Chromatography Forum

Hi,

I have question about calculation of resolution in Qual MassHunter. I tried calculate mannualy value of resolution, next I compared it with value calculated by MH but results were not comparable.

Could someone help me with this?
Any idea what is the problem?
I will be very grateful for help.

Please see below calculation.
In mannualy calculation I used two formulas

Results
Formula 1
R = (8.11-7.993)/0.5*(0.135+0.082) = 1.08
Formula 2
R = 1.18*[(8.11-7.993)/(0.023+0.027)] = 2.76
Mass Hunter Qual calculation
R = 2.3

Please see below pictures in link
https://ibb.co/dj9Fpc

Kind regards,
Ewa

Any idea what is the problem?

Yes. The equivalence of your formula 1 and formula 2 is based on the assumption that both peaks are Gaussian*, which your second peak clearly is not. I don't know how MassHunter is making its calculations, but I suspect that it's actually measuring the dispersion (σ) in both distributions and using that as the basis. No matter which way you do it, the results will be virtually meaningless and can only be improved by fixing the peak shape problem. You can't compute your way around bad chromatography.


*To be pedantic about it: the "ideal" shape of a chromatographic peak is approximated by the normal distribution ("Gaussian" or "bell-shaped-curve"). The dispersion in that distribution is measured by the variance, σ^2 (f this were a statistics course, we would be talking about the standard deviation, σ). For the normal distribution, what we think of as "baseline" width is equal to 4*σ, and the width-at-half-height is equal to 2.35*σ. In fact, I could look up the width at any fraction of the maximum height in a table of the normal distribution.

A quick look at your figure shows that the second peak is far from being Gaussian, and I suspect that the first peak also is significantly non-Gaussian. As I said, I don't know the details of how MassHunter does its calculation, but since it has access to the raw data, in principle the dispersion can be measured instead of estimated. Given the actual dispersions (σ1 and σ2) and the retention times (tr1 and tr2), the resolution can be calculated as {(tr1 - tr2)/[2*(σ1 + σ2)]}.

... As I said, I don't know the details of how MassHunter does its calculation, but since it has access to the raw data, in principle the dispersion can be measured instead of estimated. Given the actual dispersions (σ1 and σ2) and the retention times (tr1 and tr2), the resolution can be calculated as {(tr1 - tr2)/[2*(σ1 + σ2)]}.

Acording to help file from MassHunter it uses the same equation you mentioned :
(Tr,2 – Tr,1) / (2Sigma,1 + 2Sigma,2)
where
Sigma is half the peak width at inflection points.

I'm interested in why the peaks seems to be fronting. Do they also do it in lower concentrations under the same conditions?

In low concentation (5ppb) peaks not fronting.

Regards,
Ewa

Which means that you were overloaded, and so probably outside the linear range of your method.