HISTORY OF CHROMATOGRAPHY AND VARIOUS TYPES OF LIQUID CHROMATOGRAPHY CHROMATOGRAPHY IS A SET OF LABORATORY TECHNIQUES INTENDED TO SEPARATE COMPOUNDS FROM A MIXTURE IN ORDER TO EITHER PURIFY THEM OR FOR THEIR IDENTIFICATION. IT HAS HAD A POWERFUL IMPACT IN TERMS OF A SCIENTIFIC CONCEPT BEGINNING IN 1860 WITH THE WORK OF FRIEDRICH GOPPELSROEDER WHO WAS A PIONEER OF PAPER CHROMATOGRAPHY. HE DEVELOPED THE THEORY OF CAPILLARY ANALYSIS BY USING PAPER STRIPS WHILE EXAMINING WINE, MILK, ALKALOIDS, DYES AND OILS AMONG OTHER. HIS WORK WAS AN IMPROVEMENT OF THE WORK OF CHRISTIAN FRIEDRICH SCHÖNBEIN, WHO WAS HIS MENTOR ( ) 1

IN 1906 MIKHAIL TSVET, A RUSSIAN BOTANIST, DEVELOPED THE CONCEPT OF LIQUID CHROMATOGRAPHY DURING HIS ATTEMPT TO PURIFY CHLOROPHYLLS FROM PLANT EXTRACTS. THIS DISCOVERY EARNED HIM THE NICKNAME OF, “FATHER OF CHROMATOGRAPHY” MORE APPROPRIATELY FATHER OF “LIQUID” CHROMATOGRAPHY. HE CONTINUED TO WORK WITH CHROMATOGRAPHY IN THE FIRST DECADE OF THE 20TH CENTURY, PRIMARILY FOR THE SEPARATION OF PLANT PIGMENTS SUCH AS CHLOROPHYLL, CAROTENES, AND XANTHOPHYLLS. SINCE THESE COMPONENTS HAVE DIFFERENT COLORS (GREEN, ORANGE, AND YELLOW, RESPECTIVELY) THEY GAVE THE TECHNIQUE ITS NAME “CHROMATOGRAPHY” OR “COLOR WRITING”. NEW TYPES OF CHROMATOGRAPHY DEVELOPED DURING THE 1930S AND 1940S AND CONTINUED TO THIS DAY MAKING THE TECHNIQUE USEFUL AND ESSENTIAL FOR MANY SEPARATION AND PURIFICATION. 2

OVERVIEW OF LIQUID CHROMATOGRAPHY LIMITING THE OVERVIEW TO LIQUID CHROMATOGRAPHY ONLY WE CAN LIST THE FOLLOWING TYPES OF CHROMATOGRAPHY: NORMAL-PHASE LIQUID CHROMATOGRAPHY (NPLC) NORMAL-PHASE LIQUID CHROMATOGRAPHY IS A TECHNIQUE THAT USES COLUMNS PACKED WITH POLAR STATIONARY PHASES RUNNING NONPOLAR OR MODERATELY-POLAR MOBILE PHASES TO SEPARATE THE COMPONENTS OF A MIXTURE. THE POLARITY OF EACH SOLUTES IS THEREFORE THE CONTROLLING FACTOR IN THEIR MIGRATION IN THE COLUMN. THE LESS POLAR THE SOLUTE THE FASTEST ITS MIGRATION AND DETECTION FROM THE COLUMN. THEY ARE FOLLOWED BY SOLUTES OF HIGHER POLARITY. THE IMPORTANCE OF SPECIFIC SOLUTE-STATIONARY PHASE INTERACTION IN THIS MODE OF CHROMATOGRAPHY IS AN ADVANTAGE OVER REVERSED-PHASE LIQUID CHROMATOGRAPHY (RPLC) THAT RELIES ON HYDROPHOBICITY ALONE. NPLC IS VERY HELPFUL FOR THE SEPARATION OF ISOMERS, OR COMPOUNDS WITH DIFFERENT FUNCTIONAL GROUP 3

REVERSED-PHASE CHROMATOGRAPHY OR RPLC REVERSED-PHASE LIQUID CHROMATOGRAPHY IS A CHROMATOGRAPHY PROCEDURE IN WHICH THE MOBILE PHASE IS MORE POLAR THAN THE STATIONARY PHASE. THE NAME COMES FROM THE FACT THAT IN NORMAL-PHASE LIQUID CHROMATOGRAPHY THE MOBILE PHASE IS LESS POLAR THAN THE STATIONARY PHASE. HYDROPHOBIC MOLECULES IN THE MOBILE PHASE TEND TO ADSORB TO THE HYDROPHOBIC STATIONARY PHASE. THEREFORE HYDROPHILIC MOLECULES ELUTE FIRST. 4

HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY (HILIC) HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY (HILIC) IS A PARTITION CHROMATOGRAPHY THAT IS CONSIDERED AS REVERSE REVERSED-PHASE LIQUID CHROMATOGRAPHY. BOTH TECHNIQUES ARE DIFFERENT FROM NORMAL PHASE LIQUID CHROMATOGRAPHY IN WHICH WATER IS PART OF THE MOBILE PHASE, AND THUS NOT ADSORPTION CHROMATOGRAPHY. DR. ANDREW ALPERT SUGGESTED THE NAME IN HIS 1990 PAPER. HE DESCRIBED THE CHROMATOGRAPHIC MECHANISM FOR IT AS LIQUID-LIQUID PARTITION CHROMATOGRAPHY WHERE ANALYTES ELUTE IN ORDER OF INCREASING POLARITY, A CONCLUSION SUPPORTED BY PUBLISHED DATA ON THE SUBJECT. 5

ION EXCHANGE CHROMATOGRAPHY ION EXCHANGE CHROMATOGRAPHY USES AN ION EXCHANGE RESIN TO SEPARATE ANALYTES BASED ON THEIR RESPECTIVE CHARGES. ION EXCHANGE CHROMATOGRAPHY USES A CHARGED STATIONARY PHASE TO SEPARATE CHARGED COMPOUNDS. THESE ARE ANIONS AND CATIONS THAT ARE POSITIVELY OR NEGATIVELY CHARGED ENTITIES 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 POSITIVE CHARGE AND WEAK ANION EXCHANGERS WITH A VARIABLE AND ADJUSTING POSITIVE CHARGE. CATION EXCHANGERS ALSO INCLUDE TWO CATEGORIES. STRONG CATION EXCHANGER SUCH AS SULFOPROPYL AND SULFOETHYL AND WEAK CATION EXCHANGERS SUCH AS CARBOXYL. 6

HYDROPHOBIC INTERACTION CHROMATOGRAPHY OR HIC IN 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 HYDROPHOBIC GROUPS ON THE SURFACE. GROUPS SUCH AS, BUTYL, PHENYL, ETHER, T-BUTYL OR OTHER GROUP THAT ARE TETHERED TO THE SURFACE OF THE STATIONARY PHASE. A BUFFER WITH HIGH IONIC STRENGTH, USUALLY AMMONIUM SULFATE, IS APPLIED TO THE COLUMN AT THE START. THIS REDUCES THE SOLVATION OF SAMPLE SOLUTES THUS AS SOLVATION DECREASES, HYDROPHOBIC REGIONS THAT BECOME EXPOSED ARE ADSORBED BY THE MEDIUM. THE PROTEINS ARE ELUTED BY GRADUALLY DECREASING THE SALT. ELUTION CAN ALSO 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 TO ENHANCE INTERACTIONS. Na 2 SO 4, K 2 SO 4, (NH 4 ) 2 SO 4, NaCL, NH 4 CL, NaBr, NaSCN HYDROPHOBIC INTERACTION CHROMATOGRAPHY IS VERY SIMILAR TO REVERSED PHASE CHROMATOGRAPHY. HOWEVER THE LIGANDS IN REVERSED PHASE CHROMATOGRAPHY ARE MUCH MORE HYDROPHOBIC THAN THE LIGANDS IN HYDROPHOBIC INTERACTION CHROMATOGRAPHY. THIS ENABLES HYDROPHOBIC INTERACTION CHROMATOGRAPHY TO MAKE USE OF MORE MODERATE ELUTION CONDITIONS, WHICH DO NOT DISRUPT THE SAMPLE NEARLY AS MUCH SPECIALLY PROTEINS THAT ARE PRONE TO DENATURATION IN ORGANIC SOLVENTS USED IN RPLC. 7

AFFINITY CHROMATOGRAPHY AFFINITY CHROMATOGRAPHY IS A VERY SPECIFIC INTERACTION BETWEEN AN ANALYTE AND A SPECIFIC LIGAND. IT IS NOT A COVALENT INTERACTION. IT HAS WIDESPREAD USE IN BIOCHEMISTRY FOR THE PURIFICATION OF PROTEINS. PROTEINS THAT ARE LABELED WITH TAGS SUCH AS HISTIDINE, BIOTIN OR ANTIGENS THAT BINDS TO SPECIFIC SURFACES EXCLUSIVELY. THESE TAGS ARE USUALLY REMOVED AFTER THE ISOLATION OF THE PROTEINS. AFFINITY COLUMNS ARE USED AS A PREPARATIVE STEP TO WASH OUT UNWANTED BIOMOLECULES FROM THE MIXTURE AND RETAIN THE TARGET COMPOUND EXCLUSIVELY. IN THIS TECHNIQUE THE BIOMOLECULE'S 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 BEEN EXTENSIVELY 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 A TOOL 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 LIGAND ITSELF. 8

SIZE-EXCLUSION CHROMATOGRAPHY SIZE-EXCLUSION CHROMATOGRAPHY OR SEC ALSO KNOWN AS GEL PERMEATION CHROMATOGRAPHY OR GPC OR 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 THEIR ELUTION IN THE COLUMN. THE AVERAGE RESIDENCE TIME IN THE PORES DEPENDS UPON THE ACTUAL SIZE OF THE MOLECULES. HOWEVER, MOLECULES THAT ARE LARGER THAN THE AVERAGE PORE SIZE OF THE PACKING ARE NOT RETAINED AND ELUTE FIRST. IT IS GENERALLY A LOW-RESOLUTION CHROMATOGRAPHY TECHNIQUE AND THEREFORE IT IS OFTEN RESERVED FOR THE FINAL, "POLISHING" STEP OF THE PURIFICATION. IT IS ALSO USEFUL FOR DETERMINING THE TERTIARY OR QUATERNARY STRUCTURE OF PURIFIED PROTEINS, ESPECIALLY SINCE IT CAN BE RUN UNDER MILD CONDITIONS. 9

