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- Posts: 35
- Joined: Sun Apr 08, 2018 3:17 am
I have a validated method for the determination of 22 known impurities for two active's. I have them all separated with the exception of 1 impurity that co-elutes with 1 active and two other impurities that co-elute with each other. The method is a gradient method using a Phenomenex Biphenyl column, 150mm X 2.1mm, 1.7um.
I have been able to develop a secondary method that is able to separate these peaks, (however other co-elutions exist). Nevertheless worst case I can properly identify/quantitate these peaks using a secondary method if needed.
My real question, is that for my own interest I wanted to develop an Orthogonal method to the Biphenyl column method. I've tried the following:
Primary Method:
Column: Biphenyl 150mm X 2.1mm, 1.7um
Mobile phase A: 0.1% TFA
Mobile Phase B: Methanol
Temperature: 35C
Alternate Method 1:
Column: HSS T3 C18 150mm X 2.1mm, 1.7um
Mobile Phase A: 0.1% TFA
Mobile Phase B: 75:25 Acetonitrile:Water
Correlation (Comparison of K' of Primary method to K' Alternate method): 0.80
Alternate Method 2:
Column: HSS T3 C18 150mm X 2.1mm, 1.7um
Mobile Phase A: 10mm Sodium Acetate buffer pH 5 containing 0.25% TFA/L
Mobile Phase B: 25:75 10mm Sodium Acetate buffer pH 5:Acetonitrile containing 0.25% TFA/L
Correlation (Comparison of K' of Primary method to K' Alternate method): 0.80
I can't seem to find a truly orthogonal method (Correlation coefficient as low as possible)
I wanted to try a HILIC method. I've never successfully developed a HILIC method.
I've tried using a Luna Silica (2) column 250mm X 4.6mm column with a mobile phase of 100% Acetonitrile, and 97:3% ACN:Water.
I've tried using a Luna Amino column 150mm X 4.6mm same mobile phase as above - Was able to get some retention, but nothing significant.
I've tried using a Kinetex F5 column 100mm X 2.1mm, 2.5um mobile phase 95% ACN: 5% 5mm Ammonium Acetate buffer pH 3 - Peaks elute in solvent front.
I've tried using a Phenomenex cyano 150mm X 4.5mm, 4um mobile phase 95% ACN: 5% 5mm Ammonium Acetate buffer pH 3 - Peaks elute in solvent front.
Some of the peaks are in fact extremely polar which is why in my original methods above my mobile phase A is 100% Aqueous.
Why can't I get significant retention on my HILIC columns?
Thanks