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History of chromatography

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A brief history of Liquid Chromatography including its major types.

A brief history of Liquid Chromatography including its major types.

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  • 1. HISTORY OF CHROMATOGRAPHYANDVARIOUS TYPES OF LIQUID CHROMATOGRAPHYCHROMATOGRAPHY IS A SET OF LABORATORY TECHNIQUES INTENDED TO SEPARATECOMPOUNDS FROM A MIXTURE IN ORDER TO EITHER PURIFY THEM OR FOR THEIRIDENTIFICATION.IT HAS HAD A POWERFUL IMPACT IN TERMS OF A SCIENTIFIC CONCEPT BEGINNING IN1860 WITH THE WORK OF FRIEDRICH GOPPELSROEDER WHO WAS A PIONEER OF PAPERCHROMATOGRAPHY.HE DEVELOPED THE THEORY OF CAPILLARY ANALYSIS BY USING PAPER STRIPS WHILEEXAMINING WINE, MILK, ALKALOIDS, DYES AND OILS AMONG OTHER.HIS WORK WAS AN IMPROVEMENT OF THE WORK OF CHRISTIAN FRIEDRICHSCHÖNBEIN, WHO WAS HIS MENTOR (1799-1868)1www.orachrom.com/net
  • 2. IN 1906 MIKHAIL TSVET, A RUSSIAN BOTANIST, DEVELOPED THE CONCEPT OFLIQUID CHROMATOGRAPHY DURING HIS ATTEMPT TO PURIFY CHLOROPHYLLSFROM PLANT EXTRACTS. THIS DISCOVERY EARNED HIM THE NICKNAMEOF, “FATHER OF CHROMATOGRAPHY” MORE APPROPRIATELY FATHER OF“LIQUID” CHROMATOGRAPHY.HE CONTINUED TO WORK WITH CHROMATOGRAPHY IN THE FIRST DECADE OFTHE 20TH CENTURY, PRIMARILY FOR THE SEPARATION OF PLANT PIGMENTSSUCH AS CHLOROPHYLL, CAROTENES, AND XANTHOPHYLLS.SINCE THESE COMPONENTS HAVE DIFFERENT COLORS (GREEN, ORANGE, ANDYELLOW, RESPECTIVELY) THEY GAVE THE TECHNIQUE ITS NAME“CHROMATOGRAPHY” OR “COLOR WRITING”.NEW TYPES OF CHROMATOGRAPHY DEVELOPED DURING THE 1930S AND1940S AND CONTINUED TO THIS DAY MAKING THE TECHNIQUE USEFUL ANDESSENTIAL FOR MANY SEPARATION AND PURIFICATION.2www.orachrom.com/net
  • 3. OVERVIEW OF LIQUID CHROMATOGRAPHYLIMITING THE OVERVIEW TO LIQUID CHROMATOGRAPHY ONLY WE CAN LIST THE FOLLOWING TYPES OFCHROMATOGRAPHY:• NORMAL-PHASE LIQUID CHROMATOGRAPHY (NPLC)NORMAL-PHASE LIQUID CHROMATOGRAPHY IS A TECHNIQUE THAT USES COLUMNS PACKED WITH POLARSTATIONARY PHASES RUNNING NONPOLAR OR MODERATELY-POLAR MOBILE PHASES TO SEPARATE THECOMPONENTS OF A MIXTURE. THE POLARITY OF EACH SOLUTES IS THEREFORE THE CONTROLLING FACTORIN THEIR MIGRATION IN THE COLUMN. THE LESS POLAR THE SOLUTE THE FASTEST ITS MIGRATION ANDDETECTION FROM THE COLUMN. THEY ARE FOLLOWED BY SOLUTES OF HIGHER POLARITY.THE IMPORTANCE OF SPECIFIC SOLUTE-STATIONARY PHASE INTERACTION IN THIS MODE OFCHROMATOGRAPHY IS AN ADVANTAGE OVER REVERSED-PHASE LIQUID CHROMATOGRAPHY (RPLC) THATRELIES ON HYDROPHOBICITY ALONE.NPLC IS VERY HELPFUL FOR THE SEPARATION OF ISOMERS, OR COMPOUNDS WITH DIFFERENT FUNCTIONALGROUP3www.orachrom.com/net
