Analysis of racemic acid derivitives by chiral high performance liquid chromatography Tara Kuknyo Orech, Knox College
Overview <ul><li>Chiral HPLC </li></ul><ul><li>Amino and Phosphonic Acids </li></ul><ul><li>α Hydroxy-phosphonates </li></...
What is HPLC? <ul><li>High Performance Liquid Chromatography </li></ul><ul><li>P can also stand for “Pressure” </li></ul><...
Chirality <ul><li>Chiral molecules differ in “handedness”,  </li></ul><ul><li>or 3-D orientation </li></ul><ul><li>Common ...
Chiral Chromatography <ul><li>Chromatography…with chirality? </li></ul><ul><li>Affinity Chromatography </li></ul><ul><li>S...
Column Used <ul><li>Nucleosil Chiral-3, 4mm diameter,  </li></ul><ul><li>pores 100Å, made for organic solvents </li></ul><...
Disadvantages to Chiral HPLC <ul><li>Expensive ($1,000-$10,000) </li></ul><ul><li>Affinity chromatography=specific column ...
Why Chiral Chromatography? <ul><li>Analyzes enantiomeric purity </li></ul><ul><li>Optical Rotation does not work for new c...
What is a β-amino phosphonic acid? α β * * Abrams, Martha Leigh. “Progress towards the synthesis of phosphorus analogs of ...
β-Amino Phosphonic Acids <ul><li>Tyrosine and phenylalanine derivatives </li></ul><ul><li>R and S-Valine first, translate ...
FMOC-Derivative <ul><li>Eluent100:18:1 heptane, dioxane and trifluoroacetic acid, very non polar </li></ul><ul><li>Can det...
Method <ul><li>S and R Valine-FMOC separately </li></ul><ul><li>Optimize the Peak </li></ul><ul><li>Run a racemic mixture ...
First Factor: Wavelength
Optimized Factors <ul><li>Wavelength: 280 nm </li></ul><ul><li>Flow Rate: 1 mL/min </li></ul><ul><li>Output Range: 1.0 </l...
Initial Readings Problem: both at 8 minutes! S-Valine R-Valine
Mixed Example
Separation of the Peaks <ul><li>Dilute from 12 mmol to 1 mmol </li></ul><ul><li>Slow flow rate to .5 mL/min </li></ul><ul>...
Extractions of FMOC-Cl Before extractions One extraction Two extractions
Re-Optimized! <ul><li>Changes: wavelength at 265 nm, sample size  </li></ul><ul><li>5 µL </li></ul><ul><li>S-Valine at ~16...
Future Research <ul><li>Tyrosine and Phenylalanine </li></ul><ul><li>β-amino phosphonic acid derivatives </li></ul><ul><li...
Project 2: Racemic Dimethyl  1-hydroxybenzylphosphonate  Pogatchnik, D. M., Weimer, David F. “Enantioselective Synthesis o...
Optimal Settings Flow Rate 1.0 mL/min Wavelength 285 nm Output Range 1.0 Amplifier Setting 0-200mV Sample Concentration 4 ...
Separation of enantiomers
Results Total Average 69.14 : 30.86 +/- 1.39 Average of Batch 1 68.5 : 31.5 +/- 1.42 Average of Batch 2 69.95 : 30.05 +/- ...
Future Research <ul><li>Optimize further to obtain 50:50 results </li></ul><ul><li>Nucleosil Chiral-2 </li></ul><ul><li>Po...
Thank You! <ul><li>Howard Hughes Medical Internship </li></ul><ul><li>Knox College </li></ul><ul><li>Dr. Larry Welch  </li...
