High-Performance Liquid Chromatography I

High-Performance Liquid Chromatography Interfaced with Fluorescence Line-Narrowing Spectroscopy for On-Line Analysis : 

High-Performance Liquid Chromatography Interfaced with Fluorescence Line-Narrowing Spectroscopy for On-Line Analysis Kenneth P. Roberts, Ryszard Jankowiak, and Gerald J. Small* Ames Laboratory – USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 Suzana Hamdan Franklin Ifeanyichukwu Uba

Main Author and Co-Authors : 

Main Author and Co-Authors Gerald J. Small PhD Physical Chemistry (1967) University of Pennsylvania B.S. Chemistry and Mathematics (1963) University of British Columbia Research interests Molecular Polaritons Chemical Carcinogenesis and laser bioanalysis Hole burning Spectroscopy Fluorescence line-narrowing http://www.photosynthesisresearch.org/Gerry%20J%20Small%20(1941-2004).pdf Ryszard Jankowiak Ph.D. Condensed Matter Physics (1981) Technical University of Gdansk (Poland) M.S. Physics (1974) Adam Mickiewicz University (Poland) Research interests Laser Bioanalytical spectroscopy Photosynthesis Research Cancer Research Tunneling in amorphous solids at very low temperatures http://www.ksu.edu/chem/people/faculty/jankowiak.html Kenneth Paul Roberts PhD Chemistry (2001) Iowa State University B.Sc.; Chemistry, Occupational Safety and Health minor (1995) Southeastern Oklahoma State University Research interests Bioanalytical chemistry Chemical Carcinogenesis • Nanotechnology—quantum dots • Laser-Induced Fluorescence / Raman / NSOM / Confocal • Capillary Electrophoresis / HPLC • Environmental Chemistry http://okepscor.org/programs/resumes/roberts.pdf

Outline : 

Outline Goals Fluorescence Improved fluorescence techniques (Shpol’skii and FLNS) Principles of fluorescence line narrowing Polycyclic aromatic compounds and DNA adducts Experimental Results and discussion Conclusions Critiques Acknowledgments

Goals : 

Goals Understanding: Principles of Fluorescence line-narrowing Instrumentation Coupling with separation techniques Analytical Applications: Separation of PAHs mixtures Detection of DNA-adducts

Fluorescence - Jablonski Diagram : 

Fluorescence - Jablonski Diagram Murray, K., CHEM 7750, Analytical Spectroscopy. 10-15 s ms to s 10-8 s

Spectral band broadening : 

Spectral band broadening Observed in molecular systems Homogeneous broadening Uncertainty principle Electron-phonon interactions Inhomogeneous broadening Interaction with different solvent cages Ariese F., ‘Shpol’skii spectroscopy and synchronous fluorescence Spectroscopy’, 1993.

Shpol’skii spectroscopy : 

Shpol’skii spectroscopy Freezing the sample to low temperatures Using polycrystalline (n-alkane) matrixes Limited applicability: Non-suitable for polar derivatives Applicable for analytes compatible with the n-alkane solvent www.isan.troitsk.ru/.../personov/memory.htm Freek, A., et al., Trends in Anal. Chem., 2008. 27. 129

Slide 8: 

Ariese F., ‘Shpol’skii spectroscopy and synchronous fluorescence Spectroscopy’, 1993.

Slide 9: 

Roman I. Peresonov PhD Physics (1963) Moscow State Pedagogical institute B.S Physics (1955) Moscow State Pedagogical institute Fluorescence Line-Narrowing Spectroscopy

FLNS Principles : 

FLNS Principles Cryogenic technique (10 K or lower) High-resolution fluorescence mode Selective and sensitive detection of analytes Tool for probing purposes Combined to separation techniques Ryszard J., et al, J. Phys. Chem., 2004. 108. 10266-10283

FLNS Spectra : 

FLNS Spectra Jablonski diagram illustrating the range of S0-S1 energy differences, the three isochromats selected by the laser in FLNS (A), and the resulting spectrum (B). Freek, A., et al, Trends in Anal. Chem., 2008. 27. 129

Solvent effect on FLNS spectra : 

Solvent effect on FLNS spectra Solvent dependencies in FLNS spectra Freek, A., et al, Trends in Anal. Chem., 2008. 27. 130 Top: Four narrow bands in the multiplet origin band Middle: More broadening of the vibronic bands in excitation Bottom: Red shift in S1-S0 energy of the emission lines

Basic Instrumental requirements : 

Basic Instrumental requirements Spectrally narrow excitation source Excitation from S0-S1 Cooling systems – Low temperature sample chamber Dispersion device CCD detector with spectral resolution of 0.05 nm Ryszard J., et al, J. Phys. Chem., 1990. 8. 63

Slide 14: 

Excimer Laser Dye Laser Photodiode Zero drift control Delay Generator High-voltage pulse generator Photodiode array Mono- Chromator Optical multichannel analyzer Printer Sync Trigger Block diagram of FLNS instrumentation Sample Cryostat FLNS Instrumentation Ryszard J., et al, J. Anal. Chem. ACS, 1989. 61. 1026A

FLNS with separation techniques : 

