Chromatography

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mobile phase, stationary phase, supporting medium

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Chromatography

  1. 1. Expert Education Endeavor in Science A PRESENTATION BY CTECK SBS
  2. 2.  Invented by M.Tswett a botanist in 1906 He used for separation of coloured compounds Now chromatographic separation any given mixture can be done. Chromatography is greek word Chroma means colour and graphy means writing It is based on the difference in the rate at which components of the mixture move through the porous medium ( called stationary phase) under the influence of solvent ( mobile phase)
  3. 3. Definition Chromatography is a separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium.Components: mobile phase: a solvent that flows through the supporting medium stationary phase: a layer or coating on the supporting medium that interacts with the analytes supporting medium: a solid surface on which the stationary phase is bound or coated
  4. 4. The analytes interacting moststrongly with the stationaryphase will take longer to passthrough the system than thosewith weaker interactions.These interactions are usuallychemical in nature, but in somecases physical interactions canalso be used.
  5. 5.  Based on principle Classified as  Adsorption chromatography  Partition chromatographyChromatography can be classified based on the type ofmobile phase, stationar phase and support material
  6. 6. 1.) The primary division of chromatographic techniques is based on the type of mobile phase used in the system: Type of Chromatography Type of Mobile Phase 1. Gas chromatography (GC) gas 2. Liquid chromatograph (LC) liquid2.) Further divisions can be made based on the type of stationary phase used in the system: Gas Chromatography Name of GC Method Type of Stationary Phase Gas-solid chromatography solid, underivatized support Gas-liquid chromatography liquid-coated support Bonded-phase gas chromatography chemically-derivatized support
  7. 7. Types of Chromatography1. Liquid ChromatographyName of LC Method Type of Stationary PhaseAdsorption chromatography solid, underivatized supportPartition chromatography liquid-coated or derivatized supportIon-exchange chromatography support containing fixed chargesSize exclusion chromatography porous supportAffinity chromatography support with immobilized ligand
  8. 8. 3.) Chromatographic techniques may also be classified based on the type of support material used in the system:1. Packed bed (column) chromatograph2. Open tubular (capillary) chromatography3. Open bed (planar) chromatography
  9. 9. Theory of Chromatography1.) Typical response obtained by chromatography (i.e., a chromatogram): chromatogram - concentration versus elution time Wh Wb Inject Where: tR = retention time tM = void time Wb = baseline width of the peak in time units Wh = half-height width of the peak in time units
  10. 10. Note: The separation of solutes in chromatography depends on two factors: (a) a difference in the retention of solutes (i.e., a difference in their time or volume of elution (b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for the separation system) Peak width & peak position determine separation of peaks A similar plot can be made in terms of elution volume instead of elution time. If volumes are used, the volume of the mobile phase that it takes to elute a peak off of the column is referred to as the retention volume (VR) and the amount of mobile phase that it takes to elute a non-retained component is referred to as the void volume (VM).
  11. 11. 2.) Solute Retention: A solute’s retention time or retention volume in chromatography is directly related to the strength of the solute’s interaction with the mobile and stationary phases. Retention on a given column pertain to the particulars of that system: - size of the column - flow rate of the mobile phase Capacity factor (k’): more universal measure of retention, determined from tR or VR. k’ = (tR –tM)/tM or k’ = (VR –VM)/VM capacity factor is useful for comparing results obtained on different systems since it is independent on column length and flow-rate.
  12. 12. The value of the capacity factor is useful in understanding the retention mechanisms for asolute, since the fundamental definition of k’ is: moles Astationary phase k’ = moles Amobile phasek’ is directly related to the strength of the interaction between a solute with the stationaryand mobile phases.Moles Astationary phase and moles Amobile phase represents the amount of solute present in eachphase at equilibrium.Equilibrium is achieved or approached at the center of a chromatographic peak. When k is # 1.0, separation is poor When k is > 30, separation is slow When k is = 2-10, separation is optimum
  13. 13. A simple example relating k’ to the interactions of a solute in a column is illustrated forpartition chromatography: KD A (mobile phase)  A (stationary phase) where: KD = equilibrium constant for the distribution of A between the mobile phase and stationary phaseAssuming local equilibrium at the center of the chromatographic peak: [A]stationary phase Volumestationary phase k’ = [A]mobile phase Volumemobile phase Volumestationary phase k’ = KD Volumemobile phase As KD increases, interaction of the solute with the stationary phase becomes more favorable and the solute’s retention (k’) increases
  14. 14. Volumestationary phase k’ = KD Volumemobile phaseSeparation between two solutes requires different KD’s for theirinteractions with the mobile and stationary phases since G = -RT ln KDpeak separation also represents different changes in free energy
  15. 15. 4.) Measures of Solute Separation: separation factor ( ) – parameter used to describe how well two solutes are separated by a chromatographic system: = k’2/k’1 k’ = (tR –tM)/tM where: k’1 = the capacity factor of the first solute k’2 = the capacity factor of the second solute, with k’2 $ k’1 A value of $1.1 is usually indicative of a good separation Does not consider the effect of column efficiency or peak widths, only retention.
