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Titolo

Titolo. Sommario. Massa 1. Massa 2. Stato dell’arte. Sample. introduction. External. field. The Field-Flow Fractionation principle. Length. Flow. Thickness. Breadth. Detector. Thickness. Sample. Parabolic flow. HF FlFFF / perché?. A A B C D. The HF FlFFF channel. Flow.

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Titolo

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  1. Titolo

  2. Sommario

  3. Massa 1

  4. Massa 2

  5. Stato dell’arte

  6. Sample introduction External field The Field-Flow Fractionation principle Length Flow Thickness Breadth Detector Thickness Sample Parabolic flow

  7. HF FlFFF / perché? • A • A • B • C • D

  8. The HF FlFFF channel Flow Flow Field 1/8” PE fitting 1/8” PE Tee 1/8” Teflon tube cPVC / PSfHF membrane 24x0.08 ID cm 1/8” PEEK ferrule 1/8” PEEK ferrule

  9. < > v Modo normale in HF FlFFFMeccanismo di ritenzione Browniano z L rf Flusso l x U Campo

  10. Frazionamento di proteine in HF FlFFF Fase mobile: Ammonio acetato 50 mM ;pH7 Vin=0.70 ml/min ;Vrad=0.38 ml/min BSA 2% in FM ;mw 66 kDa Hrp 1% in FM ; mw40 kDa AP 1% in FM ; mw 160 kDa Fer 1% in FM ; mw 449 kDa

  11. Vin tR= — ln — Vout 8D Costruzione della retta di taratura lnD vs lnM rf2 kT D=A M-b D= 3d Y = A + B * X Parameter Value Error A -5.92967 0.30651 b -0.37513 0.02702

  12. NUOVO ASSETTO STRUMENTALE PER ACCOPPIAMENTOHF FFF ES/TOF/MS Hollow fiber M2 W POMPA II He M1 POMPA I Acquisizione ed elaborazione dati Rivelatore UV-VIS (DAD) o o o o o o Fase mobile Acquisizione ed elaborazione dati W ES/TOF/MS W

  13. +50 +45 +55 +40 +36 +60 66397.8 Peso Molecolare calcolato:66397.81±1.74 Peso Molecolare teorico: 66398.61 UV TIC BSA non denaturata

  14. Spettro HF-FFF-MS della Mioglobina A9 A8 A:17566.69±0.58 Da A7 A6 17566.70 Peso Molecolare Mioglobina:16951.51 Da Peso Molecolare Eme:615.35 Da

  15. A9 A8 A:17566.69±0.58 Da A7 A10 B11 B:16951.48±0.27 Da B10 B9 B8 B7 HF-FFF-MS UV UV LC-MS

  16. Confronto spettri ES/MS del complesso Mioglobina/Eme A9 +Na+ Introduzione Diretta +2Na+ +3Na+ A9 HF-FFF +CH3COO-

  17. UV TIC F 43243.0 D 42422.0 E 43165.0 C 42343.1 B 41600.4 A 41521.9 Spettro dell’HRP

  18. F 43244.1 E 43165.0 C 42343.1 D 42422.0 2 Ca 2+ 2 Ca 2+ 2 Ca 2+ Glicosilazione A 41521.5 B 41600.4 Glicosilazione

  19. Conclusioni e Prospettive

  20. Ringraziamenti

  21. Efficiency in HF FlFFF å = + + + + H H H H H H neq long poly inj i Focusing L0 Elution van Bruijnsvoort, M.; Kok, W.Th; Tijssen, R. Anal Chem 2001, 73, 4736

  22. +8 +7 +9 +10 +6 14304.9 Peso Molecolare misurato:14304.94±0.35 Da Peso Molecolare teorico: 14305.16 Da

  23. Spettro ES/MS del lisozima separato in FFF dalla BSA +8 Peso Molecolare misurato:14305.06±1.05 Da +7 +9 14305.1

  24. Liso+8 Liso+7 +35 +40 +30 +43 +27 66399.53 Peso Molecolare misurato:66399.53±4.38 Da Spettro ES/MS della BSA separata in FFF dal lisozima UV TIC

  25. 0.50 0.75 0.75 0.75 Pressure gauge 0.50 0.75 0.75 0.75 Pump 1 T-valve Injection port 2 2 1 1 Pump 2 3 3 0 0 The HF FlFFF instrumental scheme 4-way valve Metering valve Waste Mobile phase Pressure gauge UV-DAD Hollow fiber channel ESI/qTOF Metering valve Mobile phase Waste Splitting valve

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