Peak eluting too close to solvent front

[wan]

I am trying to run simultaneous analysis of melamine and cyanuric acid using HPLC-DAD.

The cyanuric acid peak is eluting too close to the solvent front. How should I go about increasing its RT?

Column: Kinetex HILIC 4.6mm x 150 mm, 5 um
Mobile phase: 90:5:5 ACN:IPA:100mM CH3COONH4 pH 5.80
Flow rate: 1 mL/min
Wavelength: 210, 220 nm
Injection vol: 1 uL

Sample was dissolved in mobile phase. My conditions were set according to an application note from Phenomenex, though that was for LCMSMS. I am hoping to be able to transfer the method to LCMS later.

Given the IPA is a stronger solvent compared to ACN in HILIC, I am thinking of using 95:5 ACN:Buffer, but I doubt the effect will be sufficient for the RT to increase drastically.

[Bintang]

When the FDA developed their method for this they showed that in order to do this separation in one run the complex has to be broken between Melamine and Cyanuric acid by going to low pH. The Cyanuric acid then has relatively low retention.
I think your best choice is to use the FDA method, that has been validated and is known to work. Maybe you can transfer the method to Kinetex, but this could be tricky since it is a pH gradient method and neat silicas retention characteristics change dramatically when changing pH.

http://www.fda.gov/Food/ScienceResearch ... 071637.htm

[wan]

Hi Bintang,

I did try the FDA method. However, I only have the same column in a shorter length (100 mm instead of 150 mm) and using that method, the cyanuric acid peak eluted at the solvent front, which is worse.

[Bintang]

So you have tried our ZIC-HILIC column with the FDA method.
The column length should not matter too much, it is strange you get no retention for Cyanuric acid.
Are you sure you see the cyanuric acid the UV absorbance is really low.

If you send us an e-mail at

support@mercksequant.com
or
chromatography@merck.de

We will do our best to help you.

[wan]

Hi Petrus,

Sorry I got your name wrongly. My bad.

I plan to try ZIC Hilic column again on Monday, since I have a few other ideas after a few experiments with Kinetex Hilic today. I will email after that.

Thank you.

[wan]

I re-run some experiments using ZIC Hilic and obtained retention for cyanuric acid, but the RT of melamine kept shifting between runs and I am seeing a large, broad peak when the % of A is decreasing, which therefore affects quantitation of melamine. There is also a peak that elutes before the solvent front. I am stumped, since I never see that before.

I am flushing the column and system to make sure there are no carry over from someone else's expt.

Btw, how far should the retention time for an analyte be from the solvent front for it to be considered ok?

I am often using 250 mm columns for multi analytes analysis, so I am used to seeing RT >= 5 min. We are not aiming for high throughput usually.

However, with 150 mm, 100 mm and 50 mm columns, RT are usually <=5 min. I am having a hard time convincing my superiors that the method can be used, even after validation. They have the idea that if the RT is so short, the analyte is "not retained" sufficiently.

[tom jupille]

The US FDA "suggests" that k' values should be >2 (i.e., >3 x t0). For "non-regulatory" methods, you can probably go down to 1, but I would not go any lower.

[Bintang]

If you see peaks eluting before the solvent front they are excluded from the pores in the stationary phase either due to charge (negativly charged species are repelled from a negatively charged stationary phase) this is usually not a problem with the zwitterionic ZIC-HILIC phases but it is not unheard of.
More likely is that they are size excluded if you get percipitate of melamine-cyanuric acid that are small enough to be freely mixing with the eluent but too large for the pores they could come out before the t0.
I seem to remember that our student had problems with percipitation of the complex and that under some circumstances she could see these eluting from the column but I can not remember the specifics.

In the published FDA method Cyanuric acid elutes at about 6 min and Melamine at 8 min. t0 is below 1 min.

[wan]

Tom: Thank you for that info!

Petrus: Thanks for telling me that! However, I see those peaks even when I am injecting only cyanuric acid or only melamine. I did test their solubility in the mobile phase before I inject and they are soluble at the concentration I am using. I filtered my samples, thinking there are particulates not observable to the naked eye, but I am still seeing the peaks before the solvent front.

I am using the conditions stated in LIB4421, rather than LIB4422. But I have to reduce the flow rate to 0.2 mL/min, because there are leakage problems. When I increase the flow rate up to 0.6 mL/min, the pressure is less than 400 bar (232 bar), but the capillary tubing kept getting "pushed out" of the PEEK fingertight fittings, resulting in leakage. I am using 0.17 cm diameter stainless steel tubings with PEEK fingertight fittings. Is that suitable?

I understand I have to adjust the gradient according to the column dimensions and flow rate. I am trying to troubleshoot this so I can increase the flow rate and decrease analysis time.

In addition, I see that the re-equilibration time in LIB4421 is about 2.8 min, considering column volume. I back calculate the time I should set for re-equilibration based on the adjusted flow rate, but realised I cannot get a relatively flat baseline. I had to increase the timing by almost double to obtain a satisfactory baseline for the next run. This resulted in a longer analysis time. Am I missing anything?

I hope to gather more data, so I can email with the chromatograms attached. Hopefully, I will get more instrumental time to troubleshoot.