Chromatography Forum

Hi to all,

Here i want to regenerate the column, which is having normal pressure while running the mobile phase but only thing is peak shape is not good, if i use new column peak shape is good and sharp.

So some of my collegues said that regenerate the column even though column pressure is not exceeded.

My column is 250X 4.6 mm length, C18, 5 micron column. Can anybody help me?

Regeneration and cleaning steps depend on the polarity of the impurity. Flow rate show be 25 - 50% of normal working flow.

POLAR IMPURITIES
Rinse out buffer solutions with 80 - 20 water: organic solvent. 20 column volumes.
Rinse with a mixture of high concentration of organic solvent 70:30 to 90:10 methanol: water or acetonitrile:water for at least 10 column volumes.
Most polar impurities are eluted when the buffer is rinsed out.

NONPOLAR IMPURITIES
Rinse column with a high water water ratio in order to remove buffer. NOTE do not go over 90% water in order to prevent phase collapse.

Rinse column with 100% methanol to remove polar compounds organic compounds

Rinse the column with 100% acetonitrile to remove medium polar organic compounds (temperature can be increrased to 40deg C)

Rinse column with THF to remove nonpolar organic compounds

All rinsing should be at least 10 column volumes

This and other helpful hints are found in Macherey-Nagel useful APPLICATION GUIDE book. For a FREE book go to www.mn-net.com

We often have problems with the packing on the front of our columns cavitating. We open the column, slurry some C18 stationary phase, and "top-off" the column to remove the void. Usually brings the column performance close to its original performance.

Regenration of RP Packings

Flush column with given sequence of solvents and in bracket column volume
WATER (20)
ACETONITRILE (20)
ISOPROPANOL(5)
HEPTANE (20)
ISOPROPANOL (5)
ACETONITRILE (20)