LECTURE 7
Chromatography Is a technique used to separate and identify the   components of a mixture. Works by allowing the molecul...
3
4
How separation occur? Chromatography is a powerful separation  method that is used to separate and  identify the componen...
How separation occur? In contrast, components that are weakly  held by the stationary phase travel rapidly  (fast). As a...
Classification of        Chromatographic Methods1. Based on physical means    The way stationary and mobile phases are bro...
Chromatographic Methods  based on physical means       Column                      Planar   chromatography              ch...
Chromatography based on    types of mobile phase                     Mobile                     Phase       Gas           ...
Chromatography based on   interaction of the analyte      with stationary phase1. Adsorption - of solute on surface of sta...
Chromatography based on   interaction of the analyte      with stationary phase3.Partition - based on the relative solubil...
12
Classification of           Chromatographic Methods                            Chromatography   Partition                 ...
AdsorptionChromatography                 14
Adsorption         Chromatography Components of the mixture selectively adsorb  (stick) on the surface of a finely divide...
16
PartitionChromatography                 17
Partition         Chromatography   Accomplished by selective and continuous    transfer of the components of the mixture ...
Partition            Chromatography Partitioning is a distribution (by dissolving) of the  components between 2 immiscibl...
Partition           Chromatography The stationary phase actually consists of a thin  film adsorbed (stuck) on or chemical...
21
Ion-exchangeChromatography                 22
Ion exchange           Chromatography Method for separating mixture of ions. Sample is aqueous solution of inorganic ion...
Ion exchange          Chromatography Ions that bond to the charged site on the   resin bead are separated from organic or...
Ion exchange         Chromatography The process begin with initially running the  analysis using a mobile phase with all ...
26
Size-exclusionChromatography                 27
Size exclusion        ChromatographyAlso called gel permeation chromatography.Separation technique of dissolved species ...
Size exclusion           Chromatography Separation depends on the sizes of the pores  relative to the sizes of the molecu...
30
Terminologies in chromatography                   31
 Elution  A process in which species are washed through a  chromatographic column by addition of fresh solvent. Mobile p...
Migration Rates of      Solutes   Distribution constant, K Retention time, tR Capacity factor, k’ Selectivity factor, ...
Distribution Constant In chromatography, the distribution equilibrium of  analytes between the mobile and stationary phas...
Retention TimeTime required for the sample to travel from theinjection port through the column to the detector.           ...
Dead Time, tM   Defined as time taken for the unretained    species to reach the detector.   Rate of migration of the un...
Capacity factor, k’ Term used to measure the migration rates of  analytes in columns. Also known as Retention Factor.   ...
Selectivity factoris defined as:                           distribution constantsA measure of the relative migration rates...
B    AM            39
Column EfficiencyTwo related terms widely used as quantitativemeasures of chromatographic column efficiency are    A.Plate...
Column Efficiency The relationship between H and N is given by the  formula                       Column length   Number ...
Experimentally, H and N can be approximated  from the width of the base of the chromatographic  peak.                     ...
 Another method for approximating N is to determine W1/2, the width of the peak at half its maximum height.              ...
Resolution, Rs a measure of the separation of two  chromatographic peaks. Baseline resolution is achieved when Rs = 1.5 ...
45
Rs values of less than 1.0 are considered unresolvedpeaks.                                                       46
Effect of Selectivity andCapacity Factor on ResolutionRelationship btw the resolution of a column andthe capacity factor k...
Effect of Resolution on        Retention TimeRelationship btw the resolution of a column andretention time:               ...
Example                             17.63 min                                         Length of column:                 16...
i)         Rs = 2 (17.63 min – 16.40 min)                   (1.11 min + 1.21 min)      = 1.06ii)                          ...
Variables Affecting   Column Efficiency1.   Mobile phase flow rate2.   Particle size3.   Diameter of column4.   Film thick...
Effect of mobile phase            flow (a) refer to liquid chromatography   (b) refer to gas chromatographyFrom both the p...
Effect of particle size  Effect of particle size on plate height for a packed GC column. The numbers to the right is the ...
Effect of diameter of         the column For packed column, the most important variable  that affect column efficiency is...
Effect of diameter of          the column Mass transfer coefficient is proportional to the  square of the particle diamet...
Effect of film thickness When stationary phase is an immobilized liquid,   the mass-transfer coefficient is directly   pr...
Applications of Chromatography Qualitative analysis Quantitative analysis                          57
Qualitative andquantitative analysis25.6 min           Retention time tell as about           compound identity = qualitat...
Qualitative analysis Based on retention time  Provided the sample produce the peak at the   same retention time as a sta...
