Chromatography Forum

with 60% of 75% ACN = maximum 45% ACN on your column

is this enough to flush any strongly retained compounds off the column?
Maybe something is accumulating on your column/frit.

Does it depend on number of samples injected or volume of mobile phases (no. of blank gradients)?

Maybe you could increase to 95%B (=71% ACN) with the same slope of the gradient or as a step after the last compound of interest has eluted.

What about the re-equilibration to initial after each gradient? Is it sufficient e.g. >5 column volumes; ca. 3 min?

My Gradient is as below
Time A B
Initial 99 1
4.5 99 1
25 60 40
27 40 60
32 40 60
35 99 1
40 99 1

My Gradient is as below
Time A B
Initial 99 1
4.5 99 1
25 60 40
30.5 5 95
32 5 95
35 99 1
40 99 1

maybe try something like this and check if there are any peaks eluting in the "extended" wash step (>60%B)

You are plugging frits. This is related to particulates or precipitation. Your diluent/sample solvent is strong so the likelihood of precipitation is low.

To rule out the unlikely possibility of strongly retained components affecting your chromatography you could purge all mobile phase from the column with 10 column volumes of 10% methanol in water then flush the column with 20 column volumes of 95/5 ACN/H2O.

mmehta in the beginning you said peaks were splitting after a couples of runs
then you say after several sample sets runs
so after how many injections do the columns go bad?

is it varying between batches of the columns?
can you say if one type of column is actually holding longer? maybe we can find something in it's characteristics that will help

You are plugging frits. This is related to particulates or precipitation. Your diluent/sample solvent is strong so the likelihood of precipitation is low.

maybe too strong, so something can precipitate under the initial conditions.

What happens if you mix some of your sample with initial mobile phase and centrifuge? Any pellets? (maybe to low to be seen)

Beside the increase of the ACN at the end of the gradient, also try to lower the ACN conc of your samples ->post from "DR"
A better match of sample and inital conditions can't be wrong.

Phosphate will not mix well with 75% acetonitrile, you could be percipitating phosphate at the mixing point where A meets B and this is plugging your inlet frit. Use less AcN in B and you should be OK.

Good catch bintang! That likely explains the problem especially when you consider the amount of phosphate present after titrating:

10 ml TEA in 2L Water (~35mM). pH 6.0 with Phosphoric Acid

I had these kind of problems when I used the mobile phase preheater.

Dear Mattias,
Would you please describe the case in few words. Or may I ask you via e-mail?

Its your filters!!...i just had the exact same problem...check out this article. http://www.hplc.hu/PDFs/UHPLC_MobilePhasePrep.pdf

I am seeing the exact same thing happen on my Hplc system. After about 200 inj. ,My first analyte peak splits on the standards and everything thereafter. I am using a sodium phosphate/meoh mobile phase and have a biological matrix. The assay was working fine for three months, then all of sudden the peak splitting started happening. New column and guard, no more split peaks until 200 injections. Even changing the guard at 200 inj, the peaks still split. It's sounding like my inlet frit on the column is clogging. Any ideas of what is causing this? Is it the mobile phase or bacteria in the lines?

Even changing the guard at 200 inj, the peaks still split.

It may be that by the time you see the splitting, the junk has already saturated the guard cartridge. Try changing the guard *before* the symptoms show up (perhaps every 100 injections) and see if that helps.

Tom,
I have tried changing them earlier and even doubling up, the first peak still splits after ~200 injections.