance
  • After all the above parameters calibrated, ‘calibration summary’ (Format-IV) report shall be prepared and all the raw data pertaining to the calibration shall also be attached with calibration summary report. ‘CALIBRATED’ label shall be tagged with instrument immediately.
  • In case, calibration of any of the parameter is not meeting the acceptance criteria, immediately the instrument should be identified by ‘UNDER MAINTENANCE’ label.
  • Investigate the reason for failure, shall be addressed at appropriate remarks column in respective calibration record.
• REFERENCES:

Not Applicable

• ANNEXURES: 

        ENCLOSURES: SOP Training Record.

•  DISTRIBUTION:
• Controlled Copy No. 01  :          Head Quality Assurance
• Controlled Copy No. 02  :         Head Quality Control
• Master Copy                    :          Quality Assurance Department
• ABBREVIATIONS:

• REVISION HISTORY:             

CHANGE HISTORY LOG

Annexure-I

DETECTOR REPRODUCIBILITY IN FLUORESCENCE DETECTOR

Annexure-II
DETECTOR RESPONSE LINEARITY IN FLUORESCENCE DETECTOR

Annexure-III
NOISE WITH FLUORESCENCE DETECTOR

Annexure-IV
CALIBRATION SUMMARY FOR FLUORESCENCE DETECTOR