HL Chemistry - Option A : Modern Analytical Chemistry

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 (SiO2)

5. Distribution Coefficient (Equilibrium Distribution ) 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

6. Some Types of Chromatography • Liquid Column Chromatography (Reverse Phase too) • High Pressure (performance) Liquid Chromatograph (HPLC) • Paper Chromatography • Thin-layer Chromatography (TLC) • 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. BASIC LIQUID CHROMATOGRAPHY 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)

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

13. 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 more polar component interacts more strongly with the stationary phase • The more polar component moves more slowly. • 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 isomers LSC or LLC Moderate Polarity Molecules LSC or LLC Compounds with Similar Functionality LSC or LLC Ionizable Species IEC Compounds with Differing Solubility LLC Mixture of Varying Sized Molecules GCC

23. 1. Ultraviolet Detector200-400nm 254 nm2. Reflective Index DetectorUniversal Detector Detectors

24. Liquid Chromatography Set Up

25. HPLC Chromatography • 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. • Injection System. Used to introduce small samples (0.1 to 500 µL) into the carrier stream under high pressure. • Reservoirs (Solvents). Multiple solvents are necessary for performing gradient elution's (i.e. changing the polarity of the mobil phase during a run). • 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. • 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 • To allow for separation in reasonable time frames • Flow-rates range from 0.1 to 10 mL/minute • Limited pulsing in the system • Many HPLC systems have a dual pump system to minimize pulsing • Flow control and reproducibility < 0.5% • 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 • Types • 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) • “Hyphenated” detector – LC-MS

31. Absorbance Detectors • 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. • 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.

32. Diode Array Detector

33. HPLC Detectors

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

35. Type of HPLC Depends on: • Molecular weight of solute • Water solubility of the solute • Polarity of the solute • 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. Reversed order of elution Increasing Mobil phase Polarity, Decreases Elution Time

38. Typical Applications of HPLC Chromatography

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 Column chromatography Characterization Homogenize Centrifugation

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

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

47. Separation on the basis of charge All proteins are charged. Their charges depend on the relative number of acid andbasic amino acids in their primary structure. All proteins have a pH value where they are uncharged: the isolelectic point (pI) H2N- Met Ala Asn Cys His Glu Ser Thr Glu Arg-COOH

48. Ionic amino acids

49. Separation on the basis of charge (continued) 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 H2N- Met Ala Asn Cys His Glu Ser Thr Glu Arg-COOH

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