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.
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Analytics · Impurities & related substances · continued
Named, unnamed and unspecified impurities as regulatory categories — the long version posts 91–120
This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
Coming back to post #90, because the follow-up matters more than the original answer.
Acetate content: counter-ion content. Trifluoroacetate or acetate from the salt form of the peptide. Affects mass calculations and should be stated on a complete certificate.
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s.vogel, post #72: Deletion sequences (incomplete coupling during synthesis): lower in mass by one residue. Chromatographically they usually elute earlier or later depending on the residue's hydrophobicity. They are the most common impurity in solid-phase synthesis. Go to post
post #92 answers the question as asked. The question underneath it is different.
Dimer and higher-order multimers: two or more peptide molecules bonded together. They appear at double the mass and higher. They may or may not separate from the monomer on HPLC depending on the method.
p.novak, post #1: Named, unnamed and unspecified impurities as regulatory categories — the long version — setting out what I have, and where I think it stops being reliable. A documentation question rather than an analytical one. I have a certificate in front of me that reports a purity figure, names a technique, gives a wavelength, and stops. No… Go to post
Truncation products: fragments from incomplete synthesis or from degradation. They elute quite differently from the intact peptide because they are much smaller and have different hydrophobicity. They are usually well separated.
This follows post #92 rather than contradicting it.
Oxidation at methionine and tryptophan: adds 16 per oxygen. Usually elutes earlier. Oxidation is common in storage, especially if the solution is exposed to light or if antioxidants are not present.
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I read post #94 twice before replying, because I had assumed the opposite.
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.
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Collapsed as off-topic by two members at trust level 3 or above
Incomplete deprotection: mass higher by the protecting group mass. Usually markedly later eluting. A synthesis artifact from incomplete removal of protecting groups.
m.yildiz, post #11: Deamidation at asparagine and glutamine: adds 1 approximately. Frequently appears as a close-eluting pair. It is a chemical modification that occurs during storage. Go to post
Aggregates: multiples of the monomer mass. May not elute at all under a standard reversed-phase method. A species that does not come off the column does not appear in the area percentage.
s.oyelaran, post #43: Deamidation at asparagine and glutamine: adds 1 approximately. Frequently appears as a close-eluting pair. It is a chemical modification that occurs during storage. Go to post
Deamidation at asparagine and glutamine: adds 1 approximately. Frequently appears as a close-eluting pair. It is a chemical modification that occurs during storage.
Residual solvents: traces of solvents used in purification. These are usually tested by gas chromatography, not by HPLC. A specification for residual solvents should be stated separately from the purity.
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Off-target structures: if the sequence synthesis goes wrong, a completely different amino acid can be incorporated. The resulting off-target peptide is a structural isomer with the same mass but a different sequence. No chromatographic purity method detects this without a reference standard.
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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.
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f.weiss, post #88: Residual solvents: traces of solvents used in purification. These are usually tested by gas chromatography, not by HPLC. A specification for residual solvents should be stated separately from the purity. Go to post
post #102 answers the question as asked. The question underneath it is different.
Deletion sequences (incomplete coupling during synthesis): lower in mass by one residue. Chromatographically they usually elute earlier or later depending on the residue's hydrophobicity. They are the most common impurity in solid-phase synthesis.
r.bakken, post #9: Deletion sequences (incomplete coupling during synthesis): lower in mass by one residue. Chromatographically they usually elute earlier or later depending on the residue's hydrophobicity. They are the most common impurity in solid-phase synthesis. Go to post
Acetate content: counter-ion content. Trifluoroacetate or acetate from the salt form of the peptide. Affects mass calculations and should be stated on a complete certificate.
Incomplete deprotection: mass higher by the protecting group mass. Usually markedly later eluting. A synthesis artifact from incomplete removal of protecting groups.
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I read post #104 twice before replying, because I had assumed the opposite.
Aggregates: multiples of the monomer mass. May not elute at all under a standard reversed-phase method. A species that does not come off the column does not appear in the area percentage.
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m.dumitru, post #103: post #102 answers the question as asked. The question underneath it is different. Deletion sequences (incomplete coupling during synthesis): lower in mass by one residue. Chromatographically they usually elute earlier or later depending on the residue's hydrophobicity. They are the most common impurity in solid-phase synthesis. Go to post
Oxidation at methionine and tryptophan: adds 16 per oxygen. Usually elutes earlier. Oxidation is common in storage, especially if the solution is exposed to light or if antioxidants are not present.
Picking up post #106: that is the part I would want checked first.
Residual solvents: traces of solvents used in purification. These are usually tested by gas chromatography, not by HPLC. A specification for residual solvents should be stated separately from the purity.
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Related substances: compounds chemically related to the target peptide but not the target peptide itself. The standard method separates them and reports them as area percent. How related they can be before they exceed specification is a regulatory question.
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m.dumitru, post #103: post #102 answers the question as asked. The question underneath it is different. Deletion sequences (incomplete coupling during synthesis): lower in mass by one residue. Chromatographically they usually elute earlier or later depending on the residue's hydrophobicity. They are the most common impurity in solid-phase synthesis. Go to post
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.
Coming back to post #111, because the follow-up matters more than the original answer.
Deletion sequences (incomplete coupling during synthesis): lower in mass by one residue. Chromatographically they usually elute earlier or later depending on the residue's hydrophobicity. They are the most common impurity in solid-phase synthesis.
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Picking up post #111: that is the part I would want checked first.
Off-target structures: if the sequence synthesis goes wrong, a completely different amino acid can be incorporated. The resulting off-target peptide is a structural isomer with the same mass but a different sequence. No chromatographic purity method detects this without a reference standard.
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j.restrepo, post #5: Aggregates: multiples of the monomer mass. May not elute at all under a standard reversed-phase method. A species that does not come off the column does not appear in the area percentage. Go to post
Disulfide formation: if a peptide contains cysteine, it can form disulfide bonds with itself or with other molecules. Under oxidising conditions multiple species appear. Reducing conditions (like DTT) convert them back.
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.
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I read post #115 twice before replying, because I had assumed the opposite.
Truncation products: fragments from incomplete synthesis or from degradation. They elute quite differently from the intact peptide because they are much smaller and have different hydrophobicity. They are usually well separated.
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s.oyelaran, post #43: Deamidation at asparagine and glutamine: adds 1 approximately. Frequently appears as a close-eluting pair. It is a chemical modification that occurs during storage. Go to post
Incomplete deprotection: mass higher by the protecting group mass. Usually markedly later eluting. A synthesis artifact from incomplete removal of protecting groups.
post #119 answers the question as asked. The question underneath it is different.
Oxidation at methionine and tryptophan: adds 16 per oxygen. Usually elutes earlier. Oxidation is common in storage, especially if the solution is exposed to light or if antioxidants are not present.
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