1. HL Chemistry - Option A : Modern Analytical Chemistry Chromatography

2. CHROMATOGRAPHY Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient (equilibrium distribution) of sample components between 2 different phases. One of these phases is a mobile phase and the other is a stationary phase.

3. Stationary Phase: Alumina Acidic: -Al-OH Neutral: -Al-OH + -Al-O - Basic: -Al-O -

4. Stationary Phase: Silica (SiO 2 )

5. Definition: Different affinity of these 2 components to stationary phase causes the separation. Concentration of component A in stationary phase Concentration of component A in mobile phase Distribution Coefficient (Equilibrium Distribution )

6. Some Types of Chromatography 1.Liquid Column Chromatography (Reverse Phase too) 2.High Pressure (performance) Liquid Chromatograph (HPLC) 3.Paper Chromatography 4.Thin-layer Chromatography (TLC) 5.Gas Liquid Chromatography

7. LIQUID COLUMN CHROMATOGRAPHY A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid. With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation.

8. Diagram of Simple Liquid Column Chromatography

9. The 4 basic liquid chromatography modes are named according to the mechanism involved: 1.Liquid/Solid Chromatography (adsorption chromatography) A.Normal Phase LSC B.Reverse Phase LSC 2.Liquid/Liquid Chromatography (partition chromatography) A.Normal Phase LLC B.Reverse Phase LLC 3.Ion Exchange Chromatography 4.Gel Permeation Chromatography (exclusion chromatography) BASIC LIQUID CHROMATOGRAPHY

10. LIQUID SOLID CHROMATOGRAPHY The separation mechanism in LSC is based on the competition of the components of the mixture sample for the active sites on an absorbent such as Silica Gel.

11. LIQUID SOLID CHROMATOGRAPHY

12. WATER-SOLUBLE VITAMINS

14. LIQUID-LIQUID CHROMATOGRAPHY The stationary solid surface is coated with a 2nd liquid (the Stationary Phase) which is immiscible in the solvent (Mobile) phase. Partitioning of the sample between 2 phases delays or retains some components more than others to effect separation.

15. Chromatography Schematic

16. ION-EXCHANGE CHROMATOGRAPHY Separation in Ion-exchange Chromatography is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column-packing).

17. REMEMBER… The stationary phase is POLAR The stationary phase is POLAR The more polar component interacts more strongly with the stationary phase The more polar component interacts more strongly with the stationary phase The more polar component moves more slowly. The more polar component moves more slowly. The non-polar component moves more rapidly. The non-polar component moves more rapidly.

18. MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS

19. Chromatography of Amino Acids

20. GEL-PERMEATION CHROMATOGRAPHY Gel-Permeation Chromatography is a mechanical sorting of molecules based on the size of the molecules in solution. Small molecules are able to permeate more pores and are, therefore, retained longer than large molecules.

21. SOLVENTS Polar Solvents Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile Non-polar Solvents N-Decane > N-Hexane > N-Pentane > Cyclohexane

22. SELECTING AN OPERATING MODE Sample TypeLC Mode Positional isomersLSC or LLC Moderate Polarity Molecules LSC or LLC Compounds with Similar FunctionalityLSC or LLC Ionizable SpeciesIEC Compounds with Differing SolubilityLLC Mixture of Varying Sized Molecules GCC

23. 1. Ultraviolet Detector 200-400nm 254 nm 2.Reflective Index Detector Universal Detector Detectors

24. Liquid Chromatography Set Up

25. HPLC Chromatography 1. Pump System. Mobil phase pressures up to 6000 psi are necessary to achieve reasonable column elution times (~ minutes). Typical flow rates are 0.1 to 10 mL/minute. 2. Injection System. Used to introduce small samples (0.1 to 500 µL) into the carrier stream under high pressure. 3. Reservoirs (Solvents). Multiple solvents are necessary for performing gradient elution's (i.e. changing the polarity of the mobil phase during a run). 4. Chromatographic Column. Typically 10-30 cm in length containing a packing of 5-10 µm diameter. Many types of columns are available, depending on the type of liquid chromatography desired. 5. Detector. Many types are available including UV, IR, refractive index, fluorescence, conductivity, mass spectrometry, and electrochemical. Diode array detectors are used when wavelength scans are desired.

