UV and diode-array detection
Knowing what the detector responds to, and using peak purity properly.
Purpose
Knowing what the detector responds to, and using peak purity properly.
This page exists so that a discussion about a number can start from a shared understanding of what produced it. It is written for a reader who is technically literate but not a practising analyst.
How it works
A single-wavelength ultraviolet detector reports absorbance at one wavelength. A diode-array detector records a spectrum at every time point, which allows a second kind of question: is the spectrum constant across the peak?
If a peak contains two co-eluting species with different spectra, the spectrum will change across the peak. That is what a peak purity calculation detects. If the two species have similar spectra — which closely related peptide impurities frequently do — the calculation will find nothing, and a passing peak purity result is therefore weak evidence of homogeneity.
What determines the result
Wavelength choice determines what you can see at all. At 214 nm the peptide bond dominates, so response is roughly proportional to peptide length. At 280 nm only tryptophan, tyrosine and cystine contribute meaningfully, so a peptide with none of those is nearly invisible.
Typical criteria
| Parameter | Typical value or criterion |
|---|---|
| Peptide bond detection | 205 – 220 nm, commonly 214 nm |
| Aromatic detection | 275 – 285 nm, commonly 280 nm |
| Peak purity angle | Below the threshold angle for a homogeneous peak |
| Reference wavelength | Set outside the analyte absorbance region |
These are typical values, not universal standards. The criteria that apply to a specific result are the ones in the validated method that produced it, which is why a report without a method identifier is harder to interpret than one with.
What it cannot tell you
Every analytical method answers one question. The most common error in this community's discussions is treating an answer to one question as an answer to another: a mass match read as a purity result, a purity figure read as a content determination, a sterility test read as a guarantee of safety.
When you read a report, write down which question was asked. The rest follows.
Related pages
Other method notes in this commons cover the neighbouring techniques, and the certificate interpretation page covers how these results get reported. The hplc subcategory is where the discussion happens.
Revision history
| 2026-07-02 | q.zhao_qa | Citation sweep: two references did not support the sentences attached to them and have been replaced. |
| 2026-02-14 | unit_conversion | Annual review: checked every figure against its source. Two rounded values tightened. |
| 2026-01-18 | ms_holloway | Corrected a unit label in the second table — it read mg where it should have read mg/mL. |
| 2025-11-24 | sourced_claims | Reverted an unsourced change and asked the editor to re-apply it with a citation. |
| 2025-08-19 | j.delacroix | Initial promotion from the source topic. Structure taken from the marked solution. |
Every edit to a maintained document records its author and a note explaining the change. An edit without a note may be reverted by any wiki editor under R9, and the revert is logged. Disagreements about content belong on the source topic rather than in the revision history (R10).
Related documents
- Reversed-phase HPLC for peptidesSeparating a peptide from its related substances and quantifying the result as area percent.
- Peak integration in practiceUnderstanding how much of a reported purity figure is the sample and how much is the integration.