HISTORY OF 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 (1799-1868)

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.

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

• 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.

• 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.

• 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.

• 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.

Na2SO4, K2SO4, (NH4)2SO4, NaCL, NH4CL, 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.

• 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.

• 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.

• 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.

• 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.

• SCHEMATIC OF AN SMB PROCESS

Extract

FeedRaffinate

Eluent

Direction of the

flow and column

switching

• 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.

• 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.

• 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.