HPLC High Performance Liquid Chromatography Chromatographic Separation Techniques ‘Which Separation Tacnique For Which Compound?LC * Widely used + Sensitivity + Accuracy * Easy automation + Separate non volatile species or thermally fragile ones + Aminoacids, proteins, nucleic acids, hydrocarbons, drugs, terpemoids, persticides, antibiotics, steroids, metal-organic species, inorganic subsatnces EFFECT OF PARTICLE SIZE OF PACKINGS * The size of column should improve as particle size decreases + Reduction in particle size from 45 to 6 micrometre results in tenfold decrease in plate height EFFECT OF PARTICLE SIZE OF PACKINGS * The size of column should improve as particle size decreases + Reduction in particle size from 45 to 6 micromeire results in tenfold decrease in plate heightEXTRACOLUMN BAND BROADENING IN LC + Broadening because of difference in flow rates between layers of liquid adjacent to wall and centre + The central part moves fast than wall side + Small-bore column much more problematic + Packing of 3-10 rm LC INSTRUMENTATION HPLC * High Performance LC + Analysis method + Accuracy and high speed:forcibly pumped mobile phase + Ultrafast analysis using a high-pressure-resistant apparatus ; * UHPLC (Ultra High Performance LC) -ultrafast LC method.Partitioning + Separation is based on the analyte’s relative solubility between two liquid phases HPLC - Modes +» Normal Phase. - Polar stationary phase and non-polar solvent. - Retain polar- non polar out + Reverse Phase - Non-polar stationary phase and a polar solvent - Retain non polar and release polar~—— 4. Bump: +The role of the pump is te force 9 liquid (called the mobile phase) through the liquid ‘chromatograph at a specific low rate, expressed in milters per min (mL/min) ‘ Normal flow rates in HPLC are in the 1-to 2-mLimin range: + Typical pumps can reach pressures in the range of 6000-9000 psi (400- fo 600-bar). + Duning the chromatographic expenment, a pump can deliver a constant mobile phase composition {(socratic) or an increasing mobile phase composinon (gradient) 2. Injector: + The injector serves to intreduce the liquid sample into the flow stream of the mobile phase, * Typical sample volumes are 5- to 20-micraliters (uL). + The injector must also be able to withstand the high pressures of the liquid system. + An autosampler is the automatic version for whan the user has many Samples to analyze or ‘when manual injection isnot practical2. Selumes —_—=— + Considered te ‘hart tthe chromatograph the columns statonary phase separates the ‘sano componarte of ntorostusng various piyacal and chomial paramotrs ~The smal pets Inside the cohen ae wha cause the high backpressure at imal flow rates «The pump mist ish hard to nove the mabe pss through the column and this resistance esuses ahh pressure wthin te chromotogreph 4 patector: +The detector can soo (dete he ndivdua molecules that come ou (ie) trem ne column " Adetecor serves oTmeastre te aMoUNTO fase mowcaes Sat Pe ener can enema, [PS erect REFRACTIVE INDEX DETECTORSREFRACTIVE INDEX DETECTORS + (1) universal response; + (2) low sensitivity to dirt and air bubbles in the cells; + (3) the ability to cover the entire refractive index range + (4) reliable and unaffected by flow rate DISADVANTAGES * relatively low sensitivity * General disability to easily remove and clean or replace the cell when, filming or clogging oceurs. APPLICATIONS * Sugar analy: ELECTROCHEMICAL DETECTORS secmamweatt] TII[L sesmecas (41890) LI glassy carbon) ElventELECTROCHEMICAL DETECTORS * Measure components displaying oxidation-reduction reactions, and detects electric currents generated by these reactions. * First electrod -ause oxidation or reduction + Second electrode: measures oxidation or reduction product + High selectivity and sensitivity * Measurement of catecholamine + Not compatible with gradient elution LC MS DETECTOR PERFECT FINGERPRINT Molecular mass, structural information, accurate quantity LC/MS/MS —tandem MS/MS — great resolutionAPPLICATIONS + Pharmaceutical analysis * Food analysis * Protein studies + Composition + Purity checking PARTITION CHROMATOGRAPHY + Stationary phase is liquid that is immiscible by mobile phase + Earlier liquid-liquid columns were used + Now-a-days, liquid-bonded-phase columns + In liquid-liquid: physical adsorption + Inbonded-phase _: chemical bonding ; highly stable packingCOLUMNS FOR BONDED PHASE CHROMATOGRAPHY ou at oko * Silica or silica-based LaAlALaAl * Surface of silica with silanol group frre + Uniform, porous, mechanically sturdy * Diameter : 1.5-10 jum (3-5 pm most common) + Siloxanes [organochlorosilane] + NORMAL PHASE PACKING * REVERSED PHASE PACKING: ¥ Bonded phase packing Y Mostly all HPLC separations Y Mass transfer is rapid in NP SP : Cy or Cig © Advantage : water can be used [inexpensive, nontoxic]Normal Phase / Reversed Phase Stationary | Mobile phase phase i Normal | High polarity Low polarity phase (hydrophilic) (hydrophobic) Reversed| Lowpolarity High polarity phase (hydrophobic) | — (hydrophilic) Reversed Phase Chromatography + Stationary phase: Low polarity + Octadecyl group-bonded silical gel (ODS) + Mobile phase: High polarity + Water, methanol, acetonitrile * Salt is sometimes added.Comparison of Normal Phase and Reversed Phase * Normal Phase * Reversed Phase + Effective for separation of structural + Wide range of applications isomers + Effective for separation of homologs + Offers separation selectivity not + Stationary phese has long service life available with reversed phase + Stabilizes quickly + Stabilizes slowly and is prone to fluctuations in retention time + Eluents are expensive + Eluents are inexpensive and easy to use Inereaso in chain length increases soparation COLUMN SELECTION : polarity simitar to analyte MOBILE PHASE SELECTION :N. kAPPLICATIONS DERIVATIVE FORMATION : reduce polarity, increases sensitivity, selectivity TON-PAIR CHROMATOGRAPHY : like RP LC salts; counter ion formation ; retention ionic and non ionic species separation CHIRAL CHROMATOGRAPHY mirror images [enantiomers] chiral agent is immobilized on solid support pi bonds, hydrogen bonds APPLICATIONS Field ‘Typical Miners _| Pharmacrutiess Amtiteotia, eves, sero, eres Bocremical ‘Amine acid, proteins carbo teeta Upads Fed prodacts Atiécal secetener, anion ‘dues, flan, aves Industrial chemicals Condeased aromatics, surtac: tans, propellant, dyes frinvaate Pesciden, hertiades, pheno, pobebliiantd biptecyts Foveasicsclence Drugs. poms, Mood ae acon ‘Clinical cthcenist Bale aomds, drug metabolites.