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Separation Analysis of Mixtures using Capillary Electrophoresis Laser-induced Fluorescence Detection

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Separation Analysis of Mixtures using Capillary Electrophoresis Laser-induced Fluorescence Detection

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    1. 1 Separation & Analysis of Mixtures using Capillary Electrophoresis & Laser-induced Fluorescence Detection

    2. 2 Electrophoresis: analytes are separated based on their ability to move through a conductive medium, usually an aqueous buffer, in response to an applied electric field.

    3. 3 Electrosmetic Electrophoretic

    4. 4 Why CE ? the high separation power short analysis time & lower operating cost Only small sample volume Heat dissipation by surface area of capillary Limit zone dispersion Use high E

    5. 5 Characteristics of Selected Detectors for CE

    6. 6 Advantages of Detection via Laser-induced Fluorescence Spectral Selectivity Sensitivity Time response Dynamic range attainable High spatial coherence of a laser beam allow for high efficient excitation High power laser facilitates the detection of small concentration of fluorophore

    7. 7 Common lasers in CELIF He-Cd (CW) 320 - 442 nm He- Ne (CW) 544 - 593 nm Ar+ ion (CW) 275 - 488 nm Diode (CW) >630 nm

    8. 8

    9. 9

    10. 10 Resolution of a Racemic Mixture of Amino Acids They are just different in chiral properties. Making Diasteromers by reaction the racemic mixture with a chiral reagent . Diasteromers behave as distinct substances and different in all properties.

    11. 11 Role of laser system: To induce on- column fluorescence of the dansyl fluorophore in both derivatized amino acids and in the diastereomerice complex formed between the amino acids and the Cu (II) complex of L- histidine. Useful characteristics of the laser light for this application: wavelength tunability for direct excitation of the dansyl fluorophore

    12. 12 D- & L- amino acids are labeled with 5- dimethyl -amino naphthalene -1- sulfonyl. Different interaction with a chiral support electrolyte (Cu (II) complex of L- histidine) in the capillary column. He- Cd laser (325 nm) Fused silica capillary 75 cm 75 m i. d. Collection by a 0.6 mm fused silica optical fiber. ?ex 325 nm ?em 600 nm

    13. 13 Enzymatic Assays (Analysis of -Galactosidase) Objective: To quantify the amounts of enzyme in picoliter volume of cell extracts using an enzymatic assay that generates multiple fluorescent products. Role of laser system: To enable ultrasensitive off-column laser- induced fluorescence detection of species eluting from capillary column. Useful characteristics of the laser light for this application: wavelength tunability & high power for direct excitation of a fluorophore present at low concentartion.

    14. 14 Fluorometric assay: The measurement of enzyme concentration involves the reaction of the enzyme with nonfluorescencent species to produce a phluorophore. First-order kinetics. Ratetot = [E] d[F]/dt = d[?f]/dt = [Et] FDG FMG + galactose FMG fluorecein + galactose

    15. 15 Analysis of alkaloids in plants They were analyzed with a simple, rapid & sensitive method of nanaqueous capillary electrophoresis with laser induced native fluorescence (NACE-LIF). Ar ion laser (488 nm) Fused silica capillary 30 cm 75 m i. d. ?exc 488 nm ?emi 500- 700 nm

    16. 16 Sensitive detection and separation of Biological compounds using CEILF detection with Nd:YAG Diode pumped, frequency-doubled cw Nd:YAG laser with wavelength 532 nm and power 5mW. CE, fused silica 37 350 m o. d. & 50 -70 m i. d. Power supply 10- 18 kV Injection: Electrokinetic 5kV in 5 s or hydrodynamic ?h= 2 cm, t= 30 s Injection volume 2.0 10-9 L

    17. 17 Capillary Electrophoresis with Laser-Induced Fluorescence Polarization Detection

    18. 18 Fluorescence Polarization

    19. 19 DNA sequencing using 96-capillary array electrophoresis High speed High-throughput 96 capillaries, 75 m i.d., 150 m o.d., 35 cm effective length. 96 gold-coated pins A CCD detector was used to monitor all capillaries simultaneously with laser-induced fluorescence at 1.75 frames per second No moving parts Ar+ ion laser

    20. 20 Using high power laser (excimer laser, 308 nm,30 ns pulsed width) Complete coating removal when the blue fluorescence emission isnt observed.

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