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Chromatography

Chromatography. Group 3: Jonathan Breshears Richard Hildebrand Yang Meng Amanda Steindorf. Uses of Chromatography. Chromatography methods are used to separate a large number of different compounds for quantitative and qualitative purposes

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Chromatography

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  1. Chromatography Group 3: Jonathan Breshears Richard Hildebrand Yang Meng Amanda Steindorf

  2. Uses of Chromatography • Chromatography methods are used to separate a large number of different compounds for quantitative and qualitative purposes • Chromatography can be utilized for recovery and purification of bio-molecules as well as analysis of compounds

  3. Basics of Chromatography • Chromatography involves a stationary phase and a mobile phase • Molecules begin in the mobile phase and are passed through the stationary phase • Molecules are retained within the stationary phase to different extents

  4. Chromatographic Methods • In all forms of chromatography molecules from the mobile phase are retained in the stationary phase, but how the molecules are retained depends on the method. • The main means of retention include: • Adsorption/Partitioning • Size Exclusion • Ion Exchange

  5. Adsorption/Partitioning Chromatography • By adsorption, the components of the mobile phase permeate through the stationary phase to different extents • They find themselves partitioned in layers within the stationary phase • The extent to which a molecule permeates depends on its distribution coefficient, K

  6. Adsorption/Partitioning Chromatography • Common Adsorbents (Stationary Phase)Alumina | Charcoal | Silica gel | Calcium carbonate Starch | Cellulose • Common Solvents (Mobile Phase) Petroleum ether | Carbon tetracholide | Isopropyl ether Toluene | Chloroform | Diethyl ether | Ethyl acetate Acetone | Methyl ethyl ketone | Dioxane | Acetonitrile Ethanol | Methanol | Water

  7. Size Exclusion Chromatography (SEC) • By SEC the dissolve molecules are passed through a column of porous gel beads • Molecules of different sizes pass through the column at different rates • A molecule’s size is defined by its molecular weight and Stokes radius, the approximate radius of the molecule as it tumbles through the column

  8. Ion Exchange Chromatography (IEC) • Ion Exchange Chromatography is a very powerful separation technique • It can separate proteins/bio-molecules differing by one charged amino acid • Depends on reversible adsorption of protein/bio-molecule to stationary phase of opposite charge

  9. Five stages of Ion Exchange Chromatography

  10. Ion Chromatography Columns

  11. The Chromatogram • Ordinate (y axis) is absorbance • Abscissa (x axis) is volume

  12. The Chromatogram Monitoring Module: Produces chromatogram

  13. The Chromatogram Used to determine many governing factors in Chromatography: - Resolution - Number of theoretical plates - Capacity or retention factor k - Selectivity of a system

  14. Governing factors in Chromatography: • Resolution (Rs) Rs = ¼ *(a-1)/a*sqrt(N)*k/(1+k) N is number of theoretical plates, a is selectivity of the resin, k is capacity factor of the stationary phase • Theoretical Plates (N) N = 5.54*(Vr1/wh)2 wh is peak width at half peak height, Vr1 is the volume through the chromatography method to the middle of first peak

  15. Governing factors in Chromatography: • Capacity Factor k = (Vr1-Vt)/Vt • Selectivity a = k2/k1 = (Vr2-V0)/(Vr1-V0) = Vr2/Vr1

  16. Competing MethodsHollow-Fibre Ultrafiltration • Lysozyme Separation • Polysulphone Membrane • Cost-effective • High Productivity & Purity • Compact Design & Easy Operation

  17. Competing MethodsMicromachined Electrical Field-Flow Fractionation (µ-EFFF) • Electric Field Perpendicular to Separation Direction • Based On Size and Electric Charges • Suitable for Sample Analysis

  18. An Industrial ApplicationsAnalysis of Amino Acids • Important in the determination of the composition of proteins or peptides, as well as the diagnosis of a disease • esp. the diagnosis of inborn errors of amino acid metabolism • Metabolic errors have been shown to arise from an abnormal gene which in turn encodes for mis-constructed proteins and decreased or defective enzyme activity • resulting in impaired intellectual reasoning or serious internal disorder.

  19. An Industrial ApplicationsAnalysis of Amino Acids • A blood sample is placed on the medium and the resulting growth ring is compared with that from a standard filter paper containing a known amount of the object amino acid • the method has drawbacks in that occasional false positive results are seen • HPLC analysis of amino acids is highly effective in solving this problem

  20. Conclusion • Chromatography can purify basically any soluble or volatile substance if the right adsorbent material, carrier fluid, and operating conditions are used. • A mixture of different components enters a chromatography process, and the different components are flushed through the system at different rates. These differential rates of the mixture as it moves over adsorptive materials provide separation • Even very similar components, such as proteins that may only vary by a single amino acid, can be separated with chromatography • Because chromatography is usually not performed under serious conditions it can be used to separate delicate products

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