Absorption, Ion and Molecular Chromatography

Holsorption youngon the sp.one of the oldest types of chromatography around.it utilizes a liquid or gascous mobile phase and analyteis assurbed onto the surface of a stationary solid phase.The relative polarity of solute and sp determine the rateof movement of that solute through a column or across thesurfaceSilica , a ailiin - oxygen polymer is the Sp. The surfacedroxyl groups interact with the analytes. The more theexaction , the more the analytes are retained Cy :thenger they take to pass through the column.WHOSi - OHSome analyter will bind morethan others and henceSi - OHSeparation is obtained.cture oficaParlition CheomatographyBased on the relative Soluliality of analyte in spand Mp. Both Sp and MP are liquid and solute equilibrates betweenthe MP and Stationary liquid.Solute dessolvedAn analyte is in equilibrium betweenin liq phasecoated on2 phases Amobile Cp A stationarysurface ofThe equilibrium Constant KDpartition coefficientsolid supportKDmolar concentuation of the analyte in the stationaryphase divided by the molar concertuation of the analyte in themobile phase.KD = Cs (conc in SP)cm (conc in MP)3/ on exchange chromatography:In this , a resin ( stationary Solid phase) - is used toCovalently attach anions or cations onto itIt is based on the attraction of ions of opposite charges inthe mobile phase (liquid) to the resin by electiostatic forcesused for iconic compounds , unions and cations.mixumQ=20Mobile cnions held nearacations that all attachedtoE7to2+to IP++=fCtE+CBinding of -velyP-charged aa toimmobilized cationSeparationanion oxchinge resinof -volychargedonly arian car be attracted to itsurfaceaa ItMolecular exclusion chromatographyalso known as Gel filtuation / Gel permeation.the liquid or gaseous phase passes through a porous gelwhich Separates the molecules according to its size (sieving)The pores are normally small 4exclude the larger solute molecules,(0obut allows smaller molecules toe-hagemolecules areenter the gel , causing them to0excludedflow through a larger volumesmall moleculesdistancepenitiateThis causes the larger molecules to pass throughtheColumn at a faster rate than the smallerones.Small molecules travel longer.If lacks an attractive interaction between the OP and solusmall.LargeGel particles5Affinity chromadographyMost Selective typeIt utilizes the Specific intraction between one kind ofSolute molecule and a Second molecule that is immobilizedon a Stationary phase Zone kind of molecule in complex misclure becomesattached to molecule that is covalently bound to Stationaryphase . All other molecules wash throughGprotein to be purified (eg insulir reuptor)insulin (ligard)Agalose beadExample:-The immobilized molecule may be an artibody to somespecific protein when solute containing a mixture ofproteins are paised by this molecule, only the Specificprotein is reacted to this artibody , binding it to sp.The protein is later extracted by changing the ionicStrength or pHNormal phase :hydrophilic molecules in the mobile phase.The Sp - hydrophilic , :0 has a strong affinity forthe hydrophilic molecules in the mobile phase and tobind / adsorb to the column, while the hydiophabiamdeulespass through the Column and are eluted first.In normal phase, hydrophilic molecules can be elutedfrom the Column by 1ng the polarity of the Soln inthe MP.In normal phase HPLCThe polar molecule binds / adsorbs to it and the non polarmolecules will pass through quickly the stationary phase/ / Silica is polar.MP Nunpolar sp- -Polar hydrophilic