1. 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)

2. 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.

3. 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

4. • 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.

5. • 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.

6. • 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.

7. • 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.

8. • 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.

9. • 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.

10. • 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.

11. • 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.

12. • SCHEMATIC OF AN SMB PROCESS
Extract
FeedRaffinate
Eluent
Direction of the
flow and column
switching

13. • 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.

14. • 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.

15. • 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.