Moving peak when injection volume increases

[Qui]

Hi,

I am running a RP HPLC. There are 2 peaks, suspecting to be diasteromers. This is an unknown peak. When I increase the injection volume (keeping the sample concentration constant), the former peak was eluted earlier and earlier while there is no change in RT of the latter peak.

column used: Supelco Ascentis Express RP-amide
MP A: 80:20 of 10mM ammonium acetate, pH 5.5 and ACN
MP B: 20:80 of 10mM ammonium acetate, pH 5.5 and ACN
It is a gradient elution

Why is this observed?

[kubowicz.tomasz]

Hello

Please paste chromatograms...It'd be very handy.

Regards

Tomasz Kubowicz

[Qui]

sorry, I wish to post my chromatogram here but i do not know how.

Injection volume RT of the 1st peak RT of the 2nd peak
35 ul 16.26 min 18.07 min
50 ul 15.37 min 18.08 min
80 ul 13.45 min 18.22 min
100 ul 11.84 min 18.21 min

[kubowicz.tomasz]

viewtopic.php?f=1&t=2617

[Qui]

[LiVD]

In what kind of solvent are your analytes dissolved?

[Consumer Products Guy]

Agree, think the volume of solvent (like a mostly-organic solvent) is functioning as a solvent in a mobile phase that's higher in aqueous. If that's the case, keep the injection volume small.

[Qui]

the samples are dissolved in 60% ACN: 40% buffer

are you able to elaborate? I do not quite understand the point that the solvent is behaving like a solvent in the mobile phase with higher aqueous content. why is the first peak affected and not the second pair of the pair?

[LiVD]

As a rule of thumb, your solvent should be as close as possible to the starting composition of your gradient.

If you inject a large amount of apolar solvent on your column it behaves like a plug traveling through your system. A part of your analytes will be retained immediately by your column, get out of the plug and behave normally. That's what gives you your peak at 18 min.
The rest of your analytes will travel along with the plug for a while, before retention catches up with them and they are caught up by your gradient. That's were your first peak comes from.
The larger the injected volume, the larger the plug and the stronger the effect.

There's two ways to overcome this:

Use a solvent as close as possible to the starting composition of your gradient.
If for some reason you can't, keep your injection volume as small as possible.

[Qui]

Thank you for the clear explanation.

I do understand that we try to use diluent as close to the starting composition of the gradient.
I have experienced a split peak within 1 analyte peak in such situation.

I am trying to develop an analytical method for scale up to semi prep LC by volume loading as my analyte is poorly soluble in water. Thats how I discovered the observation.

[LiVD]

Depending on the complexity of your samples, SPE might offer a solution...

[danko]

If 60% acetonitrile is absolutely necessary in order to dissolve end have your sample in solution then what do you thing will happen when your sample “meets” the 80% aqueous mobile phase upon injection?
I’m writing this because it’s a classic blunder. I’ve seen it so many times.

Best Regards

[sga]

Hi Qui,

First of all, the phenomenon that you are observing: Is it repetable.... I mean try a new set of injections for the volumes that you mention with fresh solvent.

To me this looks like a pressure variation between runs : I would suggest you to compare the presure profile between each of the runs. Once you have done that and think that this is indeed the case, then you can try the following:

1. Make sure that the column returns to the same conditions before you strart the injection for each run. I.e. you need to equilibriate enough before each run.

2. It is likely that the solvent mixing is not good enough, you better get your pump looked at.
3. It is also likely that your degassing is not good, or you simply have a bubble in the system that refuses to go away. Then degas well and pump long enough before injections.

Keep us posted on what happened ?


Regards

[danko]

There is no doubt in my mind that the problem here is the high acetonitrile concentration (60%) in the sample solution compared to the mobile phase’ acetonitrile content (20%) at the start of the run. The experiment with increasingly larger injection volume shows it clearly. And it’s – as expected - the compound with shortest Rt that is affected mostly.
The solution here is a reduction of the acetonitrile content in the sample solvent to 20% and if possible lower yet. The reason I wrote my earlier post was to draw the attention to the solubility of the sample in the mobile phase as well as the sample solvent. If 20% acetonitrile (in the mobile phase – at start conditions) is adequate to keep the sample in solution then why 60% in the sample solvent, which obviously creates problems?

Best Regards