Chromatography Forum

Between Benzene and Acetophenone, which is more polar?

If the answer is Acetophenone, would Acetophenone (in MeOH) elutes before Benzene (in MeoH)in an Reverse phase-HPLC analysis o(f C18 column) with acetonitrile as mobile phase?

If Benzene elutes first, what is/are the reasons?

The general rule in reversed-phase is that more hydrophobic compounds are more strongly retained. We tend to use the word "hydrophobic" as a synonym for "non-polar", but that's not strictly accurate. Hydrophobicity depends not only on a molecule's polarity, but on it's size, with bigger molecules having a larger hydrophobic "contact patch".

Acetophenone is more polar than benzene, but it is also larger. In many cases, it elutes after benzene, so the size effect wins.

The general rule in reversed-phase is that more hydrophobic compounds are more strongly retained. We tend to use the word "hydrophobic" as a synonym for "non-polar", but that's not strictly accurate. Hydrophobicity depends not only on a molecule's polarity, but on it's size, with bigger molecules having a larger hydrophobic "contact patch".

Acetophenone is more polar than benzene, but it is also larger. In many cases, it elutes after benzene, so the size effect wins.

Thanks.

well, it seems that Acetophenone is more polar than Benzene.
But there are many factors that can have effects on the process of elution, such as the structrue of the sample, the size, the functional group, the crystal form of the sample, the polar of the sample and so on.
Under the same mobile phase and same stationary phase, the polar of the sample often can have the main effect which can decide the "elute order" But sometimes it's difficult to judge which factor can have a more obvious influence in the process of elution.
It's not easy to judege which one will "win" unless you see the chromatomap. But I think your experience will help you.

Best Wishes
Norman

well, it seems that Acetophenone is more polar than Benzene.
But there are many factors that can have effects on the process of elution, such as the structure of the sample, the size, the functional group, the crystal form of the sample, the polar of the sample and so on.
Under the same mobile phase and same stationary phase, the polar of the sample often can have the main effect which can decide the "elute order" But sometimes it's difficult to judge which factor can have a more obvious influence in the process of elution.
It's not easy to judge which one will "win" unless you see the chromatomap. But I think your experience will help you.

Best Wishes
Norman

I am supposed to identify the compounds in the unknown sample in this Intro to HPLC and then quantify them. I have the standards for these compounds.

Since only Acetonirtile (not Acetonitrile-Water mixture) is used as mobile phase in this RHPLC analysis, it seems that the best way to determine the rention times of benzene and acetophenone, aside from running them individually, is to prepare a standard sample containing these two compounds at very different concentration. Say make one to be 40 ug and the other to be 200ug.

Say make one to be 40 ug and the other to be 200ug.

That works, but be sure to check the UV spectra to make sure they have comparable responses at whatever wavelength you are using.

Are you sure that you should be using pure acetonitrile as mobile phase ? I would expect both compounds to elute with the dead volume.

How much extra trouble is it to do two runs with one compound in each, rather than one run with one compound in each ? If you do the two runs you are completely certain which peak is which (asuming that there are no interferences, which would cause a problem in the one run scenario as well). If you do one run you have to do extra experimental work to demonstrate that any difference in peak size that you see is not due to a diffeence in response factor.

Peter

Check the UV spectra to make sure they have comparable responses at whatever wavelength you are using.

Oh..I was going to mention the wavelength we were using. The wavelength used so far are 254nm, 230nm and 205 nm.

During the analysis of a standard mixture with 6 known compounds. the peak for acetophenone appears with good resolution at all 3 wavelengths. (I can say that that peak was for acetophenone because acetophenone (alone) had been run at a much higher concentration (200 times) at those 3 wavelengths.

HPLC run for Benzene at the same much higher concentration (200 times) got messed up because the rubber septum in the cap fell inside the vial. So it needs to be rerun and I want to pick a better wavelength to include during the analysis. Any suggestion of a UV wavelength that would show both Benzene and Acetophenone?

well, I will give you some advice to choose a better wavelength.
The wavelength you chose must have a good response to both component.
If we just have one component (usually when we want to quantitate the component), we choose the maximum absorption wave length which usually have the best response to your component.
If we have many component to detect, we should choose a wavelength which can have a response to all of the components, so you can scan every of your standard from 200nm to 400 nm, to detect in which wavelength rank they all can have a good response.
Also you can have a DAD chromatomap if you have a DAD/PDA dector, which maybe helpful for you to choose a better wavelength.
Remember, the wavelenthe you choose must have a relative good response to all of your components. The wavelenth you choose if very important when you want to develop a HPLC method.

Best wishes,
Yours Norman

Do a literature search. UV spectra for both acetophenone and benzene are available.

Do a literature search. UV spectra for both acetophenone and benzene are available.

Yes, I tried and will try again but this time, not just for benzene and acetophenone but for all 6 compounds. Professor wants a better analysis of the standard mixture that shows better peak for benzene.

BTW, I did analysis of benzene standard alone with the same method for acetophenone. It elutes later than acetophenone. So, polarity was the primarily effect.

well, I will give you some advice to choose a better wavelength.
The wavelength you chose must have a good response to both component.
If we just have one component (usually when we want to quantitate the component), we choose the maximum absorption wave length which usually have the best response to your component.
If we have many component to detect, we should choose a wavelength which can have a response to all of the components, so you can scan every of your standard from 200nm to 400 nm, to detect in which wavelength rank they all can have a good response.
Also you can have a DAD chromatomap if you have a DAD/PDA dector, which maybe helpful for you to choose a better wavelength.
Remember, the wavelenthe you choose must have a relative good response to all of your components. The wavelenth you choose if very important when you want to develop a HPLC method.

Best wishes,
Yours Norman

Yes, I will search for the best wavelength for all 6 standard compounds because Professor wants a better analysis of the standard mixture that shows better peak for benzene We (my lab partner and I) were supposed to do that before trying to run analysis for quantification for our unknowns (we each have separate unknown sample). She was using the instruments for quantification runs as soon as she got her unknowns identified and so we got behind on improving separation of the standard mix. The availability of the instruments outside class hour is limited.

BTW, I did analysis of benzene standard alone with the same method for acetophenone. It elutes later than acetophenone. So, polarity was the primarily effect.