  • 4. • REVERSED-PHASE CHROMATOGRAPHY OR RPLCREVERSED-PHASE LIQUID CHROMATOGRAPHY IS A CHROMATOGRAPHY PROCEDURE INWHICH THE MOBILE PHASE IS MORE POLAR THAN THE STATIONARY PHASE.THE NAME COMES FROM THE FACT THAT IN NORMAL-PHASE LIQUID CHROMATOGRAPHY THEMOBILE PHASE IS LESS POLAR THAN THE STATIONARY PHASE.HYDROPHOBIC MOLECULES IN THE MOBILE PHASE TEND TO ADSORB TO THE HYDROPHOBICSTATIONARY PHASE. THEREFORE HYDROPHILIC MOLECULES ELUTE FIRST.4www.orachrom.com/net
  • 5. • HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY (HILIC)HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY (HILIC) IS A PARTITION CHROMATOGRAPHYTHAT IS CONSIDERED AS REVERSE REVERSED-PHASE LIQUID CHROMATOGRAPHY. BOTHTECHNIQUES ARE DIFFERENT FROM NORMAL PHASE LIQUID CHROMATOGRAPHY IN WHICH WATER ISPART OF THE MOBILE PHASE, AND THUS NOT ADSORPTION CHROMATOGRAPHY.DR. ANDREW ALPERT SUGGESTED THE NAME IN HIS 1990 PAPER. HE DESCRIBED THECHROMATOGRAPHIC MECHANISM FOR IT AS LIQUID-LIQUID PARTITION CHROMATOGRAPHY WHEREANALYTES ELUTE IN ORDER OF INCREASING POLARITY, A CONCLUSION SUPPORTED BY PUBLISHEDDATA ON THE SUBJECT.5www.orachrom.com/net
  • 6. • ION EXCHANGE CHROMATOGRAPHYION EXCHANGE CHROMATOGRAPHY USES AN ION EXCHANGE RESIN TO SEPARATE ANALYTES BASEDON THEIR RESPECTIVE CHARGES.ION EXCHANGE CHROMATOGRAPHY USES A CHARGED STATIONARY PHASE TO SEPARATE CHARGEDCOMPOUNDS. THESE ARE ANIONS AND CATIONS THAT ARE POSITIVELY OR NEGATIVELY CHARGEDENTITIES INCLUDING AMINO ACIDS, PEPTIDES OR PROTEINS AS WELL AS OTHER BIOMOLECULES.ION EXCHANGE RESINS CONSIST OF ANION EXCHANGERS AND CATION EXCHANGERS.ANION EXCHANGERS ARE DIVIDED INTO STRONG ANION EXCHANGERS WITH A CONSTANT POSITIVECHARGE AND WEAK ANION EXCHANGERS WITH A VARIABLE AND ADJUSTING POSITIVE CHARGE.CATION EXCHANGERS ALSO INCLUDE TWO CATEGORIES. STRONG CATION EXCHANGER SUCH ASSULFOPROPYL AND SULFOETHYL AND WEAK CATION EXCHANGERS SUCH AS CARBOXYL.6www.orachrom.com/net
  • 7. • HYDROPHOBIC INTERACTION CHROMATOGRAPHY OR HICIN HYDROPHOBIC INTERACTION CHROMATOGRAPHY HYDROPHOBICITY IS USED TO SEPARATE PROTEINS FROM ONE ANOTHER.PROTEINS WITH HYDROPHOBIC AMINO ACID SIDE CHAINS ON THEIR SURFACES INTERACT WITH AND BIND TO THE HYDROPHOBICGROUPS ON THE SURFACE. GROUPS SUCH AS, BUTYL, PHENYL, ETHER, T-BUTYL OR OTHER GROUP THAT ARE TETHERED TO THESURFACE OF THE STATIONARY PHASE.A BUFFER WITH HIGH IONIC STRENGTH, USUALLY AMMONIUM SULFATE, IS APPLIED TO THE COLUMN AT THE START. THISREDUCES THE SOLVATION OF SAMPLE SOLUTES THUS AS SOLVATION DECREASES, HYDROPHOBIC REGIONS THAT BECOMEEXPOSED ARE ADSORBED BY THE MEDIUM. THE PROTEINS ARE ELUTED BY GRADUALLY DECREASING THE SALT. ELUTION CANALSO BE ACHIEVED THROUGH THE USE OF MILD ORGANIC MODIFIERS OR DETERGENTS.THE FOLLOWING IS A LIST OF SALTS THAT INCREASE HYDROPHOBIC INTERACTIONS IN THE ORDER OF THEIR ABILITY TOENHANCE INTERACTIONS.Na2SO4, K2SO4, (NH4)2SO4, NaCL, NH4CL, NaBr, NaSCNHYDROPHOBIC INTERACTION CHROMATOGRAPHY IS VERY SIMILAR TO REVERSED PHASE CHROMATOGRAPHY. HOWEVER THELIGANDS IN REVERSED PHASE CHROMATOGRAPHY ARE MUCH MORE HYDROPHOBIC THAN THE LIGANDS IN HYDROPHOBICINTERACTION CHROMATOGRAPHY. THIS ENABLES HYDROPHOBIC INTERACTION CHROMATOGRAPHY TO MAKE USE OF MOREMODERATE ELUTION CONDITIONS, WHICH DO NOT DISRUPT THE SAMPLE NEARLY AS MUCH SPECIALLY PROTEINS THAT AREPRONE TO DENATURATION IN ORGANIC SOLVENTS USED IN RPLC.7www.orachrom.com/net
