HILIC separation using IPA as mobile phase

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

7 posts Page 1 of 1

I'm developing and trying to validate a HILIC method using IPA as my mobile phase with 5% Water.

I developed this method on a Waters Acquity H Class system using 1.0mL/min and had no problems. I recently tried re-visiting this and now I'm having all kinds of problems.

Obviously I washed the system with 100% Water with a union to flush the system of any residual salts.

When I try to run the chromatographic parameters listed below I'm getting these cyclical peaks and valleys now.

I read somewhere that you have to use lower flow rates for IPA due to the increase in viscosity, but even when I try that I'm still getting cyclical baseline disturbances.

Any suggestions?

Column: Luna Amino 150mm X 4.6mm, 5um
Mobile phase: 95:5 IPA:Water
Diluent: Mobile phase
Injection volume: 50uL
Detection wavelength 210nm
Flow Rate: Tried 1.0mL/min then going to 0.5mL/min and now trying 0.25mL/min
Column Teperature: Ambient.
It seems that adding 5% methanol to the mobile phase corrected the problem. Not sure if its because methanol is more miscible with IPA than with water and under high pressure doesn't cause as much bubbles to form or if it's just helps with slightly reducing the viscosity of the mobile phase.
So another update. It appears that just adding 5% MeOH didn't completely solve the problem. I have decided to move to a gradient method holding Isocratically for 10 minutes and then pump mixing in a solution of 50:50 Methanol:ACN containing 5% Water. I also changed the column temperature to 50C and the flow rate to 0.75mL/min. This appears to have fixed the problem. However, if in the future I want to use IPA as my mobile phase are there any other recommendations? Or should I just try to stay away from IPA as the mobile phase and consider using other columns?
Did you check the energy of your detector , since 210 nm is UVcut-off for most solvents.
Deuterium lamp may need replacement.
I am a bit curious why you want to use IPA, since 99% of all HILIC methods use acetonitrile. Do you get a better selectivity?
I used 95% IPA because I tried 95% ACN and my peak eluted in the solvent front. I figured I had to choose a weaker solvent, so according to what I found IPA is weaker than ACN. Even with this, my peak elutes about 4.5minutes.

So another update, the gradient appeared to work on one of my H-Class systems, I performed some additional validation work on a 2695 system with similar chromatography but I moved to another UPLC H class and I'm getting that cyclical baseline disturbance that almost looks like a peak. My gut feeling is that the H-Class systems aren't really made to handle such a large amount of IPA as mobile phase? Is this true? Is there anything inherently wrong with using IPA as my MP?
IPA is a stronger solvent than ACN in HILIC. So it is a bit strange that you get more retention with IPA.

From my experience, the column chemistry has a huge impact in HILIC. It is also quite rare that you can run HILIC without any buffer at all in the mobile phase (10 mM is usually a good start)

If your compound is positively charged, the amino column is not a good choice. Then you should try the regular Luna HILIC, and you will get tons of retention and nice peaks.
7 posts Page 1 of 1

Who is online

In total there are 13 users online :: 0 registered, 0 hidden and 13 guests (based on users active over the past 5 minutes)
Most users ever online was 599 on Tue Sep 18, 2018 9:27 am

Users browsing this forum: No registered users and 13 guests

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry