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Liquid Chromatography HPLC HPLC: High Performance Liquid Chromatography

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Liquid Chromatography HPLC HPLC: High Performance Liquid Chromatography ( liquid-chromatography-hplc-hplc-high-performance-liquid-chro )

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• Capacity Factor • Selectivity(Relativeretention) Van Deemter Equation: (Packed Column) Hcolumn  ABu1 +Cu • Efficiency N L H SM HPLC uses packed columns predominantly dp = 1-5m small particles L = 5 – 30 cm short i. d. = ~0.5 cm  Pressure: 50-350 bar (UHPLC >400bar) Higher temperatures (~10oC) lowers viscosity, lowers pressure used, lowers retention times, may increase stationary phase bleeding, needs preheating of mobile phase. uηL P= f πr2d2 The classification of chromatographic modes according to the retention mechanism... ttvv k'R MR M p Equilibration between liquid mobile phase and solid adsorbent of the solute Equilibration between liquid mobile phase and “liquid” stationary phase of the solute Molecular sieving based on the size of analyte “Lock and Key” mechanism Liquid Chromatography Size Exclusion Affinity Ion Exchange Equilibration between liquid and ionic stationary phase To the solute R tM vM tt vv k' 2M2M2 t t v v k' 1M1M1 2γD   qk' d2 col p u (1+k')2 D 96D (1+k')2  k'2d2  H=dM f p u •Resolution H R N1 k' 4  1k' 2γD qk'd k'dp M  f u H=d   u col p  2 2 222Mf u (1+k') D 96D (1+k') SM 222 2γD qk' d k'dp  =d  colp 2 2 u u (1+k') DS 96DM(1+k')  Small dp, low A Small dp df; low C-slope. uniform flow (A) and fast equilibrations because of the smaller distances analyte molecules would travel due to better packing of smaller, bonded s.p, particles (C). Larger optimum flow rate. Diffusion coefficient, Dm, is low in LC, equilibration slow; m.p. being a liquid use of capillary columns is not possible (pressure considerations). Large s.p. surface preferred, coated on inert particles, both df, dp non-zero. Smallinertparticles,d (increasesthes.p.surfaceand p minimize multiple paths and hence minimize term A) requires very high pressures to move the m.p. through the column. Lower df is preferred; faster equilibration. Adsorption Partition

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