1. Principles for HPLC Methods Development Bioanalytical Chemistry Lecture Topic 4

2. Five Stages l Define problem l Experiment with key variables l Evaluate l Optimize l Troubleshoot

3. Define l What is the purpose? –Analytical –Preparative l What are the molecular characteristics of the analyte and sample? –CHASM

4. CHASM l Charge –Positive/negative l Hydrophobicity l Affinity –“lock and key” sites l Solubility & stability –pH, ionic strength, organic solvents l Molecular weight

5. Analytical vs. Preparative l Analytical Requirements –Linearity –Precision –Accuracy –Sensitivity –Assay reproducibility –Robustness

6. Analytical vs. Preparative l Preparative Requirements l Recovery l Product purity l Capacity l Costs –Scale up –Process throughput –Speed

7. Methods Development l Select the mode l pH map l Optimize gradient/elution –gradient slope –eluent concentration l Loading study –overload: peak width and shape

8. Common Modes l Reverse phase (RPC) –Stationary phase hydrophobic and mobile phase hydrophilic column: silica, polystyrene covalently modified with alkyl chain 3-18 C’s –EX: octadecylsilane (ODS) - C18 mobile phase: buffered water + organic solvent (propanol CH 3 CN, CH 3 OH) gradient elution

9. CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 H2OH2O H2OH2O H2OH2O H2OH2O CH 3 CN Reverse Phase

10. CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 H2OH2O H2OH2O H2OH2O CH 3 OH Reverse Phase C6H6C6H6 C6H6C6H6 C6H6C6H6 CH 3 OH Non-polar polar Polarity?

11. CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 H2OH2O H2OH2O H2OH2O CH 3 OH Reverse Phase – 50/50? C6H6C6H6 C6H6C6H6 C6H6C6H6 CH 3 OH Mobile phase More/less polar? Non-polar polar

12. Common Modes l Ion-Exchange (IEC) –Ion exchange interactions between cationic or anionic analyte and stationary phase bearing opposite charge stationary phase: polystyrene, silica modified with functional groups such as quaternary amines mobile phase: buffer containing increasing concentration of salt (NaCl, MgCl 2, K 3 PO 4, NH 4 SO 4 ) gradient elution

13. Evaluation l Resolution –degree of separation between analyte and other species present in mixture –bandspreading –selectivity l Recovery –mass recovery –activity recovery l Capacity

14. Developing Your Application l Proteins l Antibodies l Peptides l Nucleic acids

15. Proteins l All modes can potentially be used l Ion exchange common first step –mobile phase less denaturing l Antibodies –Affinity

16. Peptides l amino acid chain < 30 residues (5000 MW) l reverse phase most commonly used –historical l ion exchange can be equally effective

17. Nucleic Acids l gel electrophoresis commonly used l anion exchange predominant chromatographic method

18. Ion Exchange l Sample must be ionized in order to be retained on column significantly l Anion exchange (anionic acidic proteins) X - + R + Cl - = X - R + + Cl - l Cation exchange (protonated basic proteins) X + + R - K + = X + R - + K +

19. Column Type l 4 types: strong/weak cation/anion l Strong - ionization of ionic group does not change over usual pH range –better starting point l Weak - lose charge and sample retention for certain pH ranges

20. Cation Exchangers l Strong cation exchanger (SCX) –sulfonic acid, SO 3 - l Weak cation exchanger (WCX) –carboxylic acid, COO -

21. Anion Exchangers l Strong anion exchanger (SAX) –quaternary ammonium, e.g., N(CH 3 ) 4 + l Weak anion exchanger (WAX) –diethylaminoethyl (DEAE)

22. pH Effects l Anion exchange –RCOOH = RCOO - + H + –INcrease in pH leads to greater sample ionization and retention l Cation exchange –RNH 3 + = RNH 2 + H + –DEcrease in pH leads to greater sample ionization and retention

23. Salt/Buffer Effect l Mobile phase cations/anions can displace analyte on column l All salts are NOT equal –Anions: F - < OH - < Cl - < NO 3 - < citrate 3- (strong) –Cations: Li + < H + < NH 4 + < K + < Mg 2+ < Ca 2+ (strong) –Polyvalent ions held more strongly by ion exchange column than monovalent ions

24. Salt/Buffer Effect l Need to select appropriate pH: –Anion exchange, pH > 6 used –start: pH 8.5 protein stable? extreme end of pH range binding should be tightest –Cation exchange, pH < 6 used (pH 4.0)

25. Salt/Buffer Effect l Select Salt –0.5 - 1.0 M l Gradient –0 - 100 % gradient - to determine relative retention of sample –long, shallow to start: 0 - 1 M NaCl, 50 - 100 CV’s

26. Organic Solvent Effect l Addition of organic solvents decreases retention –Be careful! Can denature biomolecules l Can be used to create changes in selectivity l EXS: methanol or acetonitrile –water miscible

27. Cytochrome c l Function: Redox protein involved in cell apoptosis and respiration l Structure: heme protein –FW 12,384 (horse) –Basic protein 3CYT3CYT: Takano, T., Dickerson, R. E.: Redox conformation changes in refined tuna cytochrome c. Proc. Natl. Acad. Sci. USA 77 pp. 6371 (1980)

28. What mode should we use?

29. Cyt c COO - K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+

30. Cyt c COO - Cyt c NH 3 + K+K+ K+K+ K+K+ K+K+

31. COO - Cyt c NH 3 + K+K+ K+K+ K+K+ K+K+ K+K+

32. COO - Cyt c NH 3 + Na +

33. Effect of pH What Does Cyt c look like at low pH?

34. COO - Cyt c NH 3 + Na +

35. Effect of pH What Does Cyt c look like at high pH?

36. COO - Cyt c NH 2 Na +

37. Effect of pH So low pH more effective for cation exchange than high pH

38. Useful References l “The Busy Researcher’s Guide to Biomolecular Chromatography,” Perspective Biosystems, publication date unknown. l Snyder, L.R.; Kirkland, J.J.; Glajch, J.L. “Practical HPLC Method Development,” 2 nd ed. John Wiley & Son: New York, 1997.