restoring my column effeciency

[fatma elzahraa]

I have a Spheri-5 PR-18, particle size is 5u, length is 220 mm, internal diameter is 4.5 mm.
This column is for our faculty researchers' work, i.e. many people work on the same column. First, this column gives excellent results with aal who wok on it. MPs usually consist of buffer (pH range 2.2-7) or water, and acetonitrile or methanol. This column was washed very well after everyday work.
Nearly after 5 months of work ( not daily...), There was a researcher used 0.1% triehylamine in buffer and acetonitrile as a mobile phase for two or three non successive times and then I made him stop this work to prevent near column damage, and I washed the column well for 12 hours with filtered water, acetonitrile, and methanol in a gradient system.
I noticed that some researchrs work continued well and others not.
One of those whose work was disturbed with splitting and reduced resolution, is working with buffer and acetonitrile as a MP but the injected samples are coloured ( containing yellow and azodye)
After a short time, Other researchers suffer from peak splitting and ghost peaks in their chromatograms even after good wash. and the injected sample chromatogram has different peak shapes when injected several times.....
Plz, I need advice and help to make this column work well again...and If you have a working regeneration system...plz tell me...

[sfe-co2]

Even a well-packed column needs careful handling and maintenance. And herein lies one problem; with so many researchers routinely sharing one column, it means each researcher must look after the column while in his/her custody. This means minimal physical and pressue shock to the column; avoid if at all possible.

We used a lot of buffers here, but would wash the column with 10/90 H2O/MeOH, and then 90/10 H2O/MeOH after each analysis. We slow the flow rate down to 0.5 mL/min for the washing cycles. This regime works fine for us most of the times, however at times, I would need to reverse flush the column using the 90/10 H2O/MeOH mobile phase overnight at about 0.2 mL/min.

If you are consistently seeing split peaks (i.e., all peaks in the same chromatogram are split), then I think the column had developed a void at the inlet end.

Opinions from the others?

[tom jupille]

As sfe-co2 implied, the column may be irreparably damaged. There is no 100% certain column regeneration technique.

If the column has been exposed to high pH and some of the silica matrix has dissolved, the column is dead. If the column is contaminated with junk from samples, it must be flushed with something that is a good solvent for that particular flavor of junk. The problem is that a good solvent for lipids (e.g., methylene chloride) is not good for proteins (e.g., iPrOH would be better) or salts (e.g., water with perhaps a smidgen of methanol). You have to make a reasonable judgement as to the nature of the contamination.

Short-term, try running the column in the reverse-flow direction. If the peak splitting is caused by particulate contamination, this may help. If the peaks are still split, you might try to open the inlet of the column and repack any void you find with packing material taken from the outlet of an old column. Be warned this is time-consuming and is not guaranteed to fix the problem.

Long-term, I would suggest that your columns always be run with appropriate guard columns or cartidges, and that each researcher have his/her own guard, for which they are responsible. That way if they foul something up, it will probably their cartridge.

[fatma elzahraa]

thanx alot.....but you give me a washing procedure after each analysis not a suitable regeneration method....
I read on the column pamphlet that I can use dioxane, tetrahydrofuran or acetonitrile to remove contaminants.... but I am afraid of using THF as it sometimes undergoes polymerization....
If hot water helps in column cleaning....as maxmimum temperature allowed is 70....
Due to the low fund here, the guard can be used by more than one researcher......do you have a good way to clean it well....as I usually sonicate it with MeOH or ACN or isopropanol.....
N.B. there is no possibility of replacing this column in the recent time.....so plz help me to restore at least 85% of the column efficiency as researchers cannot work on this column in such a state....
Best regards

[sfe-co2]

Hi Fatma,

As Tom mentioned above, the regeneration process is almost never certain. So is changing the inlet frit and filling in the void with more packing material. Great care needs to be observed here, as Tom indicated.

Using different solvents as suggested in the column pamphlet, I think it is wise to first know the history of the column. What kind of compounds and solvents have been through that column, etc.? Perhaps a mixture of H2O/Acetonitrile might do the trick.....that is, if the column is not already "dead" in the first place.

Also, during each analysis, make sure the sample solvent is not stronger than your mobile phase. This sort of incompatibility may give rise to different undesirable phenomena, including peak splitting.

I'm sorry to hear of your financial constraints (and perhaps desperation). Apart from what Tom and myself have mentioned, maybe it's time to call a local sales representative for further help/advice. I find these reps normally very kind and willing to help.

Good luck.

[Uwe Neue]

Columns do have a limited lifetime. How limited it is, depends on the details of the use, and the level of protection. As mentioned above, I also recommend guard columns. To throw away a guard column is much cheaper than to throw away an analytical column.

You mentioned that one analyst was running the column with triethyl amine in buffer. The use of triethylamine in a true buffer is not a problem, but you need to check the final pH of the mobile phase. If the pH was alkaline, the subsequent difficulties that other researchers have experienced is due to a partial dissolution of the silica, and subsequent column voiding. If this is the case, there is no way to recover the complete column performance as you had for a brandnew column, but you can potentially recover some additional life by adding some packing material manually to the inlet of the column (where I expect the void to be). You can use the packing from a guard column to fill up the space. You won't get the performance that you used to get, but hopefully the double peak will go away.

If you open the column and don't see an empty space, the problem is not related to column voiding. Close it again and continue.

[HW Mueller]

Also, there are guard columns on the market (see Upchurch) which you can easily fill yourself if you have bulk stationary phase (or one that is close to your stationary phase).
Without any more information it is about impossible to give advice byond that which you got already.

[fatma elzahraa]

Thanx alot for ur answer.....
The pH is adjusted to 3-6 after TEA is added to buffer.....so I do not mean the pH but I mean that TEA trapped in the column and reduce its performance...how to remove it well....?

[fatma elzahraa]

Generally....I need to regenerate my column....do you have a good workin procedure...?
N.B. My columns cannot be re packed.....

[Mark Tracy]

A 30 minute wash with 30:69:1 MeOH:H2O:HOAc will remove TEA. If that does not restore the column, then TEA residue is not the problem.

[ym3142]

as a summary, two things you can try to reclaim your column:
1) wash your column;
2) wash your column reversely;
3) change the frit if dirty;
4) fill the void if present.
Find a good washing procedure from the manufacture or internet. Good Luck;
If all fails, you need a fund raising compaign(to buy a new column), sorry.

[Greg O]

Hi Fatma,
I work in the pharmaceutical industry, so during one day I run at least 3 different MP's and different types of samples through a single column over the past 10 months considerable degradation has occured in efficiency. The best advice I can offer is to rinse with a 30% Acetonitrile/h2o solution on 0.5mil/min. Also there are many schools of thought on "regenerating" a column, some say it cannot be done. I have a considerable budget to work with so very seldom open the columns to clean the frits, but rinsing with the above solvent and reversing the column has produced positive results, one column was purchased in January has been reversed at least four times and it was providing excellent results even on Friday afternoon.

Good luck

[HW Mueller]

One would be inclined to think that there are good people at work if it worked on Friday afternoon?