I am facing lots of probleams during by gradient method developement , i am giving linear gradients i am getting so many peaks due to the blank gradient run ,

How to remove all this even when i start gradient with high buffer content to elutes by polar impurity in reverse phase early and after that to elute my components on isocratic one by keeping another composition i am getting too many peks evern after isocratic mobile phase stabilisation of 10 to 15 minutes .

I would like to know where i am going worng i want to discuss in general what can be the causes of this kind of happenings , and how to avoid all this , as i face this kind of problems lotsof time even when i gives linear gradient from 890% buffer to 90% acetonitrile .

with acetonitrile water and phosphate buffers and ion pair like 1- octacne also at 215 nm i am getting tooo many ,
so lets discuss in general what are the points which can impact all this and what can be the solutions .

The first problem area may be the quaility of your solvents, and most importantly the quality of your water. To prove this, do the following:
run two or three blank gradient with normal equilibration with the starting composition of the gradient. Then run the same three blank gradients with triple the equilibration time with the starting composition of the gradient.
I assume that in the second and the third gradient, you get consistent blank peaks in the gradient. If these blank peaks increase, when you do a longer column equilibration, they are from the low quality of your solvent(s), most likely from the water.
If this is indeed the case, you need to think about the sources of contamination in the water, i.e. the quality of your water.

Dear forum memebers
What is the basic criteria for a selection of a gradient profile (mobile phase)during HPLC method developement.

The first thing to try is to use a linear gradient. If the chromatogram obtained from the linear gradient indicates that there is too much resolution at the beginning of the gradient, and not enough at the end of the gradient, you can make the gradient steeper at the beginning and flatter at the end. And vice versa.

Dear Dr. Uwe Neue

Thanks
IS gradient selection depends on analyte property ?

If this question is related to the use of the gradient profile, I do not see a fundamental reason why there should be a link. However, you may get different results (meaning resolution between peaks) from one gradient profile to another.

To eluted protein with a decreasing gradient ( start with 4M NaCL), which side of linear gradient should be the higher concentration?