Chromatography Forum

Im doing a separation but the peaks are not separating. According to the inhouse method (unsure if it was vaildated) i should be using mobile phase 80% H20 and 20% IPA (isocratic) with the pH adjusted to 3.8 with formic acid. But using this mobile im not getting the separation. I found the method online (similar but not identical) and that method states 71% water, 13% mM ammonium formate (pH 3.0) and 16% IPA(isocratic). Would the fact that im using formic acid instead ammonium formate make a difference? also the column im using is a aglient 3.5um XBD Phenyl 4.6x150 and the method online is using Xterra phenyl 5um 4.6x150mm is the combination of these two things making me not achieve the separation? Bascially im flying blind given a method that there is no evidence it ever worked! i would appreciate you help thanks in advance

I think the problem is....the 2 methods i know that work on the internet are ph3.0 ammonium formate as the buffer. I think the method inhouse is wrote wrong. It excluded the buffer and had the wrong pH therefore thats why im not achieveing the separation as even if the 3.8 pH is adequate...it is not being maintained as there is no buffer!!....any body know any other buffer that can maintain a pH 3.0? i dont think we have ammonium formate in house.

Even with the same mobile phase, those different columns may well give you different selectivity.

Re the ammonium formate, simply prepare formic acid to the specified concentration and "titrate" up to pH 3.8 with concentrated ammonium hydroxide solution.

As Tom has pointed out it could be due to the difference in coumns or even the difference in organic modifier (16 vs. 20% IPA). What kind of compounds is it that you are trying to separate? Are they acids, bases or neutrals or unknowns?

im separating explosives. Nitroaromatic explosives; Niroamine explosives; HMX; 1,3,5-TNB, RDX, 1,3-DNB, 2,4,6-TNT; Tetryl; NB; 2-Am-4,6-DNT; 2,4-DNT; 4-Am-2,6-DNT; 2,6-DNT; 4-NT; 3-NT; 2-NT

I didn't think there was a point to using pH modifiers when separating explosives? I did it on C18s and specialty explosive columns (Dionex Acclaim E2) with just methanol, water, and temperature. I saw another method using a Waters NovaPak C8 and a water:isopropanol mobile phase from CRREL, but they also did not use any modifiers. Are you using UV detection or MS detection?

The typical explosives do not require buffering for chromatography. Acetate or formate will help ionzation (ESI-) if using MS. Below is a link to the analysis of 17 explosives with a C18 column and methanol water mobile phase to give you an idea of what you should be seeing:

http://www.phenomenex.com/Application/D ... RL=/Search

im using UV. The methods i have come across all use buffer for example
http://www.waters.com/webassets/cms/lib ... 2737en.pdf

I also have one explosives method already validated but i need a second one to confirm. Im using UPLC PDA

I think you don't need the formic acid or ammonium formate modifiers. You are probably just seeing differences in selectivity due to the different stationary phase (XDB phenyl versus XTerra Phenyl). Either re-develop the method on the XDB or buy the right column for your method.

Going to try the Kinetex column and see how it goes...thanks for the advice guys