Applications of Chromatographic Techniques

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Applications of Chromatographic Techniques

HPLC TYPE APPLICATIONS‐ Affinity , Ion Exchange, Gel Permeation

Affinity Chromatography

Ligand Spacer Matrix

Ligand : Site of Interaction

Spacer: what binds ligand to support

Matrix: Supporting Phase

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• Ligand is an atom, ion, or molecule that generallydonates one or more of its electrons through acoordinate covalent bond to, or shares its electronsth h l t b d ith t lthrough a covalent bond with one or more centralatoms or ions .

• Two types of Ligands are brought into use:

– Specific

– General– General

• Specific Ligands : Binds only to one species

• Group Specific Ligands: Binds to specific groups ontarget species.

Affinity ChromatographyLigand Types

Samples Types of Ligands usedEnzymes Substrate, Inhibitor, Cofactor

Antibody Antigen, Virus, Cells

Lectin Polysaccharides, Glycoprotein, Cell receptor

Nucleic Acid Complementary base sequence, p y qhistone, nucleic acid, polymerase binding protein

Hormones Receptors, Carrier proteins

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• Carbon chain interposed between Ligand and Matrix.

• Used when active site is located deep within the

SPACER

• Used when active site is located deep within thesample molecule

• If too long it can interact with sample species on itsown ( hydrophobic interactions)

• If too short the ligand is unable to reach the activesample moleculesample molecule.

• Commercial phases have spacers which are optimizedfor specific separations.

Affinity Chromatography• Should be a rigid, stable and high surface

area.• It must be insoluble in solvents and

buffers employed in the processit t b il l d t li d• it must be easily coupled to a ligand or spacerarm onto which the ligand can be attached

• must exhibit good flow properties and have arelativley large surface area for attachment

• Agarose is the most popular however,cellulose, dextrans and polyacrylamide hasalso been used frequently.

• Sepharose is a bead form of agarose gel• Sepharose is a bead form of agarose gel.• In general any matrix useful for ion

exchange or gel filtration is also good foraffinity chromatography.

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• Ligand should be bound to support to create a stablephase‐ immobilization.

• Two Steps

FORMATION OF A PHASE

–Activation of the support with a reactivecompound.

–Attachment of a ligand• How the spacer arm is attached to the ligand is

important, as it should not interfere with ligand bindingto the protein?

• But usually the best way to attach a ligand has to beworked out by trial and error, synthesizing small testmolecules with alkyl groups attached to the ligand invarious ways and determining which bind best to theprotein.



• Two Steps





• But usually the best way to attach a ligand has to beworked out by trial and error, synthesizing small testmolecules with groups attached to the ligand in variousways and determining which bind best to the targetmolecule.

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• Two Steps





• But usually the best way to attach a ligand has to beworked out by trial and error, synthesizing small testmolecules with groups attached to the ligand in variousways and determining which bind best to the targetmolecule.

Affinity ChromatographySpecific Phases

Type Specificity Protein A- Sepahrose Cl4B Fc region of IgG and relatedProtein A- Sepahrose Cl4B Fc region of IgG and related

moleculesCon A- Sepharose Terminal –D- glucopyranosyl ,

D- mannopyranosyl or similar residues

Blue Sepharose- Cl6B Broad range of enzymes hi h h l tidwhich have nucleotide

cofactors, serum albumin etc.Lysine- Sepharose 4B Plasminogen, ribosomal RNA

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BASIC STEPS

• Sample Introduction

• Adsorption of the components of interest

• Removal of impurities

• Elution of components


Sample IntroductionBASIC STEPS

Ensure that the column has adequate capacity

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Absorption BASIC STEPS

l fl f h lUse a slow flow rate of the solventso that the SAMPLE is allowed tomove through the column.

Larger duration of stay of theSAMPLE allows better interaction ofSAMPLE allows better interaction ofthe sample with unreacted sites.


Washing BASIC STEPS

i ll i h i i iBasically removing the impuritiesout of the column by passing freshvolumes of solvent in a repeatedfashion

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Elution BASIC STEPS

l f h l l fRemoval of the target molecule fromthe matrix and its collection.

Washing buffer and eluting buffers aredifferent.

After elution the column generally getsregenerated.


