High Performance Liquid Chromatography (HPLC) PDF

4/25/2020 Chapter 24 - Diffusion in liquids is 100 times slower than diffusion in gases. So it is not generally feasible to use open tubular columns in liquid chromatography • The efficiency of a packed column increases as the size of the stationary phase particles decreases. • Typical particle s...

4/25/2020 Chapter 24 - Diffusion in liquids is 100 times slower than diffusion in gases. So it is not generally feasible to use open tubular columns in liquid chromatography • The efficiency of a packed column increases as the size of the stationary phase particles decreases. • Typical particle sizes in HPLC are 1.7 to 5 mm. • Smaller particles in packed column • Decreased Plate height H increased resolution 1 4/25/2020 Decreasing particle size permits us to improve resolution or to maintain the same resolution while decreasing run time. (a and b) Chromatograms of the same sample run at the same linear velocity on 5.0-cm-long columns packed with C18-silica. Plate number increased from 2000 (a) to 7500 (c) when the particle size decreased, so the peaks are sharper with the smaller particle size At the optimum flow rate for each column (minimum plate height in Figure 24-3), the number of theoretical plates in a column of length L (cm) is approximately dp is the particle diameter in mm. The 5.0-cm-long column in Figure 24-2a with 4.0-mm-diameter particles is predicted to provide N of ~ (3 000)(5.0)/4.0=3800 plates. (The observed plate number for the second peak is 2 000. Perhaps the column was not run at optimum flow rate). When the stationary phase particle diameter is reduced to 1.7 mm, the optimum plate number is expected to be ~(3 000)(5.0)/1.7 = 8 800. The observed value is 7500. 2 4/25/2020 Why small particles give better resolution ? 1) Small particles provide more uniform flow through the column, thereby reducing the multiple path term, A, in the van Deemter equation. 2) The distance through which solute must diffuse in the mobile and stationary phases depends on the particle size. The smaller the particles, the less distance solute must diffuse. This effect decreases the C term in the van Deemter equation for finite equilibration time. Or equilibration time is achieved faster Smaller particle size leads to • higher plate number (better resolution) • higher pressure (because of particle resistrance) • shorter optimum run time • lower detection limit (because small particle size with narrow columns and high flow rate, analyte is not diluted so much as it travels through the column) Column Pressure The penalty for small particle size is resistance to solvent flow. The pressure required to drive solvent through a column is where ux is linear flow rate, h is the viscosity of the solvent, L is the length of the column, r is column radius, and dp is the particle diameter. The factor f depends on particle shape and particle packing. From the above equation, the pressure in HPLC is proportional to the flow rate and column length But inversely proportional to the square of column radius (or diameter) and the square of particle diameter. Another penalty of small particle size is the increased frictional heating as solvent is forced through the particles. The center of a column is warmer than the outer wall, and the outlet is warmer than the inlet. To avoid band broadening from temperature differences, column diameter should be ≤ 2.1 mm for 1.7-mm particles 3 4/25/2020 4 4/25/2020 Columns are expensive and easily degraded by dust or particles or impurities. To avoid introducing particulate matter into the column, samples should be centrifuged or filtered through a 0.5-mm filter before they are loaded into vials for an auto-sampler or injected manually. Structure 1. Guard column: Protect the main column • it has the same stationary phase of main column (usually 1 cm long) • It adsorb strongly Fine particles and irreversible adsorbed solutes. • periodically replaced after a set number of injections or time in service. 1. Main column: steel or plastic 5–30 cm long inner diameter 1–5 mm Titanium frits contain the stationary phase uniform flow between narrow tubing and the column 5 4/25/2020 Effect of raised temperature: 1. Decreases retention times and improves resolution because of decreased viscosity of solvent and solutes. 2. increased temperature can degrade the stationary phase and decrease column lifetime. 3. Can also degrade the analyte or the solvent used. To reduce temperature effect, use narrow column and small amount of analyte. Bare silica can be used in adsorption chromatography Bonded stationary phase on silica is used in liquid-liquid partition chromatography • pure, spherical microporous silica: most common support permeable to solvent • has a surface area of several hundred square meters per gram. • Silica cannot be used above pH 8, because it dissolves in base. Bare silica (a) : A silica surface has up to 8 mmol of silanol groups (Si-OH) per square meter. Silanol groups are protonated at pH < 2–3 and dissociate to negative Si-O- ap H > 3. Silica with ethylene bridges resist hydrolysis up to pH 12. 6 4/25/2020 Tailing of amine silica on bases Bare silica (a) : Si-O- (at pH > 3) retain strongly protonated bases (RNH3+) and lead to tailing. Metallic impurities in Type (a) silica also cause tailing. (b) Less acidic Type (b) silica with fewer Si-OH groups and less metallic impurity gives Symmetric peaks with shorter retention time Bonded stationary phase is covalently bonded to silica surface Used for liquid-liquid partition chromatography 7 4/25/2020 The siloxane (Si-O-Si-R) bond hydrolyzes below pH 2, so HPLC with a bonded phase on a silica support is generally limited to the pH range 2–8. If bulky isobutyl groups are attached to the silicon atom of the bonded phase, the stationary phase is protected from attack by H3O+ and is stable for long periods at low pH, even at elevated temperature Another type of nonpolar stationary phase has a polar embedded group such as polar amide group. Embedded polar groups provide alternate selectivities from C18 stationary phases, Improved peak shapes for bases, and compatibility with 100% aqueous phase. Other nonpolar stationary phases should not be exposed to100% aqueous phase because they become very difficult to re-equilibrate with organic phase. Nonpolar bonded phase with embedded polar amide group offers different selectivity from C18, has improved peak shape for bases, and tolerates 100% aqueous eluent. 8 4/25/2020 Superficially Porous Particles Also called fused-core particles -It consist of a 0.25-mm-thick porous silica layer on a 5-mm diameter nonporous silica core. -A stationary phase such as C18 is bonded throughout the thin porous outer layer. -Mass transfer of solute into a 0.25-mmthick layer is 10 times faster than mass transfer into a 2.5 mm fully porous silica -SPP are especially suitable for separation of macromolecules such as proteins, which diffuse more slowly than small molecules. The Elution process In adsorption chromatography, solvent molecules compete with solute molecules for adsorption sites on the stationary phase The relative abilities of the different solvents to elute a given solute is mostly related to the elution strength of the eluents Elution occurs when solvent displace solutes from the stationary phase (competition between solute and solvent). 9 4/25/2020 The Elution process In normal-phase chromatography (bare silica): we use a polar stationary phase a less polar solvent (or non aqueous solvent). A more polar solvent has a higher eluent strength. Reversed-phase chromatography nonpolar or weakly polar stationary phase the solvent is more polar (or aqueous). A less polar solvent has a higher eluent strength. More common In general, eluent strength is increased by making the mobile phase more like the stationary phase. 10 4/25/2020 But if one solvent does not provide sufficiently rapid elution of all components, we use gradient elution 11 4/25/2020 Isocratic HPLC Separation Separation becomes better as B decreases, however, it takes much longer time 12 4/25/2020 Isocratic HPLC Separation Separation becomes much better as B decreases, however, it takes too long Gradient Elution Start with Low B% and then increase B% gradually Result : Good separation and shorter time 13 4/25/2020 Hydrophilic Interaction Chromatography ( or Hydrophilic Interaction liquid Chromatography (HILIC) 14