Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 241. Introduction: Fundamentals of Distribution Equilibrium2. Gas Chromatography (Chapter 2 & 3)3. Liquid Chromatography (Chapter 4 & 5)4. Other Analytical Separations (Chapter 6-8) a. Planar chromatography b. Supercritical fluid chromatography c. Electrophoresis d. Centrifugation e. Field Flow FractionationSeparation sciencesBasic conceptsRetentionEfficiency and ResolutionDevelopment: linear, Radial, and multiple developmentKey PointsPlanar ChromatographyPlanar ChromatographyA. Introduction1. Planar chromatography is a chromatographic technique in which the supporting medium is a flat bed or plane rather than a column2. This was the first type of chromatography developed but is not currently used as much as column chromatographic techniques.3. Traditionally planar chromatography has been performed by applying a small band a sample to one end of a plate containing a layer of stationary phase. The end of the plate near the point of sample application is then placed in mobile phase and solvent is allowed to migrate up the plate by capillary action. As the solvent travels by the sample, it begins to carry solutes with it. Different solutes will have different interactions with the stationary and mobile phases, making them migrate at different speeds upon the plate.4. In modern planar chromatography, the same basic approach is used, but the mobile phase may applied by a pump or force other than capillary action. Newer techniques also tend to be more automated than traditional types of planar chromatography in terms of sample application of detection.5. Attributes of Planar chromatographyB. Theory1. As in other types of liquid chromatography, separation in planar chromatography can be based on either adsorption or partitioning.2. One difference between column and planar chromatography is the presence of a third phase in the planar system. This third phase is the atmosphere or gaseous vapors located above the surface. In order to obtain reproducible separation, it is necessary to control this phase as well as the stationary and mobile phases.3. Another difference between planar and column chromatography is that planar chromatography separates based on their different distances of travel in a set amount of time. Column chromatography separates solutes by their different times of travel along a given distance, the length of the column. This difference means that slightly different expressions for retention, efficiency and resolution must be used for planar chromatography.4. Retention: ZfZsZoRf = Zs / (Zf – Zo)a. The fundamental parameter in TLC is the retardation factor Rf:Zf: Distance traveled by the solvent front from the point of application.Zs: Distance traveled by the solute front fromthe point of application.Zo: Distance between the point of application of solvent and solute.b. The value of Rf is related to the capacity factor (k) of the solute by the following equation:k = (1- Rf)/ Rfc. By using the above equation, planar chromatography can be used to obtain estimates of k for a solute on different stationary phase and mobile phase combinations. This can be useful in screening a number of columns or mobile phase for use in column liquid chromatography.5. Efficiency(a) The efficiency of a separation in planar chromatography is described in terms of plates and plate height. N = (Zs / )2 N = 16*(Zs / Wb)2H = Zs /nN: number of theoretical plates; H: plate height.: standard deviation of the solute band (in distance units)Wb: baseline width of the solute band (in distance units)b. Note that the efficiency of a planar system is not constant, but depends on the distance that the solute has traveled, or its retention and Rf value. c. The change in efficiency of a planar chromatography system with distance and the presence of a third phase have made the derivation of exact plate height equations for planar chromatography difficult. These concurrently occur with another complicating factor: the flow rate of mobile phase through a system with capillary flow is not constant with time. d. For a system with capillary flow, the change in the mobile phase velocity with time is described by the following equation:Zf = (xt)1/2t : time required by the mobile phase to migrate distance Zf.x : the system constant.e. The plate height equation for a planar system with capillary flow is shown belowHtot = a [(Zf2/3 – Zo2/3)/(Zf-Zo)] + b (Zf + Zo)a = 3/2 A dp (dP/2D)1/3b = 2 D / (x Rf)dp: size of particles D: diffusion coefficient of solutex : system constant6. Resolution: Rs = (-1) [N/(1+k)]1/2[k/(1+k)]Rf = Zs / (Zf – Zo)The maximum resolution is achieved at Rf value between 0.2 and 0.5.C. Types of planar Chromatography 1. There are two main types of planar chromatography a. paper chromatography b. Thin-layer chromatography (TLC)2. Paper Chromatography is a special type of planar chromatography in which paper is used as both the support material and stationary phase. Although this method is the historical important, being the first type of chromatography described, it is rarely used in current practice.3. Thin-layer Chromatography (TLC) is the main type of planar chromatography used at the present. It involves the used of any non-paper materials as the supporting medium.4. Conventional TLC and high performance TLC (HPTLC): by their separation efficiency.D. Stationary and mobile phases of TLC1. The support materials used in TLC are essentially the same as used in column liquid chromatography. These can include materials used n adsorption chromatography (silica and alumina), liquid coated support or bonded-phases for use in partition chromatography (reversed-phase and normal-phase), and support for use in ion-exchange, affinity and size exclusion chromatography.2. The mobile phase used in TLC are also essentially the same as used in column liquid chromatography. The exactly type used will depend on the stationary phase and type of separation to be performed.Ion-suppression chromatographyIon-pair ChromatographyMicellar Liquid ChromatographyHydrophobic interaction Chromatography (HIC)E. Elution and Development:1. Solutes can be eluted in planar chromatography using either isocratic or gradient elution. 2. For gradient elution in TLCa. Mobile phase