Elution Methods‐ Biospecific BASIC STEPS

hibi ( f li d) i dd d i hInhibitor ( free ligand) is added into theeluting buffer ( solvent) where itcompetes with the solute (TARGETMOLECULES)

This will lead to the elution of theThis will lead to the elution of theTARGET MOLECULE

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Elution Methods‐ Non‐ specific BASIC STEPS

i dd d h d hA reagent is added that denatures thesolute (TARGET MOLECULE)(pH, KSCN, Urea, ionic strength etc.)

This will lead to the elution of theTARGET MOLECULE as it will leaveTARGET MOLECULE as it will leaveLIGAND due to a conformationalchange.

Affinity ChromatographyAffinity Chromatogram

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Affinity ChromatographyExample 1

Affinity ChromatographyExample 2

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Applications of Affinity Chromatography

Various techniques developed from Affinity Chromatography


Various techniques developed from Affinity Chromatography

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• Boronate affinity chromatography (BAC) –

– Glycated haemoglobin analysis is used in the management ofdiabetes, HbA1c provides an indication of the degree ofdiabetic control over the preceding two to three monthdiabetic control over the preceding two‐ to three‐monthperiod. BAC is the definitive method for determination ofHbA1c as it is the only interference free method presentlyavailable to the routine laboratory.

– At a pH above 8, most boronate derivatives form covalentbonds with compounds that contain cis‐diol groups in theirstructurestructure

– sugars such as glucose possess cis‐diol groups, boronates arevaluable for resolving glycoproteins (e.g., glycohemoglobin)from non‐glycoproteins (e.g., normal hemoglobin).

Applications of Affinity Chromatography• Boronate affinity chromatography (BAC) –

– Also used in the purification of Catecholamines (Hormones) ,nucleosides & Nucelotides.

• Compound which is a ligand in BAC is m‐ aminophenylCompound which is a ligand in BAC is m aminophenylboronate forming a tetrahedral boronate ion underalkaline conditions. This anion can bind to the 1,2‐ cis –diol group and the interaction is enhanced in thepresence of Mg2+ ions and inhibited by amine containingbuffers.

• HEPES and Morpholine are the basic buffers used forabsorption while Tris and Sorbitol are used as desorption( l ti ) B ff( eluting ) Buffers.

• Lectin Affinity Chromatographyl‐ Lectins are non‐immune system proteins that have the ability torecognize and bind certain types of carbohydrateresidues

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• Lectin Affinity Chromatography– Concanavalin A, which binds to ‐D‐mannose and ‐D‐

glucose residues, and wheat germ agglutinin, which binds toD‐N‐acetylglucosamine

– isolation of many carbohydrate‐containing compounds, suchas polysaccharides, glycoproteins, and glycolipids

– has been in the separation and analysis of isoenzymes likeimmobilized wheat germ agglutinin was used to distinguishbetween the liver‐ and bone‐derived isoenzymes of alkalinebetween the liver and bone derived isoenzymes of alkalinephosphatase in human serum

– Concanavalin A have been used to separate apolipoproteinA‐ and apolipoprotein B‐containing lipoproteins in humanplasma


• Protein A/G Affinity Chromatography– ligands that have been used in direct analyte detection by

affinity chromatography are antibody‐binding proteins suchas protein A and protein Gas protein A and protein G

– protein A and protein G are bacterial cell wall proteinsproduced by Staphylococcus aureus and group Gstreptococci, respectively

– Protein A and protein G bind most strongly toimmunoglobulins at or near neutral pH, but readilydissociate from these solutes when placed in a buffer with alower pH.

– The ability of protein A and protein G to bind to antibodiesmake these good ligands for the analysis ofimmunoglobulins, especially IgG‐class antibodies, inhumans.

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Applications of Affinity Chromatography• Immunoaffinity Chromatography

– The term "immunoaffinity chromatography" (IAC) is usedfor an affinity chromatographic method in which thestationary phase consists of an antibody or antibody‐relatedy p y yreagent

– IAC have been developed for anti‐idiotypic antibodies ,glucose‐containing tetrasaccharides , granulocyte colony‐stimulating factor IgG , immunoglobulin E , interferon ,tumor necrosis factor‐ , interleukins , ß2‐microglobulin andtransferrintransferrin

– determination of fibrinogen in human plasma

• the amount of fibrinogen in the retained peak was determined bythe measurement of its absorbance at 280 nm.

