HPLC

HPLC

ADVANTAGES
1. SENSITIVE
2. CAN BE AUTOMATED
3. QUANT ANALYSIS
4. GOOD AT SEPARATING VOLATILE/THERMALLY LABILE COMPOUNDS
5. WIDE RANGE OF SUBSTANCES: in/organic, metals, ions, proteins, aa,etc.
6. SEPARATE ON SIZE, CHARGE, POLARITY, SOLUBILITY
7. CHIRAL SEPARATIONS
8. + WIDE RANGE OF DETECTORS
9. RECOVER SAMPLES
DISADVANTAGES
1. EXPENSIVE, IMPURITY FREE SOLVENT USED.
2. MOVING PARTS & CONNECTIONS
3. OPTIMAL PERFORMANCE = KEEP IT RUNNING REGULARLY
Selection of LC modes
HPLC HARDWARE
1. Mixing controller
  1. Mix difft solvents in precise ratios
  2. reqd for: ISOCRATIC= constant solvent composition. GRADIENT= changing solvent composition
  3. mix diffnt solvents in precise ratios
  4. make sure that solvents are miscible
2. Injector
  1. loop & valve configuration
  2. load & inject positions
  3. flat ended syringe - manual injection
  4. ensure the loop is entirely filled - inject sample in 1 plug (~ 3 rinses)
3. Columns
  1. generally = stainless steel
  2. Analytical column: ID- 1-5mm, particle size- 3-5um, length- 35-250mm, plates/m-40000-70000
  3. typical: 4.6mm x 5um x 150m = 10000 plates/column
  4. Microbore or 'rocket'
    1. short (30-75mm), thin (<4.6mm ID), small particles (3um) = faster
  5. what's inside?
    1. normal p.: polar stat, n-polar solvent (Si stst p.), 1:9 EtOAc:hexane mob p.
    2. reverse p.: n-polar stat, polar solvent. (C18 stat p.), 15:85 MeOH:H2O mob p.
  6. Monolithic (single crystal)
    1. Polymer based, porous core, can control pore size
    2. can run under lower pressure condition
    3. better resolution, speed.
    4. useful for larger mols.
  7. How do you choose?
    1. partition Chrom.
      1. OPTIMISING CONDITIONS (3 parameters)
        N : plate number
        k : retention factor (strongly dependent upon make-up of mob. p.)
        Anmerkungen:
        ideal range of k:2-10 complex mix: 0.5 - 20 α : can be changed by choosing diff column packing
        α: selectivity factor
      2. stat. p. is a 2nd liquid immiscible w liq mob. p
      3. nonionic, polar w low/moderate mol mass. (ionic cmpds w derivatization &ion pairing)
      4. change mob. p. composition = enhance separation
      5. SOLVENT STRENGTH
        Strong solvent: interacts strongly w solutes (often polar)
        Polarity index (P') - based upon solubility in 3 separate solvents, -a measure of relative polarity, -higher P' = stronger
        Mixed solvent
        P(AB)' = *AP'(A) + *BP'(B) *A= volume fraction of solvent A
      6. Normal Phase : k2/k1 = 10 ^(P'1-P'2)/2
        Anmerkungen:
        if the ratio k2/k1 is large, need o have a big k2. if its small, - not resolved k2: final retention factor k1: initial "
      7. Rev Phase: k2/k1 = 10 ^(P'2-P'1)/2
      8. EFFECT OF MOB. P. ON α.
        manipulate chemi nature: - proton acceptor, proton donor, dipolar interactions
        Anmerkungen:
        start to add small amt of acid or base.
        in rev p. : aq solns of : THF, acetonitrile, methanol.
      9. ION PAIR CHROM. (ANALYSTES THAT MAY EASILY IONISE) -org acids, amines.
        + solvent modifier
        org salt w large org counter ion
        added in small conc
        2 xplanations
        counter ion pairs w analyte ion & this pair partition into stat. phase.
        counter ion retained by the neutral stat. phase giving it a charge & get reversible ion-pair interactions w charged analyte
      10. CHIRAL SEPARATIONS
        ceramic Mg silicate
        cyclodextrin
        Pirkle type chiral stat. phase. Works on π acceptor/ π donor interactions some H bonding.
        separate chiral compounds - enantiomers
        either mob/stat phase reqd to be chiral.
        complexation btwn chiral resolving agent w one of the isomers results in separation.
    2. adsorption chrom.
    3. Ion Chrom
