As a core instrument in fields such as biomedical testing, environmental pollutant analysis, and petrochemical component separation, the analytical liquid chromatography, with its convenient full‑touch screen and precise analytical capabilities, has become an essential tool in laboratories. However, during long‑term use, factors such as mobile phase preparation, sample handling, and equipment wear can lead to various faults, affecting detection efficiency and potentially causing data deviations. This article systematically breaks down six high‑frequency faults in analytical liquid chromatography, from cause analysis to practical step‑by‑step solutions, helping laboratory personnel quickly troubleshoot problems, ensure stable instrument operation, and improve the reliability of detection results.
I. What to Do When Analytical LC Column Backpressure Is Too High? (High‑Frequency Fault)
Excessive column backpressure is one of the most common problems in analytical LC. If not addressed promptly, it can damage the column and affect separation performance.
Cause analysis
Precipitation of buffer salts: Buffers such as phosphate and ammonium acetate, if not flushed in time or stored at low temperatures, can precipitate salts inside the column, blocking the packing pores and causing a sharp rise in backpressure.
Deposition of sample contaminants: Incomplete sample pre‑treatment (e.g., inadequate filtration or centrifugation) allows proteins, particulates, and other impurities to accumulate at the column inlet, gradually forming a contamination layer that obstructs mobile phase flow.
Practical solutions
For salt precipitation: Raise the column temperature to 40‑50 °C (stay within the column‘s tolerance to avoid heat damage). Flush forward with pure water at a low flow rate (0.2‑0.5 mL/min) for 30‑60 minutes. Once the backpressure drops significantly, reduce to room temperature and continue flushing with pure water for another hour. Finally, switch to pure methanol and flush for 30 minutes to regenerate the column.
For sample contamination: Back‑flush the column (reverse the inlet/outlet connections to prevent contaminants from entering deeper). Flush with pure water at 0.3 mL/min for 40 minutes, then with pure methanol for 30 minutes. Next, flush with a methanol‑isopropanol mixture (4:6, v/v) for 20 minutes (isopropanol dissolves strongly hydrophobic impurities), then sequentially with methanol and pure water for 20 minutes each. Restore forward connection and flush with methanol for 30 minutes before storing.
II. Bubbles Appearing in the Mobile Phase of an Analytical LC?
Bubbles in the mobile phase cause baseline drift, peak distortion, and in severe cases interrupt liquid flow, affecting detection continuity. This is often related to improper maintenance of the mobile phase filter.
Cause analysis
The mobile phase filter (typically 0.22 μm or 0.45 μm membrane) is often soaked in buffers such as ammonium acetate or potassium dihydrogen phosphate. If not cleaned promptly after experiments, microorganisms multiply in the buffer, forming fungal mats that clog the filter pores. This creates negative pressure during mobile phase flow, drawing in air and forming bubbles.
Practical solutions
Remove and clean the filter: Dismantle the filter from the flow path, immerse it in 5% nitric acid solution (nitric acid kills fungi and dissolves impurities), and sonicate for 15‑20 minutes (power 200‑300 W, taking care not to damage the membrane). For severe contamination, soak in 5% nitric acid for 12‑36 hours, shaking every 8 hours.
Restore flow path and degas: Rinse the filter repeatedly with pure water until no acid remains (check the rinsate with pH paper – neutral). Reinstall the filter. Run the instrument‘s “Purge” function at 3‑5 mL/min for 10‑15 minutes and observe whether bubbles disappear. Finally, open the purge valve and flush the filter with pure water at 1 mL/min for 1 hour to ensure unobstructed flow.
III. No Pressure Indication and No Liquid Flow in an Analytical LC? Check These Two Points
No pressure and no liquid flow constitute an urgent fault that directly interrupts experiments. Priority should be given to checking the pump body and sealing components.
Cause analysis
Worn pump seal: The pump seals (often made of fluororubber) are exposed to organic solvents (methanol, acetonitrile) and buffers for long periods, leading to ageing, deformation, or wear. This causes mobile phase leakage and failure to build pressure.
Large amount of air in the pump: Air can enter the pump when changing mobile phases without proper priming, when the mobile phase level is too low, or when the instrument has been idle for a long time. Air trapped in the pump chamber creates an “air lock” that prevents liquid delivery and pressure building.
Practical solutions
Worn seal replacement: Turn off the instrument, disassemble the pump head (refer to the instrument manual to avoid damaging precision parts), remove the old seal, wipe the pump head contact surface with a lint‑free cloth moistened with methanol, and install a new seal of the same model (ensure it is seated correctly without wrinkles). Reassemble and run the pump with pure methanol at 1 mL/min for 30 minutes, checking for leaks.
Pump degassing: Connect a 50 mL glass syringe (without needle, ensuring a tight seal) to the pump outlet. Start the pump at 0.5 mL/min and slowly pull the syringe plunger to withdraw air from the pump chamber. When no more bubbles appear in the syringe, stop the pump, remove the syringe, restart the pump, and flush the flow path with pure methanol for 20 minutes. Observe whether the pressure returns to normal.
IV. Large Pressure Fluctuations and Unstable Flow in an Analytical LC?
Pressure fluctuations and unstable flow lead to retention time drift and poor peak area repeatability. They are often related to system bubbles or check valve contamination.
