Chromatography Forum

Hi

I would like to know why the column efficiency test or calculations of theoretical plates are done only in isocratic mode?

To do the calculation in gradient mode requires input from 2 runs with different steepness gradients, and even then the math is fairly complex. The chromatography modeling programs (DryLab, ChromSword, ACD Chromatography Simulator) have the appropriate calculations built-in.

If you really want to get into it, get a copy of Snyder & Dolan's High Performance Gradient Elution book. Here's the link from Amazon

I would like to know why the column efficiency test or calculations of theoretical plates are done only in isocratic mode?

A simple answer (possibly over-simplified) is that the test for theoretical plates is only relevant in isocratic mode.

The basic theory is that you are calculating how well packed the column is. This is done by injecting a small amount of a sample, that has no interaction with the column chemistry, and measure how much it spreads out as it runs through the column. The formula is basically a ratio of the retention time to the peak width (at 50% peak height).
If you calculate HETP (Height Equivalent Theoretical Plates), then you divide the column length by the plate count.

The term “Theoretical Platesâ€

Hi Rande,

According to your post it means that you can never calculated theoretical plates for Reverse phase, normal phase column ??or does it mean that during an isocratic run in reverse phase column no interaction happens between column and the substance injected..??
Plz clarify…

thanks

It is not correct that the plate count needs to be determined from an unretained peak. As a matter of fact, most QC procedures measure the plate count with a well retained peak, to get around extra-column effects.

As Tom has pointed out, it is close to impossible for the practitioner to measure plate counts with gradients, and just to use the isocratic formula for plate count to a gradient chromatogram gives you complete nonsense.

While there are no fixed plate count test procedures between different manufacturers, there are some reasonable coincidences. Most manufacturers measure reversed-phase plate counts with a purely hydrophobic sample, such as toluene, naphthalene, or acenaphtene. Most methods measure the plate count close to the minimum of the van-Deemter curve. But this is where the similarities stop.

I will "stand corrected" in so far as testing reverse phase columns.

As I said, my previous post was over simplified.
Also, my personal experience is primarily with aqueous buffer systems.

I do want to explain my comments about the test injection “having no interaction with the columnâ€

Correct and valid plate count measurements are possible in any type of chromatography. To do the measurement with retained peaks is - for multiple reasons - easiest with reversed-phase columns.

Rande, I agree with you: in some cases, it is best to do the plate count measurement with an unretained peak. In some cases, such as in SEC, there is no way around it. However, with standard RP columns on a standard instrument, a plate count measurement using an unretained peak gives you complete junk due to extra-column bandspreading. This is why I thought it necessary to object to your first post.