Chromatography Forum

Hi everyone, I have been analyzing Ibuprofen and have some problems:
1. I use the buffer prepared with H3PO4 (0.05M) then adjusted to pH 2.0 with NaOH. My Retention time is about 23-25 minutes. The RT is very stable but Tailing Factor is too big: 2.5 whereas the required tailing factor is below 2.0.

2. To resolve the problem, I changed the buffer prepared with NaH2PO4 (0.05M) and adjusted to pH 3.0 with CH3COOH. The result is fabulous, the Tailing Factor reduce to 1,8 and the RT is about 22-24 min, the reproduction is good. But when I change pH to 2.0 with the same modifier, everything is ok except the RT which reduced to 10 min. Any reply will be appreciated!

First off, *details* about what you are doing would be extremely helpful:
- what stationary phase
- what column dimensions
- what flow rate
- what organic modifier & concentration
- purpose of the analysis (potency? content uniformity? related substances? . . . ?)

How much acetic acid did you have to use to get the pH down to 2 (the pKa of acetic acid is 4.8 )? It's quite possible that you have so much in there that it's acting as an organic modifier to decrease retention.

First off, *details* about what you are doing would be extremely helpful:
- what stationary phase
- what column dimensions
- what flow rate
- what organic modifier & concentration
- purpose of the analysis (potency? content uniformity? related substances? . . . ?)

How much acetic acid did you have to use to get the pH down to 2 (the pKa of acetic acid is 4.8 )? It's quite possible that you have so much in there that it's acting as an organic modifier to decrease retention.

Sorry for not giving you enough information.
I use the Waters C18 column, 300x3.9.
The flow rate is 1.2ml/min.
Ratio of buffer and Acetonitrile = 60:40
I do the assay only
I did put a lot CH3COOH to the buffer to get pH 2.0 and by the way can you explain the way organic modifier decrease the retention? Is it related to the IP.
I am so confused!

You are doing reversed-phase chromatography (RP).

Mobile phases in RP are generally mixtures of water (or an aqueous buffer) and a polar organic solvent (in your case, acetonitrile). Water is the "weak" solvent and acetonitrile is the "strong" solvent -- which means that the more organic solvent in your mobile phase, the shorter the retention.

Acetic acid is an organic solvent that happens to be somewhat acidic. It has a pKa of about 4.8, so in order to get the pH down to 2, you need somewhere around 30 - 40% acetic acid -- which means that about 1/3 of your "buffer" consisted of acetic acid. In effect your mobile phase is 40% water, 20% acetic acid, 40% acetonitrile.

Now, that column has a dead volume (Vm) of about 2.3 mL (you can estimate that from Vm ≈ 0.5 x L x dc^2 (where L is the column length and dc is the column internal diameter). At a flow rate of 1.2 mL/min that gives you a dead time of about 2 minutes. Which means that a retention time of 22 minutes represents a k' value of 10 (the definition is here: http://www.lcresources.com/resources/TSWiz/hs210.htm ). That is high for a single-component analysis (k' ranges generally run from about 2 to about 10).

Now for advice:

1. If you are following a compendial procedure or a validated method, follow it *exactly*. Minor adjustments are allowed, but what you did was *not* minor.

2. If it's not a validated method, then start over again. Microbondapak C18 was introduced in the early 70's and is the "VW Beetle" of HPLC columns (if you're in North America, I'm not talking about the "New Beetle", I'm talking about the original rear-engine, 60 hp, air cooled bug). It was a great column in its day, but it's day ended three decades ago. You can go to virtually any column vendor's web site and find appropriate conditions for ibuprofen on a newer (shorter, faster) column.

3. You'll be less confused if you learn more about how HPLC works. A great starting point is Mike Dong's book "Modern HPLC for Practicing Scientists". Here's the link to it on Amazon: http://tinyurl.com/cvxumad. I'll also throw in a plug for my web course "Fundamentals of HPLC" running next month:
http://www.lcresources.com/training/trfund.html

Thanks so much for your help, all your advice is much valuable to me. I should learn more before do something in practice.

Not necessarily. I can tell you from my own experience that I have learned much more from my mistakes (and I have made some whoppers!) than from any textbook! The trick is to not make the same mistake more than once.

If nothing else, trying (and failing) gives a good idea about what questions to ask.