[Question] what is a reasonable system suitability RSD for this work
Genuine question, and the title is the question: what is a reasonable system suitability RSD for this work.
UV response is not uniform across species. At 214nm you are looking at the peptide bond, which is why it is the working wavelength for this class; at 280nm you only see tryptophan, tyrosine and phenylalanine.
Resolution between two peaks depends on retention, selectivity and efficiency. A shallower gradient buys retention and usually resolution, at the cost of peak width and run time.
Area percent is the integrated area of your peak over the total integrated area at one wavelength on one gradient. Change any of those and the number changes without the sample changing.
Ask me anything specific. Anything general I will probably get wrong.
best — the order this archive was captured in
Why two honest labs report different numbers on the same vial.
Start with the gradient. A shallower slope holds compounds on the column longer and usually separates close-eluting species better. A steeper one gets you a faster run and a fatter peak. If a related substance elutes near the main peak, one method resolves it and reports it separately, the other absorbs part of it into the main peak.
Then integration. Where the baseline is drawn under a shoulder is a decision made by a person or by a piece of software configured by a person. It moves the number.
A point or two of spread between services on this assay is ordinary. Treating one lab as ground truth is how people end up in arguments with suppliers that neither side can win.
I would not blame the sample yet. Everything you have described is consistent with the column rather than the vial.
integration decisions move the number more than the sample does
reproducibility beats resolution if you only get one of them
reproducibility beats resolution if you only get one of them
Adding one practical thing — run the blank. It answers this before anyone has to argue about it.
Gave the same trace to two people I trust and got two integrations about a point apart. That was the most educational afternoon I have had here.
Same method at both labs, or two different gradients?