High-Performance Liquid Chromatography HPLC, when GC won’t cut it!!!

Components Mobile phase reservoirs HPLC Pump(s) Mixing valves Sample injector (manual or auto) Column Detector Plumming Mobile phase waste container

HPLC-UV HPLC Pump Jacket for controlling column temperature 6-port valve Mobile Phases A and B Sample loop HPLC column syringe MP waste Detector

HPLC Separations Different analytes have different equilibria between the mobile phase and stationary phase Equilibrium is dynamic; thus we can view it as a given analyte molecule spending a fraction of time dissolved in the mobile phase Since different solutes gave different fractions, a separation of the analytes occur as they are pushed through the column by the mobile phase

Types of HPLC Reverse-phase (polar mobile phase/non-polar stationary phase/somewhat polar analytes) Normal Phase (non-polar mobile phase/polar stationary phase/non-polar analytes) Adsorption (non-polar mobile phase/polar stationary phase/non-polar analytes); isomer separation Ion-Exchange (salts/ionic stationary phase) Size-exclusion (aqueous/gel for large MW solutes, >104)

Columns Length (5-15 cm); much shorter than GC column Diameter (4 mm down to 50mm) Particle size (3, 5, or 10 mm) Different phases bonded to silica Typically detection limit is decreased by decreasing the column diameter Optimal linear flow rate conserved; so optimal volumetric flow rate decreases with the square of the radius 4 mm/ 1.0 mL/min; 1 mm/60 mL/min

Reversed phase stationary phase Most common; n-octyldecyl, C18 Si-O-Si-(CH2)17-CH3 CH3 Si-O-Si-(CH2)17-CH3 CH3 Si-O-Si-(CH2)17-CH3 CH3 Si-O-Si-(CH2)17-CH3 CH3

C18 Phase designed to retain very polar compounds

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 or acetonitrile

RP-HPLC Separation of a Tryptic Digest of BSA 11.36 100 17.23 95 90 85 80 12.57 75 12.74 70 65 Relative Abundance 60 55 50 45 17.68 40 36.21 35 1.21 15.13 24.95 30 25 24.53 20 2.54 22.46 15 3.01 5.43 6.14 21.73 10 25.20 20.41 5 27.31 29.53 32.43 37.18 48.55 40.11 45.43 5 10 15 20 25 30 35 40 45 Time (min)

HPLC Method Development Isocratic, Fig 25-25 Harris Find the best methanol separation Use Table 25-25 to guide you in finding the best acetonitrile and THF separations Based on separations try binary mixtures Methanol, 38 % Acetonitrile, 30 % THF, 22 % 19 % MeOH/15% acetonitrile, 15 % acetonitrile/11% THF, 19 % MeOH/11% THF Trinary mixture, 13:10:7 Temperature/computer simulations

Gradients First step Do you need a gradient? long, simple gradient Adjust accordingly Can become complex Do you need a gradient? If Dt/tG > 0.25, then a gradient is appropriate Dt = time between first and last peak tG = time of gradient

Dt = 22-8 = 14 min tG = 22-4 min = 18 min Dt/tG = 14/18 = 0.63 > 0.25

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

Ion Exchange Ion exchange resins Bound to polystyrene support 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

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

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

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