Chromatography General. Chromatographic Process Chromatographic Systems

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Transcript of Chromatography General. Chromatographic Process Chromatographic Systems

  • ChromatographyGeneral

  • Chromatographic Process

  • Chromatographic Systems

  • Chromatographic TechniquesTLC/PCHPLCGC/SFCPC-Paper Chrom

  • Chromatography Separation Mechanism Adsorption Partition Ion - Exchange & Ion - Interaction Size Exclusion Affinity (antibody-antigen interactions; chemical interaction; attraction) Complexation - Chelation Ion exclusion (Separation of weak acids)

  • Sorption problemsABsorptionADsorption Different sorptions explained

  • Chromatograhy Mechanism of SeparationAdsorptionPartitionIon exchange

  • Chromatography Mechanism of SeparationAffinitySize Exclusion

  • Chromatogram Basic ParametertR = retention timetm = dead timeH1/2HW1/2unretained

  • Chromatographic Theories Adjusted retention time: tR = tR tM

    Plate theory distillation plate number

    N = 5.54[(tR tM)/w1/2]2

    Plate height H = L/N

    This theory did not include interaction of analytes with stationery phase

  • Chromatography Peak Broadening

  • Chromatographic Theories Rate Theory kinetic factors van Deemter

    H = B/u + Cu (+ A)

    Where:u velocity of mobile phaseB effect of molecular diffusion C Resistance to mass transferA Spreading related to different distance traveled by molecules in packed columns

  • Chromatography Packing Effect on Broadening

  • Chromatography - EquilibriumAmobile Astationary

  • Van Deemter factors:Molecular diffusion (B) in mobile phase proportional to time analyte spends in a column affected by diffusion coefficient of analyte in mobile phase affected by temperature and pressure not important in LC low diffusion coefficient inversely affected by mobile phase velocity

  • Van Deemter factors:Resistance to mass transfer (C): Mass transfer in mobile and stationary phase Lack of equilibrium moving phase Affected by thickness of liquid phase Affected inversely by the diameter of particles or inner diameter of capillary column Lower at higher temperatures (viscosity)

  • Van Deemter factors:Conclusions: Minimum value for H is achieved when: stationery phase thickness is minimal column packed with the smallest particles capillary columns have the smallest internal diameter mobile and stationary phases have low viscosity and high diffusion coefficient

  • Chromatography van Deemter PlotMobile phase velocityMultipath effectMass transferDiffusion (Longitudinal) Plate height (cm)HCuAB/u

  • Chromatography - Resolution

    ResponsetR1 tR2DtRWb1R = 2(tR1 tR2)/Wb1 Wb2Wb2Baseline resolution for Gaussianshape peaks = 1.5100%

  • Chromatography - Resolution Resolution equation where separation parameters are included:

    Rs = x (a-1/a+1) x k2/1+k2x (L/h)1/2

    Where:a selectivity factor (separation) a = tR1/tR1k migration term, capacity factor; k = ms/mmL column lengthh plate height

  • Chromatography - Resolution

  • Chromatography Qualitative Analysis Retention data RT; Rf; RRT; Kovacs Index Quantitative Analysis Peak area and height usually proportional to the amount of component Calibration Internal Standard method External Standard method Area Normalization method

  • Chromatogram Basic ParametertR = retention timetm = dead timeH1/2HW1/2unretained

  • IS - CholestaneCholesterol12345RRT1 = RT1/RTISRRT2 = RT2/RTISRRT3 = RT3/RTISAccurate to e few digits (2) at fourthDecimal Point

  • END

  • Chromatography - Methodology

  • Peaks Broadening