Principles for HPLC Methods Development

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

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

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

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

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

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

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

8. Common Modes • 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 CH3CN, CH3OH) • gradient elution

9. Reverse Phase CH2CH2CH2CH2CH2CH2CH2CH3 H2O CH3CN CH2CH2CH2CH2CH2CH2CH2CH3 H2O CH2CH2CH2CH2CH2CH2CH2CH3 CH3CN CH2CH2CH2CH2CH2CH2CH2CH3 H2O H2O CH2CH2CH2CH2CH2CH2CH2CH3

10. Reverse Phase Polarity? C6H6 CH3OH CH2CH2CH2CH2CH2CH2CH2CH3 H2O CH2CH2CH2CH2CH2CH2CH2CH3 C6H6 CH3OH CH2CH2CH2CH2CH2CH2CH2CH3 H2O H2O CH2CH2CH2CH2CH2CH2CH2CH3 C6H6 polar Non-polar

11. Reverse Phase – 50/50? Mobile phase More/less polar? C6H6 CH3OH CH2CH2CH2CH2CH2CH2CH2CH3 H2O CH2CH2CH2CH2CH2CH2CH2CH3 C6H6 CH3OH CH2CH2CH2CH2CH2CH2CH2CH3 H2O H2O CH2CH2CH2CH2CH2CH2CH2CH3 C6H6 polar Non-polar

12. Common Modes • 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, MgCl2, K3PO4, NH4SO4) • gradient elution

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

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

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

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

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

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

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

20. Cation Exchangers • Strong cation exchanger (SCX) • sulfonic acid, SO3- • Weak cation exchanger (WCX) • carboxylic acid, COO-

21. Anion Exchangers • Strong anion exchanger (SAX) • quaternary ammonium, e.g., N(CH3)4+ • Weak anion exchanger (WAX) • diethylaminoethyl (DEAE)

22. pH Effects • Anion exchange • RCOOH = RCOO- + H+ • INcrease in pH leads to greater sample ionization and retention • Cation exchange • RNH3+ = RNH2 + H+ • DEcrease in pH leads to greater sample ionization and retention

23. Salt/Buffer Effect • Mobile phase cations/anions can displace analyte on column • All salts are NOT equal • Anions: • F- < OH- < Cl- < NO3- < citrate3- (strong) • Cations: • Li+ < H+ < NH4+ < K+ < Mg2+ < Ca2+ (strong) • Polyvalent ions held more strongly by ion exchange column than monovalent ions

24. Salt/Buffer Effect • 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 • Select Salt • 0.5 - 1.0 M • 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 • Addition of organic solvents decreases retention • Be careful! Can denature biomolecules • Can be used to create changes in selectivity • EXS: methanol or acetonitrile • water miscible

27. Cytochrome c • Function: Redox protein involved in cell apoptosis and respiration • Structure: heme protein • FW 12,384 (horse) • Basic protein 3CYT: 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+ COO- K+ K+ COO- K+ K+ K+ COO- K+

30. Cyt c COO- K+ NH3+ NH3+ NH3+ COO- K+ Cyt c NH3+ NH3+ COO- K+ NH3+ NH3+ COO- K+

31. NH3+ NH3+ NH3+ COO- Cyt c NH3+ COO- NH3+ NH3+ NH3+ COO- K+ K+ K+ COO- K+ K+

32. NH3+ NH3+ NH3+ Na+ COO- Cyt c NH3+ COO- Na+ NH3+ NH3+ NH3+ COO- Na+ Na+ Na+ Na+ Na+ COO- Na+

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

34. NH3+ NH3+ NH3+ Na+ COO- Cyt c NH3+ COO- Na+ NH3+ NH3+ NH3+ COO- Na+ Na+ Na+ Na+ Na+ COO- Na+

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

36. NH2 NH2 NH2 Na+ COO- Cyt c NH2 COO- Na+ NH2 NH2 NH2 COO- Na+ Na+ Na+ Na+ Na+ COO- Na+

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

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