[2026 update] Detection wavelength and why 214 nm and 280 nm disagree

Detection wavelength and why 214 nm and 280 nm disagree — setting out what I have, and where I think it stops being reliable.

Posting the method first, because I know what the first three replies will otherwise be.

• Column: C8, 4.6 x 150 mm, 3.5 um
• Mobile phase: 0.1% TFA in water / 0.1% TFA in acetonitrile
• Gradient: 8% to 70% organic over 31 minutes
• Detection: 214 nm
• Injection: 10 uL
• Sample: tirzepatide, reconstituted to 1.0 mg/mL, injected within an hour

The main peak integrates at 97.5% of total area. There is a small feature on the trailing edge that I cannot decide is a shoulder or a baseline artefact, and that is what I am actually asking about.

Picking up post #8: that is the part I would want checked first.

Method validation is the demonstration that a method can separate the compound from its degradation products and impurities reliably. A method that cannot resolve an impurity from the parent peak will not detect that impurity.

Area percent is not mass percent. It is a proportion of absorbance, weighted by each species' extinction coefficient. For closely related impurities the approximation is usually good. For structurally dissimilar impurities it can be poor.

Before anything else: what was the gradient, and at what wavelength? Area percent at different wavelengths is not the same number even on the same sample because different species absorb differently at different wavelengths. With the method stated, I can tell you something useful. Without it, all I can say is that there is one large peak.

I read post #41 twice before replying, because I had assumed the opposite.

Detection wavelength: 214 nm detects the peptide bond and is relatively insensitive to composition. 280 nm detects aromatic residues and is strongly composition-dependent. Area percent at one wavelength is not area percent at the other.

post #49 is right about the mechanism and I think understates the practical bit.

For anyone arriving from a search: the marked solution above is the direct answer, and the replies underneath it add the caveats that make it safe to use.

post #63 is right about the mechanism and I think understates the practical bit.

This is why a purity figure without the underlying chromatogram is weaker evidence than it appears. It is also why two competent laboratories can report different numbers on the same vial without either being wrong.

On post #68 — agreed on the reasoning, with one qualification.

Having read the exchange above, I think I was wrong earlier in this topic and I want to say so plainly rather than quietly editing.

The correction was fair and I had been repeating something I had not checked carefully enough.

Detection wavelength: 214 nm detects the peptide bond and is relatively insensitive to composition. 280 nm detects aromatic residues and is strongly composition-dependent. Area percent at one wavelength is not area percent at the other.