Chromatography Forum

Hi,

I am running on a Kinetex HILIC 2.6 µm (3.0x150 mm), where I see a constant drop of plate count during the sequence. The sample is a peptide.

It runs with 10 mM ammonium acetate pH 5.2 in 90% acetonitrile

The samples are dissolved in mobile phase and filtered before injection and I see no issues with increasing pressure or drifting retention times. But the peaks are getting broader and broader...

According to the manual you should wash the column with 5% acetonitrile in 100 mM ammonium acetate pH 5.8. This I have done for two days, but I see no improvement at all!!

Does anyone have a good explanation what is going on, or a washing trick?

Hi,

What volume do you inject?

Is the method isocratic?

What is the washing solvent composition?

What kind of samples do you inject?

A dirty column is one possibility, but there are a lot more! Also check if every connection/fitting is correctly tightened.

Thanks for your reply!

Some more info on the method:
Flow: 0.6 ml/min
Temp: 40°C
MP-A: 10 mM NH4OAc pH 5.2 in 90% ACN
MP-B: 10 mM NH4OAC pH 5.2 in 50% ACN
Gradient: 0% B to 25% B in 45 minutes
Equilibration for 20 min at 0% B before next injection
Injection volume: 40 µl (of a 0.05 mg/ml solution of a decapeptide + fatty acids). Dissolved in mobile phase A with no problem.

Wash solvent: 5% ACN in 100 mM NH4OAc pH 5.2

I think you have injected a non-polar molecule on your column from your sample matrix. This molecule is completely retained by the column.

Try reversing the column and washing it with 10x column volumes of 100% IPA. Then wash with water and finally use the manufacturer's suggested washing procedure.

Thanks for your suggestion, but I get a bit confused here..

The fatty acids are very hydrophobic and my understanding is that they should have no retention on the column?

The matrix (fatty acids) dissolves easily in mobile phase A in which they are dissolved. The method starts with 100% mobile phase A.

The beauty of using HILIC for this application is the very simple sample prep!

This is the confusion. HILIC columns (have AQ at end) were developed from the amino column used to separate simple sugars or carbohydrates. However, the amino column's mode can be switched between AQ and normal phase which is 'very' non-polar. So non-polar the most common solvent is Hexane or Chloroform which do not tolerate water at all. Thus, you can dissolve a fat or oil in these solvents (or IPA) and use them to quantitate various components.

I think we still have a confusion going on!

The Kinetex HILIC column is unmodified silica, there should not be any hydrophobic interactions possible?

But for sure, I have a problem with my method and flushing with IPA will be tested!

Just missing one thing: what solvent composition do you use for needle/loop flushing? If you would use water, it could also explain the symptoms.

Is it possible to inject lower amounts like 10 or 5 µL and see if the peak shape is better and more reproducible?

Good point!

I use a Waters Alliance system and the needle wash is just 10% ACN in water (right now). But precipitation should lead to increased pressure?

I think something is sticking to my column material, but what and how to get rid of it?

Googled a bit, and found that someone used high conc of formic acid to kill the silanols (releasing all junk bound by ion-exchange).

But still.... I have stable retention times!! That should not be possible if the silica is bound to something.

Forgot to mention that I injected 20 µl of sample on a dying column, same low plate count as with 40 µl

You need to avoid using strong solvent (water in the case of HILIC) to needle wash. Try changing that bottle to 90/10 ACN/Water.

Check http://www.sigmaaldrich.com/technical-d ... -mode.html

Thanks!

You have a volatile compound in your mobile phase. If it dissapears your mobile phase is changed. How long is your sequence?

I run for about four days (continously) before the column is dead (or has lost 70% of the plats count)

Not sure what you mean with evaporation of volatile?

The mobile phase contains acetonitrile, water, ammonium acetate and acetic acid