Chromatography Forum

I have a general question about mobile phase in HPLC. How do we choose a mobile phase? Does it matter if the solution that we use for preparing the standards is different with that used for the mobile phase? For example, does it matter if the mobile phase does not contain any acetonitrile but my standards do?

Thanx in advance!

For best results, prepare your Sample and STD dilutions by using the HPLC mobile phase as the diluent. Variances in analyte peak areas and chromatography will occur with solutions that have markedly different solubility for your query analyte and stationary phase.

further to the above:

If the samples or standards are in a solvent that is more strongly eluting than the running solvent, you are likely to get peak broadening, and in the worst case, little or no retention. The reason is that the analyte is in a local solvent environment in which it won't bind to the stationary phase. It won't bind until it's been diluted out by the running solvent, so instead of collecting in a sharp band on the top of the column, it will elute a long way into the column.

For isocratic methods, the injection solvent should be equal to, or less eluting than the running solvent, and injection volumes should be small. Textbooks will suggest limits, but it's all a matter of how far you are prepared to risk before peak-shape problems cause coelutions and bad quantification.

For gradient methods, things are less fussy. Early-eluting peaks will be affected more seriously than later peaks if you use a strongly eluting injection solvent.

Many people use water/ACN to analyse library and stored compounds dissolved in DMSO. It can be used successfully to determine the concentration of the samples using LC-MC-UV-ELSD and a programme such as FRAC (available from ChemTech Software) to correct for the gradient etc effects in ELSD usage.

Anthony Squibb
Director Chemistry Products
ChemTech Software.

I am not sure what the question was. If the first question was how to find a mobile phase where the analyte elutes, I suggest to read some method development schemes. In brief, run a gradient from water to organic and - if you want an isocratic separation - figure out at which solvent composition the analyte(s) elute. If the question is how to choose acetonitriel or methanol, you will also get the answer from method development schemes.

Thank you all for the answers!

To be more specific, I want to run ochratoxin method with HPLC. My standards are diluted in acetonitrile, the manufacturer's instructions for the immunoaffinity columns recommend using only methanol for eluting the sample, and my method says that for injection volume and mobile phase I shall use acetonitrile and glacial acetic acid. I guess is not a good idea to make this combination...

Ochratoxin can be analyzed by reversed-phase - its a rather simple molecule.

You should have told us that you want to use an affinity column. All the answers above are meaningless for an affinity column. Read the instructions of the manufacturer.

You would have gotten better information if you would have told us up front what your problem is!!!

I dont have experience in HPLC so, I dont really know what is important what is not.

The manufacturer does not say much about what mobile phase is recommended to be used...!

Anyway, thank you all for your answers.

Please don´t take this personally, as you are by no means alone with this sort of thing, but I just wonder:
How do you people do HPLC without really knowing what is important?

Evik,

Affinity chromatography is very, very different from what we thought the discussion above was about.
If you’re planning to utilize this technique - which is quite handy in some situations and especially with regards to it’s specificity - you’ll need to understand it in more comprehensive manner.
Accidentally, acetonitrile will not cause too much trouble in affinity chrom, provided it’s in a reasonably low concentration. Something like 5 – 10% would begin worrying me, for instance.
It reminds me of the “philosophicalâ€

Heh...

Well, I guess the theory is just theory, when there is no practical work involved.


Anyway, all the answers are appreciated! Thank you all!