GRADIENT HPLC METHOD DEVELOPEMENT

[RAMESH JANGA]

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

[pharmason]

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

[Hollow]

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

[sgkhayade]

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!!

[RAMESH JANGA]

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.

[RAMESH JANGA]

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.

[pharmason]

Hi ramesh
Use Zorbex Eclipse XDB Phenyl column .

[tom jupille]

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.

[danko]

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€