Chromatography Forum

Dear all

can any one give suggestion for this question

i develope a method for gefitinib inetermediate by using gradient HPLC.

My problem i observed two peaks about 1.0 resolution.[RT's at nearly at 25.3min and 25.6 min]

i am using buffer as 0.1% formic acid in water and acetonitrile solvent and C18 column .

The gradient program
TIME BUFFER ACETONITRILE

0.01 90 10
5.00 90 10
20 40 60
35 50 50
37 90 10
40 90 10

so i tried different time programs and differ buffer pH's but i did not get separation for those two peaks.

please give reply

ramesh

Hi ramesh
this is MR patel , i would suggest you change your organic modifer use 70:30(Acetonotrile:Methanol) as organic modifier , if u provide some structure information of related compound you trying to seperating i can give u perfact answer.
100% methanol will give you seperation but at a same time it will increase your runtime too. so u just add methanol % up to you get resolution of 2.5 so u can pass robustnesness parameter during your validation

if not allready done, try some runs at different temperatures.

Hi
This is santosh from Bngalore
You try buffer with sodium or potassium phosphate instead of formic acid and you can play with temperature also!!

Hi ramesh
this is MR patel , i would suggest you change your organic modifer use 70:30(Acetonotrile:Methanol) as organic modifier , if u provide some structure information of related compound you trying to seperating i can give u perfact answer.
100% methanol will give you seperation but at a same time it will increase your runtime too. so u just add methanol % up to you get resolution of 2.5 so u can pass robustnesness parameter during your validation

hi patel

i am already tried with 100% methanol and 80:20((Acetonotrile:Methanol)
as organic modifier but we did not get separation.
In methanol those two peaks comletely merged.
In 80:20, i observed same resolution.
please give another suggestion.

Hi
This is santosh from Bngalore
You try buffer with sodium or potassium phosphate instead of formic acid and you can play with temperature also!!

hi santosh

i want to develope a method with LC-MS compatable.

Hi ramesh
Use Zorbex Eclipse XDB Phenyl column .

There are only six ways to control (change) the selectivity of a reversed-phase separation:
- gradient steepness
- temperature
- organic solvent type
- pH
- additives
- column type

So far, it looks like you have explored gradient steepness and solvent type. Additives are always a tricky proposition with gradients. That leaves temperature, pH, and column type.

• Temperature should be easy to change.
  You can adjust your pH up and maintain MS compatibility with ammonium formate.
  In general, the most different column selectivity to a C8 or C18 column would be an "embedded polar group" column.

Hi Ramesh Janga,

The gradient you describe looks very odd to me. Especially the step from time 20 to 35 min is quite intriguing. I guess one might call it reverse gradient for whatever reason. But I can assure you that this step is unnecessary at the best.
Now you don’t specify the column dimensions nor the flow rate, so I can not be certain of my assumption but I believe the peaks of your interest elute at the end of the gradient “the genuine oneâ€