{"id":2308,"date":"2019-04-08T08:10:43","date_gmt":"2019-04-08T06:10:43","guid":{"rendered":"https:\/\/www.kromidas.de\/hplc\/nuetzliches\/hplc-tipp-des-monats\/"},"modified":"2021-05-17T15:25:56","modified_gmt":"2021-05-17T13:25:56","slug":"hplc-tipp-des-monats","status":"publish","type":"page","link":"https:\/\/www.kromidas.de\/en\/hplc\/useful\/hplc-tipp-des-monats\/","title":{"rendered":"HPLC-Tip of the month"},"content":{"rendered":"[vc_row type=”in_container” full_screen_row_position=”middle” column_margin=”default” scene_position=”center” text_color=”dark” text_align=”left” overlay_strength=”0.3″ shape_divider_position=”bottom” bg_image_animation=”none”][vc_column column_padding=”padding-4-percent” column_padding_position=”top” background_color_opacity=”1″ background_hover_color_opacity=”1″ column_link_target=”_self” column_shadow=”none” column_border_radius=”none” width=”1\/1″ tablet_width_inherit=”default” tablet_text_alignment=”default” phone_text_alignment=”default” overlay_strength=”0.3″ column_border_width=”none” column_border_style=”solid” bg_image_animation=”none”][vc_column_text]\n

Why Is It That Peaks Appear Later With the New Column?<\/strong><\/h4>\n

Problem<\/strong><\/p>\n

The new column you ordered has just arrived. Although it has the same stationary phase and the same dimensions and you have used the same supplier, the peaks come later, however constant you kept the chromatographic conditions. Why?<\/p>\n

Answer<\/strong><\/p>\n

Suppose the chromatographic conditions (temperature, eluent incl. pH value, ionic strength etc) and sample preparation have indeed remained constant. Now check whether the breakthrough\u00a0 (front peak, dead time) is also delayed.\u00a0 If that is the case, the flow rate has probably changed. Check your pump and look out for leakages.\u00a0 Once you have made sure with the help of a stopwatch and a measuring cylinder that the flow is okay, look at your column as the likely cause for the discrepancy. The density of packing may vary from one column to the next if your supplier does not follow consistent packing procedure. You now have a column that contains a greater amount of material than its predecessor, e.g. 0.90 g instead of 0.82 g. It is no surprise that all peaks \u2013 including the front peak \u2013 elute later. Let us now look at the second case where the peaks elute later, but the dead time tM<\/sub>, remains constant. If tM<\/sub>\u00a0remains constant while tR<\/sub>\u00a0changes, so too does the retention factor k. Now this means that there is a change in the interaction between the components and the stationary phase, so a chemical change is taking place. If you are certain that the chemistry (temperature, eluent composition, pH value, ionic strength, modifier concentration) have remained constant the problem must lie with the supplier. Although the stationary phase carries the same label, its properties are not identical with the previous one, which shows in the differences between the various batches. However, it is also possible that you have unintentionally changed the surface of the previous column, perhaps by injecting a substance that was irreversibly sorbed, thus modifying the properties of the stationary phase and its interaction with the components.<\/p>\n

Conclusion<\/strong><\/p>\n

Is tM<\/sub>\u00a0constant or not? This is the detail that points you to the cause of your problem \u2013 be it physical changes in your system (flow), at the supplier\u2019s end (density of packing) or chemical problems either at your end (temperature, eluent) or at the supplier\u2019s end (properties of stationary phase) \u2212 unless you have tampered with the chemistry of the stationary phase.<\/p>\n

\u00a9\u00a0 Dr. Stavros Kromidas[\/vc_column_text][\/vc_column][\/vc_row]\n","protected":false},"excerpt":{"rendered":"

[vc_row type=”in_container” full_screen_row_position=”middle” column_margin=”default” scene_position=”center” text_color=”dark” text_align=”left” overlay_strength=”0.3″ shape_divider_position=”bottom” bg_image_animation=”none”][vc_column column_padding=”padding-4-percent” column_padding_position=”top” background_color_opacity=”1″ background_hover_color_opacity=”1″ column_link_target=”_self” column_shadow=”none” column_border_radius=”none” width=”1\/1″ tablet_width_inherit=”default” tablet_text_alignment=”default” phone_text_alignment=”default” overlay_strength=”0.3″ column_border_width=”none” column_border_style=”solid” bg_image_animation=”none”][vc_column_text] Why Is It That Peaks…<\/p>\n","protected":false},"author":1,"featured_media":0,"parent":2306,"menu_order":4,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-2308","page","type-page","status-publish"],"yoast_head":"\nHPLC-Tip of the month - Dr. Stavros Kromidas<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.kromidas.de\/en\/hplc\/useful\/hplc-tipp-des-monats\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"HPLC-Tip of the month - 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