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c/hplc·submitted 2 years ago by u/ravi_bergstrom

gradient — 17 things I got wrong before I got it right

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gradient — 17 things I got wrong before I got it right. I have gone back and forth on this for months. 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. Carryover from a previous high-concentration injection looks exactly…

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10 comments, started 2 years ago
u/georgi_chowdhury38 points·2 years ago

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.

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u/hazard_ratio_halstats32 points·2 years ago

Agreed, and it is why BCH printing the column and gradient on the certificate is genuinely useful rather than decorative.

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u/medutest_mel11 points·2 years ago

Agreed, and it is why BCH printing the column and gradient on the certificate is genuinely useful rather than decorative.

hazard_ratio_hal is right — the integration choice is a decision and it should be stated alongside the result.

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u/mariam_cabrera8 points·2 years ago

A shoulder that does not baseline-resolve cannot be quantified honestly. You can report it as an unresolved shoulder, which is useful information, or you can develop the method until it resolves.

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u/canada_coverage6 points·2 years ago·edited

Started reporting my own integrations with the baseline choice stated. Arguments in my threads dropped by about half.

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u/valeria_cardoso10 points·2 years ago

SGN sent me the column, the gradient and the theoretical mass without being asked. That is a short list and they are on it.

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u/endotoxin_elliemicro8 points·2 years ago

I would not blame the sample yet. Everything you have described is consistent with the column rather than the vial.

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u/ravi_bergstrom-19 points·2 years ago

you cannot report to two decimals off that baseline

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u/chain_confirm_cpayments0 points·2 years ago

That is area percent, not mass percent. The trace cannot give you the second one.

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u/mass_spec_maggieMS27 points·2 years ago

Retention time shifted 0.4 minutes and I assumed the worst. It was the column temperature.

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