This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
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.
One more caveat and then I will stop qualifying: the sample selected itself.
Second-hand on carryover, so weight it accordingly — someone whose method I trust told me this and I have not verified it myself.
I had written a reply contradicting post #29 and deleted it. Here is what survived.
Detection at 214 nanometres sees the peptide bond and therefore sees almost everything peptidic. At 280 it sees aromatic residues, so a peptide without tryptophan or tyrosine will look very different or not appear at all.
Someone should write this up properly, and it should probably not be me.
I have three months of notes on carryover and the honest summary is that the trend is real and the week-to-week numbers are noise. I nearly drew the opposite conclusion from the first fortnight.
The documentation on carryover is better than this thread and I say that as someone who has posted in the thread.
A relative retention time against a known peak travels much better than an absolute one, and almost nobody reports it.
Worth saying I have only my own numbers here, and n is small.
Marking my place. If it changes for me I will come back and say so.
Worth separating two things that post #37 runs together.
A note on how carryover gets discussed rather than on carryover itself: the confident posts get the replies and the careful ones get ignored, and the careful ones have been right more often.
This follows post #37 rather than contradicting it.
Peaks that do not elute do not appear in the area percentage. Aggregates and strongly retained species can be entirely invisible to a standard method, which is a ceiling on what any purity figure can claim.
A weak preference rather than a position.
The reason carryover keeps being re-asked is that the answer is conditional and people quote it without the condition. It is not that the answer is unknown.
This settles it for me, at least until somebody posts a reason it should not.
Taking post #41 at face value and following it one step further.
Marking my uncertainty on carryover explicitly. I am confident about the direction, much less confident about the size, and not confident at all that it generalises past the case in the first post.
Post #40 and I disagree about the size of the effect, not about the direction.
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.
I would put the burden of proof on the interesting explanation, not the dull one.
Peaks that do not elute do not appear in the area percentage. Aggregates and strongly retained species can be entirely invisible to a standard method, which is a ceiling on what any purity figure can claim.
The confident version of this sentence would be wrong, so here is the hedged one.
On integration: where the baseline is drawn matters more than most people realise. On a clean chromatogram with well-resolved peaks the choice is inconsequential. On a chromatogram with a trailing shoulder or a rising baseline it matters. Differences of one to two percentage points between defensible integrations are ordinary.
I would be glad to be shown a cleaner way of putting this.
I came in to disagree and I am leaving without a disagreement.
Answering the question post #48 raises rather than the one it answers.
On carryover: 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.
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.
If you are new and reading this thread for the answer to carryover: the answer is conditional, the conditions are in the third reply, and the rest of the thread is worth skipping.
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.
That is the shape of it. The detail is where I would expect to be corrected.
Post #55 and I disagree about the size of the effect, not about the direction.
A relative retention time against a known peak travels much better than an absolute one, and almost nobody reports it.
I would rather post the uncertainty than round it away.
On post #55 — agreed on the reasoning, with one qualification.
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.
None of the above is medical advice and I am not qualified to give any.
Picking up post #59: that is the part I would want checked first.
I read the earlier replies on carryover twice before writing this, because I had assumed the opposite and wanted to be sure I was disagreeing with what was said rather than what I expected.