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Analytics · HPLC & UHPLC · continued

Coming back to: Resolution and tailing factor: real acceptance criteria posts 61–90

This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.

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OstrowskiTL2Member7 Sep 2025#61

Small correction to my own earlier position on Resolution and tailing factor. I had the units the wrong way round, which changes the conclusion by an order of magnitude and therefore changes it entirely.

0 likes 11mo
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j.silvaTL27 Sep 2025#62

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.

The uncertainty is in the assumption, not in the calculation.

2 likes 11mo
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taper_shiftTL38 Sep 2025#63
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h.nwosuTL29 Sep 2025#64
dr.villanueva, post #27: Column chemistry and particle size: smaller particles (1.7 μm) give better resolution and higher efficiency than larger particles (3.5 μm or 5 μm), at the cost of higher back pressure. Newer methods increasingly use smaller particles. Go to post

Worth separating two things that post #60 runs together.

On Resolution and tailing factor: the maintained page in the documentation commons covers the general case with citations and a review date, which is more reliable than any reply here including this one.

29 likes in reply to #27 11mo
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l.oseiTL210 Sep 2025#65

Change the wavelength and the proportions change even though the sample has not. That is the reason the wavelength has to be on the document for the number to mean anything.

That is a description of practice, not a recommendation of it.

0 likes 11mo
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a.asanteTL211 Sep 2025#66

I had written a reply contradicting post #64 and deleted it. Here is what survived.

The confident answers on Resolution and tailing factor and the well-sourced answers are not the same answers, which is the most useful thing I have learned reading this category.

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ThibodeauTL3Regular12 Sep 2025#67

Bookmarking this. I will come back when I have something worth adding.

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m.restrepoTL213 Sep 2025 · edited#68
compounding_ruth, post #36: A request rather than an answer: could whoever has the primary source for Resolution and tailing factor post it? I have seen the claim three times this month and each version had lost a qualifier. Go to post

A chromatogram image at a resolution where you can see peak shape but not baseline detail is worth having and is not the same as the data. Ask for the integration table if the number matters.

Old habit: I write down the expected answer before I calculate it.

0 likes in reply to #36 10mo
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IMainwaringTL3Regular13 Sep 2025#69
n.norgaard, post #59: Post #57 put the caveat in the right place and I want to underline it. What I would check first on Resolution and tailing factor is whether the thing being measured moved or whether the way of measuring it moved. Those look identical in a graph. Go to post

Narrowing post #66, because the general version has more than one answer.

Particle size and column dimensions determine what resolution is achievable at all. A 5 micrometre 250 millimetre column and a sub-2 micrometre 100 millimetre column are different instruments in practice.

2 likes in reply to #59 10mo
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b.adeyemiTL214 Sep 2025#70

I keep a log for Resolution and tailing factor specifically because my memory of it turned out to be systematically wrong in one direction. Six weeks of notes cost nothing and settled it.

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z.okonkwoTL215 Sep 2025#71

The honest answer on Resolution and tailing factor is that it depends, and the useful part is the list of what it depends on. Four items, in rough order of how much they matter.

Most people get the first two right and then argue about the fourth.

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p.iyer_pharmdTL3Pharmacist16 Sep 2025#72
n.laurent, post #33: 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. Adding a source would improve this post and I do not have one to hand. Go to post

A shoulder on the trailing edge is most often a closely related species rather than an artefact. The way to find out is to change the gradient slope, not to argue about the integration.

It took me longer than it should have to see that.

32 likes in reply to #33 10mo
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n.ibarraTL217 Sep 2025#73

Typical suitability criteria are a replicate area relative standard deviation below about two per cent, a tailing factor inside a defined window, a resolution minimum against a specified peak, and a plate-count floor.

One of those cases where knowing the mechanism does not help the decision.

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system_suitabilityTL3Analytical chemist18 Sep 2025 · edited#74

Adding thanks rather than a view. I do not have a view worth the space.

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a.aguirreTL219 Sep 2025#75
e.ferreira, post #43: Particle size and column dimensions determine what resolution is achievable at all. A 5 micrometre 250 millimetre column and a sub-2 micrometre 100 millimetre column are different instruments in practice. One case, stated as one case. Go to post

I read post #71 twice before replying, because I had assumed the opposite.