Quantitative analysis1. Analysis based on Peak Height     The height of chromatographic peak is      obtained by connecti...
3. Calibration method (also known as external  method)   Involve preparation of series of standard    solutions that appr...
4. Internal Standard Method   Equal   amounts of an internal standard    substance is introduced into each standard and  ...
5. Area Normalization Method   In the normalization method, the areas of all    eluted peaks is normalized.   The percen...
Tailing and fronting A common cause of tailing and fronting is a  distribution constant that varies with  concentration....
Tailing and fronting                       65
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CHM260 - Separations Methods

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CHM260 - Separations Methods

  1. 1. LECTURE 7
  2. 2. Chromatography Is a technique used to separate and identify the components of a mixture. Works by allowing the molecules present in the mixture to distribute themselves between a mobile and a stationary phase. mobile phase = solvent or gas stationary phase = column packing material 2
  3. 3. 3
  4. 4. 4
  5. 5. How separation occur? Chromatography is a powerful separation method that is used to separate and identify the components of complex mixtures. Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile phase to varying degrees. Those components that are strongly retained by the stationary phase move slowly with the flow of mobile phase. 5
  6. 6. How separation occur? In contrast, components that are weakly held by the stationary phase travel rapidly (fast). As a consequence of these differences in mobility, sample components separate into discrete bands that can be analyzed qualitatively and/or quantitatively. 6
  7. 7. Classification of Chromatographic Methods1. Based on physical means The way stationary and mobile phases are brought into contact.2. Based on the types of mobile phase Either gas, liquid or supercritical fluid.1. Based on the kinds of equilibria involved in the in solute transfer between the phases Interaction of analyte between stationary and mobile phases. 7
  8. 8. Chromatographic Methods based on physical means Column Planar chromatography chromatographystationary phase is stationary phase isheld in narrow tube; supported on a flat platemobile phase moves by or in the interstices of apressure or gravity paper; mobile phase moves through capillary action or gravityExample: Example:Gas chromatography (GC) Thin-layer chromatographySupercritical-fluid (TLC)chromatography (SFC) Paper chromatography (PC) 8
  9. 9. Chromatography based on types of mobile phase Mobile Phase Gas (SupercriticalGas Chromatography fluid) Supercritical-fluid Chromatography (Liquid) Liquid Chromatography 9
  10. 10. Chromatography based on interaction of the analyte with stationary phase1. Adsorption - of solute on surface of stationary phase; for polar non-ionic compounds.2. Ion Exchange - attraction of ions of opposite charges; for ionic compounds. 1. Anion - analyte is anion; bonded phase has positive charge. 2. Cation – analyte is cation; bonded phase has negative charge. 10
  11. 11. Chromatography based on interaction of the analyte with stationary phase3.Partition - based on the relative solubility ofanalyte in mobile and stationary phases. a. Normal phase – stationary phase polar, the mobile phase nonpolar. b. Reverse phase– stationary phase nonpolar, the mobile phase polar.3.Size Exclusion – separate molecules by size;sieving- stationary phase is a porous matrix. 11
  12. 12. 12
  13. 13. Classification of Chromatographic Methods Chromatography Partition Adsorption Ion-exchange Size-exclusionLiquid- Gas-liquid Liquid-solid Gas-solid Liquid-solid Liquid-solidliquid 13
  14. 14. AdsorptionChromatography 14
  15. 15. Adsorption Chromatography Components of the mixture selectively adsorb (stick) on the surface of a finely divided solid stationary phase. As mobile phase (gas/liquid) carries the mixture through the stationary phase, the components of the mixture stick to its surface with varying degrees of strength and thus separate. Stationary phase: solid Mobile phase: gas or liquid 15
  16. 16. 16
  17. 17. PartitionChromatography 17
  18. 18. Partition Chromatography Accomplished by selective and continuous transfer of the components of the mixture back and forth between a liquid stationary phase and a liquid mobile phase as the mobile phase liquid passes through the stationary phase liquid. Stationary phase: liquid Mobile phase: liquid or gas 18
  19. 19. Partition Chromatography Partitioning is a distribution (by dissolving) of the components between 2 immiscible phases. Seperations of the components will be based on relative solubilities of the components in the mobile and stationary phase. Example of partitioning using polar stationary phase.  Polar components will retain longer than the non-polar components.  Non-polar components will move quickly through stationary phase and will elute first before the polar components and vice-versa. 19
  20. 20. Partition Chromatography The stationary phase actually consists of a thin film adsorbed (stuck) on or chemically bonded to the surface of a finely divided solid particles. If the mobile phase is gas, the volatility (vapor pressure) and solubility in stationary phase plays an important role. 20
