Cleaning a reverse-phase HPLC column is easier than you might think.
With just two steps—water first, then organic solvent—you can flush out salts, buffers, and sticky residues that drag down your separations. Follow this recipe, and your column often feels like new.
A: Flush with a water-rich mobile phase (90/10 water/organic) at 60 °C and 1 mL/min for one hour to remove salts and buffers. Then run 100% methanol or acetonitrile under the same conditions for one hour to strip away non-polar residues.
Why You Need to Clean a Reverse-Phase HPLC Column
Think of your HPLC column like a coffee filter. Run enough samples, and tiny residues build up inside. Salts from buffers lodge in the pores, and non-polar gunk—like oils from coffee, plasma, or soil extracts—slowly coat the stationary phase. If left unchecked, efficiency drops and peaks broaden.
👉 For a refresher on why separation depends so much on polarity, check out Dr. Polite’s Kool-Aid analogy in HPLC Demystified Part 1 .
Step 1: Flush with Water to Remove Salts and Buffers
- Mobile phase: 90% water / 10% methanol (or acetonitrile)
- Flow rate: 1 mL/min (for 4.6 mm ID columns)
- Temperature: 60 °C
- Time: 1 hour
Water is a weak solvent for most analytes, but it’s unbeatable for dissolving salts and polar buffer components. Running hot water-rich mobile phase through the column clears out anything polar that’s been left behind.
Step 2: Use 100% Organic Solvent for Non-Polar Contaminants
- Mobile phase: 100% methanol or acetonitrile
- Temperature: 60 °C
- Time: 1 hour
After the polar cleanup, switch to pure organic solvent. This rinse pulls off non-polar residues—like oils, fats, or other hydrophobic material—that water alone can’t touch. When both steps are complete, most columns return to peak performance.
👉 For more detail on how to deal with a dirty or clogged frit, check out Dr. Polite’s How to Clean a C18 Column video.
Quick Cleaning Protocol When You’re Short on Time
Need to rescue a column mid-day? Try the “express wash”:
- 10 minutes at high-water mix
- 20 minutes at 100% organic
It won’t be as thorough as the full two-hour protocol, but it clears most buildup so you can keep running samples.
What Cleaning Can and Can’t Fix in HPLC Columns
Cleaning works wonders for chemical contamination, but it can’t fix physical damage. If the packing bed has collapsed, if voids have formed, or if silica has been compromised, the column is beyond repair.
Also note:
- Normal-phase columns are cleaned with non-polar solvents like hexane.
- Ion-exchange columns often require high-salt washes.
Each column chemistry has its own “recipe.”
Practical Tips to Keep Your Column Performing Like New
- Flush with water-rich solvent after buffer-heavy runs.
- Use organic rinses after oily or hydrophobic samples.
- Avoid exceeding 60 °C unless your column manufacturer specifies otherwise.
- Don’t try to “fix” a physically damaged column—it’s done.
👉 Want more guidance on column care? Dr. Polite also explains how to test an HPLC column to check efficiency and decide whether cleaning helped.
