Retention time (revision 10)
Old revision·18:51, 6 Jan 2025·SatietySunniva
| Retention timeChromatography | |
|---|---|
| Symbol | t_R |
| Depends on | Column, mobile phase, gradient, flow, temperature |
| Relative measure | Relative retention time, against a reference peak |
| Topic infobox · conventions | |
Retention time is the interval between injection and the appearance of a component's peak maximum at the detector. Under fixed conditions it is characteristic of a substance and reproducible, which makes it the basis of chromatographic identification.[1]
It is not a property of the substance alone. Column chemistry and dimensions, mobile-phase composition, gradient profile, flow rate, temperature and even instrument dwell volume all change it, so a retention time is only meaningful alongside the conditions that produced it.[1]
Relative retention time — the ratio of a peak's retention to that of a reference peak in the same run — is more robust, because system variations that shift both peaks cancel.[2][3]
What it can identify
[edit]Matching a sample peak's retention to that of a reference standard run under identical conditions on the same day is the ordinary chromatographic identification. It is weak evidence on its own: many species can share a retention time on a given system.[2]
Co-injection strengthens it substantially. Spiking the sample with the standard should enlarge the existing peak without producing a shoulder; a shoulder indicates two species.
The strongest form combines retention matching with an orthogonal determination — mass confirmation or a second chromatographic mode with different selectivity. This is what pharmacopoeial identification tests specify.[1]
Drift and its causes
[edit]| Cause | Effect |
|---|---|
| Column ageing | Progressive shift, usually earlier |
| Temperature change | Shift in either direction |
| Mobile-phase preparation | Small shifts, run to run |
| Instrument dwell volume | Systematic offset between instruments |
| Gradient delay | Same, for gradient methods |
Dwell-volume differences are the reason a method transferred between instruments often needs adjustment: the delay between the pump forming a gradient and it reaching the column differs, so retention shifts even with identical settings.[1]
Because of all this, a retention time quoted on a certificate is a within-system value. It is worth reporting — it lets a repeat determination be checked for gross discrepancy — but it is not a transferable identifier.[2]
See also
References
- ^ a b c d United States Pharmacopeia, General Chapter <621>, Chromatography.
- ^ a b c United States Pharmacopeia, General Chapter <1503>, Quality Attributes of Synthetic Peptide Drug Substances.
- ^ Mant CT, Hodges RS. "Analysis of peptides by high-performance liquid chromatography." Methods in Enzymology 271:3–50 (1996). PMID 8782429.