As an efficient and precise “separation and analysis expert” in the laboratory, the high performance liquid chromatograph (HPLC) has become deeply integrated into many fields, including teaching and research, pharmaceutical development, environmental testing, food quality control, and chemical analysis. Whether you are a beginner new to the instrument or an experienced laboratory professional needing to standardise operating procedures, mastering a set of standard, meticulous methods is essential – this not only makes experimental results more accurate but also protects the long‑term stable operation of the instrument and extends its working life. Today, we will break down the complete HPLC workflow step by step, from pre‑operation preparation to shutdown and maintenance.
Pre‑operation Preparation: Good preparation makes the experiment run smoothly
Mobile phase preparation is fundamental to the experiment and directly affects subsequent analytical results. According to the experimental plan, prepare a mixed mobile phase with a methanol‑to‑water ratio of 8:2, and also have pure methanol ready. After preparation, filter the mobile phase through a membrane and then degas it by sonication. This avoids impurities and bubbles that could interfere with the detection. The prepared mobile phase is now ready for use.
Startup and System Equilibration: Adjust patiently to lay the foundation for detection
First, turn on the pump power and set the instrument pressure upper limit. For conventional HPLC, the pressure limit is typically set to 40 MPa – this is a key parameter for safe instrument operation.
Next, place the pump’s inlet filter into pure methanol, set the flow rate to 1 mL/min, and start the pump. Flush the system for 10‑20 minutes to clean the tubing and remove residual impurities. After flushing, switch the filter to the previously prepared methanol‑water mobile phase and let the pump continue running, allowing the system to gradually adapt to the mobile phase.
Then turn on the detector power, set the detection wavelength to 280 nm, and perform a zero adjustment to ensure the detector is in a stable initial state.
Workstation Connection and Baseline Stabilisation: Confirm signal connection and wait for optimal conditions
Start the chromatography workstation and check that the interface shows successful communication between the instrument and the workstation. Then carefully observe the baseline trend. Only when the baseline becomes stable, without obvious fluctuations, is the system ready for sample injection.
Sample Analysis Procedure: Operate precisely to capture every chromatographic peak
Before injection, reconfirm the baseline position. If the baseline has drifted, re‑zero if necessary to ensure an accurate detection starting point.
Set the injection valve to the “LOAD” position and inject the standard sample. Then quickly turn the injection valve to the “INJECT” position to avoid sample leakage or prolonged residence that could affect the results.
The workstation will now start data acquisition automatically. Simply wait until the chromatographic peaks appear completely on the interface, then click “Stop” to end acquisition.
Calibration Curve Establishment: Standardise parameters for more accurate quantification
First, set the quantification parameters: choose peak area quantification, external standard method, and 1‑point calibration. These parameters should match the quantitative needs of the experiment.
Then fill in the component table – for example, when analysing capsaicin, accurately enter the component name “capsaicin”, the corresponding retention time of 5.2 minutes, and the standard concentration of 100%. Every piece of information must be correct; otherwise, the calibration result will be affected.
Finally, define the calibration curve: locate the corresponding spectrum file and perform recalibration. The system will automatically generate a standard curve, which will serve as an important basis for subsequent quantitative sample analysis.
Sample Detection and Data Analysis: Batch testing can be both efficient and accurate
Repeat the injection steps for the test samples: inject the sample, wait for complete acquisition of the chromatographic peaks, and click “Stop”. To view the analysis results, simply click the “Report” button on the screen, and all the detection data will be clearly displayed. If multiple samples need to be analysed, there is no need to repeat complex settings – just perform the injection steps carefully each time.
Historical Data Retrieval: Easy access for convenient data management
If you need to review previous experimental data, go to the “Reprocessing” interface of the workstation, find and open the desired historical data file, and click “Recalculate”. A full screen report will appear immediately, making it easy to trace the experimental process and verify results.
Standard Shutdown Procedure: Careful finishing protects instrument health
After the experiment, the shutdown steps must also be performed carefully. First, turn off the detector power, then stop the pump. Next, flush the system with pure methanol for 10‑20 minutes to thoroughly clean residual mobile phase from the tubing. After the system pressure stabilises, turn off the pump power. Finally, clean the injection valve thoroughly with pure methanol or pure water, readying it for the next use.
Precautions: These details determine the life of both the experiment and the instrument
Before each experiment, always confirm that the mobile phase has been filtered and degassed – this is the foundation for avoiding tubing blockages and ensuring detection accuracy.
When operating the instrument, strictly follow the set pressure upper limit (e.g., 40 MPa) – do not operate above the pressure limit to prevent damage to the pump and tubing.
Replace the mobile phase regularly to keep the system clean and avoid accumulation of residues that could affect instrument performance.
After each use, record the usage details to establish a complete instrument log, facilitating future maintenance and problem tracing.
Mastering this standard operating procedure will not only allow you to fully utilise the analytical performance of the HPLC and obtain accurate and reliable experimental data, but also extend the instrument’s service life through proper operation. Remember, the long‑term stable operation of the instrument depends on every careful operation and dedicated maintenance.
