all the peaks were eluted at the early of chromatogram

[serenetengteng]

Hi,

Currently, i had face the problems of all the peaks tend to elute at early of chromatogram (50 min). Previously, i had run the same sample with the same condition, the peaks were separated well and major peak is eluted at 18 min. My analyst is polar compounds.

Column : C-18
Solvent (A): Water
Solvent (B): Acetonitrile
Gradient elution (start with 100 % water)

I had noticed that the pump pressure is fluctuated (30 bar -> 24 -> 17 -> 19 -> 20 -> 30 bar).
There was air bubble occurred at the tubing after 2 solvent were mixed. ( I had degas the solvent manually and the degasser of the HPLC was spoilt.)
Besides, i noticed that the pressure during condition of column (water) was lower than flushing of column (33 bar)

are these conditions cause all my peaks shifted?

Really appreciate for all of your opinion.

Thank you.

[oscarBAL]

Hi serenetengteng; I really do not know but could be a dewetting phenomena aka hydrophobic colapse. When you are running highly acueous mobil phase you will need a long time to stabilyze your columns this is due to the repultion or dificulti that water can entry into the pores of the particules; the substrate have hydrophobic caracter and tends to repell water; so mass transfer is not optimal. Waters has been talking about these things when they promete Atlantys Columns; you could read a litlle bit about it at: http://www.waters.com/webassets/cms/lib ... a20756.pdf

Nevertheless I do not know if this is your case. For how long your method was working fine?
Why you start at 100% water if your Rt is so high? you could star at 97 or 95 % water for example as soon as you reduce surface tension.

sorry for my very bad ortography english is not my first lenguage but I hope to be understandable.
Hope some one else can help you more

[annaand]

What flow rate are you using? What are the column dimensions, particle size? It looks for me that your pressures is way too low. For a column (C18): 150x4.6mm, 3um, 85/15/0.1 Water/ACN/TFA I have ~250Ba with flow rate 1mL/min.
I would look for more bubbles if I were you. The pressure fluctuations over 10Ba are usually caused by a bubble. Even small one in on of the lines, tubing.
I suspect that your peaks are coming out at the very beginning because if you have a bubble in line A (aqueous phase) the A mobile phase is not pumped properly. So you may end up with only B (ACN) being pumped which is flashing down your analytes at the beginning.

[Camisotro]

What kind of compounds are we talking about here? If retention is so poor that you have to start your method with 100% water (which as mentioned upthread can create risk of column collapse), perhaps it suggests that your retention mechanism for these polar compounds is not ideal for your needs. Perhaps you need a different mobile phase additive, or a different column.

[serenetengteng]

Very thank you for all of the feedback. Really appreaciate.


I had been used this method for more than 6 months. It was fine for me. I will look try to re-wet the column and compared with a good known column afterward.
I had start the gradient elution with 100 % water because the my sample is highly polar. It gave nicer profile if i start the elution with 100 % water compare to 97% / 95% water.

My flow rate is 1 ml/min. column is Hypersil Gold C-18, 150 x 4.6 mm, 5 um.

My sample is highly polar compounds and i know HILIC column definitely will work well with it. I had chosen C-18 in my experiment because C-18 is easy accessible in my lab. And It had gave nice profile also. This is first time i encounter this kind of problem with all peaks were eluted at the begining of chromatogram.

Thanks..

[unmgvar]

the low pressure like you say is what I think indicating the problem

from my experience it could be 3 things
wrong bottle in the solvent line
wrong changes in the method using another line
a leak in the proportion valves, introducing C or D to the system.
what solvents do you have in the other lines?
if it is ACN or MEOH then it my be your problem.
one solution to check this, is to move those lines to the water bottle as well and see how the sample behaves then.
if the peaks now move back then it is the problem

[Camisotro]

What type of polar compounds? If you are only using pure water and ACN with no additive right now, and you need to start at 100% water to get any retention at all - you may get better retention with either a buffer that keeps your analytes in neutral form, or with an ion-pairing reagent, or both. If your method works for you, great, but I guess I'd worry about reproducibility with a method that requires a 100% pure water start.

As for the question of whether column collapse has happened - are your *other* methods still working on this same C18 column, or are those showing different retention times too?

[vishnu]

hello,
I think there is a choking problem in any of the systems.
because you said there is a fluctuation in pressure.
then i think there may be a chock in the inlet capillary or at the seals present in the pump head or at the detector head.
Try cleaning by removing all of them and sonicating with appropriate solvent (You may use water).

This is a suggestion (because once i got the same problem). please don't mistake me.