26. Schematic of an HPLC System

27. HPLC System

28. Pump System Desirable Features: Must generate pressures up to 6,000 psi Must generate pressures up to 6,000 psi To allow for separation in reasonable time frames To allow for separation in reasonable time frames Flow-rates range from 0.1 to 10 mL/minute Flow-rates range from 0.1 to 10 mL/minute Limited pulsing in the system Limited pulsing in the system Many HPLC systems have a dual pump system to minimize pulsing Many HPLC systems have a dual pump system to minimize pulsing Flow control and reproducibility < 0.5% Flow control and reproducibility < 0.5% Corrosion resistance Corrosion resistance

29. Sample Injection System Used to introduce small samples (0.001 to 0.5 mL) into the carrier stream under high pressure

30. HPLC Detectors No universal or versatile detector No universal or versatile detector Types Types General – respond to mobil phase bulk properties which vary in the presence of solutes (e.g. refractive index) General – respond to mobil phase bulk properties which vary in the presence of solutes (e.g. refractive index) Specific – respond to some properties of the solute (not possessed by the mobil phase (e.g. UV adsorption) Specific – respond to some properties of the solute (not possessed by the mobil phase (e.g. UV adsorption) “Hyphenated” detector – LC-MS “Hyphenated” detector – LC-MS

31. Absorbance Detectors The UV/Vis source usually comes from a monochromator so the wavelength can be selected, or scanned. The UV/Vis source usually comes from a monochromator so the wavelength can be selected, or scanned. Absorbance increases as eluate passes through the cell. Absorbance increases as eluate passes through the cell. If wavelength scanning is desired, the flow is stopped long enough for the scan to take place. If wavelength scanning is desired, the flow is stopped long enough for the scan to take place. It’s possible to have the same setup using IR light, although not as common since many useful solvents are not IR transparent. It’s possible to have the same setup using IR light, although not as common since many useful solvents are not IR transparent.

32. Diode Array Detector

33. HPLC Detectors

34. HPLC Column Must operate in high pressure Must operate in high pressure Usually constructed of metals Usually constructed of metals Typical dimensions Typical dimensions 10-30 cm long 10-30 cm long 1-3 cm ID 1-3 cm ID Contains packing material which holds the stationary phase Contains packing material which holds the stationary phase Many types exist Many types exist Typical packing materials are 5- 10 µm in diameter Typical packing materials are 5- 10 µm in diameter Guard column used to extend life of main column Guard column used to extend life of main column

35. Type of HPLC Depends on: 1. Molecular weight of solute 2. Water solubility of the solute 3. Polarity of the solute 4. Ionic/non-ionic character of the solute

36. Separation Principles in HPLC General Rule of Thumb: Polarity of analytes ≈ polarity of stationary phase ≠ polarity of mobile phase To achieve good separation, the analytes should interact with the stationary phase, but not too strongly or the retention time will become very long

37. Increasing Mobil phase Polarity, Decreases Elution Time Reversed order of elution

38. Typical Applications of HPLC Chromatography Field of Application Separation Pharmaceuticals Antibiotics, Sedatives, Steroids, Analgesics Biochemical Amino acids, Proteins, Carbohydrates, Lipids Food Products Artificial Sweeteners, Antioxidants, Preservatives Industrical Chemicals Condensed Aromatics, Surfactants, Propellants, Dyes Forensic Chemistry Drugs, Poisons, Blood Alcohol, narcotics Clinical Medicine Bile Acids, Drug Metabolites, Urine Extracts, Estrogens Pollutants Pesticides, Herbicides, Phenols, PCBs

39. HPLC of Orange Juice Compounds

40. How to Increase HPLC Resolution 1. Increase column length 2.Decrease column diameter 3.Decrease flow-rate 4.Pack column uniformly 5.Use uniform stationary phase (packing material) 6.Decrease sample size 7.Select proper stationary phase 8.Select proper mobile phase 9.Use proper pressure 10.Use gradient elution

41. Separating Proteins from Mixtures In order to understand and study proteins it is essential to separate them from the biological fluid. Proteins can be separated from each other based on differences in physical properties Due to different amino acid sequences proteins differ in solubility, size, charge, and binding affinity and can be separated on either of these properties. The inside of a cell. White shapes are proteins (several 10s of thousands per cell).

42. Water, Chemical bonds and groups Amino acids, pH dependence Protein primary sequence, peptide bonds, secondary structures

43. Protein studies: Understanding protein structure and function relationships All proteins have a distinctive 3D structural conformation This unique structure enables its function Amino acid sequence determines structure A major goal of biochemistry is to determine how amino acid sequences specify the 3D conformations of proteins and to catalogue all proteins in cells. Characterization cell

44. Protein purification: general experimental setup Homogenize Centrifugation Column chromatography Characterization

45. Gel permeation chromatography: separating on basis of size Mixture of proteins 1.A mixture of proteins in a small volume is applied to a column filled with porous beads 2.Because large proteins cannot enter the beads, they emerge sooner than do small ones 3.A detector (e.g. UV) is used to detect protein fragments 4.Fragments are collected separately UV time

