Standard Operating Procedure (SOP): Operation of High-Performance Liquid Chromatography (HPLC)
Purpose:
The purpose of this Standard Operating Procedure (SOP) is to provide detailed instructions for the proper operation of the High-Performance Liquid Chromatography (HPLC) machine. This SOP ensures accurate and reliable analysis of samples using HPLC techniques.
Scope:
This SOP applies to all personnel involved in operating and maintaining the HPLC machine in a pharmaceutical or quality control laboratory.
Responsibility:
Laboratory personnel: Responsible for performing HPLC analysis as per this SOP.
Quality Assurance (QA): Responsible for reviewing and approving this SOP.
Procedure:
4.1. Equipment and Materials:
HPLC machine (including pumps, injector, column, detector, and data acquisition system)
HPLC columns (appropriate for the analysis)
Mobile phase(s) and solvents (as per method requirements)
Sample vials and caps
Syringes or auto-sampler (if applicable)
Standard solutions or reference materials (for calibration and system suitability)
4.2. Pre-Operational Preparations:
4.2.1. Ensure that the HPLC machine is clean and free from any residue or contaminants from previous analyses.
4.2.2. Familiarize yourself with the specific operating instructions and technical specifications provided by the manufacturer for the particular model of the HPLC machine being used.
4.2.3. Gather all necessary equipment and materials.
4.3. HPLC Method:
4.3.1. Obtain the HPLC method to be used for the analysis from a validated source, such as a pharmacopeia monograph, internal method, or method development protocol.
4.3.2. Review the method for specific details regarding column, mobile phase, flow rate, injection volume, gradient or isocratic conditions, and detector settings.
4.4. System Preparation:
4.4.1. Prime the HPLC system by flushing the solvent(s) through the system to remove air bubbles and stabilize the baseline.
4.4.2. Prepare the mobile phase(s) and ensure they are degassed (if required) to minimize baseline noise and maintain system performance.
4.5. Calibration and System Suitability:
4.5.1. Perform the necessary calibration of the HPLC system as per the method requirements, including detector wavelength verification, flow rate accuracy, and system suitability tests.
4.5.2. Use appropriate standard solutions or reference materials for calibration and system suitability.
4.6. Sample Preparation and Injection:
4.6.1. Prepare the sample solution according to the method requirements, ensuring accurate concentration and appropriate solvent compatibility.
4.6.2. Filter the sample solution using a suitable filter if necessary to remove particulate matter.
4.6.3. Load the sample into the sample vials and cap them securely.
4.6.4. Set up the sample injection parameters, including injection volume and injection sequence, either manually or through an auto-sampler system.
4.7. Analysis Run:
4.7.1. Set the HPLC method parameters on the data acquisition system or control software, including column temperature (if applicable), gradient or isocratic conditions, and detector settings.
4.7.2. Start the data acquisition system or initiate the analysis run as per the method instructions.
4.7.3. Monitor the chromatogram during the analysis run for any abnormalities or unexpected results.
4.8. Data Analysis and Interpretation:
4.8.1. Analyze the obtained chromatogram and interpret the data according to the method requirements and acceptance criteria.
4.8.2. Document the results obtained, including peak identification, retention times, peak areas or heights, and any relevant calculations or data analysis.
4.9. Post-Analysis Procedures:
4.9.1. Clean the HPLC system thoroughly after each analysis run, following the cleaning procedures specified in the standard operating procedures.
4.9.2. Properly dispose of any waste generated during the analysis, adhering to appropriate waste disposal guidelines.
4.9.3. Store the remaining samples and reference materials according to the recommended storage conditions to maintain their integrity.
Records:
5.1. Maintain a record of the calibration process, including the date, equipment used, and results obtained.
5.2. Document the details of each analysis conducted, including the date, sample identification, method used, system suitability results, and analytical results obtained.
5.3. Retain the records in a secure and organized manner as per the laboratory’s document control procedures.
Abbreviations: (if applicable)
HPLC: High-Performance Liquid Chromatography
QA: Quality Assurance
References: (if applicable)
Relevant pharmacopeia monographs or internal method protocols for HPLC analysis.
Manufacturer’s operating instructions and technical specifications for the specific model of the HPLC machine being used.
Revision History:
Version 1.0: [Date of initial creation]
Provides the initial version of the SOP for the operation of the HPLC machine.
Version 1.1: [Date of revision]
[Specify the changes made during the revision, such as updates to the procedure, clarification of steps, or addition of new sections.]
Note: Any future revisions to this SOP should be documented and approved by the appropriate personnel before implementation.