Regarding setting of Gradient profiel.

[prashant.pqa]

Dear All
I developing method for combination drugs. In this combination one drug is very polar in nature and other is somewhat non-polar. Also i am using ion pair reagent in mobile phase. In this one drug containg two amine group free and another drug one amine group free. As i did isocratic run in which when i am using 85% buffer then polar drug is eluting at 8.0 min and if i will do 55% buffer other is eluting at 12.6min. I did segmented gradient with decrease of buffer 85 to 45% buffer with segment of 20% of curve 6. but after 12 min there is big hump is there in which non-polar drugs is merging and no peak is there.
Can anyone share there experience in this aspect.

Thanking one and all.
PRASHANT

[Uwe Neue]

I have a few questions:

Did you run the gradient method more than one time, and in all cases you get the hump? If you ran it only once, then the issue could simply be an accumulation of a lot of things on the column. A second or third gradient may show less of the problem and also get you to stable retention times for your analytes. If you have run repeats, and you get the same result, the next question is the wavelength of detection. If you can select a wavelength that is able to detect your analytes and does not see the hump (which may be due to the elution of the ion-pair reagent that is adsorbed on the surface), you may be able to solve the problem.

Overall, you may be better off with a HILIC method on straight silica. Your compounds should be retained nicely, and HILIC would allow you to work without the ion-pair reagent.

[prashant.pqa]

Thank you Uwe Neue.
I have run more than 5-6 times after stabilization of column of initial condition,then also same problem is there.
I am using detection of wavelength 232 nm and Octane sulfonic acid as Ion pair reagent. mobile phase phosphate buffer with pH 3.2.
As one compound is eleuting with 8.0 min and hump will start on 12 min onwards.

Shall i change pH of mobile phase with acetate buffer ?

[Uwe Neue]

I expect that the situation will be worse with an acetate buffer. My suggestion is to go to a higher wavelength for detection.

[dave.cheng]

I think it maybe the solubility issue of the non-polar compounds in your mobile phases, which make the hump peak.
you need do a trial for testing the concentration of MP is the better solubility your non-polar compounds.
good luck!

Dave

[prashant.pqa]

Thank you very much.
As per your opinion the higher wavlength but i tried with PDA detector so i tried to extract the chromatogram with different wavelength but it is not possible. As one compound is having Lambda max 232nm and other is at 228nm . This making some whta difficult.

thank you very much

[prashant.pqa]

Dear sir
as you told that solubility may be the issue of the non-polar compound so it may be giving hump. But as i tried with isocratic with 55% buffer that time i am getting peak with rt 12.5 min. Shall i cheack solubility in mobile phase with ion pair reagent as what composition i am using.

Thanking you

[Mark Tracy]

Given the description of your method, the hump is what you should expect. Your ion-pairing mobile phase needs a couple minutes to re-establish equilibrium with the column after a sudden change. I did something similar (http://www1.dionex.com/en-us/acclaim_li ... 29722.html). A shorter ion-pairing agent will equilbrate faster (I used TFA) and move the hump earlier. You can also try raising the temperature. Of course, you will need to completely re-optimize the concentrations of ion-pairing agent and organic modifier.

[Uwe Neue]

If you can't change the wavelength, Mark's suggestion is worth a try. A flatter gradient for the transition, which will result in an overall longer run time, or a gradient method with a flat gradient without a second isocratic step might be worth trying.

However, I am not very keen on gradient methods with ion-pair reagents, since the reequilibration with the ion-pair reagent is problematic. Considering that you will need a fair amount of work with possibly very little improvement, I am going back to my other suggestion: develop a HILIC method.

[ym3142]

what is the retention of nonplar at 55% buffer? if it was 4 min, so a perfect method.

[prashant.pqa]

Thank you Mark
I am using Octanesulfonic acid 5mM in Phosphate buffer(20mM) pH3.2 with 0.2 % TEA. and 20% THF in Acetonitrile.

What is your opinion on this ? What will the nexxt step ?

Thank you once again.

[Uwe Neue]

Your hump is from the THF. Replace it with more acetonitrile.

[ym3142]

These analyte retention can be manipulated by IP. so stick to Isocratic , good luck

[Mark Tracy]

The last two posts are correct. You should be able to eliminate THF and gradients. Because one compound is doubly charged while the other is singly charged, you can use ionic strength. The 2+ compound will move much faster than the 1+ compound when you change ionic strength. As you raise ionic strength, lower the %MeCN to keep the first peak at a convenient retention.

[prashant.pqa]

Thank you Mark Sir
As you told removing of THF. I will try that also. As what run i have took gradient is necessary with Ion pair . I want to use the same method for biological matrix also. So as hrdophollicity and hydrophobicity matters lot. I am using THF is because as one drug is very much hrdophollic so due to THF it will retain while another drug is hydrophobic it may elute first. Bur with plain Acetonitrile the hydrophobic drug is not eleuting out.



can i have your mail ID so i will send you some chromatogram ?
Thanking you