Counterpoint on Resolution and tailing factor, offered without confidence: the same observation is consistent with a much duller explanation, and nobody has ruled the dull one out.

0 likes in reply to #43 10mo
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v.fontaineTL219 Sep 2025#76
dr.villanueva, post #27: Column chemistry and particle size: smaller particles (1.7 μm) give better resolution and higher efficiency than larger particles (3.5 μm or 5 μm), at the cost of higher back pressure. Newer methods increasingly use smaller particles. Go to post

Purity by chromatography answers "what proportion of what I detected is the intended species". It does not answer how much is in the vial, which is a separate assay.

If this contradicts something upthread, the upthread version may well be the better one.

24 likes in reply to #27 10mo
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n.okwuosaTL220 Sep 2025#77

Since Resolution and tailing factor keeps coming up, it should probably be a maintained page rather than a recurring thread. I am happy to draft it if someone with more direct experience will review it.

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hana.satoTL4 Moderator21 Sep 2025#78

Column chemistry and particle size: smaller particles (1.7 μm) give better resolution and higher efficiency than larger particles (3.5 μm or 5 μm), at the cost of higher back pressure. Newer methods increasingly use smaller particles.

I would rather be precise about what I do not know than vague about what I do.

1 like 10mo
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s.rasmussenTL222 Sep 2025 · edited#79
j.moreau, post #37: Post #35 put the caveat in the right place and I want to underline it. Speaking only to Resolution and tailing factor as I have actually seen it, rather than as it is usually described: the effect is real, it is smaller than the thread suggests, and the variance between people is larger than the effect. Go to post

A chromatogram image at a resolution where you can see peak shape but not baseline detail is worth having and is not the same as the data. Ask for the integration table if the number matters.

0 likes in reply to #37 10mo
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l.trevinoTL223 Sep 2025#80

Adding the measurement that post #79 says would settle it.

One more thing on Resolution and tailing factor that took me far too long to see: the two figures people quote are not measuring the same quantity. Once you notice that, the apparent contradiction disappears.

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j.silvaTL224 Sep 2025#81
e.ferreira, post #43: Particle size and column dimensions determine what resolution is achievable at all. A 5 micrometre 250 millimetre column and a sub-2 micrometre 100 millimetre column are different instruments in practice. One case, stated as one case. Go to post

Typical suitability criteria are a replicate area relative standard deviation below about two per cent, a tailing factor inside a defined window, a resolution minimum against a specified peak, and a plate-count floor.

0 likes in reply to #43 10mo
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m.stephanopoulosTL3Regular24 Sep 2025#82

I came in to disagree and I am leaving without a disagreement.

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z.iyerTL225 Sep 2025 · edited#83

The thing about Resolution and tailing factor that took me longest to accept is that a plausible mechanism is not evidence of an effect. It is a reason to look, not a result.

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l.oseiTL226 Sep 2025#84
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a.asanteTL227 Sep 2025#85
compounding_ruth, post #36: A request rather than an answer: could whoever has the primary source for Resolution and tailing factor post it? I have seen the claim three times this month and each version had lost a qualifier. Go to post

Injection volume matters because column overload distorts peak shape, and an overloaded main peak can swallow a small neighbour. A certificate without injection volume is missing something load-bearing.

29 likes in reply to #36 10mo
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d.bakkerTL228 Sep 2025#86

A relative retention time against a known peak travels much better than an absolute one, and almost nobody reports it.

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p.marchettiTL228 Sep 2025#87

Taking post #85 at face value and following it one step further.

I have been on both sides of the Resolution and tailing factor argument in this category within eighteen months, which should tell you how strong the evidence for either side is.

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b.fonsecaTL229 Sep 2025#88

Adding a small correction to the Resolution and tailing factor summary above rather than a disagreement with it. The substance holds; one of the figures is out by a factor that matters.

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a.adebayoTL230 Sep 2025#89
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r.laurentTL21 Oct 2025#90

Gradient delay volume differs between instruments and shifts the whole chromatogram. It is why a transferred method rarely reproduces retention times exactly on a different system.

That is one dataset and I would not build a rule on it.

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