{"id":493,"date":"2023-07-31T14:30:59","date_gmt":"2023-07-31T21:30:59","guid":{"rendered":"https:\/\/kingchem.org\/?page_id=493"},"modified":"2025-09-29T20:15:25","modified_gmt":"2025-09-30T03:15:25","slug":"running-a-sample-on-hplc","status":"publish","type":"page","link":"https:\/\/kingchem.org\/?page_id=493","title":{"rendered":"Running a sample on HPLC"},"content":{"rendered":"\n
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  1. Open the Software and select the Project: “Empower “software, click \u201cRun Sample\u201d, and choose the project.<\/li>\n\n\n\n
  2. Edit the instrument method: Select \u2018Instrument Method\u2019, then go to \u201cedit\u201d<\/li>\n\n\n\n
  3. Select the pump module: Click on \u201cW600\u201d (the pump control).<\/li>\n\n\n\n
  4. Access the flow settings: Navigate to the “Flow\u201d tab<\/li>\n\n\n\n
  5. Set the flow rate: For an analytical column, set the maximum flow rate to 1 ml\/min to avoid over-pressurization<\/li>\n\n\n\n
  6. Adjust solvent conditions: Set the solvent composition (e.g., A% and B%). For example, in a Carboxyfantrip analysis, use 90% methanol with 0.1%TFA (A) and 10% THF (B).<\/li>\n\n\n\n
  7. Select the save method: In the method set, choose the previously saved method and click “choose”. (Note: If it’s not listed under “Develop methods”, it won’t appear; ensure it’s properly saved.)<\/li>\n\n\n\n
  8. Select the previously saved method in the method set and click \u201cchoose\u201d (if it is not in the method set it will not be under \u201cdevelop method\u201d). <\/li>\n\n\n\n
  9. Configure the controller: Verify the controller is directed towards the analytical setup: switch from semi prep to analytical mode.<\/li>\n\n\n\n
  10. Bypass the column for Priming: Once the method is selected, turn the lever to “R”, which diverts flow around the column and allows dripping. This will remove any air bubble. Ensure the gas is flowing.<\/li>\n\n\n\n
  11. Start solvent flow: Click “Monitor” to begin pumping solvent and flushing the system.<\/li>\n\n\n\n
  12. Adjust Flow for flushing: Press Direct on the controller to manually increase the flow rate for fast flushing. <\/li>\n\n\n\n
  13. Change flow back to 1 ml\/min after bubbles are gone, then turn the lever to \u201cC\u201d to push through the column<\/li>\n\n\n\n
  14. Right click to customize channels: under 2D increments, uncheck e-SATIN Ch1 under 3D, check the new PAA spectrum. Apply changes, the chromatogram top will show pressure, and the bottom will display the UV vis spectrum<\/li>\n\n\n\n
  15. Once UV-Vis is leveled off, you can inject your sample. Meanwhile, you can increase the flow rate to 2 ml\/min until the system is fully flushed, and any residual compounds are eluted from the column<\/li>\n\n\n\n
  16. When the chromatogram is consistent, adjust the solvent ratio back to your target composition and continue flushing.<\/li>\n\n\n\n
  17. Prepare for injection: Click the “Prepare inject button”, then inject with the lever in the \u201cload\u201d position.<\/li>\n\n\n\n
  18. After injection, move lever down to \u201cinject\u201d then click inject <\/li>\n\n\n\n
  19. After removing the syringe, return the lever to the \u201cload\u201d position.<\/li>\n\n\n\n
  20. Right click to customize channels \u2192 2D increments \u2192 uncheck e-SATIN Ch1 \u2192 3D \u2192 circle new PAA spectrum and let it run for 30-40 min. (for Carboxyfantrip, 10 minutes is fine. Peak comes out around 5:30-6:30.)<\/li>\n\n\n\n
  21. Ensure to save data at the end of run.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-493","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/kingchem.org\/index.php?rest_route=\/wp\/v2\/pages\/493","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/kingchem.org\/index.php?rest_route=\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/kingchem.org\/index.php?rest_route=\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/kingchem.org\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/kingchem.org\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=493"}],"version-history":[{"count":5,"href":"https:\/\/kingchem.org\/index.php?rest_route=\/wp\/v2\/pages\/493\/revisions"}],"predecessor-version":[{"id":941,"href":"https:\/\/kingchem.org\/index.php?rest_route=\/wp\/v2\/pages\/493\/revisions\/941"}],"wp:attachment":[{"href":"https:\/\/kingchem.org\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=493"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}