ChromatographicMethods: Basics Advanced Chromatographic Methods: Basics, Advanced HPLC MethodsLecture of Seema Mishra in the advanced course Bioinorganic Chemistry & Biophysics of Plants 2012Chromatography: Basics Chromatography a physical method for the separation of mixture g p y p y pbased on the concept of partition coefficientChromatographyinvolves two phases Chromatography involves two phasesMobile phase: a liquid/ gas which caries the mixture to be separated Stationary phase: through which the mixture is carried bymobile Phase it can be solid/ liquid Phase, it can be solid/ liquidSeparations are carried out based on differences in physical andCh i l ti f tit t f i t h Chemical properties of constituents of a mixture such assize, shape, mass, charge, boiling point, polarity or chemical affinity Chromatography: TerminologyChromatogram: The visual output of the Chromatogram: The visual output of the chromatographRetention time: The characteristic time Retention time:The characteristic time a particular analyte takes to pass throughthe system i.e. from column inlet to the k i peak maximaPeak height: The distance between thepeak maximum and the base linePeak width: The distance between each side of a peak measured at certain height of the peak of the peakx-axis=the time and y-axis =a signalChromatography: principle/Theory Chromatography: principle/Theorythe solutes will elute in order of their increasing distribution coefficients with respect to the stationary phasePlate theory: column is considered to be divided into a number of platesN =5 55* t2/ w2N = 5.55tR/ w equilibrium must exist in each plateXs = KXm(Xm) ; concentration of solute in the mobile phase(m) ; p(Xs) ; concentration of solute in the stationary phase(K); distribution coefficient of the solute between the two phases with reference to the stationaryphase phases with reference to the stationary phase K X /X K = Xs/XmChromatography: principle/Theory Chromatography: principle/Theorythe change of mass of solute (dm) in plate (p) will be d =(X X )dV dm= (Xm(p-1)- Xm(p))dVAt equilibriumdm= vsdXs(p) + vmdXm(p) Chromatography: principle/Theory g p y p p yX = X e-vvn/ n ! Xm(n)=X0. e v / n !Basic elution curve equation it shows that if (n= no. of theoretical plates) is large, the function tends to the Gaussian function.Vr =Vm+KVSVr =Vm KVSThe retention volume depends solely on the distribution coefficient and the volumes of the two phases that are present in the column the two phases that are present in the column. K(A) K(B)orVS(A) VS(B) The separation of two solutes depends exclusively on the magnitude of their distribution coefficients (K(A)) and (K(B)) and the amount of stationary phase available to them, (V(A)) and (V ) and (V(B)).ChromatographyElution modeIsocratic elution : The composition of the mobilephase kept constant through out elutionIsocratic elution Gradient elution : The composition of the mobile phasevaried duringelution varied during elutionLinear gradient Linear gradient% BLinear gradient Step gradientStep gradientStep gradient minChromatography g p yElution modePAH analysis through HPLC y gI ti l tiGradient elutionIsocratic elutionACN/H2O ACN/H O70/30 ACN/H2O0-550/505-20 100/0ACN/H2O 70/3020-30100/0Chromatography: Types Chromatography: TypesBased on shape of chromatography (stationary phase)Paper chromatography: Paper chromatography:- A paper serves as stationary phase - Separating and identifyingmixtures by colourThin layer chromatography: - Thin layer of silica gel, alumina or cellulose adsorbed on an inert substrate adsorbed on an inert substrateColumn chromatography: Th t ti h i k d i l - The stationary phase is packed in a columnChromatography: Types g p y ypBased on physical state of mobile phaseGas chromatography: -Mobile phase is gas like He p gApplications:Analytical chemistry, petrochemical environmental monitoring petrochemical, environmental monitoring Not good for bimolecules e.g. protein due to high heat heatLiquid chromatography : Mobile phase is liquide.g. high performance liquid chromatogrephyg g p q g p yHi h f li id h t hChromatography: TypesHigh performance liquid chromatography Optimized for rapid high resolution separations-Very high efficiency HPLC columns with inert packing materials- Fine particle packing (5m) providing larger surface for interaction- HPLC high pressure pumps with very constant flow (6000-10000 psi) -Unique high accuracy, lowdispersion, HPLC sample valves Unique high accuracy, low dispersion, HPLC sample valves (sub l - few l )- Extremely precise gradient mixers (optional).High sensitivitylowdispersionHPLC detectors - High sensitivity low dispersion HPLC detectorsA li ti Applicationsquality control, process control, forensic analysis, environmentalmonitoring and clinical testingChromatography: TypesHigh performance liquid chromatography Chromatography: TypesHigh performance liquid chromatography Normal phase (NP-HPLC): Polar stationary phase e.g. silica Non polar mobile phase e.g. Toluene Polar interactionNon polarPolar pReverse phase (RP-HPLC): Non polar stationaryphase e.g. C18 Reverse phase (RP HPLC):Non polar stationary phase e.g. C18Polar