HPLC when GC wont cut it

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HPLCwhen GC wont cut it!!!
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Types of HPLC

• Reverse-phase (water/MeOH-soluble)
• Normal Phase (very polar)
• Adsorption (very non-polar)
• Ion-Exchange (ionic)
• Size-exclusion (large MW, gt104)

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C18 Phase designed to retain very polar compounds
CH3
Si-O-Si-(CH2)17-CH3
CH3
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Reverse-phase mobile phases

• Water
• Methanol
• Acetonitrile
• THF
• Additives, salts, acids, bases
• Ion pairing

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Gradients in reverse-phase

• For complex mixtures
• Polar non-polar
• Buffer A 100 H2O
• Buffer B 100 MeOH

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Separation Efficiency

• Van Deempter H a b/v cv
• H plate height
• A multiple paths
• B longitudinal diffusion
• C equilibrium
• Ideal v

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Key Variables effecting HPLC separation and
sensitivity

• Minimizing H
• Smaller ID of packing beads (3-10 mm)
• Maximizing N - number of extraction events
• Longer columns N L/H
• Maximizing sensitivity
• Smaller ID columns
• Band concentration vs. sample capacity

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Parameters used to describe retention of analyte

• Capacity factor - k
• (tr-tm)/tm, where tr is the retention time of the
  solute and tm is the void retention independent
  of column length
• Selectivity factor a ka/ kb tra/trb
• Selectivity of a column for the separation of
  component A and B
• Resolution (tra trb)/W
• N1/2/4(a/(a-1))2 ((1kb)/kb)2

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Normal Phase

• Bare silica
• Mobile phases, (ethyl acetate/ hexane)
• HILIC columns
• Attach polar groups to silica
• Methanol to water

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Ion Exchange

• Ion exchange resins
• Strong cation, -SO3-H
• Weak cation, - COO-H
• Strong anion, - N(CH3)3OH-
• Weak anion, - NH3OH-
• Bound to polystyrene support
• Mechanism
• RSO3-H P RSO3-P H

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Ion Exchange Gradients

• Mobile Phase A H2O
• Mobile Phase B 500 mM NaAc

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Single ion chromatography

• Separation of small ionic species
• PO43, SO42-, BrO3-, NO2-, F-, Cl-, ect
• Mg2, Na, Ca2, Li, Ba2, ect
• -Detected by differences in conductivity

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Size Exclusion Chromatography

• Stationary phase is a gel
• Fractionates sample on the basis of size
• Elution volume vs. molecular weight
• Pore size of the gel defines the MW range
• Exclusion limit (10 6), permeation limit (103)
• Ve V0 KVi
• Large molecules can not diffuse into the pore, Ve
  V0

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Stationary and Mobile phases

• Gel filtration hydrophilic packing (styrene and
  divinylbenzene) and aqueous mobile phase
• Gel permeation hydrophobic packing (sulfanated
  divinylbenzenes and polyacrylamides) and
  non-polar organic mobile phases

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Affinity Chromatography

• A handle is attached to a solid support, which
  is packed into a column
• This handle selectively binds to a certain
  analyte or group of analytes
• Examples
• Antibodies to capture specific proteins
• avidin binds to biotin

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ICAT reagent

• Selectively capture cysteine-containing peptides

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TLC

• Glass plates coated with thin layer of coated
  particles
• Apply sample with capillary tube or syringe or
  fancy applicators
• Develop plate
• Rf dr /dm, retardation factor