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HPLC elution order

Been on a basic HPLC course past couple of days, and had the exam today. All went fine, apart from one question that we were all a little confused about, everyone was coming up with different reasoning, most of which sounded about right, but I'd like a definitive answer from someone a bit more experienced with it!

Q: A test sample is run on a C18 column. The sample contains a mixture of the following:

Benzoic acid

Aniline

Toluene

Phenol

The mobile phase being used was a 60:40 mixture of MeOH :s-smilie:

odium Citrate buffer (pH 3.2). Aniline eluted first. In what order did the other compounds elute?

Many thanks :smile:

Reply 1

EierVonSatan

Since it's a reverse phase column I would expect aniline -> benzoic acid -> toluene. But I may be wrong I don't do that much HPLC afterall :tongue:

Reply 2

Revd. Mike

And phenol after benzoic acid but before toluene? I think that may have been what I put...

Reply 3

EierVonSatan

oh wait I didn't see phenol in there (the image isn't showing up) :tongue:

I would guess that phenol is more polar than PhCO2H and so come off before (as it dissolves in water at room temp, whilst PhCO2H doesn't)

Reply 4

Revd. Mike

Aah, seems to be working for me!

Hmm, yeah, it was phenol/benzoic acid issue that was confusing me! I know I got toluene in the right place hehe.

Reply 5

EierVonSatan

I honestly wouldn't like to call it :nah:

I'm soo going to TLC them tommorrow

Reply 6

Revd. Mike

Haha, have I peaked your curiousity that much? :biggrin:

Reply 7

EierVonSatan

it just sounds like it's something I should know, but invariably don't

Reply 8

Revd. Mike

EierVonSatan

it just sounds like it's something I should know, but invariably don't

Fair enough xD God knows I've had enough of those moments lol.

Let me know if your TLCing answers the question :biggrin:

Reply 9

Revd. Mike

If it helps, I have some pKa data!

Toluene - 41
Aniline - 27
Phenol - 9.95
Benzoic Acid - 4.21

Reply 10

Revd. Mike

Bump! Surely there must be someone good with HPLC who can help me? It's really bugging me >_<

Reply 11

EierVonSatan

Sorry I didn't get round to TLCing those :tongue:

pKa's won't help I don't think

Reply 12

Revd. Mike

Hehe, no worries :p:

I dug out the pKa stuff because a couple of scientists at work (who also couldn't figure it out!) suggested they might help lol xD

Reply 13

earendil2k

I would say the elution order is aniline; benzoic acid; phenol; toluene.
At pH 3.2 (the pH of the citrate buffer) aniline is totally protonated, thus becoming the most polar compound. Benzoic acid is only partially dissociated into benzoate ion at pH 3.2, and is the second most polar compound considering the ammount of benzoate ion coexisting with benzoic acid at pH 3.2. Phenol is totally undissociated but is more polar than toluene thus being the third eluted compound. Toluene being nonpolar elutes last.