mobile phase such as waterHydrophobic interactionPolar Non polar PolarNon polarChromatography: TypesHydrophilic interaction chromatography (HILIC-HPLC)-A kind of partition chromatography - solute equilibrate between a liquid stationary phase and eluent-Separationbased on polar differences Separation based on polar differences-Can separate acid, base and neutral molecule in single chromatogram -Good for separation of very polar compounds such as amino acids, glycopeptides oligonucleotides and highly polar natural products glycopeptides, oligonucleotides, and highly polar natural products Ion exchange chromatographyChromatography: TypesIon exchange chromatographyPrinciple: highly charged proteins bind stronger to the column material, so that they elute at higher salt concentrations in the buffer than less charged proteinsFrom: www.ucl.ac.uk/~ucbcdab/enzpur/ionX.htmfoto of phycobiliprotein purification in the lab of H. Kpper on a MonoQ anion exchange columnIonpair chromatographyChromatography: TypesIon pair chromatography - Ion Pair Chromatography is a method for improving the separation of charged analytes g p y p g p g y-The ion pair reagents comprise of an alkyl chain with an ionizable terminusAdvantages over ion exchange- Simple preparation of buffers- Wide choice of carbon chain lengths for i d t ti d ti improved retention and separation- Significantly reduced separation time-Simultaneous separation of both ionizedand nonionized solutes- Highly reproducible results- Improved peak shape p p pi l i h t hChromatography: Typessize exclusion chromatographyPrinciple: Small proteins can enter more of the pores in the column material than large proteins, so that small proteins migrate slowerFrom: elchem.kaist.ac.kr From: http://en.wikipedia.org/wikiChromatography: TypesThe higher-affinity solutes are preferentiallyDisplacement chromatographyg y p yretained near the head of the column, withthe lower-affinity solutes moving fartherdownstreameach makingpure zones downstream each making pure zonesA displacer having higher affinity than Sample components will push other Sample components will push other Components downstream making a displacementtrainAdvantage:Comparatively more material can bep yseparated High-retention conditions can achieved without gradient operation without gradient operationDisadvantage:Overlapof zone may occur Overlap of zone may occur Difficulty in interpretation on chromatogramColumn regeneration AffinitychromatographyChromatography: TypesAffinity chromatography Based on a highly specific interactionbetween analyte and stationary phasedolly.biochem.arizona.edu/.../methods.html foto of TcHMA4 purification in the lab of H. Kpper on an IMAC columnChromatography: TypesS i l t h i Special techniques Fast protein liquid chromatography Fast protein liquid chromatography-Also called as fast performance liquid chromatographyOften used for protein purification - Often used for protein purification- Operates at low pressure typically less than 5 bar- flow rate is relatively high, typically 1-5 ml/min.-Relatively low cost-Pressure is not a limitation Supercritical fluid chromatography(SFC)- Mobile phase is carbon dioxide-To separate thermallylabile molecules To separate thermally labile molecules- Separation of chiral coumpoundsChiral column chromatography:- Chiral stationary phase F ti f ti - For separation of enantiomersChromatography: TypesSpecial techniques Special techniquesTwo-dimensional chromatography:-Two columns with different physicochemical properties are used- Increased peak capacityHyphenated techniquesHyphenated Techniques combinechromatographic and spectral methods tol it th d t f b th exploit the advantages of both.Chromatography - Produces pure or nearly pure fractions of chemical components in a mixture components in a mixture.Spectroscopy Produces selective information for identification usingp py gstandards or library spectra.Hyphenated techniques yp qLC-Absorbance dataJ amesK HardyandTheUniversityof Akronhttp://ull chemistryuakronedu/chemsep/hyphen/ J ames K. Hardy and The University of Akron http://ull.chemistry.uakron.edu/chemsep/hyphen/Hyphenated techniques Hyphenated techniquesLiquid chromatography- mass spectrometry (LC-MS) ESI-MSHPLCQTOF-MSHyphenated techniques yp qHPLC-ICP-MSInductively Coupled Plasma Mass Spectrometry (ICP-MS)argongasFrom: Perkin Elmer teaching file (top); LC-ICP-MS experiment at UFZ Leipzig (bottom)Hyphenated techniquesHPLC-ICP-MSS i ti f A i Pl t t t th h RP HPLC (C18) l d t ICP MS1Speciation of As in Plant extract through RP-HPLC (C18) coupled to ICP-MS9761023 541114138 12 15Hyphenated techniquesHPLC-ICP-MS-ESI-MS(II)(II)Hyphenated techniquesHPLC-ESI-MS-ICP-MSESI MSICP-MSESI-MSHPLCHyphenated techniquesHPLC ESI MS ICP MS HPLC-ESI-MS-ICP-MSHyphenated techniquesHPLC-ESI-MS-MSAnalytical LabUFZ, Leipzig UFZ, LeipzigAnalytical LabUFZ Leipzig UFZ, LeipzigThe slides can be downloaded from the workgroup hhomepage ni konstan de Department of Biolog Workgro ps Kpper lab www.uni-konstanz.de Department of Biology Workgroups Kpper lab,or directlyyhttp://www.uni-konstanz.de/FuF/Bio/kuepper/Homepage/AG_Kuepper_Homepage.html