The operating details may vary slightly between different brands and models of HPLC, but the core ideas and operating logic are the same. Remember the old rule: “Flush before starting, clean thoroughly after finishing.”
I. Preparation Before Analysis
Mobile Phase Preparation
Solvent selection: Use HPLC‑grade or purer solvents; otherwise impurities may cause baseline disturbances.
Water purity: Use 18.2 MΩ·cm ultrapure water, preferably freshly prepared, to avoid microbial growth that could damage the column and affect detection.
Filtration: All mobile phases must be vacuum‑filtered through a 0.45 μm or 0.22 μm membrane before use to remove fine particles; otherwise the system or column may become blocked.
Degassing: Bubbles in the mobile phase must be removed, otherwise they can cause pressure fluctuations and baseline disturbances in the pump or detector. Common methods include online degassing, sonication, helium sparging, and vacuum filtration.
Sample Preparation
The sample must be completely dissolved; the resulting solution should be clear and free of solid particles.
It is recommended to dissolve the sample in a solvent that matches the initial mobile phase composition to avoid solvent effects that can distort peak shapes.
Special reminder: Before injection, the sample solution must be filtered through a 0.22 μm syringe filter.
Column Check
Confirm that the column type (C18, C8, amino column, etc.) matches the analytical method and the sample properties.
Check the column performance certificate; the column efficiency and pressure should be within acceptable ranges.
Check Laboratory Environment
Ensure sufficient space in the waste bottle, and that the power supply and any required gases are functioning properly.
II. Starting Up, Flushing, and Equilibrating the System (“Warm‑up”)
Correct Start‑up Sequence
Turn on the computer → turn on the instrument modules (power on) → launch the workstation software.
This ensures the software correctly recognises and connects to each module, reducing errors.
Install Column and Flush the System
Connect the column into the flow path, paying attention that the arrow on the column label aligns with the mobile phase flow direction.
Key step: Start the pump at a low flow rate (e.g., 0.2–0.5 mL/min) using the mobile phase ratio required for the method, then gradually increase to the working flow rate. This avoids high‑pressure shock that could damage the column and system.
Check all connections for leaks.
System Equilibration
Maintain the working flow rate and allow the mobile phase to continuously flush the column and the entire flow path until:
Baseline stable: The signal line on the workstation is straight, with no drift or spikes.
Pressure stable: System pressure fluctuations are generally less than ±1%.
Equilibration usually takes 10–30 minutes; gradient methods may take longer.
III. Method Setting and Sample Analysis
Create / Recall Method
Set the parameters accurately in the chromatography software, including flow rate, mobile phase ratio, column oven temperature, detection wavelength, run time, injection volume, etc.
Observe Baseline
Confirm that the baseline is stable before preparing for injection.
Injection Operation (for manual injection without an injection‑triggered start)
Using a suitable syringe, rinse it several times with the sample solution, then draw a volume slightly larger than the injection volume.
Turn the injection valve to the “Load” position. Fully insert the needle into the port, quickly inject the sample, and then withdraw the needle.
Immediately turn the valve to the “Inject” position, and simultaneously click “Start” on the workstation to begin data acquisition.
Note: The action must be fast and smooth; do not pause too long between “Load” and “Inject”.
IV. Shutdown and Maintenance (“Proper Finishing Work”)
Flush the System (important step)
After the analysis is complete, do not stop the pump directly.
Choose an appropriate solvent to thoroughly flush the system, depending on the type of mobile phase used.
If a buffer salt was used: First flush with a high‑water‑content mobile phase (e.g., 90% water) for 30–60 minutes to remove salt, then flush with a high‑organic‑phase mobile phase (e.g., 90% methanol or acetonitrile) for 30–60 minutes to replace the water, preventing microbial growth or salt precipitation.
If no buffer salt was used: Flush directly with a high organic phase (e.g., 90% methanol or acetonitrile) for at least 30 minutes.
Correct Shutdown Sequence
Stop the pump → turn off the detector lamp (especially the UV lamp, to prolong its life) → close the workstation software → turn off instrument power → finally turn off the computer.
Column Storage
Remove the column, cap both ends, and store according to the manufacturer’s instructions.
Log Book Entry
Record the instrument usage details, including the user’s name, sample, method, instrument status, etc., for future reference.