46. Affinity Chromatography: separating on the basis of affinity X XX X X X X X To separate proteins that recognize a chemical group X 1.X is covalently attached to beads that are packed in a column 2.Sample of proteins is added 3.Washed with buffer to remove non specifically bound protein 4.Eluted with high concentration of soluble X X X X X X X

47. Separation on the basis of charge All proteins are charged. Their charges depend on the relative number of acid and basic amino acids in their primary structure. All proteins have a pH value where they are uncharged: the isolelectic point (pI) H 2 N - Met Ala Asn Cys His Glu Ser Thr Glu Arg- COOH

48. Ionic amino acids

49. Separation on the basis of charge (continued) H 2 N - Met Ala Asn Cys His Glu Ser Thr Glu Arg- COOH His: 6.0 Glu: 4.1 Arg: 12.5 N-terminal amine: 8.0 C-terminal acid: 3.1 For this peptide: pI=  pKa/N= 6.3 Positively charged at pH < 6.3 Negatively charged at pH > 6.3

50. Ion Exchange Chromatography: separation on basis of net charge + ++ + + + + + -- - + - + - - + + + - - - - - - - - - 1.Positive or negatively charged resin can be used for separation of positive or negatively charged proteins 2.Sample of proteins is added 3.Washed with buffer to remove non specifically bound protein 4.Elute with increasing concentration of salt 5.Proteins with highest net charge come of last

51. Why hydrogels are used for protein separations 1.Correct protein folding in aqueous environment 2.Proteins can denature on surfaces 3.Hydrogels are >90% water, good environment for proteins 1.2. 3.

52. In Normal Phase Liquid Chromatography: The column packing in the column is very polar! Polar compounds are going to be attracted to the polar column packing by hydrogen bonding or dipole-dipole attractions. Polar compounds are going to move slowly! Non-polar compounds are going to come off the column first, while the polar compounds are going to come off column last. Usually, one starts will a less polar solvent to remove the less polar compounds, and then you slowly increase the polarity of the solvent to remove the more polar compounds. Compare Reverse Phase to Normal Phase Column Chromatography

53. Reverse Phase Column Chromatography The stationary phase (column packing) is now NON-POLAR The stationary phase (column packing) is now NON-POLAR Non-polar compounds will move more slowly because they are attracted to the column packing. 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. The more polar component moves more quickly down the column. Polar solvents, such as water and methanol are used in reverse phase chromatography Polar solvents, such as water and methanol are used in reverse phase chromatography Used mainly in columns, such as HPLC Used mainly in columns, such as HPLC

54. Reverse phase chromatography Silica is alkylated with long chain hydrocarbon groups, using 18 carbons long. This is usually referred to as C-18 silica.

55. Summary of Methodology One of the main aims of biochemistry is to characterize and catalogue all proteins in the cell We have discussed some important tools for separating proteins based on physical properties such as size, affinity, charge. Chromatography methods: ion exchange, affinity, gel permeation chromatography Electrophoresis: iso electric focusing, SDS PAGE, 2D gels (in the Biochemistry lecture series)

56. Overview of Paper Chromatography Works on principle of Partition. Works on principle of Partition. Separates dried liquid samples. Separates dried liquid samples. Mobile phase is solvent used. Mobile phase is solvent used. Stationary phase is water bound to surface of paper. Stationary phase is water bound to surface of paper. Advantage : its cheap! Advantage : its cheap!

57. Important Concepts in Paper Chromatography Capillary Action – the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity. Capillary Action – the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity. Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents. Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents. Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase.

58. Paper/TLC Chromatography Animation

59. Simple Example of Paper Chromatography using “Sharpie” Pens

60. Dye Separation in a Black “Sharpie” Concentration of Isopropanol 0%20%50%70%100% 1. Dyes separated – purple and black 2. Not soluble in low concentrations of isopropanol 3. Partially soluble in concentrations of isopropanol >20%

61. Thin Layer Chromatography Works mainly on principle of adsorption. Works mainly on principle of adsorption. Mobile phase is the solvent Mobile phase is the solvent Stationary phase is the solid on the plate. Stationary phase is the solid on the plate.

62. TLC vs. Column Chromatography Thin-layer chromatography and column chromatography and are different types of liquid chromatography. 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 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! The principle of operation is the same!

63. Thin Layer Chromatography The surface of a plate consists of a very thin layer of silica on a plastic or aluminum backing. The silica is very polar. This is the stationary phase. Material is spotted at the origin (bottom) of the TLC plate. The plate is placed into a glass jar with a small amount of a solvent in a glass jar. This solvent acts as the moving phase. The plate is removed from the bottle when the solvent is close to the top of the plate. The spots are visualized (explanation to follow). Non-polar compounds will be less strongly attracted to the plate and will spend more time in the moving phase. This compound will move faster and will appear closer to the top of the plate. Polar compounds will be more strongly attracted to the plate and will spend less time in the moving phase and appear lower on the plate.

