Calculation of capacity factor from KNO3 peak/solvent peak

[Ramachandra]

I have devoloped a gradient method for analysis of pharmaceutical product containing mobile phase 2mM triethyl amine(pH:4.0):acetonitrile. In that, solvent peak eluted at 0.5 min. For calculation of dead time KNO3 was injected, it was eluted at 0.56 min with mobile phase water:acetonitrile(70:30). But, software automatically calulated capacity factor with reference to solvent peak (0.5min). Now, what unretained peak retention time(0.5 or 0.56 min) I have to consider for calculating capacity factor ?

[Uwe Neue]

1. Why do you need to calculate a capacity factor?
2. Since you are not doing science, but rather want reproducible results, a separate injection of a well defined marker such as KNO3 is more suitable than relying on an undefined "solvent peak".

[Ramachandra]

Thank you Uwe Neue, Actually, i am studying retention mechanism of a set of drugs, with different mobile phase additives. So,to study the effect of additives on stationary phase, i need to calculate capacity factor. So, shall i consider dead time of KNO3 for calculation of capacity factor?, and one more doubt, what are the factors effect the dead time of a column? i mean % of organic modifiers, column length, mobile phase pH-----ext? how those will effect?

[danko]

Hi Ramachandra,
Since you’re comparing the effect of different mobile phase compositions, you don’t actually need to pay too much attention to the exact retention time for the dead volume. What you can do is just use the same value (e.g 0.5 min or wathever) all the time. It is after all dead volume (i.e. no retention) you’re after. And the theoretical value should be good enough for your study’s purpose.

Regarding factors affecting the dead volume/time, it is the physical ones that are relevant – i.e. column dimentions, packing density and particle porousity. What does not matter in this context (or it shouldn’t matter) is the chemical parameters, like pH etc. You’ll have to remember that only not retained compounds can be concidered as a dead time markers, which implies that the observed retention is only due to the time the compound is using to travel through the column without experiencing any kind of attraction.

Best Regards

[Ramachandra]

Thank you danko, for your useful information.

[Uwe Neue]

If you are doing a theory study, you need to familiarize yourself with all the intricacies of the determination of the dead volume. In my opinion, a marker that is unretained and has the same size of your analyte is the best choice.

I disagree with danko that you do not need to pay attention how the dead volume is changing with mobile phase composition. This is all part of the game of defining the retention factor.

[danko]

Hi Uwew,
I agree that the dead volume determination is important, but that's where the consensus stops. If the value changes with alteration of the mobile phase composition then the value does not represent the dead volume/time - i.e. the marker is not valid.

Best Regards

[Uwe Neue]

Hi Danko

From a fundamental standpoint, and for theory purposes, the determination of the dead volume is a complicated beast. People have written lengthy papers about this problem, and ripped each others hairs out (figuratively speaking).

The question is the definition of the beginning and the end of the stationary phase. You can determine the liquid volume inside a column by a gravimetric method, for example by measuring the column weight after filling it with methanol and methylene chloride. This gives you the volume of everything that is not attached to the particle. Or, in a reversed-phase column, you can determine the volume that is reached by water by injecting D2O with a RI detector. This method counts the amount of adsorbed organic as part of the stationary phase. Or, you can use what us bloody amateurs are doing, i.e. a polar neutral marker that does not interact with the stationary phase in most solvent compositions, such as uracil. This follows for the most part the D2O results, and is convenient, because you can see uracil in the UV. Or, you can use a marker of the same size as your molecule, but which does not interact with the stationary phase. Such things become important when you want to determine t0 for a large molecule, such as a protein. Or you can do other things (that I personally regard as nonsense most of the time) such as using an ionic marker such as KNO3. Such a molecule will suffer from ion-exclusion effects, and may be problematic for non-ionic molecules, or for molecules with the opposite charge. Or you can do even worse nonsense by looking for the first baseline disturbance in your chromatogram, which is completely undefined since you have no clue what is causing this disturbance.

So, now you see why this is such a mess, and one needs to think through this carefully, if one wants to do real chromatography theory. On the other hand, for most practical problems, such as simply running an analysis and having something to put your hat on, it does not make a difference.

Best regards
Uwe

[HW Mueller]

Those of you who have been with this forum a long time might remember that I have mentioned the use of T2O, which I thought to be an ideal tm marker for a long time. A closer look then revealed that its position can also be dependent on mobile phase composition (H2O based mobile phases).
Even though I pointed out, several times, that determination of tm is not so simple, I never thought that its definition is dependent on analyte and mobile phase.