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Analysis of racemic acid derivitives by chiral high performance liquid chromatography

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Analysis of racemic acid derivitives by chiral high performance liquid chromatography

  1. 1. Analysis of racemic acid derivitives by chiral high performance liquid chromatography Tara Kuknyo Orech, Knox College
  2. 2. Overview <ul><li>Chiral HPLC </li></ul><ul><li>Amino and Phosphonic Acids </li></ul><ul><li>α Hydroxy-phosphonates </li></ul><ul><li>Results and Future Research </li></ul>
  3. 3. What is HPLC? <ul><li>High Performance Liquid Chromatography </li></ul><ul><li>P can also stand for “Pressure” </li></ul><ul><li>Fast! </li></ul><ul><li>Large number of plates </li></ul><ul><li>Detected by UV-Vis </li></ul>Nelson, David L., Albert L. Lehninger, and Michael M. Cox. &quot;3.3 Working with Proteins.&quot; Lehninger Principles of Biochemistry . New York: W.H. Freeman, 2008. Print. http://www.cnwtech.eu/HPLC-CNW.png http://www.onu.edu/files/images/chemistry/HPLC.jpg
  4. 4. Chirality <ul><li>Chiral molecules differ in “handedness”, </li></ul><ul><li>or 3-D orientation </li></ul><ul><li>Common in organic chemistry and biology </li></ul><ul><li>Very difficult to separate because they have identical physical properties, except for light rotation </li></ul>http://www.answersingenesis.org/images/chirality-rgb.jpg
  5. 5. Chiral Chromatography <ul><li>Chromatography…with chirality? </li></ul><ul><li>Affinity Chromatography </li></ul><ul><li>Separated by three chiral binding site or chiral pocket stationary phases </li></ul>&quot;Online Guide to Chiral HPLC.&quot; Mark Earll's Homepage . Web. 20 Aug. 2009. <http://www.raell.demon.co.uk/chem/CHIbook/chiral.htm>.
  6. 6. Column Used <ul><li>Nucleosil Chiral-3, 4mm diameter, </li></ul><ul><li>pores 100Å, made for organic solvents </li></ul><ul><li>Family of columns that uses Nucleosil silica </li></ul><ul><li>Charge-transfer interactions, hydrogen bonds, dipole-dipole intereactions and steric effects </li></ul>Chiral Selector: N(3,5-dinitrobenzoyl)-L-phenylglycine Nucleosil Silica &quot;NUCLEOSIL CHIRAL.&quot; MACHEREY-NAGEL Homepage . 2011. Web. 13 Apr. 2011. <http://www.mn-net.com/tabid/6150/default.aspx>.
  7. 7. Disadvantages to Chiral HPLC <ul><li>Expensive ($1,000-$10,000) </li></ul><ul><li>Affinity chromatography=specific column for different molecules </li></ul><ul><li>Interactions between stationary phase and chiral molecule are fairly weak, making it difficult to separate </li></ul>&quot;Online Guide to Chiral HPLC.&quot; Mark Earll's Homepage . Web. 20 Aug. 2009. <http://www.raell.demon.co.uk/chem/CHIbook/chiral.htm>.
  8. 8. Why Chiral Chromatography? <ul><li>Analyzes enantiomeric purity </li></ul><ul><li>Optical Rotation does not work for new compounds </li></ul><ul><li>Less sensitive to impurities than opt. rot. </li></ul><ul><li>Allows separation of enantiomers </li></ul>
  9. 9. What is a β-amino phosphonic acid? α β * * Abrams, Martha Leigh. “Progress towards the synthesis of phosphorus analogs of β -amino acids.” Knox College Honors Thesis, May 2008.