• The sample was a 20‐µL aliquot of plasma diluted 1:10

Applications of Affinity Chromatography• an immobilized heparin column is used for the

determination of antithrombin III in human plasma• Octylglutathione has been used as a ligand for the

separation and analysis of glutathione S‐transferasep y gisoenzymes in human lung and liver samples.

• "Immobilized metal ion affinity chromatography(MIAC)", also known as "metal chelate affinitychromatography", is another method that has beenwidely used in purification processes– The affinity ligand is a metal ion that is complexed with an

i bili d h l i I i di i id i himmobilized chelating agent. Iminodiacetic acid is the mostcommon chelating agent used, but carboxymethylasparticacid,tris‐carboxymethylethylenediamine,tris(2‐aminoethyl)amine, or dipicolylamine sometimes are also used. The metalions placed within these chelating groups are Cu2+, Zn2+, Ni2+,Co2+, or Fe3+.

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Applications of Affinity Chromatography• MIAC separates proteins and peptides on the basis of

interactions between certain amino acid residues (such ashistidine, tryptophan, or cysteine) and the metal ionswithin the immobilized metal chelate and has been used

i l ifi i f l id i dcommercial purification of several peptides, proteins, andamino acids have been purified commercially since itsdiscovery

• The demand for pharmaceutical grade plasmid DNA(pDNA) is expected to grow in the future, placing pressureon industry to produce the required volumes of pDNA

ff h h ff l h bl• Affinity chromatography offers a solution to the problemof purifying pDNA from E.coli for gene therapy andvaccine applications without co‐ purification ofundesirbale products like C‐DNA, RNA and endotoxins.

Applications of Gel permeation Chromatography• It has applications in PROTEOMICS wherein the protein in their

quaternary structures could be separated.

• It is also used to assess the size and polydispersity of theh d lsynthesized polymer.

• It is also used for characterization of food polysaccharides asthey are typically polydisperse compounds with widedistribution in molecular weight, sequence and structure.

• food industry uses native and modified starches, dextrins,dextrans, glucans, pullulans, modified celluloses, pectins,carrageenans, and gums from both microbial and plant seedsources. In foods and beverages, polysaccharides may find useas thickening agents, emulsifiers, emulsion stabilizers, or to addstructure to solids.

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Applications of Gel permeation Chromatography

• They are also important for their ability to modify fatand water holding properties and to control aromaand water‐holding properties, and to control aromaand/or flavor release

• GPC has also been employed for detection ofPesticide contamination in lanolin ( a base materialfor cosmetics & pharmaceutical application) – GLCapplicationapplication.

IEC & Applications

• Ion‐exchange chromatography is a process thatallows the separation of ions and polar moleculesp pbased on the charge properties of the molecules.

• Used for almost any kind of charged moleculeincluding large proteins, small nucleotides andamino acids .

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IEC

Ion exchange chromatography retains analytemolecules based on coulombic (ionic) interactions.S i h f di l i i f i l

PRINCIPLE

Stationary phase surface displays ionic functionalgroups that interact with analyte ions of oppositecharge

TYPESCation exchange : positively charged cations becausethe stationary phase displays a negatively chargedfunctional group such as a phosphonic acid ( NETfunctional group such as a phosphonic acid ( NETNEGATIVE CHARGE)Anion exchange : negatively charged anions usingpositively charged functional group such as aquaternary ammonium cation ( NET POSITIVE CHARGE)

Column ChromatographyNet Charge on the Ion exchanger‐measure to retainopposite charge ion clearly depends upon the pH ofthe mobile phase with which it is contact.

Net charge is also a function of pKa of the ion groupinvolved.

(R+ X‐) + P‐ ( R+P‐) + X‐ ( Absorption)

( R+P‐) + S‐ (R+S‐) + P ( Desorption)

Example of Anion Exchanger‐ Diethylaminoethyl(DEAE) and Cation Exchangers is Carboxymethyl (CM).

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Ion Exchange ChromatographyWide range of materials have been used for formingion exchangers‐ cross linked polystyrene; cross linkedpolydextrans, cellulose and silica.

Silica Gel based ion exchangers are not widely used asthey have poor stability in extreme pH conditions.

Branch of Ion exchange chromatography involvingseparation of simple inorganic cations and anions isknown as Ion chromatography.

