Chromatography Forum

iam working on reverse phase C18 column with mobile phase
Methanol:Sodium lauryl sulphate pH=3 (65:35)+ 0.1% triethylamine.
My work was runnig well, but now there is no good peaks appear, and I wash my column for several days and the peaks are widened
Can You advise me with a good wash To regenerate my column and get sharp peaks again

There is usually a method for column cleaning in the small manual supplied with the column.

If you can not find a suitable method, come back and I will try to help!

really, i tried too much and peak fronting and broadening are bit reduced, but there is still slight peak fronting and broadening and i cannot take these peaks in my results...\
can you help me with a suitable procedure for cleaning my column?

For cleaning reversed-phase column you can try following procedure:
a) mobile ohase without buffer
b) methanol/acetonitrile (50+50)
c) acetonitrile/2-propanol (75+25)
d) 2-propanol
e) dichlormethane
f) 2-propanol
g) methanol

Use 10 - 20 column volumes for each solvent.
In case of metal contamination you can use flush with aqueous 0,05 M EDTA followed by water. Then go on from pont a)
I don´t known if it is enough performance.

There have been other threads about column regeneration, but I would like to make a couple of points:

1. There is no "universal" regeneration technique. The idea is to wash the column with something which will dissolve "garbage" precipitated or adsorbed on the column. You have to have some idea of what is contaminating the column (what works for salts will not work for fats or proteins).

2. If the problem is caused by loss of bonded phase or by collapse of the packing bed (development of a void at the head of the column), then no amount of "regeneration" will help. No column lasts forever.

We have found a lot of our columns "die" by generating a void in the front of the column. Often we have regenerated by topping them off with a slurry of an appropriate packing. Just take off the front nut and filter and take a look. At least you might determine the mode of failure..

Depends on how much you value your time..

James makes a very valid point. Most businesses are very conscious of how much money they spend these days but very few remember to factor in the time taken to solve these types of problems. Even for the lowest paid science jobs (in Europe at least) its not worth spending more than half a day trying to save a column its simply cheaper to throw it out and fit a new one.
I actually saw a presentation yesterday where the company employed someone whose main job was to replace columns. They had 30 systems each with 8 columns and they changed the columns every day. It simply wasnt worth waiting for a column to fail and the associated time taken to re-run samples. This may be an extreme example from a CRO running 24/7 but there is a lesson there for everyone to consider.

absolutely, I don't have the time to be trying to salvage columns that are failing. Replacement is the quickest way to fix a deteriorating column.

absolutely, I don't have the time to be trying to salvage columns that are failing. Replacement is the quickest way to fix a deteriorating column.

Quickest but more expensive if the column is failed in a few injections.

well thats pushing it a little. I have never had one go in that kind of time frame!

As they say in US automobile advertising claims "your mileage may vary". Each person/lab has to look at the relative cost of time vs. replacement.

Hi guys,

Who says the problem is the column? Fronting is usually due to the strong solvent effect or partly precipitation. By the way, void in the front of the column, shows as tailing or peak- splitting.
In any case I’ll change the column with a corresponding one, to see if it eliminates the problem.
If it doesn’t help I’ll change it back and then concentrate my efforts on the mobile phase. That is, if I had the problem. Do not start with throwing things away, only to find out, that the problem was something else.

B.R. Danko