Hello

I have a gradient HPLC method:
Flow: 2.5 mL/min
Column: C18
From AcN/H2O 25+75 to AcN 100% within 40 Minutes.

A peak is obtained with this system after 38 Minutes.

When will this peak appear using an isocratic system AcN/H2O 30+70, Flow = 1.0 mL/Min., C18-Column ? I know, it will take a long time, but I'd like to have a rough estimation.

How can I calculate this ?

Thanks
Florian

From a first look I would say that it is going take hours and hours (if it is not stick for ever in your column...). I'm sure that Drylab users could potentially help you, and of course you'll get a theoretical retention factor by extrapolation...

What you need to have is retention information for your compound at a few different ACN concentrations, then you can get a rough idea of its retention at your point of interest based upon the linear relationship between log k and %B (% ACN). Plot %B on the X axis, log k on the Y axis, and you should have a straight line with a negative slope. From this, you can interpolate the retention of your compound at whatever %B you're interested in. I tend to agree with Kostas, tho'. It'll take a dogs age.

FYI, this does not work in any way, shape, or form with proteins being separated by RP - that's a whole different beast.

To a first approximation: never.

To a slightly more precise (but no more accurate) approximation: 60 hours or so.

Depending on the dwell volume of your system, the %ACN at elution is something in the vicinity of 90%. Assuming that you would have a reasonably low k' (let's say 2) at this composition, you can apply the "rule of three": a 10% change in organic results in a 3x change in retention. You're dropping by 60%, so your retention would increase by a factor of 3^6 (call it 700). Assuming a 15-cm x 4.6-mm column, k'=2 would mean a retention time of about 5 minutes, so the isocratic retention at 30% would be in the vicinity of 3500 minutes.

As Kostas pointed out, even if you used DryLab (which will let you predict isocratic retention from gradient calibration runs), extrapolating this far is fantasy rather than prediction.

And as juddc pointed out, this assumes small molecules ("rule of three" does not apply for peptides or proteins).

-- Tom Jupille / LC Resources Inc.

Hi Florian,

Tom beat me to it, but our answers are not dissimilar. Here is my calculation.

You did not give us all the information that one needs, but it is a reasonable start.
I assume that your column is a 15 cm column, with a dead volume around 1.5 mL. I also assume that you have a modern HPLC, with a gradient delay volume of around 1 mL.
What I need to know first, is the solvent composition at the column outlet at the time when your peak leaves the column. It is the solvent composition 2.5 mL earlier than the composition at 38 minutes, with other words the composition at 37 minutes. If all these assumptions were correct, your peak leaves the column at a solvent composition of 94% organic (actually, either your instrument has a much higher gradient delay volume than I assumed, or this compound is very hydrophobic).
Under normal gradient conditions, the peak has a retention factor of about 2 to 3 at the moment when it leaves the column. Also, for a normal small molecule, the natural logarithm of the retention factor changes by about 10 from 0 to 100% organic. You want to know what it will be at a solvent composition at 94%-30% = 64% less than where it elutes in the gradient. This means that the natural logarithm of the retention factor will change by a factor of 0.64*10 = 6.4, i.e from between ln(2) and ln(3) to between ln(4.5) and ln (7). So your retention factor in 30% is somewhere between 100 and 1000.

All of these things change, if this is not as small molecule, but a peptide, or if your gradient delay volume is much larger.

Anyway, if you buy Drylab, it will do this for you.

Uwe Neue