HPLC

HPLC

1. ADVANTAGES

   • SENSITIVE
   • CAN BE AUTOMATED
   • QUANT ANALYSIS
   • GOOD AT SEPARATING VOLATILE/THERMALLY LABILE COMPOUNDS
   • WIDE RANGE OF SUBSTANCES: in/organic, metals, ions, proteins, aa,etc.
   • SEPARATE ON SIZE, CHARGE, POLARITY, SOLUBILITY
   • CHIRAL SEPARATIONS
   • + WIDE RANGE OF DETECTORS
   • RECOVER SAMPLES

2. DISADVANTAGES

   • EXPENSIVE, IMPURITY FREE SOLVENT USED.
   • MOVING PARTS & CONNECTIONS
   • OPTIMAL PERFORMANCE = KEEP IT RUNNING REGULARLY

3. Selection of LC modes

4. HPLC HARDWARE

   • Mixing controller
     • Mix difft solvents in precise ratios
     • reqd for: ISOCRATIC= constant solvent composition. GRADIENT= changing solvent composition
     • mix diffnt solvents in precise ratios
     • make sure that solvents are miscible
   • Injector
     • loop & valve configuration
     • load & inject positions
     • flat ended syringe - manual injection
     • ensure the loop is entirely filled - inject sample in 1 plug (~ 3 rinses)
   • Columns
     • generally = stainless steel
     • Analytical column: ID- 1-5mm, particle size- 3-5um, length- 35-250mm, plates/m-40000-70000
     • typical: 4.6mm x 5um x 150m = 10000 plates/column
     • Microbore or 'rocket'
       • short (30-75mm), thin (<4.6mm ID), small particles (3um) = faster
     • what's inside?
       • normal p.: polar stat, n-polar solvent (Si stst p.), 1:9 EtOAc:hexane mob p.
       • reverse p.: n-polar stat, polar solvent. (C18 stat p.), 15:85 MeOH:H2O mob p.
     • Monolithic (single crystal)
       • Polymer based, porous core, can control pore size
       • can run under lower pressure condition
       • better resolution, speed.
       • useful for larger mols.
     • How do you choose?
       • partition Chrom.
         • OPTIMISING CONDITIONS (3 parameters)
           • N : plate number
           • k : retention factor (strongly dependent upon make-up of mob. p.)
             • ideal range of k:2-10 complex mix: 0.5 - 20 &nbsp;&nbsp;α : can be changed by choosing diff column packing&nbsp;
           • α: selectivity factor
         • stat. p. is a 2nd liquid immiscible w liq mob. p
         • nonionic, polar w low/moderate mol mass. (ionic cmpds w derivatization &ion pairing)
         • change mob. p. composition = enhance separation
         • 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
         • Normal Phase : k2/k1 = 10 ^(P'1-P'2)/2
           • if the ratio k2/k1 is large, need o have a big k2. if its small, - not resolved k2: final retention factor k1: initial "
         • Rev Phase: k2/k1 = 10 ^(P'2-P'1)/2
         • EFFECT OF MOB. P. ON α.
           • manipulate chemi nature: - proton acceptor, proton donor, dipolar interactions
             • start to add small amt of acid or base.
           • in rev p. : aq solns of : THF, acetonitrile, methanol.
         • 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
         • 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.
       • adsorption chrom.
       • Ion Chrom
         • active sites for cation exchange: sulfonate (strong acid) & carboxylic acid gp (weak)
         • Anionic exchangers - strongly basic tertiary amine gps / weak basic primary amine groups.
         • sulfonate based cationic resin equilibrated w NaOAc -injection of amines & alcohol
         • base materials: silica/zirconium/ organic polymer
         • with polymer columns: -know the pressure limitations of the packing, - packing may swell/contract in different solvents.
       • Size exclusion C. (gel permeation c.)
         • high mol mass sp.
         • packings (~10um) silica/polymer particles - a network of uniform pores
         • most stat. phases are crosslinked polymers
         • polymers may expand/contract in diffnt solvents.
         • Vtot = Vg + Vi + Vo
           • 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)
         • Vo = largest mol, Vi + Vo = smallest mols, Ve=Vo + KVi = intermediate mols (assume theres no mixing)
           • KVi : measure of th efraction of mols in the pores (K = Cs/Cm)
         • get finer separation by connecting columns in series. problems??
       • Affinity
         • bond an affinity ligand to a neutral solid support
         • ligand reversibly and selectively bind to analyte (Ab, enz inhibitor)
         • mobile phase hv dual roles:
           • promote strong binding (analyte & affinity ligand)
           • release bound sp. when other cmpds hv been eluted
         • can wash out by changing ionic strength or changing pH
       • Bonded Phases
         • PHENYL ETHER (extremely polar aromatics)
         • SILICA
         • NITRO (aromatics & double bonds)
         • PHENYL HEXYL (aromatics)
         • SULFONIC ACID (weak bases)
         • QUATERNARY AMINE (nucleotides, nucleosides, organic acids)
         • DIOL (rev: gel filtration of peptides & proteins) (normal: = to silica, x deactivated by H2O)
         • CYANO (slightly polar)
         • AMINO (normal: alternate selectivity to silica) (rev: carbs) (ion xchange: anions & org'c acids)

5. GENERAL STRUCTURE

6. DETECTORS

   • IDEAL REQMNTS
     • adequate sensitivity
     • stable & reproducible
     • linear response over several orders of magnitude
     • short response time, independent of flow rate
     • similarity in response to all possible analytes
     • non-destructive
     • minimum internal volume
     • compatible w liquid flow
   • 3 basic types: optical, electrochemical, mass
   • 1) REFRACTIVE INDEX
     • changes in DENSITY when analytes comesthrough cell
     • sensitive to turbulence, temp, solvent changes
     • used w isocratic systems only
     • fragile flow cells
   • 2) EVAPORATIVE LIGHT SCATTERING DETECTORS (ELSD)
     • effluent nebulised into fine mist -- through laser & scattered radiation detected at right angle to laser
     • volatile mob. phase.
     • evaportn tube is heated, but temp kept low - labile cmpds not degraded
   • 3) FLUORESCENCE
     • sample excited by radiation from source (Hg, Xe, laser)
     • resulting fluorescence detected at 90° to excitn beam
     • can derivatise sample (add fluoro tag)
   • 4) UV & UV-VISIBLE (190-900nm)
     • filter variable, fixed wavelength, wavelength variable
     • UV-VIS needs 2 lamps: a UV lamp - Hg, a visible lamp - tungsten
   • 5) PHOTODIODE ARRAY (PDA)
     • individual photosensitive elements : small silicon photodiodes (consist of a reversed-biased pn junction
   • 6) ELECTROCHEMICAL
     • 4 electrochemical techniques: amperometry, voltammetry, coulometry, conductometry
     • Specific applications
     • respond to sp that can be red/ox
     • output is electron flow generated
     • 3 electrodes : working, auxillary, reference
   • 7) MASS SPEC
     • common ionisation sources : electrospray ionisation (ESI), Atmospheric Pressure Chemical Ionisation (APCI)

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