1. CHEMILUMINESCENCE DETECTION IN FIELD-FLOW FRACTIONATION P. Reschiglian, A. Zattoni, B. Roda, D. Melucci, Department of Chemistry “G. Ciamician”, University of Bologna Via Selmi 2, 40126 Bologna (Italy) M. Guardigli, A. Roda Department of Pharmaceutical Sciences, University of Bologna Via Belmeloro 6, 40126 Bologna (Italy)

2. Outline FFF basics –The FFF principle –Some historical applications –The channels –Basics on retention Off-line FlFFF-CL –Instrumental layout –Methods On-line GFFF-CL –Instrumental layout –Methods Fractionation of enzyme-coated microspheres –Comparison with UV/Vis detection –High CL specificity –High CL sensitivity –Free/bonded enzyme determination Conclusions Perspectives

3. The Field-Flow Fractionation principle Thickness Externalfield Sample introduction Flow Detector Length Breadth Parabolic flow Thickness Sample

4. Some applications of FFF

5. The GFFF channel Sample outlet Clamping frame Spacer Channel wall Injection valve Sample

6. The Flow FFF channel Cross-flow Outlet Spacer Frit Membrane Plexiglas Top Bar Plexiglas Bottom Bar Cross-flow Inlet Longitudinal flow Inlet (Sample inlet) Longitudinal flow Outlet (to the detector)

7. Reversed FFF Steric/Hyperlayer mechanism w x Lift forces d Field Flow Accumulation wall

8. Retention in steric/hyperlayer FFF Retention

9. Improving detectionin FFF Improving detection in FFF UV/Vis turbidity Low sensitivity Low specificity Complex dependence on particle features –Advanced methods required for quantitative analysis CL detection High sensitivity High specificity Independence on particle features, analytical signal from the dark –Direct quantitative analysis Limits of detection at zepto moles level

10. Off-line FlFFF-CL 1234. 56 1234.5 0.75 Pumps Flow through UV/Vis detector FlFFF channel Injection valve 1234. 56 0.1234 Mobile phase 0.75 4.00 0.75 0.50 Fraction collection Cross flow Addition of CL substrate and measurement Microtiter plate luminometer

11. SamplesSamples –Carboxylate-functionalized, polystyrene beads (PS/), 6.10±0.57, 3.00±0.14 µm coated with: Horseradish peroxidase (HRP) EC 1.11.1.7, Type VI-A (Sigma)Horseradish peroxidase (HRP) EC 1.11.1.7, Type VI-A (Sigma) Alkaline phosphatase (AP) EC 3.1.3.1 (Roche)Alkaline phosphatase (AP) EC 3.1.3.1 (Roche) Mobile phaseMobile phase –SDS 0.05 % w/v TRIS/HNO 3 @ pH = 8.7 Flow ratesFlow rates –Injection: 1.4 mL/min; 6 s –Channel flow: 4-5 mL/min –Cross-flow: 0.5 mL/min FlFFF channelFlFFF channel –Model F-1000 Universal Fractionator (FFFractionation LLC) UV/Vis detectionUV/Vis detection –On-line Dynamax Model UV-1 (Varian) @400 nm CL detectionCL detection –Off-line microtiter plate luminometer Luminoskan Ascent, Labsystem Oy –200 uL fraction/well –Luminol-based (ECL ® ) substrate for PS/HRP –Dioxetane-based (Lumiphos ® Plus) substrate for PS/AP Off-line FlFFF-CL

12. Off-line FlFFF-CL Increase in detection sensitivity Injected sample: 1  g PS/HRP 6  m + 2.5  g PS/HRP 3  m Injected sample: 0.1  g PS/HRP 6  m + 0.25  g PS/HRP 3  m 2.5 s

13. Off-line FlFFF-CL Increase in detection specificity Injected sample: mixture of PS/HRP 6/3  m and PS 10/4  m Injected sample: 5  g PS/HRP 6  m + 5  g PS/AP 3  m

14. On-line GFFF-CL Pump Flow-through UV/Vis detector Flow-through CL detector GFFF channel Injection valve 1234.56 0.12345 1234.56 Mobile phase with CL substrate 0.75

15. SamplesSamples –Carboxylate-functionalized, coatedpolystyrene beads (PS/), 6.10±0.57, 3.00±0.14 µm –Carboxylate-functionalized, coated polystyrene beads (PS/), 6.10±0.57, 3.00±0.14 µm –Horseradish peroxidase (HRP) EC 1.11.1.7, Type VI-A (Sigma) GFFF systemGFFF system –Home-built PC/PVC channel 30x2x0.014 cm –HPLC pump Model 9010 (Varian) –Rheodyne Model 7125 injector, 5 µL loop Flow ratesFlow rates –Injection flow: 0.2 mL/min; 15 s –Stop flow: 5 min –Elution flow: 0.75 mL/min UV/Vis detectionUV/Vis detection –On-line Model 2550 detector (Varian) @400 nm CL detectionCL detection –On-line photomultiplier- based flow-cell luminometer (Lumiflow, Immunotek, Moscow, Russia) CL-inducing mobile phaseCL-inducing mobile phase –SDS 0.05 % w/v TRIS/HNO 3 @ pH = 8.6, 1 mM luminol (Sigma), 10 µM p-iodophenol (Sigma), H 2 0 2 1mM On-line GFFF-CL

16. On-line GFFF-CL Increase in detection sensitivity Injected sample: 10  g PS/HRP 6  m Injected sample: 1.25 µg PS/HRP 6 µm 1 s

17. On-line CL vs. UV/Vis detection in GFFF Limit of detection of micron-sized PS Limit of detection based on S/N values Comparison between CL and UV/Vis

18. Conclusions Off-line FlFFF-CL –Fast, flow-assisted ELISA for multiple analytes –Trace-level fractionation for Ag, Ag-Ab and viruses On-line GFFF-CL –Probing FFF mechanisms by real-time imaging –Development of flow- assisted, competitive immunoassays –Trace-level fractionation of bacteria and cells Off-line FlFFF-CL –Increase in detection specificity –High size-based separation performance –Fast analysis time –Drastic reduction in LoD On-line GFFF-CL –No need of post-column reactions –Double, real-time detection: UV/Vis+CL –Drastic reduction in LoD

19. Reactor GFFF channel CL detector CarrierReagentsSample Antibody coated spheres Analyte HRP labelled analyte derivative Immunoreaction GFFF size-based fractionation CL detection Perspectives Towards a GFFF-CL competitive immunoassay

20. Off-line FlFFF-CL free-AP retention and adsorption on free-PS Injected sample: free AP 1.4 10 -4 µg Injected sample: free AP, 0.01 µg; uncoated carboxylated PS 6 µm, 5 µg; uncoated (carboxylated PS 3 µm, 5 µg