{"id":51842,"date":"2025-03-24T00:00:00","date_gmt":"2025-03-24T00:00:00","guid":{"rendered":"https:\/\/discover.restek.com\/uncategorized\/using-the-ezlc-modeler-for-cannabinoid-separations-part-2\/"},"modified":"2026-02-05T20:27:07","modified_gmt":"2026-02-05T20:27:07","slug":"using-the-ezlc-modeler-for-cannabinoid-separations-part-2","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/de\/blogs\/gnbl4849\/using-the-ezlc-modeler-for-cannabinoid-separations-part-2","title":{"rendered":"Using the EZ<\/em>LC Modeler for Cannabinoid Separations\u2013Part 2: Using EZ<\/em>LC Software to Monitor Effects of Ammonium Formate Concentrations on Cannabinoid Separations"},"content":{"rendered":"\n

Using the EZ<\/em>LC chromatogram modeler is a fast and easy way to see how different variables can affect separations. Part one<\/a> of this series discussed how to use EZ<\/em>LC software as well as how to optimize an existing method. For cannabinoid analysis, Restek elected to use formic acid and ammonium formate as preferred mobile phase additives. This blog will demonstrate how ammonium formate buffer concentrations can impact cannabinoid separations.<\/p>\n\n\n\n

Cannabinoid separations can be challenging. They can often be difficult due to overlapping hydrophobicity. However, the presence of acidic cannabinoids allows us to utilize an additional lever: pH. The pKa range of these acidic cannabinoids falls between 2.90\u20134.75. Depending on the pH of the mobile phase, the retention time, and sometimes the elution order, of cannabinoids can change. The neutral cannabinoids will remain the same, however the acidic cannabinoids can change retention time because they often fall on the pKa slope.<\/p>\n\n\n\n

To dive a little deeper into this phenomenon, let\u2019s discuss pKa. A low pKa indicates a strong acid, while a high pKa indicates a weak acid. When developing a chromatographic method, it is ideal to have a mobile phase with a pH 2 units away from the pKa value. This causes the analyte to become either fully protonated or deprotonated. When the pH is above the pKa value, the compound is deprotonated, and when the pH is lower than the pKa the compound will be fully protonated.<\/p>\n\n\n

Figure 1:<\/strong>\u00a0Retention behavior of acids and bases on a standard reversed-phase column<\/div>
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\"\"<\/figure>\n\n<\/div><\/div><\/div>\n\n\n
<\/div>\n\n\n\n

 As the pH is lowered, the portion of the total molecules present in their protonated form increases and the number of deprotonated molecules decreases, so the overall polarity of the acid decreases, resulting in increased retention times. In relation to cannabinoids, most common cannabinoids are either neutral or acidic. By adding buffer to the mobile phase, the acidic cannabinoids can become deprotonated, depending on the pH of the buffer, resulting in higher affinity for the mobile phase and reduced retention.<\/p>\n\n\n\n

Using EZ<\/em>LC software and the same method parameters from above, we can demonstrate the movement of these analytes by changing the concentration of the ammonium formate in the mobile phase. In the following examples, you will see the highlighted compounds show changes in retention times along with some changes in elution order as well. CBNA switches with \u03949-THC while THCA-A switches with CBC.<\/p>\n\n\n

Figure 2:<\/strong>\u00a00 mM ammonium formate + 0.1% formic acid<\/div>
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Figure 3:<\/strong>\u00a04 mM ammonium formate + 0.1% formic acid<\/div>
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<\/div>\n\n\n
Figure 4:<\/strong>\u00a08 mM ammonium formate + 0.1% formic acid<\/div>
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Figure 5:<\/strong>\u00a012 mM ammonium formate + 0.1% formic acid<\/div>
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\"\"<\/figure>\n\n<\/div><\/div><\/div>\n\n\n
<\/div>\n\n\n\n

 As you can see, adjusting the buffer concentration is another tool that can be utilized when developing methods for compounds that are ionizable. It is important that mobile phases are prepared fresh to ensure that the pH remains constant, so the method remains reproducible.<\/p>\n\n\n\n

Tune in for part 3 of this series to learn why cannabinoids that are not in your panel still matter!<\/p>\n\n\n\n

