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.
28 likes 16mo
Analytics · HPLC & UHPLC · continued
Carryover and the ghost peak from last week's standard posts 31–60
This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
Collapsed as off-topic by two members at trust level 3 or above
System suitability testing: injections run before and during the sample run to establish whether the instrument, column and method were performing when the sample was analysed. If suitability did not pass, the sample results from that run are uninterpretable.
On post #29 — agreed on the reasoning, with one qualification.
Method validation is the demonstration that a method can separate the compound from its degradation products and impurities reliably. A method that cannot resolve an impurity from the parent peak will not detect that impurity.
5 likes 16mo
crossover_review, post #10: Coming back to post #8, because the follow-up matters more than the original answer. Reversed-phase separates on hydrophobicity. A peptide is retained on a non-polar stationary phase and eluted by increasing organic solvent. For peptides the mobile phase almost always contains an ion-pairing acid, typically 0.1% TFA, which suppresses… Go to post
Thank you for the correction. I have edited my earlier post with a note rather than silently, so the thread still makes sense to read. The error was mine and it was the kind that comes from remembering a figure instead of looking it up.
Practical note that does not fit anywhere else. Whatever you conclude from this topic, write down what you did and when. The single most useful thing in your own records is not any individual result; it is that they are dated and consecutive.
21 likes 16mo
This follows post #33 rather than contradicting it.
Gradient slope is the single biggest driver of apparent purity differences. A shallower gradient over a longer run resolves more impurities and gives a higher purity figure. A steep gradient produces a tidier-looking chromatogram with fewer visible peaks and gives a lower purity figure. Both are legitimate methods and they will not produce the same number.
9 likes 16mo
nl_translator, post #23: 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. Go to post
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.
plateau_notes, post #16: For anyone arriving from a search: the marked solution above is the direct answer, and the replies underneath it add the caveats that make it safe to use. Go to post
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.
Collapsed as off-topic by two members at trust level 3 or above
Coming back to post #37, because the follow-up matters more than the original answer.
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.
Picking up post #37: that is the part I would want checked first.
Thank you for the correction. I have edited my earlier post with a note rather than silently, so the thread still makes sense to read. The error was mine and it was the kind that comes from remembering a figure instead of looking it up.
27 likes 16mo
j.moreau, post #3: 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. Go to post
I disagree with the reply above, and I think the disagreement is substantive rather than terminological.
The distinction being drawn does not survive when you look at the published data for this specific question. I would be glad to be shown wrong on this, because the version I am arguing against is more convenient.
This is why a purity figure without the underlying chromatogram is weaker evidence than it appears. It is also why two competent laboratories can report different numbers on the same vial without either being wrong.
0 likes 16mo
post #42 is right about the mechanism and I think understates the practical bit.
Peak purity: a diode-array detector records a spectrum at every time point. If a peak contains two co-eluting species with different spectra, the spectrum changes across the peak. A passing peak-purity result says the spectrum is constant; it is weak evidence of homogeneity if the impurities have similar spectra.
2 likes 16mo
Worth separating two things that post #40 runs together.
Before anything else: what was the gradient, and at what wavelength? Area percent at different wavelengths is not the same number even on the same sample because different species absorb differently at different wavelengths. With the method stated, I can tell you something useful. Without it, all I can say is that there is one large peak.
8 likes 16mo
a.eriksen, post #43: post #42 is right about the mechanism and I think understates the practical bit. Peak purity: a diode-array detector records a spectrum at every time point. If a peak contains two co-eluting species with different spectra, the spectrum changes across the peak. A passing peak-purity result says the spectrum is constant; it is weak… Go to post
Before anything else: what was the gradient, and at what wavelength? Area percent at different wavelengths is not the same number even on the same sample because different species absorb differently at different wavelengths. With the method stated, I can tell you something useful. Without it, all I can say is that there is one large peak.
Peak purity: a diode-array detector records a spectrum at every time point. If a peak contains two co-eluting species with different spectra, the spectrum changes across the peak. A passing peak-purity result says the spectrum is constant; it is weak evidence of homogeneity if the impurities have similar spectra.
0 likes 16mo
Gradient slope is the single biggest driver of apparent purity differences. A shallower gradient over a longer run resolves more impurities and gives a higher purity figure. A steep gradient produces a tidier-looking chromatogram with fewer visible peaks and gives a lower purity figure. Both are legitimate methods and they will not produce the same number.
4 likes 16mo
On post #44 — agreed on the reasoning, with one qualification.
Reversed-phase separates on hydrophobicity. A peptide is retained on a non-polar stationary phase and eluted by increasing organic solvent. For peptides the mobile phase almost always contains an ion-pairing acid, typically 0.1% TFA, which suppresses secondary interactions and sharpens peaks.
13 likes 16mo
This follows post #46 rather than contradicting it.
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.
0 likes 16mo
q.zhao_qa, post #25: post #24 answers the question as asked. The question underneath it is different. Before anything else: what was the gradient, and at what wavelength? Area percent at different wavelengths is not the same number even on the same sample because different species absorb differently at different wavelengths. With the method stated, I can… Go to post
I read post #48 twice before replying, because I had assumed the opposite.
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.
n.achebe, post #47: Gradient slope is the single biggest driver of apparent purity differences. A shallower gradient over a longer run resolves more impurities and gives a higher purity figure. A steep gradient produces a tidier-looking chromatogram with fewer visible peaks and gives a lower purity figure. Both are legitimate methods and they will not… Go to post
Having read the exchange above, I think I was wrong earlier in this topic and I want to say so plainly rather than quietly editing.
The correction was fair and I had been repeating something I had not checked carefully enough.
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.
31 likes 16mo
This follows post #51 rather than contradicting it.
This is why a purity figure without the underlying chromatogram is weaker evidence than it appears. It is also why two competent laboratories can report different numbers on the same vial without either being wrong.
16 likes 16mo
j.moreau, post #3: 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. Go to post
System suitability testing: injections run before and during the sample run to establish whether the instrument, column and method were performing when the sample was analysed. If suitability did not pass, the sample results from that run are uninterpretable.
Coming back to post #55, because the follow-up matters more than the original answer.
Method validation is the demonstration that a method can separate the compound from its degradation products and impurities reliably. A method that cannot resolve an impurity from the parent peak will not detect that impurity.
23 likes 16mo
Picking up post #55: that is the part I would want checked first.
Method validation is the demonstration that a method can separate the compound from its degradation products and impurities reliably. A method that cannot resolve an impurity from the parent peak will not detect that impurity.
10 likes 16mo
Worth separating two things that post #55 runs together.
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.
1 like 16mo
post #59 is right about the mechanism and I think understates the practical bit.
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.
0 likes 16mo