Baseline width calculation

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Hi all,

Is it possible given:
- specs of the chromatography instrument
- peptide information (e.g. sequence)
- retention time
to calculate(estimate) the baseline width?
What I found so far is:
N = 16* (tr/w)^2 eq . (1)
since I have the retention times I wondered how to determine the value N so I can determine the baseline width w.
For N I found two different formula's that can make an estimate:
N = L/2dp, with L the column length and dp the particle diameter
and
N = L/H, with L again the column width and H the height of the plate.
Is it correct to calculate N based on one of these formula's and then filling it in equation (1), or am I on a completely wrong path?
Sorry, but you're on a completely wrong path.

The values of N that you get from either of those formulas are *estimates* (and very crude ones at that). Using those values to predict actual peak widths is like using EPA automobile mileage estimates (if you're in the US) to predict *exactly* how far you can go on a tank of gas: as they say in the car ads "Your mileage may vary.)! In real life, the actual plate heights are often around half of the theoretical estimate.

To make things worse, your equation (1) assumes isocratic data. Most peptide separations are carried out using gradients. As it happens, you cannot measure the plate count from a single gradient run; you need data from two runs with two different gradient rates (I'm assuming linear gradients here) and even then the math is complex. In general, the plate count is measured isocratically and the same value imputed to the gradient separation. The various modeling programs like DryLab or ChromSword can do the calculation from two gradients, but require the actual peak widths as input.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
I found a link (http://www.sepscience.com/docs/Bespoke/ ... Csol92.pdf) where they actuallly do it this way. My problem is that the retention time is calculated in silico, so I do not have data about the gradients(except that it is assumed to be linear). Is there any other way you know of (without using plate counts) plate to get the baseline width based on peptide info, retention time and/or instrument specs? Any hints where to look for might be helpful!
I found a link (http://www.sepscience.com/docs/Bespoke/ ... Csol92.pdf) where they actuallly do it this way. My problem is that the retention time is calculated in silico, so I do not have data about the gradients(except that it is assumed to be linear).
No, it was actually isocratic.

Any hints where to look for might be helpful!
Get hold of "High Performance Gradient Elution" by Snyder & Dolan (here's a link to it on Amazon: http://tinyurl.com/ct68wxz
) and read pages 31-39.

If you assume a plate number and you know the k* (average k' during elution), then you can estimate the width:
σ ≈ t0 *(1 + k*/2)/(N^0.5)

The catch is that in order to get k*, you have to know the S-value (the slope of the plot of log[k'] versus fraction strong solvent) -- which requires at least two experiments with different gradient steepness. S-values do have a molecular weight dependence (S can be interpreted as the number of strong solvent molecules required to displace one analyte from the stationary phase -- analogous to the Z-value in ion exchange), but it's pretty rough -- peptides probably have S values in the range from about 5 to about 15 -- so unless you do the experiments, the results are little better than guesswork.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
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