64. Thin-Layer Chromatography: A Two-Component Mixture More polar! Less polar! solvent front origin mixture solvent front component B component A origin solvent front component B component A origin Increasing Development Time

65. Thin-Layer Chromatography: Determination of R f Values R f of component A = d A d S R f of component B = d B d S The R f value is a decimal fraction, generally only reported to two decimal places

66. Thin-Layer Chromatography: Qualitative Analysis

67. Visualization Method The previous slide shows colored spots. Most of the time, the spots won’t show unless they are visualized! 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. Vizualization is a method that is used to render the TLC spots visible. A visualization method can be: A visualization method can be: Ultraviolet light Ultraviolet light Iodine vapors to stain spots Iodine vapors to stain spots Colored reagents to stain spots Colored reagents to stain spots Reagents that selectively stain spots while leaving others unaffected. Reagents that selectively stain spots while leaving others unaffected.

68. TLC Advantages Advantages over paper: Its faster Its faster It gives a better separation. It gives a better separation. It is more versatile as the solid on the plate can be varied. It is more versatile as the solid on the plate can be varied.

69. Uses of TLC To determine how many components there are in a mixture (is it really pure?) 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 determine the best solvent conditions for separation on a column To identify the substances being studied To identify the substances being studied To monitor the composition of fractions collected from column chromatography To monitor the composition of fractions collected from column chromatography To monitor the progress of a reaction To monitor the progress of a reaction

70. Gas-Liquid Chromatography Works on principle of Partition. Works on principle of Partition. Mobile phase is the carrier gas. Mobile phase is the carrier gas. Stationary phase is oil bound to surface of beads within the column. Stationary phase is oil bound to surface of beads within the column.

71. Most Common Stationary Phases 1.Separation of mixture of polar compounds Carbowax 20M (polyethylene glycol) 2.Separation of mixtures of non-polar compounds OV101 or SE-30 (polymer of methylsilicone) 3.Methylester of fatty acids DEGS (diethylene glycol succinate)

72. Gas-Liquid Chromatography

73. Retention time is used to identify a component of a mixture. It depends on:- Retention time is used to identify a component of a mixture. It depends on:- The temperature of the column. The temperature of the column. The length of the column. The length of the column. The material used to pack the column. The material used to pack the column. The gas pressure. The gas pressure.

74. Gas –Liquid Chromatography The area under a peak is proportional to the amount of substance present. The area under a peak is proportional to the amount of substance present.

75. Filters/Traps Air Hydrogen Gas Carrier Column Gas Chromatography gas system gas system inlet inlet column column detector detector data system data system Data system Syringe/Sampler Inlets Detectors Regulators H RESET

76. Schematic Diagram of Flame Ionization Detector Collector Jet Flame Detector electronics  - 220 volts Column Chassis ground Signal output

77. Gas-Liquid Chromatography Gas-Liquid Chromatography is often combined with mass spectroscopy. The GC separates the components then the MS analyses them. Gas-Liquid Chromatography is often combined with mass spectroscopy. The GC separates the components then the MS analyses them.

78. One possible Use of GC: SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS

79. Gas Chromatogram of Methyl Esters of Fatty Acids

80. Another GC Use: TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS

81. GLC ADVANTAGES 1.Very good separation 2.Time (analysis is short) 3.Small sample is needed -  l 4.Good detection system 5.Quantitatively analyzed

82. DISADVANTAGES OF GAS CHROMATOGRAPHY Material has to be volatilized at 250º C without decomposition !

83. Summary of Some Chromatographic Techniques Technique Stationary Phase Mobile Phase Typical Application Paper Trapped water in the paper Organic Solvent amino acid mixtures amino acid mixtures food colors or dyes food colors or dyes Thin Layer Oxide Coating Organic Solvent detect amino acids detect amino acids composition of dyes and food colors composition of dyes and food colors Column Oxide packing or resin Organic Solvent preparative preparative separation of plant pigments separation of plant pigments Gas-Liquid Oxide or volatile liquid on a solid support Gas analysis of oil mixtures analysis of oil mixtures detect drugs & steroids detect drugs & steroids fruit esters fruit esters High Performance Liquid Oxide Packing or Resin or Molecular Sieve Liquid analyze foods, pesticides, etc analyze foods, pesticides, etc detect iron in body fluids detect iron in body fluids detect blood alcohol detect blood alcohol