Ch.28 HPLC 28.1 Basic types of Liquid Chromatography Partition (LLC) Adsorption (LSC) Ion Exchange (IC) Size Exclusion (SEC or Gel Chromatography) High Performance (Pressure) LC Glass column st.steel (high pressure) 28A. Scope of HPLC 28.2 HPLC becomes popular : high sensitivity : possible for quantitative analysis : suitable for non-volatile, thermally fragile : wide applicability amino acids, proteins, nucleic acids hydrocarbons, carbohydrates, dugs terpenoids, pesticides, steroids, antibiotics metal-organic compounds
28.3 28B. Column Efficiency in LC 28.4 two additional sources of zone broadenings 1. Effects of particle size of packings H A B / u (C C M d 2 p S C M )u
28B. Column Efficiency in LC 28.5 2. Extra-column band broadening In injection system detector region connecting parts differences in flowrate inside flow conduits In GC, diffusion is so fast, E-C band broadening minimized, but in LC, it becomes serious. H ex 2 r u 24D M u: cm/s r: radius of tube D M : diffusion in MP 28B. Column Efficiency in LC 28.6 3. Effect of Sample Size
28C. Instruments for LC 28.7 Flow through particle size 2~10 m in modern HPLC. : pump is critical, ~ several thousand PSI. 1. Mobile-Phase reservoirs & Solvent Treatment 28C. Instruments for LC 28.8 Type of mobile phase usage 1) Isocratic 2) Gradient elution : two or more solvents vary their compositions during run. 50/50 MeOH/water 40/60 MeOH/water then raise MeOH at 8%/min
28C. Instruments for LC 28.9 2. Pumping Systems ~6000PSI, pulse-free output 0.1~10mL/min, flow reproducibility 0.5% Corrosion resistant components Reciprocating pumps : ~90% Pneumatic pumps 28C. Instruments for LC 28.10 4. Liquid Chromatographic Columns Stainless steel tubing Heavy-walled glass tubings (<6000PSI) Analytical Columns length: 10~30 cm (typical) id: 4~10 mm packing materials: 5~10 m typical : 4.6mm~25 cm (40,000 ~ 60,000 plates/meter) Guard Columns : protect the analytical column by removing particulates, contaminants from solvents or samples : similar packings to AC but usually larger particles
28C. Instruments for LC 28.11 5. Types of Column Packings Pellicular type : spherical, nonporous, glass or polymer beads 30~40 m, thin porous layer silica, alumina, PS-DVB resin, ion exchange resin. Porous type : porous microparticles (3~10 m) silica (most common), alumina, synthetic resins (PS-DVB) organic films by chemical or physical bondings 28C. Instruments for LC 28.12 6. Detectors No universal detector ideal detectors : similar requirements to GC : except temperature range : minimum internal volume band broadening Types of detector Bulk property detectors : rely on mobile phase bulk properties such as refractive index, dielectric constant, density Solute property detectors : UV absorption - 71% fluorescence - 15% electrochemical detectors - 4.3%
28C. Instruments for LC 28.13 28C. Instruments for LC 28.14 Absorbance detectors Fluorescence detectors UV absorption first then detect the emitted Fluorescence at 90 degree to the excitation beam Hg or Xe : excitation -Minimize volume (1~10 L) < 600 psi -Double beam -Wavelength fixed or variable
28C. Instruments for LC 28.15 RI detectors : applicable to nearly all solutes general to all types of solute but nit sensitive 28C. Instruments for LC 28.16 Evaporative light scattering detector (ELSD) Column effluent mist fine analyte particles nebulizer drying LASER : light scattered at right angles and detected by a silicon photodiode. Advantages: response is app. same for all non volatile solutes DL: 5ng/25 L
28C. Instruments for LC 28.17 Electrochemical detectors : based on amperometry, polarography, coulometry, conductometry Advantages over optical detectors highly sensitive simple convenient widely applicable cell volume: 1~5 L 28C. Instruments for LC 28.18
28C. Instruments for LC 28.19 Mass Spectrometric Detectors (MSD) Relatively large solvent volumes split flow or thermospray Vacuum required Thermospray : vaporize and ionize sample only applicable to polar solute requires salt (ammonium acetate) in solvent 1~10 picogram. 28D. Partition Chromatography 28.20 Widely used (3,000Da) for nonionic polar compounds. Liquid-liquid chromatography : Liq. SP on the surface of packings by physical adsorption - loss of SP, solubility problem in gradient elution Bonded-phase chromatography : SP is chemically bonded to the support surface. 1) Columns in bonded-phase chromatography : silica or silica based particles (3~10 m) hydrolyzed surface by heating with 0.1M HCl for 1 day.
