PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Liquid 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 aspectsSize exclusive chromatography for polymer and bio-polymer Standard entropy effectK =1-2adc2Retention of a solute is dependent on Standard entropy effect.Diameter of the pore is very important for solute selectivity.Multiple pore sizes should be used for separate solutes with different sizes. GPC: Gel permeation Chromatography (polymer scientists)GFC: Gel filtration Chromatography (biochemists)Linear range for separating solutes with different molecularweight.Separation of Block Co-PolymersSize, and functions of blocksGradient polymer elution chromatography (GPEC)Mechanisms: (a) precipitation/re-dissolution, (b) adsorption/de-sorption, (c) size exclusion effect. (1) Precipitation chromatography: non-solvent/good solute elution(2) Sudden-transition gradient polymer elution chromatography: two of non-solvents / one of good solute elutionLC Method DevelopmentProblem DefinitionMode selectionSelectivity OptimizationSystem optimizationMethod ValidationWhat type of LC should be used? Stationary phase and mobile phase,TemperatureColumn length, particle size, flow rate, instrument configuration, sample injection…Accuracy, sensitivity, specificity, detectionLimit, quantification limit, linearityMode SelectionProblem DefinitionMode selectionSystem optimizationMethod ValidationSelectivity Optimization Stationary phase and mobile phase, Temperature….log k = c + mVx + rR2 + sπ2 + aΣα2 + b Σβ2HHH(Liquid chromatography)1. Solvatochromic solvent selectivity parameters2. Snyder solvent triangle classification methodNitromethane(Xn)Ethanol (Xe)Dioxane (Xd)a. solvent strength: a parameter for estimating the solvent’s abilityTo cause migration in a chromatography system. b. solvent selectivity: the factor to distinguish the solvents that have suitable solvent strength for a separation.Different types of solvents can be selected by using liquid fromDifferent solvent. kb/ka = 10(P’A – P’B)/2For NPLC (capacity factors): kb/ka = 10(S’A – S’B)/2For RPLC (capacity factors):Solvent Strength for Mixtures:ST = 1 S1 + 2 S2 + … PT = 1 P1 + 2 P2 + … For NPLC,For RPLC,Volume fraction of solvents 1, 2 and …By varying the types of solvents used in mobile phase mixturesand keeping the total strength constant, the overall retention of solutes remains about the same, but changes of solvent selectivity will lead to the the change of resolution between two adjacent peaks (i.e. changing will affect resolution) Rs = [N1/2/4][(α -1)/(α)][k2/(1+ k2)]Solvent Strength for Mixtures:SM = 3.0STHF = 4.4SW = 0Selection of Isocratic elution method using solvent mixturesFrom strong solvents to weak solvents!Search strategies for Optimizing Isocratic Separation (I)Direct SearchInterpretive methodLC Method DevelopmentProblem DefinitionMode selectionSelectivity OptimizationSystem optimizationMethod ValidationWhat type of LC should be used? Stationary phase and mobile phase,TemperatureColumn length, particle size, flow rate, instrument configuration, sample injection…Accuracy, sensitivity, specificity, detectionLimit, quantification limit, linearityLiquid 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 aspects1. Recently, a 40-year health statistical report has shown that excess vitamin E could cause a bad long-term consequence to human health. A scientist wishes to analyze vitamin E in various food samples by a normal-phase chromatography. (a) A standard sample of vitamin E is injected onto a 4.1 mm ID x 10 cm normal-phase LC column using a mobile phase of 2% methanol: 98% ethyl ether. At a flow-rate of 2.0 ml/min, vitamin E elutes at 19.8 min. The void time under these conditions is 1.20 min. What mixture of methanol and ethyl ether must be used in order to achieve an elution time of 9.5 min for vitamin E on this system? (P’methanol = 5.1, and P’ethyl ether = 2.8) (b) When the elusion time of vitamin E is 9.5 min, another peak from the sample appears at 9.3 min that interferes with the vitamin E’s detection. Suggest a new mobile phase mixture that could be used to help improve this separation. (c) In the case where the new mobile phase in (b) does not help the separation, suggest one other specific factor that could be changed to improve the resolution of vitamin E from the interfering