{"id":2016,"date":"2026-02-13T11:17:12","date_gmt":"2026-02-13T10:17:12","guid":{"rendered":"https:\/\/bioeducator.eu\/?page_id=2016"},"modified":"2026-02-13T11:17:13","modified_gmt":"2026-02-13T10:17:13","slug":"reversed-phase-chromatography","status":"publish","type":"page","link":"https:\/\/bioeducator.eu\/?page_id=2016&lang=en","title":{"rendered":"Reversed-phase Chromatography"},"content":{"rendered":"\n

Reversed-phase chromatography (RP-HPLC) is the foundation of modern chemical analysis, accounting for over 80% of all separations performed in R&D, quality control and clinical diagnostics laboratories. Its dominance stems from its versatility, the stability of stationary phases and its excellent compatibility with mass detection (LC-MS).<\/p>\n\n\n\n

\"Reversed-Phase<\/figure>\n\n\n\n

Physicochemical retention mechanism<\/h2>\n\n\n\n

In the article „Fundamentals of chromatography<\/a>„, we have already explained the difference between normal phase and reversed phase chromatography. However, it is worth returning to this topic. It concerns the arrangement of phases (mobile and stationary). In normal phase chromatography, the stationary phase is polar and the mobile phase is non-polar (strong organic solvents). As the name suggests, in reverse phase chromatography, it is exactly the opposite. The stationary phase is non-polar (hydrophobic) and the mobile phase is polar.<\/p>\n\n\n\n

Interestingly, inverted phases are the most common today. This is because they are much more versatile.<\/p>\n\n\n\n

Mechanism: Why do substances „escape” into the column?<\/h3>\n\n\n\n

Most people think that the column (stationary phase) acts like a magnet that captures molecules. In reality, the opposite is true:<\/p>\n\n\n\n

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  1. Water likes water: <\/strong>Water molecules in the liquid flowing through the column (mobile phase) are very strongly bonded to each other (forming a network of hydrogen bonds).<\/li>\n\n\n\n
  2. The analyte is an intruder:<\/strong> When we inject a substance (analyte) that does not like water, it \u2018pushes\u2019 the water molecules apart. This is very tiring and energetically disadvantageous for the system.<\/li>\n\n\n\n
  3. Pushing out:<\/strong> Water wants to regain its calm as quickly as possible, so it literally pushes the intruder towards the \u201cfat\u201d chains lining the column. The substance hides there, just to have as little contact with water as possible.<\/li>\n<\/ol>\n\n\n\n

    Three main forces within the resin<\/h3>\n\n\n\n

    Once molecule has been pushed out of the water and reaches the hydrophobic groups of the stationary phase, three types of interactions begin to take place:<\/p>\n\n\n\n

    1. London forces (the most important)<\/strong><\/p>\n\n\n\n

    It is a kind of \u2018molecular glue\u2019. It occurs between the hydrophobic parts of the molecule and the hydrophobic chains in the column. The more \u2018fatty\u2019 (non-polar) the molecule is, the more strongly it sticks to the column and the slower it flows out of it.<\/p>\n\n\n\n

    2. Dipole-Dipole Interactions<\/strong><\/p>\n\n\n\n

    They act a bit like small magnets. If the substance and the column have specific chemical groups (e.g. phenyl or cyanide), they can additionally \u201cstick\u201d to each other.<\/p>\n\n\n\n

    3. Electrostatic Interference (Often problematic)<\/strong><\/p>\n\n\n\n

    It’s pure electricity. Acid groups (e.g. silane groups) are sometimes exposed on the surface of the column. If the analyte has a positive charge, it will „stick” to them too strongly. Chemists usually try to avoid this because it causes peak tailing.<\/p>\n\n\n\n

    In short:<\/strong> Water does not like non-polar analytes, so it pushes them to the side (to the column). How long the substance stays there depends on how much it likes the hydrophobic column filling and how we control the polarity of the mobile phase.<\/p>\n\n\n\n

    Characteristics of stationary phases and column construction<\/h2>\n\n\n\n

    Wyb\u00f3r odpowiedniej kolumny jest krytycznym etapem projektowania metody. Wydajno\u015b\u0107 separacji zale\u017cy zar\u00f3wno od chemii powierzchni, jak i fizycznej struktury no\u015bnika.<\/p>\n\n\n\n

    No\u015bnik krzemionkowy i jego modyfikacje<\/h3>\n\n\n\n

    Most RP-HPLC columns are based on ultra-pure type B silica (with low metal content). Key parameters are:<\/p>\n\n\n\n

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