retention time

[offroad]

hello dear friends...

these are very basic questions, but I just can't figure out how to answer them with confidence.

If I have an HPLC column with known especifications, (size, int diameter etc)... could I say that for a given solvent (mobile phase), component A will always have the same retention time no matter what mixture it is in?

If I am making a calibration curve, and my standard is not pure as expected, let's say 97%, would that difference make a considerable difference in the retention time at the chromatogram? I suppose a simpler question would be: how do we go about not so pure "standards"?

What happens to the retention time of compound A if one of the solvent in the mobile phase is also part of the mixture that A is in?

Thanks.

[lmh]

Question 1:
Given exactly the same column and solvents, is the retention time constant?

In theory, it ought to be, but there are some other factors that can affect retention time. Most obviously, there is the dead volume of the system. Any dead volume between the pump mixer and the column delays the arrival of a gradient at the column, and in isocratic chromatography, any dead volume between the injector and the column delays the arrival of the sample at the column. Dead volume at the other end of the column delays the arrival of a peak at the detector. If you really want the same retention time, you need to use not only exactly the same column and solvents, you need to make sure your hplc is also identical. Generally columns age, life isn't perfect, solvents are never quite the same, and it is really quite hard work to make a method so resilient that it can be transferred from lab to lab without any change in retention time.

Question 2
How does an impure standard affect my retention time?

In theory, it doesn't. You just get 2 peaks, one at the correct retention time for the product, and another one somewhere which corresponds to the impurity. The problem with impure standards is how to interpret the calibration curve. If your standard is only 90% pure on a molar basis then instead of injecting 100pmoles, you have only injected 90, so if you entered the calibration level as 100, you would have a wrong curve and you will consistently overestimate your samples. It's fine if you know how impure your standard is, but if you don't know, it can be hard to tell. Just because the impurity peak is 5% of total peak area, that doesn't mean your standard is 95% pure. The impurity may have a lower or higher signal per pmole.

Question 3
What happens to retention time if the standard or sample are dissolved in the mobile phase?

If you are doing isocratic chromatography, it's probably ideal to dissolve the sample in the mobile phase. If you dissolve the sample in a less-eluting solvent than the mobile phase, there will be little or no change in retention time. If you dissolve the sample in a more eluting phase, the worst-case scenario is that it will elute early (because it is carried into the column in a "bubble" of high-strength solvent before the solvent mixes with the real mobile phase and dilutes it sufficiently for the analyte to bind to the column). This also messes up peak-shape.

[offroad]

Thanks

[JGK]

"If I have an HPLC column with known especifications, (size, int diameter etc)... could I say that for a given solvent (mobile phase), component A will always have the same retention time no matter what mixture it is in? "

If the sample solvent is radically different from the mobile phase you may affect the retention time. For example in a highly aqueous Mobile phase if you introduce a sample in a highly organic matrix the sample (in the organic matix) may produce localised column effects which could affect retention (and retention time).