  • 8. • AFFINITY CHROMATOGRAPHYAFFINITY CHROMATOGRAPHY IS A VERY SPECIFIC INTERACTION BETWEEN AN ANALYTE AND A SPECIFIC LIGAND. IT IS NOT ACOVALENT INTERACTION.IT HAS WIDESPREAD USE IN BIOCHEMISTRY FOR THE PURIFICATION OF PROTEINS. PROTEINS THAT ARE LABELED WITH TAGSSUCH AS HISTIDINE, BIOTIN OR ANTIGENS THAT BINDS TO SPECIFIC SURFACES EXCLUSIVELY. THESE TAGS ARE USUALLYREMOVED AFTER THE ISOLATION OF THE PROTEINS.AFFINITY COLUMNS ARE USED AS A PREPARATIVE STEP TO WASH OUT UNWANTED BIOMOLECULES FROM THE MIXTURE ANDRETAIN THE TARGET COMPOUND EXCLUSIVELY.IN THIS TECHNIQUE THE BIOMOLECULES AFFINITY FOR A METAL (ZN, CU, FE, NI ETC.) IS TAKEN ADVANTAGE OF.PROTEIN A FROM STAPHYLOCOCCUS AUREUS IS ONE OF THE FIRST IMMUNOGLOBULIN BINDING MOLECULES THAT HAS BEENEXTENSIVELY USED DURING THE PAST 20 YEARS IN “PROTEIN A” RESINS.BASED ON ITS AFFINITY TO IMMUNOGLOBULINS, PROTEIN A AFFINITY CHROMATOGRAPHY HAS FOUND WIDESPREAD USE AS ATOOL IN THE DETECTION AND PURIFICATION OF ANTIBODIES.THE BIODEGRADABLE NATURE OF THE MATRIXES TO WHICH PROTEIN A LIGAND IS ATTACHED (AGAROSE DERIVED MATRICES)MAKES IT NECESSARY TO ADD MULTIPLE “POLISHING” STEPS TO REMOVE THE LEACHABLES INCLUDING PROTEIN A LIGANDITSELF.8www.orachrom.com/net
  • 9. • SIZE-EXCLUSION CHROMATOGRAPHYSIZE-EXCLUSION CHROMATOGRAPHY OR SEC ALSO KNOWN AS GEL PERMEATION CHROMATOGRAPHY OR GPCOR GEL FILTRATION CHROMATOGRAPHY OR GFC SEPARATES MOLECULES ACCORDING TO THEIR SIZES.SMALLER MOLECULES CAN ENTER THE PORES OF THE MEDIA AND THEREFORE CAN BE SLOWED IN THEIRELUTION IN THE COLUMN. THE AVERAGE RESIDENCE TIME IN THE PORES DEPENDS UPON THE ACTUAL SIZE OFTHE MOLECULES. HOWEVER, MOLECULES THAT ARE LARGER THAN THE AVERAGE PORE SIZE OF THE PACKINGARE NOT RETAINED AND ELUTE FIRST.IT IS GENERALLY A LOW-RESOLUTION CHROMATOGRAPHY TECHNIQUE AND THEREFORE IT IS OFTENRESERVED FOR THE FINAL, "POLISHING" STEP OF THE PURIFICATION. IT IS ALSO USEFUL FOR DETERMININGTHE TERTIARY OR QUATERNARY STRUCTURE OF PURIFIED PROTEINS, ESPECIALLY SINCE IT CAN BE RUNUNDER MILD CONDITIONS.9www.orachrom.com/net
  • 10. • TWO-DIMENSIONAL CHROMATOGRAPHYAT TIMES A SINGLE COLUMN IS NOT SUFFICIENT TO SEPARATE ALL THE COMPOUNDS TO BEANALYZED. IT THEN BECOMES NECESSARY TO DIRECT THE UNRESOLVED PEAKS INTO A SECONDCOLUMN WITH A DIFFERENT PHASE AND PROPERTY.SINCE THE MECHANISM OF SEPARATION IN THE SECOND COLUMN IS DIFFERENT FROM THE FIRSTONE IT THEN BECOMES POSSIBLE TO SEPARATE THE COMPOUNDS THAT WERE INDISTINGUISHABLEIN THE FIRST DIMENSION, THUS THE NEED FOR TWO-DIMENSIONAL CHROMATOGRAPHY.10www.orachrom.com/net
