HPLC when GC won’t cut it!!!

HPLCwhen 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, >104)

P P C18 Phase designed to retain very polar compounds CH3 Si-O-Si-(CH2)17-CH3 CH3

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 % H2O • 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 mm) • 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 – a = k`a/ k`b = tr`a/tr`b • Selectivity of a column for the separation of component A and B • Resolution = (tra – trb)/W = N1/2/4(a/(a-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, -SO3-H+ • Weak cation, - COO-H+ • Strong anion, - N(CH3)3+OH- • Weak anion, - NH3+OH- • Bound to polystyrene support • Mechanism • RSO3-H+ + P RSO3-P+ + H+

Ion Exchange Gradients • Mobile Phase A – H2O • Mobile Phase B – 500 mM NaAc

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

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

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

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

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

HPLC when GC won’t cut it!!!