April 11, 2001

Exam: report on status of grading (Friday?)

What's Next ?

• Separation Methods-- Chromatography
  • GC
  • HPLC
    • supercritical fluids (briefly)
    • electrophoresis (briefly)
• Electrochemistry
  • potentiometric methods
  • voltametric methods
• Magnetic Resonance
• Misc.
  • thermal methods
  • flow methods
  • array methods, new trends

Paper or Project-- ???

• One paper (Analytical Chemistry) on specific instrument or method
• Web search -- additional information on same method
• Needs to be narrow... not, say, NMR or GC

SEPARATIONS

• distillation
• solvent extraction
• adsorption

All characterized by two phases and an equilibrium

• useful if equilibrium for components varies enough
• exploit those differences
  • sometimes, very clear cut separations
    • can completely separate fats and oils from aqueous foods by hexane extraction
    • can capture most organic vapors on charcoal
    • useful, of course, only if other species are not extracted also
  • often, the separation is only slightly preferential
    • could improve results by painstaking repetition
      • fractional distillation-- each cycle improves the separation

Example-- repeat solvent extraction

• Assume K = conc(upper) / conc(lower) = 2.0
  • that is, 2/3 of solute goes into upper layer
  • assuming we use equal volumes.
• Start with 100 mg of solute A
• Perform one extraction
  • upper layer=67 mg, lower layer=33 mg
• Transfer lower layer to separatory funnel #2 (33 mg)
  • replace the missing solvent in both funnels
  • shake and regain equilibrium
• Funnel #1 has 67 mg ( 44 mg upper, 23 mg lower)
• Funnel #2 has 33 mg (22 mg upper , 11 mg lower)
  • transfer lower layer to next funnel
    • funnel 1 keeps 44 mg
    • funnel 2 keeps 22 and adds 23 = 44 mg
    • funnel 3 gets 11 mg
• Replace the missing solvents and extract again
  • funnel 1 has 44.4 mg ( 29.6 upper, 14.8 mg lower)
  • funnel 2 has 44.4 mg (same distribution)
  • funnel 3 has 11.2 mg (7.5 mg upper, 3.7 mg lower)
  • can show that amount in each funnel will follow a binomial distribution
  • after many cycles, this distribution will approximate a Gaussian
• Original Penicillin was purified from fermentation mixture by countercurrent fractional extraction--
  • a rack of 200-400 tubes, 100 ml volume, interconnected.
  • shake, settle, transfer, repeated hundreds of times in 24-48 hrs
  • Penicillin would emerge separated into perhaps 25 tubes
• Chromatography Shares a Similar Behavior
• sample is distributed between two phases
  • one phase is stationary and stays put
  • the other phase is mobile and passes over or through the stationary phase
    • the process is continuous
    • the process ideally is slow enough that equilibrium is continually achieved everywhere

• In GC the mobile phase is vapor + carrier gas
  • the other is liquid (coated on particles or walls of column)
• In LC and HPLC the mobile phase is a solvent
  • the stationary phase is a solid

• Analysis is generally done using a non-continuum model
  • consider the column divided into tiny equilibrium segments
    • by analog with fractional distillation, called plates
  • each plate achieves equilibrium distribution
    • then passes on some sample by transfer of mobile phase
    • each plate receives from on neighbor, passes on to next plate
  • sample occurs (or emerges) as a peak
    • retention time tracks the k value
    • peak width increase with retention time
    • peaks narrows as the number of theoretical plates increases
  • N- the number of plates or HETP is a fixed value
    • corresponds roughly to distance required to approximate equilibrium
    • influenced by particle size, flow rate
    • N can be determined from peak shape
    • N = 16 (t_r'/w)²
• Chromatography Instrumentation (generic)
  • Column
    • packed
    • coated capillary
      • smaller diameter particles = better resolution
  • Mobile phase
    • GC-- carrier gas (He, light gases best)
    • HPLC-- fluids. pumps, mixers
  • sample injection
    • vaporizer in GC / syringe
    • alternative GC (gas samples, desorbed samples)
    • mechanical injector for HPLC
    • auto-injectors
  • Programming Options
    • temperature programming GC
    • isocratic vs. gradients in HPLC
    • column switching methods
    • to deal with sample mixture, extremely varied components
  • Detectors (many options)
    • Somewhat universal response type detectors
      • GC: TCD, flame ionization, light scattering (evap. solvent)
      • HPLC: UV absorption
      • Ion Chromatography: conductance
      • reliance on retention time to identify species ?
        • ok for controlled mixtures
        • often useless for real mixtures
        • of limited forensic value
    • Somewhat more selective
      • GC: Photoionization, Electron Capture
      • HPLC: electrochemical
    • Species Specific Detectors
      • GC/MS, LC/MS
      • GC/IR (gas phase)
      • Typically unambiguous identification of species
    • Data systems
      • retention time
      • peak area
      • deal with slight overlap, drifting baseline
      • computations (internal standards)