Ghost peak in my separation

[sara allen]

Hi freinds,
I try to simultaneous determination of Chrorpheniramine maleate, Pseudoephedrine and acetaminophen.
I use:
C18 column
mobil phase: 0.05 molar KH2PO4,Methanol, Triethylamine(85:15:0.02)
medium:water
in my chromatogram exist 2 peaks under chlorpheniramine and acetaminophen that erea of these peaks are somtimes low and sometimes high and sometimes disappear.
I'm sure from my solvent and I wash the injectore and column very well.
I omit triethylamine and use NaH2PO4 instead of KH2PO4, but this problem persist.
Is these peak, oxygen?
I have to use water for medium.
Thanks,

[Celiagg]

Hello!!

What kind of detector do you use? I need to know to help you better..

Perhaps your reactives are not pure. It may be an isomer of the analytes.

It's probable that in your "mobile phase", your analytes are not stables. You have to be sure that they are not reacting between them.

You have to be sure that with pH you are useing, some of the molecules are not dissociated in two forms, protonated and not.

Frist at all, I would know the pKa of your analytes, and would be sure that in your mobile phase pH is enough to make molecules be not ionized(neutral), or that are just in only one form of ionization.

Second , I would inject them one by one separately, to determine the retention time and look for one ore more peak.

It will give you a clue of what is happening..


If you had bubles of oxygen (air), the peak shape would be really narrow and spike .

Regards,

Celiagg.


If you want, You can write to me to : cegrunder@yahoo.es

[sara allen]

thanks for your suggestion,
this peak be appeared in my placebo,but the wonderful point is that: this peak is sometimes low, somtimes high and sometimes disappear.
I try some system and some mobil phase composition and I this peak exists sometimes in all of my systems.

Thanks,

[HW Mueller]

Could indeed be dissolved air, which usually gives broad peaks, not spikes.

[Celiagg]

Triethilamine makes a strong bond with C18 columns. When you clean the colunm at the end of the day , can you monitorize what is going out of the column? Perhaps are rests of TEA.


I hope not to offend you whit this I'm going to tell you, but it happend to me once: Are you sure that you have elute everything from the previous run before to inject again? Try a run with an hour or more of time viewing what is going out and after, inject the next sample. Perhaps your solvents ore materials are not so pure and you can see something that is dissolved on it.

I'll try to inject just water, just solvent like blank to see what can be.

Do you prepare you KH2O4 solutions everyday? It must be fesh not to permit growth of mycroorganisms...It's important; and Water must be fresh deionized too.

regards

[sara allen]

Thanks Cellia,

I try water, ultered water,oxygened water and I dot get any peak.I examine my mobile phase,too.
I forget to say that flow gradient 0.5-1. is the pressure effective in this case?

[Celiagg]

Hello Sara,

I did not understand you very well...I've never read anything about working with flow gradients. I t's better to work with organic modiffier gradient at constant flow. So pressure changes with viscosity, but you can fix flow and I think it's going to be better.

The optimal flow depends on the column that you're using. If the column is 4.6 mm I.D., normal flow is 1 mL/min more or less. But you have to be sure that cell of detector is not very small: you could damage it.

If column is microbore, flow not may excedd 0.2, 0.3 mL/min.

Well, we know your solvents are not the problem if you can not see peaks in its runs.

I think it's a component of your standars that elute very late. Try to run an injection for 1 hour or 2 hours of runtime, start at High % of organic solvent at the end of the normal time of a run. Try to see if you find something.

If you detect something , try to buy a new fresh standard . It may be contaminated or degradated.

Tell me please your experimental conditions and perhaps i could see what is the probem better.


Regards

[firework1]

Does this ghost peak show up in your blank injection?

[vyncyi]

sorry to budge in on this thread but i'm also having some problems of this sort. i work with methanol, acetonitrile & water system, and ghost peaks keep on appearing.
i've tried it without a column just to test out whether it's my hardware problem. the problem is, it only happens when the gradient mixture consist of water. i've tried to sonicate and purge the system but it just won't work. the funny thing is, with an isocratic water flow, it won't appear.
can anyone suggest anything? thank you very much

[philippem]

To answer the last question. most probably due to impurities in the water, which elutes during gradient.
you concentrate the impurity at high water content and than by gradually increasing the organic solvent % the impurities elutes from the column, this can be very (ir)reproducible (

for teh first "problem" , think it has to do indeed with the fact that not all componetns are eluted frothe column under the above mentionned conditions, so a cleannup with higher% of methanol would help to get rid of these gost peaks.

anyhow I always start with a gradient to see what is in the sample and than you can go for isocratic analysis. Flow gradient, can only be done if you first did a solvent gradient (according to me)


regards

philippe

[sara allen]

I try blank and mobile phase and this ghost peak is not appeare. erea of chlorpheniramine is very low and I have to decrease my flow