      1. active sites for cation exchange: sulfonate (strong acid) & carboxylic acid gp (weak)
      2. Anionic exchangers - strongly basic tertiary amine gps / weak basic primary amine groups.
      3. sulfonate based cationic resin equilibrated w NaOAc -injection of amines & alcohol
      4. base materials: silica/zirconium/ organic polymer
      5. with polymer columns: -know the pressure limitations of the packing, - packing may swell/contract in different solvents.
    4. Size exclusion C. (gel permeation c.)
      1. high mol mass sp.
      2. packings (~10um) silica/polymer particles - a network of uniform pores
      3. most stat. phases are crosslinked polymers
      4. polymers may expand/contract in diffnt solvents.
      5. Vtot = Vg + Vi + Vo
        Anmerkungen:
        Vg= vol occupied by stat p. Vi= vol of solvent held in pores Vo= exclusion vol (vol solvent req to elute cmpnds too big for pores)
      6. Vo = largest mol, Vi + Vo = smallest mols, Ve=Vo + KVi = intermediate mols (assume theres no mixing)
        Anmerkungen:
        KVi : measure of th efraction of mols in the pores (K = Cs/Cm)
      7. get finer separation by connecting columns in series. problems??
    5. Affinity
      1. bond an affinity ligand to a neutral solid support
      2. ligand reversibly and selectively bind to analyte (Ab, enz inhibitor)
      3. mobile phase hv dual roles:
        promote strong binding (analyte & affinity ligand)
        release bound sp. when other cmpds hv been eluted
      4. can wash out by changing ionic strength or changing pH
    6. Bonded Phases
      1. PHENYL ETHER (extremely polar aromatics)
      2. SILICA
      3. NITRO (aromatics & double bonds)
      4. PHENYL HEXYL (aromatics)
      5. SULFONIC ACID (weak bases)
      6. QUATERNARY AMINE (nucleotides, nucleosides, organic acids)
      7. DIOL (rev: gel filtration of peptides & proteins) (normal: = to silica, x deactivated by H2O)
      8. CYANO (slightly polar)
      9. AMINO (normal: alternate selectivity to silica) (rev: carbs) (ion xchange: anions & org'c acids)
GENERAL STRUCTURE
DETECTORS
1. IDEAL REQMNTS
  1. adequate sensitivity
  2. stable & reproducible
  3. linear response over several orders of magnitude
  4. short response time, independent of flow rate
  5. similarity in response to all possible analytes
  6. non-destructive
  7. minimum internal volume
  8. compatible w liquid flow
2. 3 basic types: optical, electrochemical, mass
3. 1) REFRACTIVE INDEX
  1. changes in DENSITY when analytes comesthrough cell
  2. sensitive to turbulence, temp, solvent changes
  3. used w isocratic systems only
  4. fragile flow cells
4. 2) EVAPORATIVE LIGHT SCATTERING DETECTORS (ELSD)
  1. effluent nebulised into fine mist -- through laser & scattered radiation detected at right angle to laser
  2. volatile mob. phase.
  3. evaportn tube is heated, but temp kept low - labile cmpds not degraded
5. 3) FLUORESCENCE
  1. sample excited by radiation from source (Hg, Xe, laser)
  2. resulting fluorescence detected at 90° to excitn beam
  3. can derivatise sample (add fluoro tag)
6. 4) UV & UV-VISIBLE (190-900nm)
  1. filter variable, fixed wavelength, wavelength variable
  2. UV-VIS needs 2 lamps: a UV lamp - Hg, a visible lamp - tungsten
7. 5) PHOTODIODE ARRAY (PDA)
  1. individual photosensitive elements : small silicon photodiodes (consist of a reversed-biased pn junction
8. 6) ELECTROCHEMICAL
  1. 4 electrochemical techniques: amperometry, voltammetry, coulometry, conductometry
  2. Specific applications
  3. respond to sp that can be red/ox
  4. output is electron flow generated
  5. 3 electrodes : working, auxillary, reference
9. 7) MASS SPEC
  1. common ionisation sources : electrospray ionisation (ESI), Atmospheric Pressure Chemical Ionisation (APCI)

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