  21. 21. 21
  22. 22. Ion-exchangeChromatography 22
  23. 23. Ion exchange Chromatography Method for separating mixture of ions. Sample is aqueous solution of inorganic ions or organic ions Stationary phase are small polymer resin “beads” usually packed in a glass tube. a. These beads have ionic bonding sites on their surfaces which selectively exchange ions with certain mobile phase compositions as the mobile phase penetrates through it. 23
  24. 24. Ion exchange Chromatography Ions that bond to the charged site on the resin bead are separated from organic or inorganic ions aqueous solution. The process is repeated several times by changing of the mobile phase composition. 24
  25. 25. Ion exchange Chromatography The process begin with initially running the analysis using a mobile phase with all the ions in the mixture. The mobile phase is then change for several times in a stepwise fashion so that one kind of ion at a time is removed. The process is repeated until complete separation achieved. 25
  26. 26. 26
  27. 27. Size-exclusionChromatography 27
  28. 28. Size exclusion ChromatographyAlso called gel permeation chromatography.Separation technique of dissolved species is based on the size of the components.Stationary phase: porous polymer resin particles (molecular sieves).The components to be separated enter the pores of these particles and are slowed down from progressing through this stationary phase. 28
  29. 29. Size exclusion Chromatography Separation depends on the sizes of the pores relative to the sizes of the molecules to be separated. Small particles are retarded to a greater extent than large particles (some of which may not enter the pores at all) and separation occurs. Particles with size bigger than the pore size will be eluted first from the column. 29
  30. 30. 30
  31. 31. Terminologies in chromatography 31
  32. 32.  Elution A process in which species are washed through a chromatographic column by addition of fresh solvent. Mobile phase Is one that moves over or through an immobilized phase that is fixed in place in a column or on the surface of flat plate. Stationary phase A solid or liquid that is fixed in place. A mobile phase then passes over or through the stationary phase. Retention time Time required for the sample to travel from the injection port through the column to the detector. 32
  33. 33. Migration Rates of Solutes Distribution constant, K Retention time, tR Capacity factor, k’ Selectivity factor, α 33
  34. 34. Distribution Constant In chromatography, the distribution equilibrium of analytes between the mobile and stationary phases can often be described quite simple. Let say, we have analyte A. The distribution equilibrium is written as: Therefore, the equilibrium constant K is called distribution constant and is defined as: 34
  35. 35. Retention TimeTime required for the sample to travel from theinjection port through the column to the detector. AA typical chromatogram for a two-component mixture.The small peak on the left represents a species that is not retained on thecolumn & so reaches the detector almost immediately after elution isstarted. 35
  36. 36. Dead Time, tM Defined as time taken for the unretained species to reach the detector. Rate of migration of the unretained species is SIMILAR as the average rate of motion of mobile phase molecules. So, tM also can be expressed as the time required for an average molecule of the mobile phase to pass through the column. 36
  37. 37. Capacity factor, k’ Term used to measure the migration rates of analytes in columns. Also known as Retention Factor. 37
  38. 38. Selectivity factoris defined as: distribution constantsA measure of the relative migration rates of species A and Bwith a stationary phase material in chromatography. 38
  39. 39. B AM 39
  40. 40. Column EfficiencyTwo related terms widely used as quantitativemeasures of chromatographic column efficiency are A.Plate height, H B.Number of theoretical plates, N 40
  41. 41. Column Efficiency The relationship between H and N is given by the formula Column length Number of theoretical plates Plate height The efficiency of chromatographic columns increases as the number of plates becomes greater and plate height become smaller. 41
  42. 42. Experimentally, H and N can be approximated from the width of the base of the chromatographic peak. The equation: N can be calculated using tR and W. To obtain H, the length of the column must be known. 42
  43. 43.  Another method for approximating N is to determine W1/2, the width of the peak at half its maximum height. 2 N = 5.54 tR W1/2 43
  44. 44. Resolution, Rs a measure of the separation of two chromatographic peaks. Baseline resolution is achieved when Rs = 1.5 44
  45. 45. 45
  46. 46. Rs values of less than 1.0 are considered unresolvedpeaks. 46
  47. 47. Effect of Selectivity andCapacity Factor on ResolutionRelationship btw the resolution of a column andthe capacity factor k’, selectivity factor α and thenumber of plates N is given by this equation: Rs = √N α–1 k’ 4 α 1 + k’ Simplified: Rs = √ N 47
  48. 48. Effect of Resolution on Retention TimeRelationship btw the resolution of a column andretention time: 2 3 tR = 16Rs H 2 α 1 + k’ u α–1 (k’)2 Simplified: tR = Rs2 48
