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.
     • Guard Column
       • function
         • remove impurities from reach analytical column
       • Particle size is larger to minimize pressure drop

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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