  10. 10. β-Amino Phosphonic Acids <ul><li>Tyrosine and phenylalanine derivatives </li></ul><ul><li>R and S-Valine first, translate techniques </li></ul>S-Phenylalanine S-Tyrosine S-Valine
  11. 11. FMOC-Derivative <ul><li>Eluent100:18:1 heptane, dioxane and trifluoroacetic acid, very non polar </li></ul><ul><li>Can detect all amino acids with FMOC at similar wavelengths (~280 nm) </li></ul>Kortenaar, Paul B.W. et al.&quot;&quot;Rapid and Efficient Method for the Preparation of F-moc-amino Acids Starting from 9-fluorenylmethanol.&quot;&quot;  International Journal of Peptide and Protein Research  26.4 (1996): 398-400. Web. 12 Jan. 2009
  12. 12. Method <ul><li>S and R Valine-FMOC separately </li></ul><ul><li>Optimize the Peak </li></ul><ul><li>Run a racemic mixture </li></ul>
  13. 13. First Factor: Wavelength
  14. 14. Optimized Factors <ul><li>Wavelength: 280 nm </li></ul><ul><li>Flow Rate: 1 mL/min </li></ul><ul><li>Output Range: 1.0 </li></ul><ul><li>Amplifier Setting: 0-1V </li></ul><ul><li>Sample Concentration: 12 mmol </li></ul><ul><li>Sample Size: 20 μ L </li></ul><ul><li>Solvent: Acetone:Acetonitrile, Buffer </li></ul><ul><li>Eluent: 100:18:1 heptane, dioxane and trifluoroacetic acid </li></ul>
  15. 15. Initial Readings Problem: both at 8 minutes! S-Valine R-Valine
  16. 16. Mixed Example
  17. 17. Separation of the Peaks <ul><li>Dilute from 12 mmol to 1 mmol </li></ul><ul><li>Slow flow rate to .5 mL/min </li></ul><ul><li>Alter eluent polarity </li></ul><ul><li>All methods diluted peaks, even if put them further downfield </li></ul><ul><li>FMOC-Cl? 8 mins! </li></ul>L-Valine with x5 TFA
  18. 18. Extractions of FMOC-Cl Before extractions One extraction Two extractions
  19. 19. Re-Optimized! <ul><li>Changes: wavelength at 265 nm, sample size </li></ul><ul><li>5 µL </li></ul><ul><li>S-Valine at ~16 mins </li></ul><ul><li>R-Valine at ~13 mins </li></ul>S-valine
  20. 20. Future Research <ul><li>Tyrosine and Phenylalanine </li></ul><ul><li>β-amino phosphonic acid derivatives </li></ul><ul><li>α-hydroxy phosphonates (racemic) </li></ul>
  21. 21. Project 2: Racemic Dimethyl 1-hydroxybenzylphosphonate Pogatchnik, D. M., Weimer, David F. “Enantioselective Synthesis of α -Hydroxy Phosphonates via Oxidation with (Camphor Sulfonyl). University of Iowa Department of Chemistry . 1997.
  22. 22. Optimal Settings Flow Rate 1.0 mL/min Wavelength 285 nm Output Range 1.0 Amplifier Setting 0-200mV Sample Concentration 4 mmol Sample Size 5 μ L Solvent Heptane Dioxane TFA 100:20:.1 Eluent Heptane Dioxane TFA 100:20:.1
  23. 23. Separation of enantiomers
  24. 24. Results Total Average 69.14 : 30.86 +/- 1.39 Average of Batch 1 68.5 : 31.5 +/- 1.42 Average of Batch 2 69.95 : 30.05 +/- .94
  25. 25. Future Research <ul><li>Optimize further to obtain 50:50 results </li></ul><ul><li>Nucleosil Chiral-2 </li></ul><ul><li>Polarimeter to determine on S vs R </li></ul><ul><li>Complex products from HPLC to form diasteromers, determine S vs R by NMR </li></ul><ul><li>Another factor…temperature? </li></ul>N(3,5-dinitrobenzoyl)-L-phenylglycine N(3,5-dinitrobenzoyl)-D-phenylglycine
  26. 26. Thank You! <ul><li>Howard Hughes Medical Internship </li></ul><ul><li>Knox College </li></ul><ul><li>Dr. Larry Welch </li></ul><ul><li>Dr. Diana Cermak </li></ul><ul><li>American Chemical Society </li></ul><ul><li>Illinois State Academe of Science </li></ul>
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