Chromatography Forum

Using the following condition, a blank solution is injected; a mysterious peak always shows up. What is the peak? Can anyone give a clue?

HPLC condition:
Column: C18 150x4.6 mm, 3.5 um
Mobile phase: phosphate buffer pH3/Acetonitril
Gradient: B% 25 to 80% at 0 to 45 min
Column temp: 30 C
Wavelength: 238 nm
Solution injected: 10 ul H2O/Acetontrile

Question:
-One peak shows up at the end of the run. What is the peak? The column is cleaned with H20/Acetontile 5:95 for enough time. The similar peak also shows up with phenyl column 50x4.6mm.

Any reply is appreciated!!!.

What happens when you inject a phosphate/ACN solution or if you just run the gradient?

I injected Water (no buffer)/ACN. There was not siginificant peak for the whole run, only some baseline noise was found. But one peak always appeared at the end of the elution.

e.g For 20 min run, the peak will elute at ~18min. Peak Area is ~ 12000.

Is it something eluted from the column?

Thanks.

It is something eluting from the column, but the source is not the column.

Try this - run at your initial conditions for ~20 minutes, then run the gradient twice without actually injecting anything. If the peak is larger in the first of these chromatograms, then the source is probably either the water used to prepare the mobile phase, or in the salt (less likely if you're using LC grade salt). The peak would be larger in the forst chromatogram because you have allowed more of it to build up on the column than in the second run.

To eliminate the contaminant (usually something from the water polishing system's resin bed - styrene monomers, dimers) you can:
-try bottled LC grade water
-install a small C-18 column between the A pump and mixing chamber if you're running a 2 pump system (remember to clean this occasionally)
-use a Empore Extraction disk to pretreat your water before making the A phase (a 3M product available from Varian & probably other vendors). Empore disks are available in a 47mm size to fit common filtration set-ups. You filter a liter or two of water, rinse the disk with MeOH (catching that in something other than your filtered water container), repeat. This is the best long-term solution to the problem that has plagued gradient chromatographers, especially at lower wave lengths.

Do you fush the column back to 25%B before the end of the run-time?
I also see a large "peak" when flushing back with a steep gradient at the end of a run. Might be some refraction-effects? (If anyone knows, I'm glad to learn )

Further: What is the concentration of your phosphate buffer? High ACN contents can cause precipitation...

We still don´t know whether you see this only when you inject something.

Thanks everyone for all the suggestions!

The mobile phase is prepared with HPLC grade water.

Actually, no matter sample solution or diluent (HPLC grade water/acetonitrile 50:50) is injected, as long as the very steep gradient method (i.e. B% 20-80% @ 0 - 20 min) is run, this late eluted peak is found.

In the past I have seen problems with poor quality AcN causing the peak as described. It may be an impurity in the AcN that will build up on the column as the mobile phase is pumped through it.

The mobile phase will build up this impurity on the RP column until the concentration of AcN is high enough to release it as a somewhat broad peak.

Try different suppliers or lot numbers of the AcN HPLC grade solvent and see if the peak disappears.

I tried several columns of different dimension, some columns are new. This late eluted peak is still found.

Have you tried DR's suggestion? What was the result?

LucyUS: Just to make sure, that you did not misunderstand me: With "steep gradient" I mean the flushing back from 80 to 20%B at the end of the run, not your actual gradient (which is not very steep )

We are discussing here without knowing whether lucy is seeing that peak without an injection.

Hello everyone,

Washing the column with mobile phase buffer/ACN 80: 20 for 20 minutes, without injection, the gradient runs were performed.

For three consecutive gradient runs, the first run had the biggest peak area, the second and third run had almost similar peak area.



I appreciate all your help

lucyUS,

This implies that DR's first assumptions are right.

If the peak is larger in the first of these chromatograms, then the source is probably either the water used to prepare the mobile phase, or in the salt (less likely if you're using LC grade salt). The peak would be larger in the forst chromatogram because you have allowed more of it to build up on the column than in the second run.

It is either the water or the phosphate salt. HPLC grade water you use may be contaminated or alternatively the salt. You may further isolate the problem by running same blank gradients without phosphate.

bulent

Or it could be buffer from a leaking pH electrode,
or it could be residual detergent on glassware.