July 02, 2020, 01:11:47 AM
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Topic: Explain the elution order of RP-HPLC for aspirin, acetaminophen and caffeine  (Read 9055 times)

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Offline confusedstud

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A reverse phase HPLC is used to separate a sample containing aspirin, acetaminophen and caffeine. The elution order is as follows 1. Acetaminophen 2. Caffeine and 3. Aspirin

So this trend seems to suggest that acetaminophen is the most polar, followed by caffeine and lastly aspirin. However from many online sites, they stated that the Polarity order is Caffeine>acetaminophen>aspirin. So according to this the elution order should be 1. caffeine 2. acetaminophen and 3. aspirin which is not the case.

What is the reason for this elution order?

Offline MOTOBALL

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You need to consider what a reverse-phase column is. 

1. What is the support ? Is that polar or non-polar ?
2. What is the chemical nature of the reverse-phase that is chemically bonded to the support ?
3. What variety is there in the chemical nature ?
4. There are RP columns and then there are RP columns; they are not all created equal !!

Offline confusedstud

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You need to consider what a reverse-phase column is. 

1. What is the support ? Is that polar or non-polar ?
2. What is the chemical nature of the reverse-phase that is chemically bonded to the support ?
3. What variety is there in the chemical nature ?
4. There are RP columns and then there are RP columns; they are not all created equal !!

1. The support I assume is the stationary phase is C18 which is octadecylsilyl a non-polar stationary phase.
2. I'm not very sure what you mean by chemical nature, I suppose it is a non-polar chemical nature?
3. Again I'm not too sure what you are referring to here.
4. The column is non-polar C18 while the solute is also non-polar in general?

I'm still unable to understand the elution order though..

Offline MOTOBALL

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1. The support I assume is the stationary phase is C18 which is octadecylsilyl a non-polar stationary phase.
2. I'm not very sure what you mean by chemical nature, I suppose it is a non-polar chemical nature?
3. Again I'm not too sure what you are referring to here.
4. The column is non-polar C18 while the solute is also non-polar in general?


1. I think that you need to do some background reading, because a support is only a support while a stationary phase is only a stationary phase; they are not the same.

2. Correct

3. What chemical changes can you make to the reverse-phase, such that it is still non-polar ??
Again, some background reading---look at different reverse-phase chemistries offered by, e.g. Agilent, Thermo, Waters and other manufacturers of HPLC columns.  How do they make their columns ?  What is the support ?

4. Different stationary phases, different carbon loading on the support, different surface coverage ?

Keep going, this is not rocket science---you just need a bit more background knowledge of RP-HPLC, all readily available via the internet.

Offline Arkcon

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Since you're trying to figure this out entirely by polarity, and not considering the nuances of reverse phase HPLC that MOTOBALL: is trying to get you to work with, lets just ask:  Which is more polar, and which is least.  More importantly, how do you know?  No, the answer isn't "some book said ..."  You've said: Caffeine is more polar than acetaminophen is more polar than aspirin.  Why is this so?  Structurally, what makes caffeine more polar?  What group, and under what conditions?  Considering this will help you understand what MOTOBALL: is trying to get across ... the mobile phase environment will change the polarity of a molecule and its interaction with a "typical" reverse phase column.
Hey, I'm not judging.  I just like to shoot straight.  I'm a man of science.

Offline Babcock_Hall

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I am going to take a different tack from my esteemed colleagues.  I once tried to look up "polarity" in a number of textbooks, and I found very few, if any, that even tried to define it.  I am tempted to define polarity operationally, for example by the order of retention times themselves.

Offline MOTOBALL

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Arkcon: I must admit that the mobile phase environment changing polarity of an analyte was a nuance that escaped me.

BabcockHall: Must admit that I would not care to rank caffeine/acetaminophen/aspirin in order of polarity off the top of my head.  Like you, I would define by an experiment. [as an aside: could it be defined theoretically by dipole moment ?]

Going back to Arkcon's comment; order of elution (apparent polarity) can be changed by the mobile phase composition, pH, and the chemical structure of the RP.

Confusedstud: Can you post the various RP-HPLC conditions that have reversed the caffeine/acetaminophen elution order ??

Then look at chemical structures of these two analytes and think about the mobile phase interactions with each.  Again, you can do this---we'll wring the truth out of you sooner or later !!!!  The effort that you put into this will be well worth it, in terms of critical thinking.

Offline Babcock_Hall

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Motoball,

I certainly agree that one component of polarity is dipole moment.  For some molecules I suspect that a good correlation exists.  However, other factors contribute, such as being a hydrogen bond acceptor or donor.  Also consider acetic acid versus palmitic acid.  Same functional group, but very different polarities.  I'll stop now before I get too far off topic.

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