In my PhD, I applied several mobile phases. An isocratic method applied made use of a 87.5% water (0.1% formic acid) - 12.5% acetonitril mobile phase on a C18 HPLC column for HPLC-DAD. By making the mobile phase acidic wih formic acid (pH around 2.8), the pH was not near pKa values. By using acetonitril, no esterification of ciprofloxacin could occur. Retention time was highly sensitive to the amount of acetonitril: using 10 or 15% of acetonitril had a large impact on the retention time.
Tank you Bavo.
What was your column stability in this pH (pH around 2.8)?
The column manufacturer gave a pH range of 1.5-10 between which the column could be used. Column stability was not recorded in detail during my PhD. As I remember well, no large shifts in plate count or assymetry factor were noticed during my PhD.
Tank you bavo, I have used the mobile phase that you suggested. this mobile phase is good but marbofloxacin and norfloxacin are my new problems, they have same retention
time with cipro and enrofloxacin.
I try to find good gradient program.
I suggest to slowly increase the acetonitrile-amount at about 5-20% acetonitrile, dependent on the degree of endcapping of your C18 column. Once over 20%, retention will be limited.
If this not works out, you can also check literature, their are certainly methods described for separating the compounds you mention. In my PhD, the scope was different. I never had to analyse the quinolones mentioned in 1 run.
I have checked literature before but I think mobile phase consist of acetonitril or methanol with acidic water by formic or acetic acid is better than bufferic mobile phase.
I used many different acidic mobile phase (formic, citric, acetic and phosphoric acid) with different gradient and isostatic system but separation of danofloxacin and enrofloxacin is very bad....
different columns also checked.
If you keep on having problems, and their are methods known which can separate your compounds applying buffers, I would suggest to try these methods. I don't see a reason why you shouldn't apply non-volatile buffers since you are not using an MS-detector.
I would transfer the mobile phase to LC/MS, I know that in LC/MS separation is not very important but it will help me to determine other compounds.
The strange part of this work is very well separation of some reports that I have used their method, for example Vazquez et al separate 8 quinolones by using H3PO4 in mobile phase, but when I used their method it was not really good ( I used exactly same materials...)!
I used buffer as mobile phase its worked well, Tank you Bavo.
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