HPLC when GC won’t cut it!!!

Types of HPLC Reverse-phase (water/MeOH-soluble) Normal Phase (very polar) Adsorption (very non-polar) Ion-Exchange (ionic) Size-exclusion (large MW, >10 4 )

P P C18 Phase designed to retain very polar compounds Si-O-Si-(CH 2 ) 17 -CH 3 CH 3

Reverse-phase mobile phases Water Methanol Acetonitrile THF Additives, salts, acids, bases Ion pairing

Gradients in reverse-phase For complex mixtures Polar non-polar –Buffer A 100 % H 2 O –Buffer B 100 % MeOH

Separation Efficiency Van Deempter H = a + b/v + cv –H – plate height –A – multiple paths –B – longitudinal diffusion –C – equilibrium Ideal v

Key Variables effecting HPLC separation and sensitivity Minimizing H –Smaller ID of packing beads (3-10  m) Maximizing N - number of extraction events –Longer columns N = L/H Maximizing sensitivity –Smaller ID columns –Band concentration vs. sample capacity

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 –  = k` a / k` b = tr` a /tr` b –Selectivity of a column for the separation of component A and B Resolution = (tra – trb)/W = N 1/2 /4(  /(  -1)) 2 ((1+k` b )/k` b ) 2

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

Ion Exchange Ion exchange resins –Strong cation, -SO 3 - H + –Weak cation, - COO - H + –Strong anion, - N(CH 3 ) 3 + OH - –Weak anion, - NH 3 + OH - Bound to polystyrene support Mechanism –RSO 3 - H + + P RSO 3 - P + + H +

Ion Exchange Gradients Mobile Phase A – H 2 O Mobile Phase B – 500 mM NaAc

Single ion chromatography Separation of small ionic species –PO 4 3+, SO 4 2-, BrO 3-, NO 2-, F -, Cl -, ect –Mg 2+, Na +, Ca 2+, Li +, Ba 2+, ect – -Detected by differences in conductivity

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 (10 3 ) V e = V 0 + KV i Large molecules can not diffuse into the pore, V e = V 0

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

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

ICAT reagent Selectively capture cysteine-containing peptides Wall of column avidin biotin linker iodoacetamide C S A TW M P A

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