Controlling the elution order of insulin and its analogs in sub-/supercritical fluid chromatography using methanesulfonic acid and 18-crown-6 as mobile phase additives
© 2023 The Authors. Journal of Separation Science published by Wiley-VCH GmbH..
The purity analysis of therapeutic peptides can often be challenging, demanding the application of more than a single analytical technique. Supercritical fluid chromatography nowadays is a promising alternative to reversed-phase liquid chromatography, providing orthogonal and complementary information. This study investigated its applicability for the separation of human insulin, its analogs and degradation products. A previously published method development protocol for peptides up to 2000 Da was successfully applied to the higher molecular weight insulins (6 kDa). A single gradient method was optimized for all insulins using a Torus DEA column (100 × 3.0 mm, 1.7 μm), carbon dioxide and a modifier consisting of methanol/acetonitrile/water/methanesulfonic acid (65:35:2:0.1, v/v/v/v). Consecutively, the crown ether 18-crown-6, which is well known to complex charged lysine sidechains and other amino functionalities, was added to the modifier to evaluate its impact on selectivity. A decreased retention and a shift in the elution order for the insulins were observed. An inverse effect on retention was found when combined with a neutral stationary phase chemistry (Viridis BEH).
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:46 |
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| Enthalten in: |
Journal of separation science - 46(2023), 22 vom: 02. Nov., Seite e2300520 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Neumann, Jonas [VerfasserIn] |
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| Links: |
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| Themen: |
12EH9M7279 |
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| Anmerkungen: |
Date Completed 27.11.2023 Date Revised 27.11.2023 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1002/jssc.202300520 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM362713871 |
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| 245 | 1 | 0 | |a Controlling the elution order of insulin and its analogs in sub-/supercritical fluid chromatography using methanesulfonic acid and 18-crown-6 as mobile phase additives |
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| 520 | |a © 2023 The Authors. Journal of Separation Science published by Wiley-VCH GmbH. | ||
| 520 | |a The purity analysis of therapeutic peptides can often be challenging, demanding the application of more than a single analytical technique. Supercritical fluid chromatography nowadays is a promising alternative to reversed-phase liquid chromatography, providing orthogonal and complementary information. This study investigated its applicability for the separation of human insulin, its analogs and degradation products. A previously published method development protocol for peptides up to 2000 Da was successfully applied to the higher molecular weight insulins (6 kDa). A single gradient method was optimized for all insulins using a Torus DEA column (100 × 3.0 mm, 1.7 μm), carbon dioxide and a modifier consisting of methanol/acetonitrile/water/methanesulfonic acid (65:35:2:0.1, v/v/v/v). Consecutively, the crown ether 18-crown-6, which is well known to complex charged lysine sidechains and other amino functionalities, was added to the modifier to evaluate its impact on selectivity. A decreased retention and a shift in the elution order for the insulins were observed. An inverse effect on retention was found when combined with a neutral stationary phase chemistry (Viridis BEH) | ||
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