[vijayak]

Hello all,
I am using a column ACE C18 4.6 X150mm 3µm. And i use water 0.1% TFA and acetonitrie as MP with in 200 injection the column gone bad and started giving tailling.  I had discussed with manufacturer and some people and they suggest <5µm columns not to be reversed while regeneration. And they say if regeneration done by reversing the column it Could well affect the pore size. Can any one suggest Is it really true? if yes what is the way to regenerae the column which have <5µm particle size and what may be the procedure.

[JGK]

Some modern HPLC columns do not have a truly symmetrical design which may lead to problems if they are reversed.

My copy of "Practical HPLC" (V. K Meyer) suggests the following for regenerating C18, C8, Phenyl and Nitrile columns:

Flow in the range 0.5 - 2 mL/min

75 mL water + 4 x 100 µL injections of DMSO during this cycle;
75 mL methanol;
75 mL Chloroform;

Water  0.1 M sulphuric acid  water (is another option).

[Chromguy]

Hi,

Reversing the flow on a well packed column should not damage the column.  It will have no effect on the pore size.  The washing procedure suggested is good but can you be more specific as to what you are injecting.  Some columns are adversely affected by exposure to acidic eluents.  TFA can sometimes strip bonded phase which will result in increased column activity and tailing for active compounds.  This also results in shorter retention times.

[vijayak]

Hi, Thank you very much for the suggestion. I inject the methanol layer and my sample is extract of fermentation broth and relatively my chromatogram is very crude. Yes i agree use of TFA will cause problem over the period infact i came accross the corrosion of the mixer(MCGV in agilent) in HPLC due to prolong use of TFA. but as a matter of fact in our company it is prefered one of the best Mass spec compactable ion pairing agent and  all our methods have TFA in the mobile phase.

Any suggestion which can replace TFA?