28D. Partition Chromatography 28.21 organochlorosilane : in case of unreacted silanol groups tailing of chromatographic peaks remedy: end capping with chloromethylsilane 28D. Partition Chromatography 28.22 RP-NP packings Stationary P Mobile P elution order NP polar nonpolar nonpolar first (triethylene glycerol hexane or supported on silica, alumina) isopropyl ether RP nonpolar polar polar first (C18) water, methanol
28D. Partition Chromatography 28.23 28D. Partition Chromatography 28.24 Maximum sample size for C18 ~ 2 C4
28D. Partition Chromatography 28.25 2. Method development in Partition Chromatography MP in partition sample interacts with MP & SP MP in GC only carries sample out. column selection polarity order: hydrocarbons < ethers < esters < ketones < aldehydes < amides < amines < alcohols MP selection k' MP(2 ~ 5) Consider N, k, depends composition. (optimum) can be changed by MP composition (with different packings) 28D. Partition Chromatography 28.26 Effect of solvent strength on retention factors Strong solvent : solvent that interacts with sample strongly (polar solvents) Polarity index P (by Snyder) -2~10.2
28D. Partition Chromatography 28.27 28D. Partition Chromatography 28.28 Polarity for mixed solvents P ' AB A P ' A B P ' B Change polarity of MP change in k 2 units 10 fold change k k k k ' ' ' 2 (P1 P2 ' 1 ' ' 1 10 ' ' 2 (P2 1 10 ) / 2 P ) / 2 Check example 28-1. A : volume fraction of A For normal phase 1: initial, 2: final For reversed phase
28.29 28D. Partition Chromatography 28.30 Effect of MP on selectivity three compatible solvents for adjustment of values MeOH, CH 3 CN, THF Fig 28-16-b-c-d, same k =10 water level adjusted with diff. solvents.
28D. Partition Chromatography 28.31 28D. Partition Chromatography 28.32
28D. Partition Chromatography 28.33 Derivative formation : derivatization before or after chromatogr. : to reduce the polarity to increase the detector response (sensitivity) to selectively enhance the response for certain components Automatic pre-column reaction with orthophthalaldehyde formed as isoindoles fluorescence 425nm Rather than IEC and postcolumn der. with Ninhydrin 28D. Partition Chromatography 28.34 Ion-Pair Chromatography MP : aqueous buffer containing organic solvent MeOH or CH 3 CN ionic compound containing a counter ion of opposite charge to the analyte. counter ion: ion that combines with the analyte ion to form an ion pair, neutral species. Applications : for small inorganic & organic ions. (in case of poor resolution in IC) Chromatography with Chiral SP (CSP) For optically active isomers (enatiomers) (GC is useful but at elevated T, difference in K for diastereomers become smaller and racemization of CSP) HPLC needs various CSP.
28F. Ion-Exchange Chromatography 28.35 Mid 1970s. IEC or IC : based on ion exchange eq. between ions in solution and ions of like sign on the solid surface. Natural ion exchangers : clays, zeolites Synthetic ion exchangers in 1930 : for water softening, deionization, solution purification For cation exchange : - SO 3 H + (strong) -CO 2 H + (weak) For anion exchange : - N(CH 3 ) 3+ OH - (strong) -NH 3+ OH - (weak) Detection : conductivity detector Problems in high eluent conductance in IC 28.36 suppressor column Micromembrane suppressor
28G. Size-Exclusion Chromatography 28.37 Gel permeation or gel filtration chromatography Packings: small (~10 m) silica or polymer particles containing a network of uniform pores into which solute diffuse. Larger molecules are excluded and no retention. elutes first. Smaller molecules can penetrate through pores entrapped and eluted last. : cross-linked styrene-divinylbenzene copolymers : porous glasses or silica particles (40~200A) 28.38