TFA at 215nm [August 26, 2004]

[HW Mueller]

Unless this Fluofix was changed quite a little one would be inclined to think that your treatment of the columns was different. Chris already indicated that you might have been close to the limit, a small change of concentration, organic additive, temp., and you might have gone overboard. Might it also be possible that the old column was initially used completely differently and, maybe, conditioned differently toward TFA??

[Newman768]

Yes, the older Fluofix was/were treated different.
Before I run the TFA-method, I had tested the column with other samples and mobile phase. Neithertheless this column is used since '03 only with this method. Guess the numbers of injections under these conditions could be around or higher than 500. That seems to be high enough to exclude (reversible) conditioning effects. And the endcapped version of the column was not treated as the not-endcapped column.
The batch-to-batch reproduciblity test (for the newer columns from vendor B) was made with the same mobile phase on the same hplc system and at the same day. Both columns failt the test. Both columns showed no baseline seperation and both had different rt's for the compond. Normally I would expect that degeneration for the same batch of column material should be at the same scale. And after all the conditions were compareble to conditions posted from Neos.

[Newman768]

Guess I should add that the seperation for each column were reprodicible.

[Uwe Neue]

With columns that are as old as you are describing, one always suspects that they have changed with age. This is especially true for your 00 column, which is the one that you are most happy with.
If the column was stored in an aqueous mobile phase, I am quite certain that it has changed over time. You may be able to check this for yourself. Ask the vendor, if they can supply you with a fresh column from the same batch of material as your 00 column. If they can, you can see for yourself, if the old column has changed or not. If it has changed, you may not be in a pleasant situation, since you will be able to reproduce your results only, if the newer columns have been "aged" in the same way as the 00 column.
If my interpretation is correct, you may be better off to redevelop your method under condtions, where the column is more stable, or even better, to go to a C18 column or something similar, like a column with an embedded polar phase.

[Newman768]

Hello Uwe Neue,

the columns were purged after every sequense and stored in pure acetonitril. This makes sure that (nearly) no TFA remains on the column. If sample matrix demands it the after the compound of interest has eluated a column flushing steps occurs. BTW: RT seems to be totally unimpressed of this step.

The problem with this application is: I moved away from C8/C18 because sample matrix is too variating and messing up seperation. Next thing is that so far only the fluorinated phase/TFA method gaves results which equals with results from gc. But gc can't candle all samples because of the different matrix. With this hplc method I can analyse samples from ppm to technical grade (without the problem of carry over) and quantitate different possible impurities if necessary. The method is suitible not only for this one product but also for a lot of comounds which gave a hard time if the seperation is tried with normal rp.
(maybe you remember talking with me about fluorinated phases/TFA at Mannheim in 2002 or 2003?)
The mechanism itself is so powerfull that I'd like to keep the fluorinated phase/TFA system and want to improve long time stability.
Right now it seems there is no way around buying (and testing) a new column from vendor A. Nethertheless I try to figure out what is killing 3 of 4 columns.

One more question to the pro's:
What about shipping a fluorinated phase with methanol? Could it be that methanol is changing the column or making it more sensitive to an aggresive mobile phase? I had observed that the seperation was enhanced (from very bad to bad) when purging the failing columns with ACN for a hour or two.

[HW Mueller]

There seem to be some aspects which I didn´t catch:
The old column and two of the new ones are endcapped, and you only mention comparisons of endcapped with endcapped? The new columns were bad from the beginning and are getting worse with TFA and somewhat better again after ACN? How do the RT change and how do the new columns´ RT differ from the old? You treated all columns the same after 2003?
Are your analytes ionizeable?

[Newman768]

Hello HW Mueller,

> Are these columns endcapped?
all columns used for the special method are not endcapped.
In 2000 I'd ordered from Vendor A one uncapped (NW) and one endcapped (EW) Fluofix. Both columns were used sometimes for testing and were flushed afterwards and stored in ACN.

> Are the new columns bad from beginning?
The new columns (from vendor B) were bad from the beginning. After flushing them with ACN they were getting better but not good enough. End of the story for those columns.

> How do the RT change?
RT for the vendor A column (the old one) is around 4.5 min with baseline seperation. Automatic integration is fine.
RTs for vendor B columns differ from 3.5 to 4 min without baseline seperation from the last negative peak. No automatic integration is pssible.
To add some facts: column dimension is 150 x 4.6 mm, flow is 0.8 ml/min, all columns shows stable RT for double (or more) injections.

> Treadment of columns
Purging step and final purge with ACN to store the columns is always the same. Maybe the times differ if the purge is done manually on another hplc system.

> Compound chemistry
weak acidic. Matrix can be anything.

[HW Mueller]

Looks like Uwe had the answer for you.
Maybe the old column was changed to the effect that the surface bears (more) + charges, at this low pH, which hold lots of trifluoroacetate?

Also, it could be that you are operating to close to tm so this may be a problem of robustness?

[Newman768]

The facts that:
a. both old columns (the endcapped and the not endcapped) from vendor A manages baseline seperation.
b. the endcapped version wasn't used often
c. all new columns from vendor B failed baseline seperation and shows different RT for the compound (each column reproducible for itself, but no trace of batch to batch reproducible)
speaks against the suggestion of "positiv" column changing through long time conditioning. Column changing caused by TFA and low pH should degrease RT, not increase. After all the system acts as an ion pair.

I agree that column surface modification is playing a role for this problem. I agree too, that robustness is correlated with the pH and I think it is very importent to increase the pH of the mobile phase to gain more robustness. I guess that the quality of column packing may play a role too with this seperation.
Next thing I do is ordering a new column from vendor A (who sold the good old one).

[Uwe Neue]

Andreas,

The facts that you stored the columns in acetonitrile and that both the old endcapped and the old unendcapped column do the same separation indicate that the problem is not associated with column age and loss of bonded phase. This is not necessarily good news either.

When you go back to vendor A, I recommend that you do not only ask for a column from current stock, but also inquire, if the packing material used for his current stock is from a different batch then the old column. If this is the case, ask them, if they can still pack you another column from the old stock. If this is possible, you have now all the pieces in your hand to compare different batches, the difference that may have occurred during method development etc. I am sure that the vendor will do his best to satisfy your needs.

[HW Mueller]

Agreed (with what your next step should be, but bot with the "theotretical" RT shifts).
It would be a lot less tortuous, though, if the necessary info would be in the first post.....
Anyway it would be interesting to find out what causes the difference, especially (for me) whether TFA has a role in it.

[Newman768]

to HW Mueller:

sorry for giving all the necessary info's not in one big block.
The hole thing started with "I have just a question" and I hadn't expected such a discussion.
Thanks to all who provided me with their answers. You were a great help.

[Newman768]

Information update:

After talking to the vendor who sold the good old column told I knew that ALL columns were packed by the same vendor (the one who produce this special material).
That means that either there were some problems with column packing (not very likely) or the material itself has changed a little (nothing unusual).