  • 11. • SIMULATED MOVING-BED CHROMATOGRAPHYTHIS TECHNIQUE IS A VARIANT OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY. IT IS USED TO SEPARATECOMPOUNDS THAT ARE DIFFICULT TO RESOLVE BY A SINGLE COLUMN WITH LIMITED LENGTH.IN THIS PROCESS THE SEPARATION IS ACHIEVED BY USING MULTIPLE SMALLER COLUMNS CONNECTED TOEACH OTHER WITH VALVE IN ORDER TO INCREASE THE LENGTH OF THE EFFECTIVE COLUMN.IN ITS USE FOR PREPARATIVE CHROMATOGRAPHY RATHER THAN MOVING THE BED, THE SAMPLE INLET ANDTHE ANALYTE EXIT POSITIONS VALVES ARE CONTINUOUSLY AND RHYTHMICALLY SWITCHED SIMULATING AMOVING BED PHENOMENON.THERE IS THEREFORE A COMPLEX VALVE ARRANGEMENT THAT PROVIDES A SAMPLE AND SOLVENT FEED ASWELL AS A WASTE AND ANALYTE OUTLET.THE SAMPLE ENTRY GOES IN ONE DIRECTION WHILE THE SOLVENT IS ENTERED IN THE OPPOSITEDIRECTION. SAME GOES FOR THE ANALYTE AND WASTE OUTLET AS WELL.THIS TECHNIQUE IS MEANT FOR BINARY COMPOUNDS OR A SINGLE COMPOUND OUT OF A GROUP OF OTHERCOMPOUNDS IN THE MIXTURE.IT IS CONSIDERABLY FASTER AS IT IS CONTINUOUS AS COMPARED WITH BATCH CHROMATOGRAPHY.11www.orachrom.com/net
  • 12. • SCHEMATIC OF AN SMB PROCESSExtractFeedRaffinateEluentDirection of theflow and columnswitching12www.orachrom.com/net
  • 13. • FAST PROTEIN LIQUID CHROMATOGRAPHY OR FPLCTHIS TERM IS USED TO DESCRIBE A NUMBER OF CHROMATOGRAPHY TECHNIQUES THAT ARE USEDTO PURIFY PROTEINS.THESE TECHNIQUES ARE SIMILAR TO THOSE USED FOR HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY (HPLC) WITH THE DISTINCTION THAT FPLC IS OFTEN USED IN THE PREPARATIONOF LARGE SCALE BATCHES OF A PURIFIED PRODUCT.IT HAS BEEN USED WITH SOFT GEL MEDIA AND LARGE BORE COLUMNS AND THEREFORE OPERATESAT LOW LINEAR FLOW RATES AS WELL AS LOW PRESSURES.13www.orachrom.com/net
  • 14. • CHIRAL CHROMATOGRAPHYCHIRAL CHROMATOGRAPHY CONSISTS OF SEPARATING STEREOISOMERS. THE STATIONARY PHASEHAS AN OPTICALLY ACTIVE LIGAND ATTACHED TO IT IN ORDER TO GENERATE A CHIRAL STATIONARYPHASE (CSP).THE ENANTIOMERS OR OPTICAL ISOMERS DISPLAY DIFFERENT AFFINITY TOWARDS THE CHIRALSTATIONARY PHASE AND THEREFORE ARE DIFFERENTLY RETAINED BY THE COLUMN. THISCONSTITUTES THE BASIS FOR THEIR SEPARATION.14www.orachrom.com/net
  • 15. • MONOLITHIC HPLC COLUMNS FOR LIQUID CHROMATOGRAPHY.THESE COLUMNS ARE SPECIAL TYPE COLUMNS WITH POROUS CHANNELS RATHER THAN BEADS. ITELIMINATES THE INTERSTITIAL SPACES BETWEEN BEADS AND REPLACES IT WITH THROUGH PORESMAKING THE SIZE OF BEAD OBSOLETE.THEIR PRIMARY USE IS IN HPLC INSTRUMENTS THAT ARE THE MOST USED LABORATORYINSTRUMENTS AFTER ANALYTICAL BALANCES AND pH METERS (AS OF 2011).THE NEED FOR IMPROVED TECHNOLOGY IN CHROMATOGRAPHY MEDIA AND PARTICULARLY HPLCCOLUMNS IS THEREFORE VERY CLEAR.ALTHOUGH THERE HAS BEEN ADVANCES IN THIS AREA IT HAS BEEN RATHER INCREMENTAL DURING ALONG PERIOD OF TIME.SEPARATION IN COLUMN IS DEPENDENT ON THE CHEMISTRY AND STRUCTURE OF THE COLUMNTHUS THE IMPORTANCE OF CHROMATOGRAPHY COLUMNS AND MEDIA.15www.orachrom.com/net

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