I am currently working on the bile acid separations by HPLC. I have developed the method on Halo column 100 x 4.6 mm 2.7 µm. Three bile acids are separated with good resolution. But a small tailing peak is coming at the end of glycocholic acid. I have tried with gradient programs, temperature effect and also tried to change the composition of my mobile phase B i.e. organic phase. None of them are working. My current mobile phase is buffer (pH 6.9) phosphate buffer and acetonitrile as B.
Kindly provide me suggestions for the problem I mentioned. Can i use a organic modifier like Tri ethyl amine. Will it work ?
Bile acids pKa ranges from 4.5-5.0
It is difficult to guess, but I think that (sodium?) phosphate pH 6.9 is not appropriate. I would try an acidic hydrophilic phase with low salt concentration provided that bile acids are soluble. On the other hand, I do not see any trouble to test tri-ethylamine, but basic pHs are not good for column stability. Good luck
My buffer is Monobasic phosphate and dibasic phosphate in 25mM concentrations. Any trouble in that. I think 25mM is good enough. If I add 0.1% TEA to Mobile phase B I.e. organic phase. What would be effect of pH on it. I hope it does not get too basic.
TEA will not help. Try adding more water to your ACN or even a Methanol/ACN blend. Also review the Agilent, Waters, and Phenomenex websites. Be sure your not reinventing the wheel.
If added, TEA should also be dissolved in the aqueous buffer. As I said before it is easier to use phosphate buffer at pH 2.0-3.0
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