20 ”Unwritten Rules” for Liquid Chromatography Instruments
**Author:Orginal from Ms Xiaoyue Lü | HAMAG
Liquid chromatography instruments may look robust, but they are actually quite delicate. Many faults are not caused by ”wearing out”, but by “misuse”—certain operational habits that we take for granted are quietly shortening the instrument’s lifespan. Each of the following 20 ”unwritten rules”represents a lesson learnt the hard way by those who came before us.
01-Mobile Phase
1. The mobile phase must be filtered—using a 0.22 μm or 0.45 μm filter membrane; failing to do so is tantamount to wearing out the pump.
2. Measure the pH in the aqueous phase—adjust it before adding the organic phase, as the organic phase will interfere with the electrode reading.
3. Use buffer salts within the correct pH range — each salt is only effective within a specific pH range; using it incorrectly is a waste of time.
4. Ultrasonic degassing alone is not sufficient — use it in conjunction with a vacuum degasser, otherwise the bubbles will make you question your sanity.
5. Do not start gradient elution at 0% organic phase — the risk of salt precipitation and bubbles is extremely high; take particular care with phosphates. (Applies only to salt-containing systems.)
02-Pumps and Valves
6. Never allow the mobile phase to run dry—running the pump dry causes direct wear to the plunger and seal rings, and pump repairs are expensive.
7. Rinse immediately after using a saline mobile phase—first with pure water, then with methanol; salt crystals will wear down the pump like a scouring pad.
8. Maintain a gentle flow rate and perform gentle injections—sudden changes shock the column bed, causing irreversible damage to the packing.
03-Chromatography Columns
9. Before using a new column, check whether the storage solution and the mobile phase are miscible—if they are not, discard the column immediately.
10. The column must be flushed with a transition phase both before and after using salt—flush with 10% methanol/water for 10 column volumes; skipping this step will ruin the column.
11. Do not backflush the column indiscriminately—unless explicitly permitted in the manual, this will cause a sharp decline in column efficiency.
12. A guard column must be fitted — a small investment that saves a lot in the long run; replace it when it becomes contaminated.
13. Columns must not be allowed to dry out — for columns not in use, screw the caps tightly onto both ends; if they dry out, they will become clogged.
04-Samples
14. Samples must be filtered—filter all samples containing particles; use a 0.22 μm hydrophilic PTFE filter membrane for polysaccharides and proteins.
15. The sample solvent must be compatible with the mobile phase—excessive elution capacity will cause solvent effects and result in poor peak shape.
05-Methodology
16. Always run a blank before gradient elution—otherwise, impurity peaks will masquerade as sample peaks, rendering all data useless.
17. Assess impurity interference in quantitation methods too—don’t take anything for granted; it will come back to haunt you during stability testing.
18. When transferring methods, be mindful of differences in instrument dead volume—the same method may yield completely different results on different instruments.
06-Daily Habits
19. Use your senses to ‘check’ the instrument daily — listen for unusual sounds, watch for bubbles, and feel for leaks; early detection means early resolution.
20. Use only chromatographic-grade reagents — impurities in analytical-grade reagents will drive you to despair as you chase down phantom peaks.
Note:90 per cent of HPLC faults are caused by operational habits; prevention is always cheaper than repairs. :)