Ion Exchange ChromatographyFIVE BASIC STEPS OF ION EXCHANGE CHROMATOGRAPHY

Starting Conditions

Adsorption Of Sample

Start ofDesorption

End ofDesorption regeneration

+ ++ +

+ + ++ +

+ + ++ +

+ + ++ +

++ +

+ +

+

Staring Buffer Counter ions

Substances to be separated Gradient Ions

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Ion exchange chromatography• The first stage is equilibration in which the ion

exchanger is brought to a starting state, in terms of pHand ionic strength, which allows the binding of thedesired solute molecules.

• The exchanger groups are associated at this time withexchangeable counter‐ions (usually simple anions orcations, such as chloride or sodium).

• The second stage is sample application andadsorption, in which solute molecules carrying theappropriate charge displace counter‐ions and bindappropriate charge displace counter ions and bindreversibly to the gel.

• Unbound substances can be washed out from theexchanger bed using starting buffer.

Ion exchange chromatography• Third stage: substances are removed from the column

by changing to elution conditions unfavourable forionic bonding of the solute molecules. This normallyinvolves increasing the ionic strength of the elutingbuffer or changing its pH.

• The fourth and fifth stages are the removal from thecolumn of substances not eluted under the previousexperimental conditions and re‐equilibration at thestarting conditions for the next purification.

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Ion exchange chromatography• Separation is obtained since different substances have

different degrees of interaction with the ion exchangerdue to differences in their charges, charge densitiesand distribution of charge on their surfacesand distribution of charge on their surfaces.

• These interactions can be controlled by varyingconditions such as ionic strength and pH.

• In ion exchange chromatography one can choosewhether to bind the substances of interest and allowthe contaminants to pass through the column or tothe contaminants to pass through the column, or tobind the contaminants and allow the substance ofinterest to pass through.

Ion exchange chromatography

• Ion exchanger consists of an insoluble matrix to whichcharged groups have been covalently bound. Thecharged groups are associated with mobile counter ions

MATRIX OF ION EXCHANGE COLUMN CHROMATOGRAPH

charged groups are associated with mobile counter ions.• These counter‐ions can be reversibly exchanged with

other ions of the same charge without altering thematrix.

• Positively charged exchangers have negatively chargedcounter‐ions (anions) available for exchange and arecalled anion exchangers.

• Negatively charged exchangers have positively chargedcounter‐ions (cations) and are termed cationexchangers.

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• The matrix may be based on inorganic compounds,synthetic resins or polysaccharides.Th h t i ti f th t i d t i it


• The characteristics of the matrix determine itschromatographic properties such as efficiency, capacityand recovery as well as its chemical stability,mechanical strength and flow properties. The nature ofthe matrix will also affect its behavior towardsbiological substances and the maintenance of biological

i iactivity.• The first ion exchangers designed for use with biological

substances were the cellulose ion exchangersdeveloped by Peterson and Sober


• Many cellulose ion exchangers had low capacities(otherwise the cellulose became soluble in water) andh d fl d h l h


had poor flow properties due to their irregular shape.

• Ion exchangers based on dextran (Sephadex), followedby those based on agarose (Sepharose CL‐6B) andcross‐linked cellulose (DEAE Sephacel) were the first ionexchange matrices to combine a spherical form withhigh porosity, leading to improved flow properties andhigh capacities for macromolecules.

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Ion exchange chromatographyMATRIX OF ION EXCHANGE COLUMN CHROMATOGRAPH

Function Group Used on Ion Exchangers

Ion exchange chromatographyAPPLICATIONS

• Purification of Biologically Active proteins‐ ENZYMESlike Creatine Kinase from Chicken Breast Muscle isrecovered by 90%.

• Purification of Immunoglobulins like IgG from Cellculture preparations ( Eli Lilly, USA)

• Nucleic acid separations – Purification of PlasmidHB101 (pBR322) has been separated using anionHB101 (pBR322) has been separated using anionexchange chromatography on Q Sepharose Highperformance. Time required for preparation throughIEC – 1 hr. and traditional CsCl density gradientcentrifugation – 8 hours.

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Ion exchange chromatographyAPPLICATIONS

• Separation of peptides from Cyanogen bromidefragments from Collagen.

• Peptide Mapping

• Separation of Oligonucleotides

• Cation exchange chromatography used for fermentativeproduction of enzyme β‐ galactosidase

• Purification of recombinant P. aeruginosa exotoxin A (MW 55,000) expressed in E. coli for use as a vaccine.Exotoxin was captured directly by 4 chromatographysteps using DEAE adsorbent.

Applications of Chromatographic Techniques

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