Operation and Calibration of High Performance Liquid Chromatography

-

STANDARD OPERATING PROCEDURE

OPERATION AND CALIBRATION OF HPLC

USP <621> | USP <1058> | Ph. Eur. 2.2.46 | WHO GMP | EU GMP Annex 15

1. Purpose

This SOP provides detailed step-by-step instructions for the operation and calibration of HPLC systems to ensure accurate, precise, and reproducible analytical results in compliance with GMP and pharmacopeial requirements.

2. Scope

Applicable to all HPLC systems (isocratic or gradient, UV or PDA detectors) used for routine QC, stability studies, and method validation in pharmaceutical laboratories.

3. Responsibilities

  • Analyst: Perform HPLC operation, calibration, and record results
  • QC Supervisor: Verify calibration data and approve operation
  • QA: Ensure compliance, review deviations, approve final records

4. Safety Precautions

  • Wear PPE (lab coat, gloves, goggles)
  • Handle solvents in a fume hood
  • Follow MSDS for chemicals and waste disposal procedures
  • Ensure proper ventilation and electrical safety

5. HPLC Operation Procedure

5.1 System Start-Up

  1. Switch on the HPLC modules and workstation.
  2. Check solvent reservoirs and degasser function.
  3. Purge solvent lines to remove air bubbles (run at 1–2 mL/min for 5–10 min).
  4. Install column and allow equilibration for at least 30 min at method-specified conditions.
  5. Check baseline stability on the detector.

5.2 Sample Analysis

  1. Load the approved analytical method on the software.
  2. Run blank and system suitability solutions.
  3. Inject samples in the sequence as per method requirements.
  4. Monitor chromatograms for peak shape, retention time, and baseline stability.

5.3 Shutdown Procedure

  1. Flush column with appropriate solvent for at least 10 column volumes.
  2. Turn off pump and detector lamps.
  3. Close solvent reservoirs and clean waste containers.
  4. Switch off the HPLC modules and workstation.

6. Calibration Procedure

6.1 Pump Calibration

  1. Set pump to deliver a known flow rate (e.g., 1.0 mL/min).
  2. Collect the output for a fixed time (e.g., 10 min) in a tared volumetric flask.
  3. Weigh collected volume to calculate actual flow rate (density correction if needed).
  4. Repeat three times and calculate mean and %RSD.
  5. Check gradient accuracy by running a known gradient program and collecting fractions to verify composition using UV absorbance.
  6. Verify pressure stability during flow and gradient operation.
  7. Acceptance: Flow accuracy ±2%, %RSD ≤2%, gradient deviation ≤2%.

6.2 Degasser Calibration

  1. Run pump with solvent and monitor baseline for air bubbles.
  2. Observe baseline drift and noise for at least 10 minutes.
  3. Acceptance: No bubbles, baseline drift <0.5% of full scale, noise <0.5 mAU.

6.3 Autosampler Calibration

  1. Set the injection volume (e.g., 10 μL) and prepare a standard solution.
  2. Inject the standard 6 times sequentially.
  3. Calculate mean peak area and %RSD for injection precision.
  4. For injection accuracy, weigh the volume injected into a tared vial or use a standard dilution.
  5. Check carryover by injecting blank after standard; acceptance: carryover ≤0.1%.
  6. Acceptance: Precision %RSD ≤2%, volume accuracy ±2%.

6.4 Detector Calibration (UV/PDA)

  1. Set detector to a known wavelength (e.g., 254 nm).
  2. Measure wavelength accuracy using standard holmium oxide or didymium filter.
  3. Check photometric linearity by injecting solutions of known concentrations.
  4. Monitor baseline noise and drift for at least 10 min.
  5. Verify response time by switching flow on/off.
  6. Acceptance: Wavelength accuracy ±1 nm, photometric linearity R² ≥0.999, noise <0.5 mAU, drift <0.5 mAU/10 min.

6.5 Column Oven Calibration

  1. Set oven to method-specified temperature (e.g., 30°C).
  2. Measure actual column temperature using calibrated thermocouple at column inlet and outlet.
  3. Verify stability over 30 min.
  4. Acceptance: Temperature accuracy ±2°C, uniformity ±1°C, stability ±1°C over 30 min.

