Source of Retention time

{{Infobox concept | name = Retention time | subtitle = Chromatography | image = chromatogram.svg | Symbol = t_R | Depends on = Column, mobile phase, gradient, flow, temperature | Relative measure = Relative retention time, against a reference peak }} '''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.{{r|usp621}} 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.{{r|usp621}} 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.{{r|usp1503,mant1996}} == What it can identify == Matching a sample peak's retention to that of a [[Reference standard|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.{{r|usp1503}} 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 spectrometry|mass confirmation]] or a second chromatographic mode with different selectivity. This is what pharmacopoeial identification tests specify.{{r|usp621}} == Drift and its causes == | 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.{{r|usp621}} 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.{{r|usp1503}} == Reporting on certificates == Naming the retention time of the principal peak is a small addition to a certificate and a useful one: it makes the document more checkable without requiring any additional analysis.{{r|usp1503}} Certificates that report retention alongside the full method conditions allow a subsequent laboratory to reproduce the separation. Those that report neither report a number that cannot be examined. This is a documentary observation about which fields make a determination checkable, and it applies equally to a supplier certificate and to a [[Third-party testing|third-party]] report.{{r|reports}} == References == {{reflist}} <ref name="usp621">United States Pharmacopeia, General Chapter <621>, ''Chromatography''.</ref> <ref name="usp1503">United States Pharmacopeia, General Chapter <1503>, ''Quality Attributes of Synthetic Peptide Drug Substances''.</ref> <ref name="reports">PeptidePedia Wiki community test-report tally, 2024–2026 (self-reported; see [[Project:Sourcing_guidelines]]).</ref> <ref name="mant1996">Mant CT, Hodges RS. "Analysis of peptides by high-performance liquid chromatography." ''Methods in Enzymology'' 271:3–50 (1996). PMID 8782429.</ref> == See also == * [[Reverse-phase HPLC]] * [[High-performance liquid chromatography]] * [[Reference standard]] * [[Area percent purity]] * [[Certificate of analysis]] {{DEFAULTSORT:Retention time}} [[Category:Chromatography]] [[Category:Analytical methods]] [[Category:Analytical science]]