Chromatography Forum

Dear All,

I developed a HPLC condition by using the neutral buffer. The mobile phases are:
A: 50 mM phosphate, pH 7.2 (not filtered),
B: 10% tert-butanol in dioxane

Gradient starts at A/B = 90/10, ends at A/B = 35/65 in 55 mins. Then go back to starting condition and equilibrate 10 min.
Temperature at 45 degree
Samples are very clean (USP standards dissolved in water/acetonitrile = 1/1)
Column used: MAC-MOD ACE 3 C18, 150X4.6 mm.

The separation was great at the first 50-60 injections. However, after that I can see all peak heights decrease gradually as injection number increases. Around 80 injections, I can see peak splitting. I have seen this phenomenon on several new columns. However, retention time of all peaks ar unchanged and system pressure only increased about 80 psi (from 1950 to 2030)

Can anybody tell what might be the problem? I guess it is due to the clogging of the inlet frit of the column. I have try to back flush the column with 50/50/0.1 water/acetonitrile/acetic acid. But I cannot get the performance back. I open the nut on the column head, the inlet frit is embedded in a ferrule (seems like one piece). Can anybody tell me where to purchase this kind of unit to replace the old one.

If you have other ideas about what might be the cause, please reply my post

Thank you very much!

i would firstly stick to good chromatography practices

first filter your buffers, preferably always
second use lower concentrations if you can, i never needed more than 20mM
third there are reasons why dioxan is no longer used in HPLC (or maybe it is sometimes in NP but I have never seen a bottle of dioxane in our lab), why not use methanol for instance
forth high temperature and high phosphate content can make your column useless - I would think that you maybe experiencing loss of theoretical plates here - check if peaks are wider

i recomend to go back and do a method development again, that's what i would do if i were in your place

I believe the relatively high pH, combined with high temperature causes the problem. Void volume at the column-head is a typical reason for peak broadening and even splitting.

Best Regards

Hi,
Is phosfate at this concetration and temperature soluable in dioxane?
(I know it is problem with phospahte pH 7.2 buffers and acetonitrile.)