  49. 49. Example 17.63 min Length of column: 16.40 min 30 cm Peak widths for 1.30 min A = 1.11 min B = 1.21 min.Calculate: i) column resolution, Rs ii) the average number of plates, N iii) the average plate height, H iv) length of column to achieve Rs 1.5 49
  50. 50. i) Rs = 2 (17.63 min – 16.40 min) (1.11 min + 1.21 min) = 1.06ii) 2N = 16 16.40 min = 3.49 x 103 Therefore, calculate the N average 1.11 min Nave = 3.44 x 103 2N = 16 17.63 min = 3.40 x 103 1.21 min 50
  51. 51. Variables Affecting Column Efficiency1. Mobile phase flow rate2. Particle size3. Diameter of column4. Film thickness 51
  52. 52. Effect of mobile phase flow (a) refer to liquid chromatography (b) refer to gas chromatographyFrom both the plots for LC and GC, we can see thatboth show a minimum in H at low linear flow rates.H increases as the mobile phase flow rateincreases. 52
  53. 53. Effect of particle size Effect of particle size on plate height for a packed GC column. The numbers to the right is the particle diameters. The smaller the particle size, the more uniform the column packing, then the more tolerant to the change in mobile-phase velocity. H increases as the particle size increases. 53
  54. 54. Effect of diameter of the column For packed column, the most important variable that affect column efficiency is the diameter of the particles that making up the packing. While for open tubular column, the diameter of the column itself is an important variable. The mobile-phase mass-transfer coefficient is known to be inversely proportional to the diffusion coefficient of the analyte in the mobile phase. 54
  55. 55. Effect of diameter of the column Mass transfer coefficient is proportional to the square of the particle diameter of the packing material, d2p (packed column). Mass transfer coefficient is proportional to the square of the column diameter, d2c (open tubular column). As a conclusion, the bigger the column diameter, the smaller the diffusion coefficient. Therefore, we can say that increase in column diameter will increase the plate height. 55
  56. 56. Effect of film thickness When stationary phase is an immobilized liquid, the mass-transfer coefficient is directly proportional to the square of the thickness of the film on the support particles d2f and inversely proportional to the diffusion coefficient of the solute in the film. With thick films and smaller diffusion coefficient, analyte molecule travel slower. As a result, thick films will reduce the mass transfer rate and increase in plate height. 56
  57. 57. Applications of Chromatography Qualitative analysis Quantitative analysis 57
  58. 58. Qualitative andquantitative analysis25.6 min Retention time tell as about compound identity = qualitative Peak Area or height tell us how much of compound is there = quantitative 58
  59. 59. Qualitative analysis Based on retention time  Provided the sample produce the peak at the same retention time as a standard under identical conditions. 59
  60. 60. Quantitative analysis1. Analysis based on Peak Height  The height of chromatographic peak is obtained by connecting the base lines on either side of the peak by a straight line and measuring the perpendicular distance from this line to the peak.2. Analysis based on Peak Area  Peak areas are usually the preferred method of quantitation since peak areas are independent of broadening effects.  Most modern chromatographic instruments are equipped with computer or digital electronic integrator that permit precise estimation of peak areas. 60
  61. 61. 3. Calibration method (also known as external method)  Involve preparation of series of standard solutions that approximate the composition of the unknown.  The peak heights or areas are plotted as a function of concentration.  The concentration of the component(s) to be analysed is determined by comparing the response(s) (peak(s)) obtained with the standard solutions. 61
  62. 62. 4. Internal Standard Method  Equal amounts of an internal standard substance is introduced into each standard and sample.  The internal standard should not react with the substance to be examined; it must be stable and must not contain impurities. The retention time must be similar to that of the substance to be examined.  The concentration of the substance to be examined is determined by comparing the ratio of the peak areas (or heights) due to the substance to be examined and the internal standard in the test solution with the ratio of the peak areas (or heights) due to the substance to be examined and the internal standard in the standard solution. 62
  63. 63. 5. Area Normalization Method  In the normalization method, the areas of all eluted peaks is normalized.  The percentage content of one or more components of the substance to be examined is calculated by determining the area of the peak(s) as a percentage of the total area of all the peaks, excluding those due to solvents or any added reagents and those below the disregard limit. 63
  64. 64. Tailing and fronting A common cause of tailing and fronting is a distribution constant that varies with concentration. Fronting also arises when the amount of sample introduced onto a column is too large. 64
  65. 65. Tailing and fronting 65

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