does chromatogram actually matter or is it forum lore at this point
Genuine question, and the title is the question: does chromatogram actually matter or is it forum lore at this point.
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
Reading a trace posted here, in the order I actually look at things.
Axes first — if the wavelength and the time axis are not labelled, I stop. Then the baseline: where has it been drawn, and does the drawing absorb anything. Then peak shape: fronting, tailing, a shoulder that never resolves. Then the blank, if one was run, for carryover.
Only after all of that do I look at the percentage, and by then I usually know how much weight it deserves. The number is the last thing on the page and the first thing everybody argues about, which is exactly backwards.
Cosigning the wavelength point. Half the disagreements in this board are two people looking at different detectors.
Ran a blank after a high-concentration injection and found the carryover I had been calling an impurity.
integration decisions move the number more than the sample does
a blank injection between samples costs four minutes and settles most arguments
report the method or do not report the number
Retitled: the original claimed a comparison the post does not actually make.
This. A shoulder that does not baseline-resolve is a question, not a quantity.
Bought a second-hand instrument and learned more in six months of fixing it than in three years of reading traces.
Started reporting my own integrations with the baseline choice stated. Arguments in my threads dropped by about half.
- 1Reading a trace posted here, in the order I actually look at things. Axes…6 comments in this branch · started by u/b12_baseline