Cause analysis
Air in the system: Insufficient degassing of the mobile phase (e.g., no online degasser, too short sonication time), loose flow path connections, or a low mobile phase level can allow air to enter the system continuously, causing pressure to fluctuate.
Contaminated or stuck check valve: The ruby ball and seat in the check valve control unidirectional flow. Fine particles or salt impurities trapped between them cause poor sealing, liquid backflow, and resultant pressure fluctuations and unstable flow.
Practical solutions
System degassing and inspection: Ensure the mobile phase has been sonicated for 20‑30 minutes (or the online degasser is on) and that the solvent bottle level is above 1/2. Check all flow path connections (e.g., tubing fittings, injector connections) and gently tighten with a wrench (avoid overtightening). Run the pump at 2 mL/min and observe the pressure curve. If fluctuations persist, proceed to the next step.
Check valve cleaning: Turn off the pump power, remove the check valves (note their inlet/outlet orientation and mark them), place them in a beaker of acetone (acetone dissolves most organic impurities and salts), and sonicate for 10‑15 minutes. Rinse thoroughly with pure methanol, air dry, and reinstall in the correct orientation. Restart the pump and run with pure methanol for 30 minutes, checking whether the pressure becomes stable.
V. Split Peaks in Analytical LC?
Split peaks are a common problem affecting detection accuracy, leading to incorrect qualitative identification and quantitative偏差. They are primarily related to the condition of the column.
Cause analysis
Severe column contamination: After many injections, strongly adsorbed impurities (e.g., pigments, macromolecular organics) that are not flushed away form a fixed contamination zone inside the column. Some components then partition between the contaminated and normal packing areas, causing peak splitting.
Column inlet bed collapse: Prolonged use or pressure shocks (e.g., sudden flow rate increase, excessive column backpressure) can loosen or collapse the packing at the column inlet. The resulting void creates a “dead volume”, causing peak splitting.
Practical solutions
Remediation of column contamination: For reversed‑phase columns (e.g., C18), flush with pure methanol for 30 minutes, then with methanol‑water (10:90) for 40 minutes, followed by methanol‑acetonitrile (50:50) for 30 minutes, and finally with pure methanol for 30 minutes. For severe contamination, add 0.1% formic acid to the flush (enhances solubility of impurities), but ensure the column is acid‑compatible.
Repair of inlet bed collapse: Work in a clean environment. Unscrew the column inlet nut. Gently wipe the inlet surface with a lint‑free swab to remove loose, contaminated packing. Prepare a paste of a small amount of the same type of column packing (same particle size) using methanol. Carefully drop the paste onto the depressed area, tamping it gently with a flat‑ended stainless steel rod until the packing is level with the original bed. After filling, flush the column inlet with pure methanol for 10 minutes, reinstall the inlet nut, and flush the column forward with pure methanol for 1 hour. Once peak shape recovers, the column can be used again.
VI. Poor Peak Area Repeatability in Analytical LC? Key Checkpoints
Poor peak area repeatability leads to unreliable quantitative results, undermining the credibility of experimental data. It is often related to injection system operation or component wear.
Cause analysis
Injector leakage: The injector seal (e.g., rotor seal) may become worn after long‑term use, or improper injection technique (e.g., too fast or forceful valve switching) can cause sample leakage during injection, resulting in inconsistent sample volumes actually entering the column.
Improper injection technique: The injection needle may not be fully inserted into the sample loop port (i.e., not seated properly), so not all the sample enters the loop. Alternatively, insufficient sample volume (e.g., only 10 μL loaded into a 20 μL loop without “overfilling”) leads to variability in the actual injected volume.
Practical solutions
Injector maintenance: Turn off the instrument, disassemble the rotor seal from the injector, clean the injector interior with methanol, and install a new seal of the same specification (ensure correct positioning to avoid misalignment). Reassemble, draw pure methanol with a microsyringe, inject into the injector, switch the valve to the “INJECT” position, and check for leaks. If no leakage, proceed to verification.
Standardised injection procedure: Ensure the needle is fully inserted into the injector port (a slight resistance is normal; do not force it). When using a sample loop, the sample volume should be 2‑3 times the loop volume (e.g., 40‑60 μL for a 20 μL loop) to ensure the loop is completely filled. After injecting the sample, switch the valve from “LOAD” to “INJECT” smoothly within 1 second to minimise sample diffusion. Also, avoid using excessively high sample concentrations (stay within the detector‘s linear range) to prevent adsorption inside the loop, which could affect repeatability.
The stable operation of an analytical liquid chromatograph depends on routine standardised procedures and regular maintenance. The six common faults described above can mostly be avoided through “prevention first + timely correction”. It is recommended that laboratories establish an instrument usage log, recording mobile phase composition, sample treatment methods, pressure changes, etc., for each experiment. After each run, flush the flow path and column promptly with appropriate solvents, and regularly inspect wear parts such as pump seals, check valves, and injector seals to extend the instrument‘s service life and ensure the accuracy and reliability of test data. If a fault cannot be resolved by the methods described, it is advisable to contact the instrument manufacturer’s professional service personnel to avoid secondary damage caused by disassembling precision components.