  16. 16. resolution (RS) – resolution between two peaks is a second measure of how well twopeaks are separated: tr2 – tr1 RS = (Wb2 + Wb1)/2 where: tr1, Wb1 = retention time and baseline width for the first eluting peak tr2, Wb2 = retention time and baseline width for the second eluting peak Rs is preferred over since both retention (tr) and column efficiency (Wb) are considered in defining peak separation. Rs $ 1.5 represents baseline resolution, or complete separation of two neighboring solutes  ideal case. Rs $ 1.0 considered adequate for most separations.
  17. 17.  Thin-layer chromatography and column chromatography and are different types of liquid chromatography. The mobile (moving) phase is a liquid. The stationary phase is usually silica or alumina. This phase is very polar. The principle of operation is the same!
  18. 18.  simple equipments Shorter developing time Wide choice of stationary phase Early recovery of separated components Superior separation Easy visualization of separated components Sensitivity Chemically inert stationary phase
  19. 19.  Coating materialsSilica gel, Alumina, Kieselger, Cellulose powder Preparation of thin layer in plates1. Dipping2. Pouring3. Spraying4. Spreading
  20. 20.  Activation of Adsorbent Sample application Developing Tanks Solvent Systems Development methods Detection of components1. Visual Assessments2. Determination of measuring the spot areas3. Direct spectrophotometry
  21. 21. The surface of the plate consists of a very thin layer of silica on a plastic oraluminum backing. The silica is very polar. This is the stationary phase. Spotthe material at the origin (bottom) of the TLC plate.Place the plate into a glass jar with a small amount of a solvent in the glass jar.This solvent acts as the moving phase.Remove the plate from the bottle when the solvent is close to the top of the plate.Visualize the spots.Non-polar compounds will be less strongly attracted to the plate and will spendmore time in the moving phase. This compound will move faster and will appearcloser to the top of the plate.Polar compounds will be more strongly attracted to the plate and will spend lesstime in the moving phase and appear lower on the plate.
  22. 22. DEVELOPING CHAMBER SPOT SOLVENT Solvent Front 1.1 cm 5.5 cm Origin Distance from starting origin to center of zoneRf = Distance from starting origin to solvent front 5.5 = 0.5 = 11
  23. 23. OH OH OH OH OH Si Si O Si O Si O O Si O O O OO O O Si Si O O Si Si O O O Si O O O O O O Si Si O O O O O
  24. 24. O OH OH OH OH Al Al Al Al Al O O O O O OAcidic: -Al-OHNeutral: -Al-OH + -Al-O-Basic: -Al-O-
  25. 25. solvent front component B Less polar! solvent front component B component A More polar! component A origin mixture origin origin solvent frontIncreasing Development Time
  26. 26.  The previous slide shows colored spots. Most of the time, the spots won’t show unless they are visualized! Vizualization is a method that is used to render the TLC spots visible. A visualization method can be:  Ultraviolet light  Iodine vapors to stain spots  Colored reagents to stain spots  Reagents that selectively stain spots while leaving others unaffected.
  27. 27. solvent frontRf of component A = dA component B dS dS dBRf of component B = dB component A dS dA originThe Rf value is a decimalfraction, generally onlyreported to two decimalplaces
  28. 28. A B unknown
  29. 29.  To determine how many components there are in a mixture (is it really pure?) To determine the best solvent conditions for separation on a column To identify the substances being studied To monitor the composition of fractions collected from column chromatography To monitor the progress of a reaction
  30. 30. The stationary phase (column packing) in the column isvery polar!Polar compounds are going to be attracted to the polarcolumn packing by hydrogen bonding or dipole-dipoleattractions. Polar compounds are going to move slowly!Non-polar compounds are going to come off the columnfirst, while the polar compounds are going to come offthe column last.Usually, one starts will a less polar solvent to removethe less polar compounds, and then you slowlyincrease the polarity of the solvent to remove the morepolar compounds.
  31. 31.  The stationary phase is POLAR The more polar component interacts more strongly with the stationary phase The more polar component moves more slowly. The non-polar component moves more rapidly.
  32. 32. Silica is alkylated with long chain hydrocarbon groups, using 18carbons long. This is usually referred to as C-18 silica. CH3 CH2 CH3 17 CH3 Si CH2 SiCH3)3 CH3 17 CH3 SiCH3)3 SiCH3)3 O Si O CH3 O O O Si Si O Si O Si O O Si O O O O O O O Si Si O Si O Si O O Si O O O O O O O Si Si O O O O O
  33. 33.  The stationary phase (column packing) is now NON-POLAR Non-polar compounds will move more slowly because they are attracted to the column packing. The more polar component moves more quickly down the column. Polar solvents, such as water and methanol are used in reverse phase chromatography Used mainly in columns, such as HPLC
  34. 34. The column packing in the column is very polar!Polar compounds are going to be attracted to the polarcolumn packing by hydrogen bonding or dipole-dipoleattractions. Polar compounds are going to move slowly!Non-polar compounds are going to come off the columnfirst, while the polar compounds are going to come offcolumn last.Usually, one starts will a less polar solvent to removethe less polar compounds, and then you slowlyincrease the polarity of the solvent to remove the morepolar compounds.
  35. 35.  Partition Chromatography Fibers of paper is stationary phase RF value
  36. 36.  Descending chromatography Ascending Chromatography Ascending Descending chromatography Radial Chromatography Two dimensional Chromatography
  37. 37.  Whatmann Filter paper 31 Schull 2045 Machery Nagel 261Rate of Flow of Whatmann filter papersThin Papers 4,54,540 (fast ) 2,20 ( slow)Thick papers 31,17 ( fast) 3 ( medium)
  38. 38.  Proper Developing solvents Preparation of samples Spotting Drying the chromatography Visualization
  39. 39. Thanks for going through the PowerPoint Presentation. Please feel free to watch more related videos and Mock Test on our website New Release of CTECK-SbS E-NET Reviser for CSIR & GATE For INR 2500/- call +91 8095110042 www.cteck-sbs.org Expert Education Endeavor in Science

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