PFChrom’s Complete Solution
In order to model the chromatographic separation, instrument and measurement effects must be accounted in some manner.
A complete solution consists first of a comprehensive and effective fitting of the chromatographic model with IRF, the full convolution integral. When such fits are possible, this will maximize the accuracy, but at a computational cost where the fitting time for a large data set can require up to several minutes, depending on the power of your machine and the number of peaks being fitted within the data.
PFChrom also offers the option to independently estimate the IRF, the instrument response function, for a given instrument, column, and prep, using either nonlinear fitting or Fourier procedures, typically with well-behaved standards. Once the IRF has been accurately estimated, you can preprocess all data sets for which this IRF is applicable in the Fourier domain prior to fitting, even the messiest of data sets where peaks are not baseline-resolved. You can then fit up to 25 preprocessed data sets in a single step. By first removing all instrument effects, the fitting will involve only closed-form models and will be exceptionally fast, on the order of a second or so for each data set fitted.
Fourier Estimates of IRF Parameters
PFChrom uses its own specialized genetic algorithm for estimating the IRF parameters directly from a standard. The algorithm can be quite accurate, and further it is both simple and exceedingly fast.
Fitted Estimates of IRF Parameters
A more rigorous and accurate IRF estimation involves fitting an appropriate standard to determine an average IRF for a given instrument, column, and prep. This will involve the fitting of the IRF-bearing chromatographic models, the convolution integrals.
The IRF procedure in PFChrom offers left-sided, right-sided, and symmetric instrument response models, and both convolution and deconvolution with a dozen different IRFs built into the program. The GA algorithm is available for optionally estimating each IRF prior to fitting.
When an IRF has been determined by nonlinear fitting of standards, its parameters can be directly copied from one or more fits which can be averaged or individually selected. A sophisticated Fourier filter can then be optionally used to remove noise introduced by the deconvolution.
Since Fourier deconvolution will add some noise to the data, even with a filter, and since in general, averaged IRF parameters will not precisely match a given data set, this preprocessing step of removing an IRF in the Fourier domain will not generate as low an error in the fitting. For those who want the swiftest possible processing, however, this is a methodology which will turn minutes into seconds since fits are nearly immediate when closed-form models can be used.
PFChrom thus supports those who have a large amount of production data to process, where speed of processing is important, where an average IRF suffices, and where an absolute optimization of each data set’s specific IRF is not needed. There is also the instance of especially messy data sets with overlapping or hidden peaks where the IRF cannot be accurately fit in this single step using an IRF-bearing convolution model.
For the R&D scientists who want to model peaks to the absolute minimum of error, who have peaks amenable to this single step convolution model fitting, and who have no issue with a peak fit which may require a few minutes of computation, PFChrom offers the maximum of accuracy and the ability to characterize every part of the modeling.
PFChrom does more than accurately quantify the moments of the eluted solute peaks and their specific deconvolutions. You also model the measure of distortion in the instrument, flow path, and detector, and you quantify the non-ideality in the chromatographic model describing the separation.
Further details can be found in the IRF Deconvolution and Fitting tutorial.