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Chromatography

Gas. Liquid. GSC. GLC. LSC. LLC. IEC. GPC. AC. Chromatography. GC: volatile solutes LC: any mobile phase soluble solutes. pre-column. guard-column. pump. injector value. analytical column. inlet filter. detector. Recorder / integrator. Eluent reservoir. IC. ionic.

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Chromatography

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  1. Gas Liquid GSC GLC LSC LLC IEC GPC AC Chromatography GC: volatile solutes LC: any mobile phase soluble solutes

  2. pre-column guard-column pump injector value analytical column inlet filter detector Recorder / integrator Eluent reservoir

  3. IC ionic HPLC ion pair water soluble HPLC normal phase non-ionic HPLC reversed phase M<2000 HPLC normal phase polar HPLC reversed phase organic soluble HPLC normal phase non-polar sample HPLC reversed phase SEC gel fitration water soluble HPLC reverse phase M>2000 IC organic soluble SEC gel fitration

  4. solute S.P M.P Interaction between molecules

  5. Pump • Motor driven syringe • Piston • Reciprocating • Multiple reciprocating • Advantage and disadvantage • Solvent reservoir limited? • Pressure pulse produced? • Gradient can be produced?

  6. %B %B %B 100 100 100 linear curve segmented 50 50 50 tR tR tR 20 20 20 10 10 10 沖提模式：isocratic elution (A% B%固定) gradient elution Gradient Elution Application (1) sample with a wide k’ range (2) sample composed of large molecule (3) samples containing late-eluting interferences (4) maximizing detection sensitivity (&前濃縮)

  7. α k’ phy. pro. 使用HPLC須注意： 1. 氣泡→壓力、流速不穩(常為溶液混合所產生) 2. 顆粒→阻塞，壓力上升 Eluent 考慮(1) sample (2) separation mode Eluent之選擇

  8. solute S.P M.P (1) phy. properties b.p.、viscosity、UV cutoff、毒性、純度、價格 UV cutoff： (2) 1 ≤ k’ ≤ 10 for simple components 0.5 ≤ k’ ≤ 25 for multiple components solute/eluent interaction↑ k’↓

  9. s x 4 major interaction (a) dispersion interaction (b) dipole interaction (c) H-bonding interaction (d) dielectric interaction dispersion force： (非極性分子) Temporacy dipole moment from x will polarize the e in adjacent molecule s (R.I.值大表示分散力大)

  10. eluent strength solvent polarity N.P. R.P. N.P. solvent polarity ↑ k’↓ R.P. solvent polarity ↑ k’↓ P’：polarity

  11. ：volume ratio lf solvent a,b solvent mixture 30% MeOH 70% H2O 40% MeOH 60% H2O (3) solvent selectivity proton donor 由proton acceptor 將solvent分類 dipole moment

  12. column 由physical size分類 同樣濃度，peak大小： A>B>C

  13. column A：1 mm i.d. and 50μL/min solvent flow column B：2.1 mm i.d. and 200μL/min solvent flow column C：4.6 mm i.d. and 1000μL/min solvent flow

  14. structure of silica gel depicting the various types of bonds and silanol groups present

  15. column 材料 bonded-stationary phase anion exchange cation exchange

  16. PS-DVB ( polystyrene – divinyl benzene) pH=1~13

  17. silica： residual silanol end-capped 2 < pH < 8 if pH > 8 Si-O-Si →SiO- pH < 2 Si-R可能被破壞 pre-column的材料常為silica

  18. ( ( polar polar hybrid (silicon-C)： pH=1~12

  19. Elution order most polar being eluted 1st 靜 相 ：C1，C8，C18 tR：C1 < C8 < C18 polarity：C1 < C8 < C18

  20. HPLC detectors • Bulk property detectors • respond to a mobile phase bulk property • refractive index, dielectric constant, or density • Solute property detectors • respond to some property of solutes • UV absorbance, fluorescence, or diffusion current

  21. Light path in a micro flow cell of a spectrophotometric detector. Cells that have a 0.5-cm pathlength and contain only 8μL of liquid are common.

  22. Refractive Index Detector A refractive index detector responds to almost every solute, but its detection limit is about 1000 times poorer than that of the ultraviolet detector. Refractive index detectors are useless in gradient elution because it is impossible to match exactly the sample and the reference while the solvent composition is changing.

  23. Fluorescence detectors Fluorescence detectors excite the eluant with a laser and measure the fluorescence. These detectors are very sensitive but respond only to the few analytes that fluoresce.

  24. Evaporative Light-Scattering Detector An evaporative light-scattering detector responds to any analyte that is significantly less volatile than the mobile phase.

  25. IC ionic HPLC ion pair water soluble HPLC normal phase non-ionic HPLC reversed phase M<2000 HPLC normal phase polar HPLC reversed phase organic soluble HPLC normal phase non-polar sample HPLC reversed phase SEC gel fitration water soluble HPLC reverse phase M>2000 IC organic soluble SEC gel fitration

  26. Size-Exclusion Chromatography (SEC) ‧固定相︰ silica or polymer particles with pore size 102-106 Å ‧Type︰ -Gel Filtration Chromatography (GFC) stationary phase: hydrophilic mobile phase: aqueous 適用: protein, amino acid, peptide. -Gel Permeation Chromatography (GPC) stationary phase: hydrophobic mobile phase: nonpolar solvent 適用: oligomers

  27. VR = V0 + KVi

  28. Applications: 1. Molecular weight determination 2. Group fraction

  29. IC ionic HPLC ion pair water soluble HPLC normal phase non-ionic HPLC reversed phase M<2000 HPLC normal phase polar HPLC reversed phase organic soluble HPLC normal phase non-polar sample HPLC reversed phase SEC gel fitration water soluble HPLC reverse phase M>2000 IC organic soluble SEC gel fitration

  30. Y- + R+X- X- + R+Y- Ion-exchange chromatography sample ion：X mobile phase：Y charged stationary phase：R anion exchange cation exchange Y+ + R-X+ X+ + R-Y+ SCX：strong cation exchange –SO3-H+ WCX：weak cation exchange –COOH SAX：strong anion exchange –NR3+OH- WAX：weak anion exchange –NH2

  31. SCX exch. capacity WCX exch. capacity COOH COO- SAX pH WAX pH

  32. R-SO3-H+(R)+ Na+ (S)+ Cl- R-SO3-Na+(R)+ H+ (S)+ Cl- selectivity coefficient： selectivity

  33. Ba2+< Pb2+ < Sr2+ < Ca2+ < Ni2+ < Cd2+ < Cu2+ < Zn2+ < Mg2+ < Tl+ < Ag+ < Cs+ < K+ < NH4+< Na+ < H+ < L+

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