6.6 Data System & Software Verification

  1. Verify user access levels and password protection.
  2. Check audit trail and electronic signature functionality.
  3. Confirm date and time accuracy.
  4. Perform backup and restore test with sample data.
  5. Acceptance: 21 CFR Part 11 compliance, audit trail functional, backup restored without error.

7. System Suitability Testing

  1. Inject standard solution and check:
    • Retention time reproducibility (%RSD ≤2%)
    • Peak area reproducibility (%RSD ≤2%)
    • Theoretical plates (≥ method-specified)
    • Tailing factor (≤2.0)
    • Resolution (≥2.0)
  2. Document results in SST log.

8. Documentation and Records

  • Maintain calibration worksheets, instrument logbooks, and SST results.
  • QA review and approval required for all calibration and operation records.
  • Keep records for the defined retention period per company policy.

9. Troubleshooting & Preventive Checks

  • Check for leaks, unusual noise, or pressure spikes in the pump.
  • Inspect solvent lines and degasser for air bubbles.
  • Ensure needle wash and sample tray function properly.
  • Monitor detector lamp intensity and replace if degraded.
  • Clean column and prevent particulate buildup.
  • Verify software updates and backup schedules.

Prepared By: ____________________

Reviewed By: ____________________

Approved By: ____________________

Effective Date: ____________________

SOP No.: __________    Revision No.: _____

Comments

Post a Comment

Popular posts from this blog

-
⚠️SOP for Risk Management and Risk Assessment (FMEA, RPN, HAZOP, SWOT) This Standard Operating Procedure (SOP) describes a comprehensive risk management and risk assessment framework using FMEA, RPN, HAZOP, and SWOT methodologies. It supports pharmaceutical quality systems in accordance with ICH Q9 and ISO 31000 for effective identification, evaluation, mitigation, and monitoring of risks. 1. Introduction Risk Management is a structured approach to identify, assess, mitigate, and monitor risks in laboratory, R&D, QC, manufacturing, and quality systems. This SOP provides step-by-step instructions for multiple risk assessment techniques, including FMEA, RPN, HAZOP, and SWOT analysis, ensuring robust evaluation, prioritization, and mitigation of risks. 2. Purpose To establish a standardized framework for risk identification, analysis, evaluation, mitigation, monitoring, and continuous improvement using multiple validated risk assessment techniques, ens...
Read more

SOP for Operation and Calibration of UV-Visible Spectrophotometer

-
SOP for UV-Visible Spectrophotometer Operation & Calibration SOP for UV-Visible Spectrophotometer Operation & Calibration SOP Code: [Assign Code] Version: 1.0 Effective Date: [Insert Date] Review Date: [Insert Date] Prepared by: [Name / Designation] Approved by: [Name / Designation] 1. Purpose To provide detailed instructions for safe operation, calibration, and verification of UV-Visible spectrophotometers in compliance with IP, USP, BP, and EP standards to ensure accurate and reliable analytical results. 2. Scope This SOP applies to all analysts and QC personnel performing routine or validation analysis using UV-Visible spectrophotometers. 3. Responsibilities Role Responsibility Analyst / Technician Operate the instrument, prepare standards, perform calibration, and record results. QC / QA Officer Review and approve calibration logs, ensure compliance with pharmacopeial requirements. Maintenance Personnel Perform preventiv...
Read more

Operation and Calibration of Tap Density Apparatus

-
Operation and Calibration of Tap Density Apparatus 1. Purpose To establish a standardized and detailed procedure for the operation, calibration, and maintenance of the Tap Density Apparatus used for determination of bulk density and tapped density of powders, granules, and solid materials in accordance with pharmacopeial and GMP requirements. 2. Scope Quality Control laboratories Research and Development laboratories Production and in-process testing laboratories Any department performing bulk and tap density analysis 3. Definitions Bulk Density: Mass of powder divided by its untapped volume. Tapped Density: Mass of powder divided by the volume after mechanical tapping. Tap Density Apparatus: Instrument that mechanically taps a graduated cylinder to obtain reproducible tapped volume. Carr’s Index: Measure of compressibility calculated from bulk and tapped density. Hausner Ratio: Ratio of tapped density to bulk density. ...
Read more