High-performance liquid chromatography: difference between revisions
Diff·revision 10 → 11·23:12, 16 Oct 2024
Difference between revision 10 and revision 11 of High-performance liquid chromatography. 11 lines changed; the page grew by 840 bytes.
| Revision 10 — 12:40, 2 Oct 2024 Areapercent_Ayo (talk) expand §Why method conditions must be stated 3,326 bytes ±0 | Revision 11 — 23:12, 16 Oct 2024 ResolutionRoo (talk) add the system-suitability criteria the method actually specifies 4,166 bytes +840 | ||
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| 12 | | Flow rate = Typically 0.2–1.5 mL/min | 12 | | Flow rate = Typically 0.2–1.5 mL/min |
| 13 | }} | 13 | }} |
| + | 14 | {{hatnote|For the specific mode used for almost all peptide work, see [[Reverse-phase HPLC]].}} | |
| 14 | 15 | ||
| 15 | '''High-performance liquid chromatography''' ('''HPLC''') separates the components of a mixture by passing a solution through a column packed with fine particles. Components that interact more strongly with the packing move more slowly and emerge later; the detector records what emerges against time, producing a chromatogram.{{r|usp621}} | 16 | '''High-performance liquid chromatography''' ('''HPLC''') separates the components of a mixture by passing a solution through a column packed with fine particles. Components that interact more strongly with the packing move more slowly and emerge later; the detector records what emerges against time, producing a chromatogram.{{r|usp621}} |
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| 25 | 26 | ||
| 26 | Resolution between two peaks depends on their retention difference, on peak width, and on the efficiency of the column. Efficiency improves with smaller particles and with longer columns, at the cost of higher back-pressure — the constraint that drove the development of sub-2-micron particles and the instruments capable of running them.{{r|snyder2010}} | 27 | Resolution between two peaks depends on their retention difference, on peak width, and on the efficiency of the column. Efficiency improves with smaller particles and with longer columns, at the cost of higher back-pressure — the constraint that drove the development of sub-2-micron particles and the instruments capable of running them.{{r|snyder2010}} |
| + | 28 | ||
| + | 29 | == What the chromatogram reports == | |
| + | 30 | The chromatogram is detector response against time. Peak area is proportional to the quantity of the eluting species multiplied by its response factor at the detection wavelength; peak position identifies it only by comparison with a known.{{r|usp621}} | |
| + | 31 | ||
| + | 32 | | Quantity | Obtained from | Caveat | | |
| + | 33 | |---|---|---| | |
| + | 34 | | [[Area percent purity]] | Main peak area over total area | Normalised; independent of how much was injected | | |
| + | 35 | | Identity | Retention matched to a [[Reference standard]] | Retention alone is weak identification | | |
| + | 36 | | Content | Area against a calibrated standard | Requires a standard and a validated method | | |
| + | 37 | | Impurity profile | Areas of minor peaks | Only species that elute and absorb are counted | | |
| 27 | 38 | ||
| 28 | == References == | 39 | == References == |