<\/div>\n\n\n\n

Resources and Further Reading<\/strong><\/p>\n\n\n\n

Dolan, J. (n.d.). Back to basics: The role of pH in retention and selectivity. Chromatography Online. https:\/\/www.chromatographyonline.com\/view\/back-basics-role-ph-retention-and-selectivity<\/a><\/p>\n\n\n\n

<\/div>\n\n\n\n

Check Out the Full Blog Series in the Related Resources below. <\/strong> <\/p>\n\n\n\n

<\/div>\n","protected":false},"excerpt":{"rendered":"

The second in a four-part series, this blog demonstrates how to use Restek’s free EZ<\/em>LC chromatogram modeler to predict the effects of mobile phase additives on cannabinoid chromatography.<\/p>\n","protected":false},"author":39,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_kadence_starter_templates_imported_post":false,"_kad_post_transparent":"","_kad_post_title":"","_kad_post_layout":"","_kad_post_sidebar_id":"","_kad_post_content_style":"","_kad_post_vertical_padding":"","_kad_post_feature":"","_kad_post_feature_position":"","_kad_post_header":false,"_kad_post_footer":false,"footnotes":""},"categories":[9],"tags":[],"industries-application":[2263,2282],"post-badge":[],"resource-type":[],"product-library":[],"resource-technique":[2343],"hf_cat_post":[623],"ppma_author":[459],"class_list":["post-51842","post","type-post","status-publish","format-standard","hentry","category-blogs","industries-application-cannabis-ja","industries-application-medical-recreational-cannabis-ja","resource-technique-liquid-chromatography-ja"],"acf":[],"taxonomy_info":{"category":[{"value":9,"label":"Blogs"}],"industries-application":[{"value":2263,"label":"\u30ab\u30f3\u30ca\u30d3\u30b9"},{"value":2282,"label":"\u533b\u7642\u7528\uff5c\u55dc\u597d\u7528 \u30ab\u30f3\u30ca\u30d3\u30b9"}],"resource-technique":[{"value":2343,"label":"\u6db2\u4f53\u30af\u30ed\u30de\u30c8\u30b0\u30e9\u30d5\u30a3\u30fc"}]},"featured_image_src_large":false,"author_info":{"display_name":"Melinda Urich","author_link":"https:\/\/discover.restek.com\/de\/author\/melinda-urich\/"},"comment_info":0,"category_info":[{"term_id":9,"name":"Blogs","slug":"blogs","term_group":0,"term_taxonomy_id":9,"taxonomy":"category","description":"","parent":0,"count":442,"filter":"raw","cat_ID":9,"category_count":442,"category_description":"","cat_name":"Blogs","category_nicename":"blogs","category_parent":0}],"tag_info":false,"authors":[{"term_id":459,"user_id":39,"is_guest":0,"slug":"melinda-urich","display_name":"Melinda Urich","avatar_url":{"url":"https:\/\/discover.restek.com\/wp-content\/uploads\/people-ulrich-melinda.jpg","url2x":"https:\/\/discover.restek.com\/wp-content\/uploads\/people-ulrich-melinda.jpg"},"0":null,"1":"","2":"","3":"","4":"","5":"","6":"","7":"","8":""}],"_links":{"self":[{"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/posts\/51842","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/users\/39"}],"replies":[{"embeddable":true,"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/comments?post=51842"}],"version-history":[{"count":9,"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/posts\/51842\/revisions"}],"predecessor-version":[{"id":87098,"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/posts\/51842\/revisions\/87098"}],"wp:attachment":[{"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/media?parent=51842"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/categories?post=51842"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/tags?post=51842"},{"taxonomy":"industries-application","embeddable":true,"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/industries-application?post=51842"},{"taxonomy":"post-badge","embeddable":true,"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/post-badge?post=51842"},{"taxonomy":"resource-type","embeddable":true,"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/resource-type?post=51842"},{"taxonomy":"product-library","embeddable":true,"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/product-library?post=51842"},{"taxonomy":"resource-technique","embeddable":true,"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/resource-technique?post=51842"},{"taxonomy":"hf_cat_post","embeddable":true,"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/hf_cat_post?post=51842"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/discover.restek.com\/de\/wp-json\/wp\/v2\/ppma_author?post=51842"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}