When running experiments, the pressure of your HPLC system is like its "heartbeat" – when it’s stable, you feel at ease; once it misbehaves, either your data drifts or the instrument gets cranky. It can be frustrating to see the pressure either spike sharply or fluctuate wildly upon startup. This guide explains how to identify the cause from the symptom, how to resolve it step by step, and how to maintain your instrument to prevent future issues – all from Elite, helping you avoid detours and keep your instrument working reliably.
A normal instrument "heartbeat" is typically between 5–20 MPa (a new column may run slightly higher, then stabilises with use). Judgement depends on both the pressure value and its stability.
Pressure too high
Obvious sign: Shortly after startup, pressure soars more than 20% above normal, sometimes even exceeding 40 MPa and triggering a red alarm.
Worrying consequences: The pump may be overstrained, column packing might be crushed, and tubing could burst.
Pressure too low
Clearly wrong: Pressure is significantly lower than usual (e.g., drops below 3 MPa) and continues falling, never reaching the required level.
Unreliable data: Insufficient mobile phase flow leads to shifting retention times, fluctuating peak areas, and irreproducible experiments no matter how many times you repeat them.
Pressure fluctuating up and down
Annoying to watch: The pressure jumps around within a short time (amplitude > 1 MPa), and the baseline becomes noisy and drifts.
Unreliable quantitation: Calculated results are far off, and instrument parts suffer increased wear, leading to premature failure.
Core logic: Pressure too high usually means increased system resistance.
Troubleshoot stepwise: Mobile phase → Column → Tubing → Instrument parameters.
Mobile phase issues
Cause 1: High viscosity (e.g., pure aqueous phase, high proportion of buffer salts) – especially at low temperatures. In winter, when the lab is cold, the mobile phase becomes as thick as a slow‑motion syrup, and pressure naturally climbs.
Solution: Increase the column temperature (e.g., from 30 °C to 40 °C) or raise the organic solvent proportion (e.g., from 5% methanol to 20%) to make the mobile phase “lighter”. Of course, check whether regulations allow such changes.
Cause 2: Buffer salt precipitation (e.g., phosphate salts crystallise in high‑organic mobile phase). If you add organic solvent first and then salt when preparing the mobile phase, the salt may settle at the bottom like sand in syrup, then enter the instrument and cause blockages.
Solution: Stop the pump immediately, flush slowly with pure water (0.2 mL/min for 30 min), then gradually switch back to the original mobile phase.
Prevention: Always dissolve the salt completely in water first, then slowly add the organic solvent while stirring thoroughly.
Blockage issues
Cause 1: Blocked column/guard column (common) → If pressure drops after removing the guard column, the guard column is almost certainly blocked. Impurities in samples slowly accumulate like sediment clogging a pipe, and pressure keeps rising.
Solution:
Guard column: replace directly (it acts like a filter – it is advisable to replace it every 100 injections).
Analytical column: if the column allows back‑flushing (e.g., C18 columns usually do), reverse‑flush with 50% methanol‑water at 0.5 mL/min for 2 hours. If that fails to clear the blockage, replace the column.
Cause 2: Blocked in‑line filter or check valve (particles or microbial contamination). After prolonged use, the filter may accumulate black debris, and the check valve may be coated with residues – just like scale blocking a water pipe.
Solution:
In‑line filter: remove and sonicate in filtered methanol for 10 minutes, or replace with a new one (keeping spares is recommended).
Check valve: disassemble and sonicate in isopropanol for 15 minutes (to remove oils), then in water for 5 minutes (to wash away salts) before reinstalling.
Incorrect instrument parameters
Flow rate set too high (e.g., mistakenly set to 2 mL/min, far above the column’s tolerance) – like forcing a large flow through a narrow pipe, pressure will certainly exceed the limit. Return to the flow rate prescribed by the method (e.g., 1 mL/min).
Column oven not set to temperature (low temperature increases mobile phase viscosity) – when it is cold, the mobile phase thickens and pressure rises, just like engine oil in winter causing a high idle speed. Allow the column to reach the set temperature (e.g., 30 °C) before starting the run; pressure will normalise.
Core logic: Pressure too low usually means insufficient mobile phase delivery. Troubleshoot in order: Leak → Mobile phase → Pump components.
Troubleshooting sequence: First check obvious spots like tubing connections, injection valve, and pump head for wetness, then examine less accessible parts like the flow cell and check valve. A loose connection is like an un‑tightened tap – liquid leaks out and pressure never builds up.
Solutions:
Loose fittings: gently tighten with a wrench (PEEK fittings are delicate – do not overtighten to avoid stripping the thread).
Worn pump seals: replace the seal (these are like rubber rings that age over time – it is recommended to replace them every 6 months to prevent leakage and corrosion of the pump head).
Leaking injection valve: replace the rotor seal (the seal wears after many injections – watch for dripping liquid during injection).
Solvent bottle level too low (the inlet tube sucks air) – when the mobile phase runs out, the tube draws air, just like a straw above the liquid – no suction, no pressure. Refill the bottle and ensure the tube is fully immersed.
Incorrect mobile phase composition (e.g., too much organic solvent, causing a sharp drop in viscosity) – too much organic phase makes the mobile phase too “thin”, as if switching from honey to water – pressure naturally falls. Prepare a fresh batch according to the method.
Blocked inlet filter (clogged by particles or microbial growth) – after long immersion, the filter may be covered with flocculent material, like a clogged aquarium filter, unable to draw liquid. Remove the filter, soak it in 10% dilute nitric acid for 30 minutes, then rinse thoroughly with water.
Check valve malfunction (cannot draw liquid properly) – a contaminated check valve acts like a failed one‑way valve, unable to pull in liquid. Clean it as described under “Pressure too high”; if cleaning does not work, replace the valve.
Proportional valve failure (gradient mixing abnormal, leading to insufficient actual flow) – test with pure methanol; if pressure still does not build up, the proportional valve may be defective and require service by an engineer.
Core logic: Pressure fluctuation usually means unstable mobile phase delivery. Troubleshoot in order: Bubbles → Pump components → Environmental interference.
Cause: Mobile phase not degassed (especially solvent mixtures that tend to form bubbles), or air has entered the pump head. Bubbles in the mobile phase act like air in a water pipe – they bounce around and make the pressure swing.
Solution:
Mobile phase: degas by sonication for 20 minutes in advance, or keep the online degasser running (ensure its vacuum is ≥ 0.2 MPa).
Purge the pump head: press the “purge” button on the instrument, or use a syringe to slowly withdraw air from the purge port until no bubbles are visible.
Worn seals / plunger (causes uneven aspiration by the pump) – worn seals are like leaky pistons – aspiration becomes erratic and pressure fluctuates. Replace the seal (if you also see leakage, the seal is certainly the culprit). In severe cases, the plunger may also need replacement.
Foreign matter inside the pump chamber (e.g., small particles causing sticking) – tiny debris inside the pump chamber is like sand in a bearing – the motion becomes rough. Flush with 50% methanol‑water at 0.5 mL/min for 30 minutes to wash the impurities out.
Unstable voltage (causes pump current to fluctuate) – in a lab with unstable voltage, the pump runs unevenly like an engine gasping for power. Connect a voltage stabiliser (ensure 220 V ± 5 %) to keep power steady.
Ambient temperature fluctuations (e.g., air conditioner blowing directly on the instrument) – when the air conditioner blows hot or cold air onto the instrument, the mobile phase viscosity changes and pressure fluctuates. Keep the lab temperature stable (20–25 °C, with variation no more than 2 °C/h) so the instrument can work peacefully.