TWO-DIMENSIONAL CHROMATOGRAPHY AT TIMES A SINGLE COLUMN IS NOT SUFFICIENT TO SEPARATE ALL THE COMPOUNDS TO BE ANALYZED. IT THEN BECOMES NECESSARY TO DIRECT THE UNRESOLVED PEAKS INTO A SECOND COLUMN WITH A DIFFERENT PHASE AND PROPERTY. SINCE THE MECHANISM OF SEPARATION IN THE SECOND COLUMN IS DIFFERENT FROM THE FIRST ONE IT THEN BECOMES POSSIBLE TO SEPARATE THE COMPOUNDS THAT WERE INDISTINGUISHABLE IN THE FIRST DIMENSION, THUS THE NEED FOR TWO-DIMENSIONAL CHROMATOGRAPHY. 10

SIMULATED MOVING-BED CHROMATOGRAPHY THIS TECHNIQUE IS A VARIANT OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY. IT IS USED TO SEPARATE COMPOUNDS 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 TO EACH 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 AND THE ANALYTE EXIT POSITIONS VALVES ARE CONTINUOUSLY AND RHYTHMICALLY SWITCHED SIMULATING A MOVING BED PHENOMENON. THERE IS THEREFORE A COMPLEX VALVE ARRANGEMENT THAT PROVIDES A SAMPLE AND SOLVENT FEED AS WELL AS A WASTE AND ANALYTE OUTLET. THE SAMPLE ENTRY GOES IN ONE DIRECTION WHILE THE SOLVENT IS ENTERED IN THE OPPOSITE DIRECTION. 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 OTHER COMPOUNDS IN THE MIXTURE. IT IS CONSIDERABLY FASTER AS IT IS CONTINUOUS AS COMPARED WITH BATCH CHROMATOGRAPHY. 11

SCHEMATIC OF AN SMB PROCESS SCHEMATIC OF AN SMB PROCESS Extract Feed Raffinate Eluent Direction of the flow and column switching 12

FAST PROTEIN LIQUID CHROMATOGRAPHY OR FPLC THIS TERM IS USED TO DESCRIBE A NUMBER OF CHROMATOGRAPHY TECHNIQUES THAT ARE USED TO PURIFY PROTEINS. THESE TECHNIQUES ARE SIMILAR TO THOSE USED FOR HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) WITH THE DISTINCTION THAT FPLC IS OFTEN USED IN THE PREPARATION OF LARGE SCALE BATCHES OF A PURIFIED PRODUCT. IT HAS BEEN USED WITH SOFT GEL MEDIA AND LARGE BORE COLUMNS AND THEREFORE OPERATES AT LOW LINEAR FLOW RATES AS WELL AS LOW PRESSURES. 13

CHIRAL CHROMATOGRAPHY CHIRAL CHROMATOGRAPHY CONSISTS OF SEPARATING STEREOISOMERS. THE STATIONARY PHASE HAS AN OPTICALLY ACTIVE LIGAND ATTACHED TO IT IN ORDER TO GENERATE A CHIRAL STATIONARY PHASE (CSP). THE ENANTIOMERS OR OPTICAL ISOMERS DISPLAY DIFFERENT AFFINITY TOWARDS THE CHIRAL STATIONARY PHASE AND THEREFORE ARE DIFFERENTLY RETAINED BY THE COLUMN. THIS CONSTITUTES THE BASIS FOR THEIR SEPARATION. 14

MONOLITHIC HPLC COLUMNS FOR LIQUID CHROMATOGRAPHY. THESE COLUMNS ARE SPECIAL TYPE COLUMNS WITH POROUS CHANNELS RATHER THAN BEADS. IT ELIMINATES THE INTERSTITIAL SPACES BETWEEN BEADS AND REPLACES IT WITH THROUGH PORES MAKING THE SIZE OF BEAD OBSOLETE. THEIR PRIMARY USE IS IN HPLC INSTRUMENTS THAT ARE THE MOST USED LABORATORY INSTRUMENTS AFTER ANALYTICAL BALANCES AND pH METERS (AS OF 2011). THE NEED FOR IMPROVED TECHNOLOGY IN CHROMATOGRAPHY MEDIA AND PARTICULARLY HPLC COLUMNS IS THEREFORE VERY CLEAR. ALTHOUGH THERE HAS BEEN ADVANCES IN THIS AREA IT HAS BEEN RATHER INCREMENTAL DURING A LONG PERIOD OF TIME. SEPARATION IN COLUMN IS DEPENDENT ON THE CHEMISTRY AND STRUCTURE OF THE COLUMN THUS THE IMPORTANCE OF CHROMATOGRAPHY COLUMNS AND MEDIA. 15