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Analysis of racemic acid derivitives by chiral high performance liquid chromatography

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  • 1. Analysis of racemic acid derivitives by chiral high performance liquid chromatography Tara Kuknyo Orech, Knox College
  • 2. Overview
    • Chiral HPLC
    • Amino and Phosphonic Acids
    • α Hydroxy-phosphonates
    • Results and Future Research
  • 3. What is HPLC?
    • High Performance Liquid Chromatography
    • P can also stand for “Pressure”
    • Fast!
    • Large number of plates
    • Detected by UV-Vis
    Nelson, David L., Albert L. Lehninger, and Michael M. Cox. "3.3 Working with Proteins." 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. Chirality
    • Chiral molecules differ in “handedness”,
    • or 3-D orientation
    • Common in organic chemistry and biology
    • Very difficult to separate because they have identical physical properties, except for light rotation
    http://www.answersingenesis.org/images/chirality-rgb.jpg
  • 5. Chiral Chromatography
    • Chromatography…with chirality?
    • Affinity Chromatography
    • Separated by three chiral binding site or chiral pocket stationary phases
    &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. Column Used
    • Nucleosil Chiral-3, 4mm diameter,
    • pores 100Å, made for organic solvents
    • Family of columns that uses Nucleosil silica
    • Charge-transfer interactions, hydrogen bonds, dipole-dipole intereactions and steric effects
    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. Disadvantages to Chiral HPLC
    • Expensive ($1,000-$10,000)
    • Affinity chromatography=specific column for different molecules
    • Interactions between stationary phase and chiral molecule are fairly weak, making it difficult to separate
    &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. Why Chiral Chromatography?
    • Analyzes enantiomeric purity
    • Optical Rotation does not work for new compounds
    • Less sensitive to impurities than opt. rot.
    • Allows separation of enantiomers
  • 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. β-Amino Phosphonic Acids
    • Tyrosine and phenylalanine derivatives
    • R and S-Valine first, translate techniques
    S-Phenylalanine S-Tyrosine S-Valine
  • 11. FMOC-Derivative
    • Eluent100:18:1 heptane, dioxane and trifluoroacetic acid, very non polar
    • Can detect all amino acids with FMOC at similar wavelengths (~280 nm)
    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. Method
    • S and R Valine-FMOC separately
    • Optimize the Peak
    • Run a racemic mixture
  • 13. First Factor: Wavelength
  • 14. Optimized Factors
    • Wavelength: 280 nm
    • Flow Rate: 1 mL/min
    • Output Range: 1.0
    • Amplifier Setting: 0-1V
    • Sample Concentration: 12 mmol
    • Sample Size: 20 μ L
    • Solvent: Acetone:Acetonitrile, Buffer
    • Eluent: 100:18:1 heptane, dioxane and trifluoroacetic acid
  • 15. Initial Readings Problem: both at 8 minutes! S-Valine R-Valine
  • 16. Mixed Example
  • 17. Separation of the Peaks
    • Dilute from 12 mmol to 1 mmol
    • Slow flow rate to .5 mL/min
    • Alter eluent polarity
    • All methods diluted peaks, even if put them further downfield
    • FMOC-Cl? 8 mins!
    L-Valine with x5 TFA
  • 18. Extractions of FMOC-Cl Before extractions One extraction Two extractions
  • 19. Re-Optimized!
    • Changes: wavelength at 265 nm, sample size
    • 5 µL
    • S-Valine at ~16 mins
    • R-Valine at ~13 mins
    S-valine
  • 20. Future Research
    • Tyrosine and Phenylalanine
    • β-amino phosphonic acid derivatives
    • α-hydroxy phosphonates (racemic)
  • 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. 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. Separation of enantiomers
  • 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. Future Research
    • Optimize further to obtain 50:50 results
    • Nucleosil Chiral-2
    • Polarimeter to determine on S vs R
    • Complex products from HPLC to form diasteromers, determine S vs R by NMR
    • Another factor…temperature?
    N(3,5-dinitrobenzoyl)-L-phenylglycine N(3,5-dinitrobenzoyl)-D-phenylglycine
  • 26. Thank You!
    • Howard Hughes Medical Internship
    • Knox College
    • Dr. Larry Welch
    • Dr. Diana Cermak
    • American Chemical Society
    • Illinois State Academe of Science

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