FLNS with separation techniques FLNS with thin-layer chromatography (TLC) FLNS with polyacrylamide gel electrophoresis (PAGE) FLNS with capillary electrophoresis/ electrochromatography (CE/CEC) FLNS with high-performance liquid chromatography (HPLC) Kenneth. P., et al., Anal. Chem. 2001. 73. 953

Slide 16: 

High-Performance Liquid Chromatography http://www.waters.com/waters/nav.htm?locale=zh_TW&cid=10049055

HPLC-FLN Instrumentation : 

HPLC-FLN Instrumentation HPLC - FLN System Kenneth. P., et al., Anal. Chem. 2001. 73. 953

Slide 18: 

Polycyclic Aromatic Hydrocarbons http://www.inchem.org/documents/ehc/ehc/v202eh03.gif

Sources of PAHs : 

Sources of PAHs Constituents of crude oils and petrochemical products Inefficient combustion of fossil fuels Sediment erosion, organic eruptions and forest fires Automobile exhaust (gaseous or aerosol-bound) Ariese F., ‘Shpol’skii spectroscopy and synchronous fluorescence Spectroscopy, 1993. http://www.envimed.com/emb03.shtml

Slide 20: 

http://www.scientificamerican.com/article.cfm?id=children-of-smoke-from-polution-to-disease Children of smoke Health Implications Carcinogenic implications (Benzo [a]pyrene) Reproductive problems Damage of the organ systems (lungs, liver, skin, kidney) http://dhs.wi.gov/eh/ChemFS/fs/PAH.htm

DNA adducts : 

DNA adducts Covalent binding of chemical carcinogens to DNA, RNA, and protein. http://en.wikipedia.org/wiki/Dna

Mechanisms : 

Mechanisms PAH activation mechanisms One electron oxidation – Radical cations Mono-oxygenation – Bay-region diol epoxides Benzo[a]pyrene (BP) – three adducts each Five depurinating and one stable adduct. Ryszard J., et al, J. Phys. Chem., 2004. 108. 10266-10283

Slide 23: 

Metabolic activation of BP Ryszard J., et al, J. Phys. Chem., 2004. 108. 10267

Slide 24: 

Formation of Stable and Depurinating DNA-Adducts Ryszard J., et al, J. Phys. Chem., 2004. 108. 10268

DNA adducts and mutations : 

DNA adducts and mutations Correlation of Depurinating Adducts with H-ras Mutations in Mouse Skin Papillomas Ryszard J., et al, J. Phys. Chem., 2004. 108. 10267

Experimental : 

Experimental Structurally similar PAHs B[a]P and B[e]P Trans-7,8-dihydrodiolbenzo[a]pyrene Trans-9,10-dihydrodiolbenzo[a]pyrene PAH-DNA adduct (carcinogenesis) Dibenzo [a,l] pyrene {Mouse Skin treated with (DB[a,l]P)} Kenneth. P., et al., Anal. Chem. 2001. 73. 952

Results : 

Results Room temperature fluorescence Chromatogram for PAH mixtures C 18 column, Mobile phase, EtOH/H2O (85:15 v/v %) λex = 351.1nm.

HPLC-FLNS Spectra : 

HPLC-FLNS Spectra On-line HPLC-FLN Spectrum for the ≈ 11 min peak (solid line) and off-line reference spectrum for trans-9,10-B[a]P-diol standard (dashed line) T = 4.2 K, λex = 394.2 nm, 200-ns gate width and 40-ns delay. (Vibrational frequencies in cm-1)

Slide 29: 

On-line HPLC-FLN Spectra for the second half of the ≈ 11 min peak. λex = 381.9 nm λex = 383.1 nm 200-ns gate width and 60-ns delay. (Vibrational frequencies in cm-1)

Slide 30: 

On-line HPLC-FLN Spectrum for the ≈ 25 min peak. λex = 368.9 nm, 200-ns gate width and 0-ns delay. (Vibrational frequencies in cm-1)

Slide 31: 

On-line HPLC-FLN Spectrum for the ≈ 27 min peak. λex = 395.7 nm, 200-ns gate width and 20-ns delay. (Vibrational frequencies in cm-1)

Fluorescence and DNA adducts : 

Fluorescence and DNA adducts At Room temperature  bad resolution At 77 K  improvement of sensitivity At 4.2 K  no improvement of selectivity due to inhomogeneous broadening Solutions Shpol’skii spectroscopy Fluorescence line narrowing Ryszard J., et al, J. Phys. Chem., 2004. 108. 10267

Slide 33: 

Ryszard J., et al, J. Phys. Chem., 2004. 108. 10275 DNA adducts

Slide 34: 

Off-line spectrum (solid line)- containing depurinating DB[a,l]P-10-N3Ade adduct. FLN reference spectrum of adduct standard (dahsed line) λex = 416 nm, 200-ns gate width and 40-ns delay time.

Biosensor chips : 

Biosensor chips

Conclusions for HPLC-FLNS : 

Conclusions for HPLC-FLNS High resolution technique Low detection limit Analysis of complex biological mixtures

Critique : 

Critique Explanation/Spectral conflict No information about the source of the reference spectra

Acknowledgments : 

Acknowledgments Dr. Kermit Murray Classmates I am innocent !! Why would you claim innocence when you left me at the crime scene ?!!