Significant Decrease in retention time

[letsdoit]

Hello friends,
I am running a Peptide with following condtns
RP HPLC C-18 column
Mob ph A 5% ACN in Water (0.1% TFA)
Mob ph B 20% water in ACN (0.1% TFA)
gradient from 15-30 in 18 minutes
I have a HP 1050 system with DAD

The retention time for the first few samples was abt 8.5 minutes . by the end of the run when I ran the samples in Triplicate the retention timesstarted decreasing and went from 8.5 to 6 to 5 to 3 and infact almost merged with the solvent front.
Am I doing something wrong ???

Also When after abt 35 injections the column pressure started increasing and it keeps on increasing...
Why is this happening ?
Any suggestions please ....

[syx]

What is your RP-18 column?
What do you mean with 'gradient from 15-30 in 18 minutes'?

My initial hypothesis is collapsed-column phenomena...

[Kostas Petritis]

I guess that from 15-30 in 18 min it means from 15% B to 30%B...

Syx, if by collapsed-column you mean dewetting, I do not agree with you as the % of organic solvent is quite high...

It seems to me that either you junked your column or you deteriorateed it. At this point I would ask what your matrix is, what is your peak shapes of the peak of interest and if the retention times of your peptide can be recovered after column regeneration...

[syx]

I have been confused by 15-30...
You are right, there is no reason for dewetting in this case.

[HW Mueller]

letsdoit, are you sure that the peak you observed is due to the peptide? How are you sure?

[letsdoit]

I am sorry for not being specific.
it means Mob phase B from 15%-30% in 18 minutes.

I guess that the peak I see is the peak of my drug as these samples were actually prepared with just the drug in Phosphate buffered saline in increasing concentrations . (nothing else in it.. Drug in PBS )
there is only one peak that appears and which increases with increasing concentrations.
As to the peak shape, I can say that they are fairly good in shape (on the borderline of being fat peaks

Please help

[sadsal123]

An increase in column pressure usually indicates a blockage. To check if your column is ok, try turning it around, dont connect the end to the detector and let the solvent run through for a few minutes (10mins or so) then turn it back round and connect everything up again and see if there is a difference in pressure (i.e. has the pressure come down?). Do you a guard column? This is an excellent way of protecting your column.

[letsdoit]

I am using a C-18 micro u Bondapak Column from Waters 3.9* 150 mm

[HW Mueller]

letsdoit, didn´t you at least take a spec of the peak with your DAD? Do you know how big (area) your peak should be? (easy to find out if you do a test injection without the column, same flow rate)
I have had it happen with peptides that a peak was something other than the standard peptide (highly pure) I injected. Especially when using TFA, strange things happened.

[letsdoit]

I will try it now and then get back to u immediately.

I hope I get to know where the problem is...
This is driving me crazy..

[Albany-12303]

Maybe the peak does not come out during the first run at all but in coming out as 'carry over' during the next run. I have had this problem before and found that the RT of 'carry over' peaks can seem to become shorter and shorter.

That's probably the only thing I could think of (besides crap wrecking the efficiency of the column) that would cause these symptoms.

[Kostas Petritis]

Albany,

This would be the case if he does not see the peptide in the first sample analysis (i.e. first injection of his sample). But your theory might be valid especially as he describes his peak as "fat"... although I would expect it to cycle around and not at the end just co-elute with the front peak for several injections...

[tom jupille]

Has this method been running reliably in the past, or are you just setting it up?

micro-Bondapack C-18 is a fine example of 1970s vintage column technology, but I would not be surprised to find that 0.1% TFA is below the recommended pH operating range.

[letsdoit]

I dont know abt the past...But the manufacturer I got the peptide from did it the same way
My HPLC pump is down .... so will run my samples again after it gets back working...(probably soon......):(
Any sugesstions for the drift in peak retention time?

[letsdoit]

I got the same results again
When I ran my peptide on another C18 column , the first 3 runs had Rt time of 17.5 min which changed to about 16 min in third run and the 4th run gave me Rt time of 4.5 minutes

Also the baseline gets a lot wavy


here is the gradient
Ph A 0.1% TFA in water
Ph B 0.1%TFA in (10% water and 90% ACN)
Gradient starts at 85% A and 15% B
0-5 min 85% A and 15% B
5-25min 60% A and 40% B
25-26min 85% A and 15% B
26-31min 85% A and 15% B


any suggestions why this is happening
this time the column was C 18 novapak column

any help is welcome