PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Liquid Chromatography1. Introduction and Column Packing Material2. Retention Mechanisms in Liquid Chromatography4. Column Preparation6. Detectors(Chapter 4 and 5 in The essence of chromatography)3. Method Development5. General Instrumental aspectsRetention Mechanisms in Liquid ChromatographyA. Adsorption Chromatography1. A LC technique which separates solutes based on their adsorption to an un-derivatized solid particles is known as adsorption chromatography, or liquid-solid chromatography. 2. Adsorption chromatography was the first type of column liquid chromatography developed (Tsweet, 1903). However, it is currently not widely used as other LC methods.3. Like gas-solid chromatography, supports in adsorption chromatography have the potential disadvantages of having very strong retention of some solutes and may be even cause catalytic changes in solutes. However, this is not as a big problem in LC as it is in since the mobile phase composition can be varied to control solute retention and lower operating temperature of LC make catalytic reactions less likely than in GC.4. One advantages of adsorption chromatography, as is also true for GSC, is that it is able to retain and separate some compounds that can not be separated by other methods. One such application is in the separation of geometrical isomers.5. Mechanism (a) Retention of solute in adsorption chromatography can be viewed as solute A displacing n moles of solvent M from a surface.Amp + nMsp Asp + nMmp(b) Based on this model, the value of k for solute A can be given bylog(k) = α’ (S0 – Asε0) + log(VaWs/Vm)Where: Va = Volume of adsorbed solvent in column per gram of supportWs = Weight of support in columnVm = Volume of bulk mobile phase in column, or void volumeAs = Area on surface occupied by solute Aε0 = adsorption energy of M per unit area of supportα’=Adsorption activity parameter ( α’ as support polarity)S0=Adsorption energy of A on supportSolvent strength ε0 can be tuned using a two-solvent strategy6. Solid SupportsB. Partition Chromatography(1) Partition chromatography, or liquid-liquid chromatography is a Chromatographic technique in which solute are separated based on their partition between a liquid mobile phase and a liquid stationary phase coated on a solid support. Phase 2Phase 1Phase 2Phase 1(2) The support material used in partition chromatography is usually silica. Un-bonded and banded stationary phase. (3) Mechanism: The retention of solute in partition chromatography is given by: k = KD (Vs/Vm)(4) Applications of partition Chromatography(5) Bonded stationary phaseNormal-phase LC (stationary phase is more polar that mobile phase)Reversed-phase LC (stationary phase is less polar that mobile phase)(a) Normal phase liquid Chromatography (NPLC) i. Since NPLC has a polar stationary phase, it retains polar compoundsMost strongly. However,It may be used for Separation of non-polarAs well as polar compounds(b) Reversed-phased liquid chromatography (RPLC) is another type of partition chromatography.ii. Strong mobile phase in NPLC is polar liquids, such as water or methanol. Weak mobile phase in NPLC is non-polar liquids, such as an organic solvent.i. Effect of hydrocarbon chain lengthii. Strong mobile phase in RPLC is non-polar liquid weak mobile phase in RPLC is polar liquid iii. Differences in retention between NPLC and RPLCphenolnitrobenzeneTolueneSoluteslog k = c + mVx + rR2 + sπ2 + aΣα2 + b Σβ2HHH(Liquid chromatography)(6) Retention mechanisms: (solvation parameter model/ Kamlet-Taft parameters)log k = c + mVx + rR2 + sπ2 + aΣα2 + b Σβ2HHHMobile phases effects on system constants (I)log k = c + mVx + rR2 + sπ2 + aΣα2 + b Σβ2HHHMobile phases effects on system constants (II)Solvent-programmed LCRetention Mechanisms in Liquid