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c/hplc·posted 2 years ago by u/anya_erdogan

[Lab] ERP cagri — PeptideMeter came back 98.8% against a claimed 98.0%

Lab Receipts ×9 Long Haul ×1 Well Actually ×1

Result first, context after: ERP cagri — PeptideMeter came back 98.8% against a claimed 98.0%. Everything below is how it was ordered, stored and sent.

Numbers, in the order they matter: 98.8% and 98.0%.

Chased a ghost peak for three weeks. It was the plumbing. It is always the plumbing.

Carryover from a previous high-concentration injection looks exactly like a small impurity. The blank injection is four minutes and it removes the ambiguity entirely.

Screenshot none of this. Read the whole thread, including the parts where I am told I am wrong.

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28 comments

12 in this archive, depth 3

best — the order this archive was captured in

u/aksel_kjaer1.2k points·2 years ago

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.

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[removed]471 points·2 years ago

[removed by moderator]

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u/sig_figs_samMOD1.2k points·2 years ago

Left up. The integration disagreement in this thread is the most useful thing on the board this week.

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u/peak_area_peteanalytical354 points·2 years ago

Correction: that is a tailing factor, not a plate count. Different diagnostics for different problems.

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u/anya_erdoganOP268 points·2 years ago

Is that baseline drawn by the software or by hand?

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u/mariam_cabrera757 points·2 years ago

Ran a blank after a high-concentration injection and found the carryover I had been calling an impurity.

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u/mateusz_mensah889 points·2 years ago·edited

the column has a history and it shows in the peak shape

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u/fatima_yildiz726 points·2 years ago

This. A shoulder that does not baseline-resolve is a question, not a quantity.

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u/dead_space_doug447 points·2 years ago

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

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u/canada_coverage555 points·2 years ago

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.

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u/marit_mensa279 points·2 years ago

Cosigning the wavelength point. Half the disagreements in this board are two people looking at different detectors.

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About c/hplc

The analytical deep end: RP-HPLC methods, gradient design, integration decisions, LC-MS confirmation, amino-acid analysis, water content, and why two labs can report 98.1% and 96.4% on the same vial and both be right. Post the chromatogram, not the summary line.

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