Chromatography Forum

Hi everyone, I'm student and doing a essay of HPLC. I have got some difficulties with it. I hope to receive your help.
1. If I am using column 250x4.6 C18 to assay Ibuprofen with H3PO4 buffer adjust to pH 2.0. Ratio between Buffer and ACN is 85:15, Flow rate: 2.0ml/min. Retention time of Ibuprofen is about 22 to 25 minute. My teacher wants to shorten time analysis by using shorter column and keep all other conditions. But he also asked me to predict the retention of Ibuprofen before running HPLC. Are there any equations that can help me predict the RT? I just need it 80% correction.

2. I also wants to reduce RT by increase pH to 7.0. But we run of out NaH2PO4. So I wonder if i use H3PO4 and adjust to 7.0 with NaOH to prepare my buffer instead of using NaH2PO4. Will I get the same result?

Thanks in advance for your answers.

What level of student (helps to formulate an appropriate reply)? And what course (instrumentation? Organic chem? Physical chem? Analytical chem?)

I am a student in college of industry. My major is analytical chemistry. The questions is a part of my thesis.
If you want me research is myself , please just give me name of books, documents or articles. I need to seize the matters before running HPLC because it costs much money with a student. Thanks for your concern.

My reason for asking was to be able to frame an answer appropriate to your level of experience ("student" could be anything from secondary school to post-graduate).

Regarding the phosphate buffer: inorganic salts essentially dissociate completely in aqueous solution. As long as you end up with the correct ratio of Na+ to PO4=, you will have the correct pH. It doesn't matter whether you start with H3PO4 and NaOH or NaH2PO4 and NaOH (or, for that matter, NaH2PO4 and Na2HPO4), once in solution, it's just Na+, H+,and PO4=.

Regarding retention: Other things being equal, retention time is directly proportional to column length (if you think about it, it takes twice as long to go twice as far) and inversely proportional to flow rate (again, if you're flowing twice as fast, it will take half as long).

There is an old (and by today's standards, ugly) tutorial for new chromatographers buried on our web site:
http://www.lcresources.com/resources/ge ... dex-2.html

The only thing I'd add to Tom's answer is that as well as pH, you may need to consider the molarity of the buffer. If you are mixing Na2HPO4 and NaH2PO4 and want a 100mM buffer, you might define 100mM as meaning 100mM total phosphate, in which case you could make solutions of 100mM of each buffer and mix. The final concentration of sodium ions will be somewhere between 100 and 200mM because Na2HPO4 contains two sodiums.

If you make the same buffer by mixing phosphoric acid and NaOH, you would have to take a different approach: use enough phosphoric acid that, when made up to volume, it will be 100mM, but prepare it at the moment in less than the full volume. Then add NaOH to pH, and finally make up to the correct volume. If you were to mix 100mM phosphoric acid and 100mM NaOH, you'd obviously end up with a more dilute buffer than if you mix 100mM Na2HPO4 and 100mM NaH2PO4.

This may seem obvious, but I'm always frightened at the range of different approaches that professional analysts come up with when faced with the task of making a buffer. Many articles in respectable journals are flawed because it's not possible to be sure what the buffers were.

Thanks for your answers. With those clues you gave me. I am reaching to my goal. Wish you all the best! Thanks very much!