Chromatography

Chromatography Basics of chromatography<ul><li>Capture<ul><li>Analytes captured from liquid mix</li></ul></li><li>Elute<ul><li>Captured analytes are eluted with solvent</li></ul></li><li>Detect<ul><li>Eluted sample is detected by absorbance</li></ul></li></ul> Common features of chromatography<ul><li> all have stationary phase</li><li>All have mobile phase</li><li>Different components travel at different rates dependent on attraction to mobile and stationary phase (higher affinity to stationary phase = longer travel time)</li></ul> Common type for pharmaceutical applications:<ul><li>Column chromatography</li><li>Thin layer chromatography</li><li>Gel filtration</li><li>Ion exchange</li><li>High pressure liquid chromatography</li></ul> Normal phase column chromatography:<ul><li>Column loaded with slurry of stationary and mobile phase together</li><li>Or (less common) column loaded dry and mobile flushed through column</li></ul>Stationary phases<ul><li>Silica gel (common)</li><li>Alumina (less common)</li><li>Polar molecules bind to silica and elute slowly while non-polar molecules bind weakly and elute quickly</li><li><img alt="Elution The expected elution order of organic classes. increasing polarity (move more slowly) saturated hydrocarbons unsaturated hydrocarbons ethers esters halides kctones aldehydes amines alcohols acids and bases 11 " src="https://knowt-user-attachments.s3.amazonaws.com/377836f3-3b48-4f52-bb8c-0855f4952ce5.png" /></li></ul>Purification tool:<ul><li>Collect fractions, determine components by methods of colour (TLC, ultraviolet, fluorometry)</li><li>Pool fractions</li><li>Columns washed by eluting with solvent - can be reused or discarded</li></ul> Thin layer chromatography:<ul><li>Stationary phase: silica (common), alumina and cellulose (uncommon)</li><li>Mobile phase: single solvent</li><li>Mobile phase moves through stationary phase by capillary action</li><li>Affinity: polarity</li></ul>Weak polar interactions:<ul><li>F254 is used to detect purity of substance as it illuminates place, allowing to see where sample is present</li><li><img alt="Weak Polar interactions Same principle as column chromatography using silica but on a smaller scale (thin layer) O—Si O —Si Eluotropic series to optimise resolution Silica gel G, F254to detect Analytical Preparative Reproducibility CF2 •o I (H3C)2C/H -o Silica Gel R Dipoles in O-H bond and polar analyte align to cancel Proton binds to hydrogen bond acceptor in analyte Separation depends on polarity of solute and solvent and stationary phase 14 " src="https://knowt-user-attachments.s3.amazonaws.com/82536a65-00cc-4c00-9d84-b9fe16024f51.png" /></li><li><img alt="THlN LAYER CHROMATOGRAPHY О О kthemtie$ase (sdvent) ир theplateeach is саж atatffnntrate, t&thesdvent teachesthetopend oftheplate,the$e is=vedfromthe beaker artthe sotvent alowed О 15 " src="https://knowt-user-attachments.s3.amazonaws.com/8eddcd9b-69b5-4d1e-a25f-763e1f45203b.png" /></li><li>Solvent is at the bottom of container - it will begin to travel up</li></ul>The will move up depending on their affinity for silica or partitioning ability into the solvent<ul><li>One which partition more into solvent will travel higher</li></ul>Detecting colourless components<ul><li>UV light is used to detect<ul><li>Once separation has been completed if you put it under UV light the fluorescent marker (F254) will cause whole plate to fluoresce.</li><li>Where it doesn't glow (remains as dark spots) shows where drugs contain aromatics/ double bonds which absorb UV light.</li></ul></li><li>Spraying the sheet with ninhydrin<ul><li>Detect amino acids and drugs which contain amine groups</li><li>Will appear pink/purple in presence of amines</li></ul></li><li>Iodine can be used for detection<ul><li>It will saturate double bonds and show up as brown spots</li></ul></li><li>Potassium permanganate<ul><li>Used to detect sugars and sugar like molecules</li></ul></li><li>Alkaline tetrazolium<ul><li>Used to detect corticosteroids</li><li>Blue spots appear in presence </li></ul></li><li><img alt="Detecting colourless components UV Light Or chemical treatment (dip, spray) Ninhydrin Or chemical vapour e.g. Iodine b a 16 " src="https://knowt-user-attachments.s3.amazonaws.com/2c56332c-0643-4633-82d0-1ab6153db63f.png" /></li></ul> RF values:<ul><li><img src="https://knowt-user-attachments.s3.amazonaws.com/e4c5c690-f752-409a-b1b3-76d7b4be0a7b.png" alt="" /></li></ul> Gel filtration chromatography<ul><li>Aka size exclusion</li><li>Large molecules are separated from small molecules by their size (difference in size need be fairly large)</li></ul>Uses:<ul><li>Purification of large molecules</li><li>Remove salts from proteins</li><li>Follow aggregation of proteins and peptides</li><li>Determine molecular weight of unknown protein</li></ul>Stationary phase<ul><li>Most common are dextran, dextran-polyacrylamide and agarose - inert compounds</li><li>Each is available in variety of pore sizes</li><li>Large molecules are eluted in void volume</li></ul>How does it work:<ul><li>Peaks elute in order of higher to lowest molecular weight</li></ul>To find molecular mass of unknown substance:<ul><li>Calibrate the column by having different known samples of molecular weight</li><li>Make a calibration curve</li><li>Run unknown sample - gather elution volume</li><li>From elution volume can use curve to find out likely molecular weight of unknown sample</li><li>Desalt/ purify sample:</li><li>Salt has a smaller molecular weight compared to proteins so will have a longer retention time allowing protein to be purified</li></ul> Ion exchange chromatography:<ul><li>Separation based on charge properties</li><li>Popular for proteins and peptides</li></ul>Stationary phase properties<ul><li>Cross linked polymers (cellulose/ agarose resins)</li><li>Has positive or negative functional groups which binds to oppositely charged ions in sample/ aqueous buffer</li><li>Cation exchange- positive charged molecules are attracted to negatively charged solid support</li><li>Anion exchange - negatively charged molecules attracted to positively charged solid support</li><li>Linear salt gradient is sued so molecules with weakest ionic interactions elute first</li></ul>Factors effecting elution:<ul><li>Size of charge</li><li>Intensity of charge</li><li>Concentration of ions</li><li>Application:</li><li>Bio pressing/ protein purification</li><li>Separate neutral compounds from mixture of anionic and cationic resins</li><li>Isolation of metabolites from biological fluids</li></ul>