Chromatography Forum

hi,
I am working on a molecule,My HPLC conditions are luna C18(2), 30 x 4.6mm, 5um with mobile phase of 0.05% TFA in H2O:0.05% TFA in ACN(78:22).and col Temp 40C.I did online mixing of pump and my Retention was shifting from 10 min to 9.6 min after 25 injections.
To get rid of probs,i did premixed mobile phase,but its same problem.
If 40C is evaporating my ACN,then it should move further,but its reverse phenomenon.could any one plz suggest or help me to get constant RT

Thank u for help

Assuming that the flow rate is ok, maybe TFA evaporation, that is a pH change, is the cause?

Your shift in retention time is not huge. How long did you leave the column to stabilize before making the injections? How long is each run? If the runs are short, it might just be that you did not leave the column to settle down for long enough before making an injection. You might solve the problem just by purging for longer.

HI, Banala,

How is your RT going?

We will appreciate if you can share your progress,

My total run time is 20 min,I am equilibrating my column for almost an hr.
My RT is starting from 9.5 min and after 50 injections it is art 9.1 min.
If ACN ot TFA is evaporating for 40C column temperature,then my RT should move to right,but its reverse phenomenon.
I think i need to optimise the method with ambient column temperature.My peak is broad and a has tailing of 1.8,so i have to keep 40C temp to make it sharp,It has tailing of 1.6 now.
any comments or suggestions are welcome

thanks

Banala,

Luna is a robust column. something is not right with your method if you have Tailing of 1.6-1.8.
I wish you could tell us more about your structure and sample preparation for further diagnosis.

Why are you so sure about relationship of the content of TFA and the RT?

Any way, thanks for sharing.

For isocratic HPLC, you should increase the column length to 100 or 150mm.

What is k'? (assuming 1mL/min, 10min using 30x4.6mm seems a bit long).

You should adjust organic so k' is around 5.
Increasing TFA to 0.1% might also help (or you could try 0.1% H3P04)

Dear banala_b:
I just want to add another possibility: What if your column heater can't hold a steady temp? Did you check it as part of a periodic PQ program?

Also, are your column dimensions 4.6x300mm? If so, this Luna C18 column should last >100 injections with stable results.

banala_b,

i tend to agree with ym3142, can you give more details for your method?
it looks also like the RT only goes sooner.
is it the case, or the RT also goes back sometimes?

if not then most probably something in your sample is staying inside the column and is blocking the "retaining sites" of the column. the more you inject the more stays in it and the less retention you get for your compound.

maybe you can flush it out, best to get ride of it before you inject it

Hi all,
Thank u for sharing ur valuable suggestions.
The method is for running dissolution samples,( so we have chosen short 30mm column,and my RT is set 9.5 min with total run time of 20min.)
The sample is in 0.1N HCl(dissolution media).I injected freshly prepared sample everytime.
My column heater is perfect in maintaining the sample temp.Everytime the RT is moving left(decreasing RT with time),its never moving in reverse direction.
I searched for pKa of molecule ,i did not get the value.I want to make sure that i am away from pKa zone(may be one of reason for RT shifts).
I want to try with 0.1 % TFA as one of our guys suggested.

all suggestions are welcome