chips for isoelectric focusing and zone electrophoresis in the free-flow mode

3014362bc816c0e34f9bb270d226e31c?s=47 andreas manz
February 02, 2003

chips for isoelectric focusing and zone electrophoresis in the free-flow mode

... talk given at MSB 2003, New Orleans.

3014362bc816c0e34f9bb270d226e31c?s=128

andreas manz

February 02, 2003
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Transcript

  1. chips for isoelectric focusing and zone electrophoresis in the free-

    flow mode Yi Xu, Chao-Xuan Zhang, Andreas Manz Imperial College, Dept. Chemistry, London UK
  2. What should I show you? Electrophoresis scaling laws !

  3. separation efficiency • Number of theoretical plates is proportional to

    voltage drop U N 
  4. heating problem •Power generated per unit length should be a

    constant const L I U  
  5. separation efficiency •Number of theoretical plates is proportional to length

    / diameter of capillary d L N 
  6. separation time •Analysis time is proportional to length * diameter

    of capillary d L t  
  7. 10 fold miniaturization 100 x faster separation 1000 x smaller

    volume 10 x lower reagent consumption identical quality of separation
  8. fluorescence [arb. units] time [s] 0 40 80 120 160

    1 2 3 4 5 6 cycle # 7 8 t 7 s synchr. fluorescence [arb. units] time [s] 0 40 80 120 160 1 2 3 4 5 6 cycle # 7 8 t 7 s synchr. fluorescence [arb. units] time [s] 0 40 80 120 160 1 2 3 4 5 6 cycle # 7 8 t 7 s synchr. electrophoresis by Franz von Heeren
  9. electrophoresis

  10. Agilent 2100 Bioanalyzer electrophoresis

  11. FFE principle

  12. FFE problem

  13. FFE problem el. current much larger heat dissipation worse

  14. FFE chip history 1994 by Dan Raymond

  15. FFE chip history by Dan Raymond

  16. FFE chip history by Dan Raymond

  17. FFE chip history by Dan Raymond

  18. latest design + -

  19. None
  20. latest design • volume 240 nL plus micro wells •

    36 x 20um inlet channels • 72 x 20um outlet channels • each side 108 x 4um channels • separation bed 12.2 x 4.1 mm – 15,552 posts – 30 x 30 um
  21. None
  22. None
  23. None
  24. diffusion…

  25. electrophoresis happens…

  26. very fast electrophoresis

  27. problem with posts

  28. 111 ms 41 ms 53 ms 64 ms 76 ms

    88 ms 99 ms integrating over rectangular area
  29. influence of electric field separation of 2 amino acids

  30. 0 200 400 600 800 0 100 200 300 400

    500 electric field [V/cm] migration distance [um] migration distance = f (E)
  31. isoelectric focusing (IEF) • establish pH gradient • establish electric

    field • charge of protein depends on pH • mobility is nil at isoelectric point • focusing
  32. IEF proof of principle 12 mm 0 mm 4 mm

    4 mm = 500 ms angiotensin I, 1.75 kV, 10 uL/min
  33. IEF – two peptides

  34. IEF - IGF-1 sample concentration 10-7 M at inlet >10-5

    M here
  35. IEF – IGF-1 • sample introduced at 4.1 mm width

    • distance post-post on structure 40 um • apparent streamline 10 um • preconcentration factor is around 100x to 400x
  36. IEF chip specific problem • side channels contribute >80% of

    voltage drop • part of the pH gradient will therefore be lost • buffer reservoirs have to be at extreme pH • some proteins will not stay in the separation area
  37. fluorescence [arb. units] time [s] 0 40 80 120 160

    1 2 3 4 5 6 cycle # 7 8 t 7 s synchr. fluorescence [arb. units] time [s] 0 40 80 120 160 1 2 3 4 5 6 cycle # 7 8 t 7 s synchr. fluorescence [arb. units] time [s] 0 40 80 120 160 1 2 3 4 5 6 cycle # 7 8 t 7 s synchr. comparison FFE CE
  38. conclusions • IEF is possible and very fast in small

    FFE system • preconcentration of over 100x is possible • problems with pH gradient and side channels • fraction collection has to be done • detector has to be attached
  39. Acknowledgment EU funding BBSRC clean room