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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Affinity Chromatography • Ligand is an atom, ion, or molecule that generally donates one or more of its electrons through a coordinate covalent bond to, or shares its electrons th through h a covalent l t bond b d with ith one or more central t l atoms or ions . • Two types of Ligands are brought into use: –

Specific

– General • Specific Ligands : Binds only to one species • Group Specific Ligands: Binds to specific groups on target species.

Affinity Chromatography Ligand Types Samples Enzymes

Substrate, Inhibitor, Cofactor

Antibody

Antigen, Virus, Cells

Lectin

Polysaccharides, Glycoprotein, Cell receptor Complementary p y base sequence, q histone, nucleic acid, polymerase binding protein Receptors, Carrier proteins

Nucleic Acid Hormones

Types of Ligands used

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Affinity Chromatography SPACER • Carbon chain interposed between Ligand and Matrix. • Used when active site is located deep within the sample molecule • If too long it can interact with sample species on its own ( hydrophobic interactions) • If too short the ligand is unable to reach the active sample molecule. molecule • Commercial phases have spacers which are optimized for specific separations.

Affinity Chromatography • Should be a rigid, stable and high surface area. • It must be insoluble in solvents and buffers employed in the process • it mustt be b easily il coupled l d to t a ligand li d or spacer arm onto which the ligand can be attached • must exhibit good flow properties and have a relativley large surface area for attachment

• Agarose is the most popular however, cellulose, dextrans and polyacrylamide has also been used frequently. • Sepharose is a bead form of agarose gel. gel • In general any matrix useful for ion exchange or gel filtration is also good for affinity chromatography.

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Affinity Chromatography FORMATION OF A PHASE • Ligand should be bound to support to create a stable phase‐ immobilization. • Two Steps –Activation of the support with a reactive compound. –Attachment of a ligand • How the spacer arm is attached to the ligand is important, as it should not interfere with ligand binding to the protein? • But usually the best way to attach a ligand has to be worked out by trial and error, synthesizing small test molecules with alkyl groups attached to the ligand in various ways and determining which bind best to the protein.

Affinity Chromatography FORMATION OF A PHASE • Ligand should be bound to support to create a stable phase‐ immobilization. • Two Steps –Activation of the support with a reactive compound. –Attachment of a ligand • How the spacer arm is attached to the ligand is important, as it should not interfere with ligand binding to the protein? • But usually the best way to attach a ligand has to be worked out by trial and error, synthesizing small test molecules with groups attached to the ligand in various ways and determining which bind best to the target molecule.

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Affinity Chromatography FORMATION OF A PHASE • Ligand should be bound to support to create a stable phase‐ immobilization. • Two Steps –Activation of the support with a reactive compound. –Attachment of a ligand • How the spacer arm is attached to the ligand is important, as it should not interfere with ligand binding to the protein? • But usually the best way to attach a ligand has to be worked out by trial and error, synthesizing small test molecules with groups attached to the ligand in various ways and determining which bind best to the target molecule.

Affinity Chromatography Specific Phases Type

Specificity

Protein A- Sepahrose Cl4B Fc region of IgG and related molecules Con A- Sepharose Terminal –D- glucopyranosyl , D- mannopyranosyl or similar residues Blue Sepharose- Cl6B Broad range of enzymes which hi h h have nucleotide l tid cofactors, serum albumin etc. Lysine- Sepharose 4B Plasminogen, ribosomal RNA

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Affinity Chromatography BASIC STEPS • Sample Introduction • Adsorption of the components of interest • Removal of impurities • Elution of components

Affinity Chromatography BASIC STEPS Sample Introduction

Ensure that the column has adequate capacity

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Affinity Chromatography BASIC STEPS Absorption Use a slow l fl flow rate off the h solvent l so that the SAMPLE is allowed to move through the column. Larger duration of stay of the SAMPLE allows better interaction of the sample with unreacted sites.

Affinity Chromatography BASIC STEPS Washing Basically i ll removing i the h impurities i ii out of the column by passing fresh volumes of solvent in a repeated fashion

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Affinity Chromatography BASIC STEPS Elution Removall off the h target molecule l l from f the matrix and its collection. Washing buffer and eluting buffers are different. After elution the column generally gets regenerated.

Affinity Chromatography BASIC STEPS Elution Methods‐ Biospecific Inhibitor hibi ( free f li d) is ligand) i added dd d into i the h eluting buffer ( solvent) where it competes with the solute (TARGET MOLECULES) This will lead to the elution of the TARGET MOLECULE

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Affinity Chromatography BASIC STEPS Elution Methods‐ Non‐ specific A reagent is i added dd d that h denatures d the h solute (TARGET MOLECULE) (pH, KSCN, Urea, ionic strength etc.) This will lead to the elution of the TARGET MOLECULE as it will leave LIGAND due to a conformational change.

Affinity Chromatography Affinity Chromatogram

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Affinity Chromatography Example 1

Affinity Chromatography Example 2

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Applications of Affinity Chromatography Various techniques developed from Affinity Chromatography

Applications of Affinity Chromatography Various techniques developed from Affinity Chromatography

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Applications of Affinity Chromatography • Boronate affinity chromatography (BAC) – – Glycated haemoglobin analysis is used in the management of diabetes, HbA1c provides an indication of the degree of diabetic control over the preceding two‐ two to three‐month three month period. BAC is the definitive method for determination of HbA1c as it is the only interference free method presently available to the routine laboratory. – At a pH above 8, most boronate derivatives form covalent bonds with compounds that contain cis‐diol groups in their structure – sugars such as glucose possess cis‐diol groups, boronates are valuable 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 m‐ aminophenyl boronate forming a tetrahedral boronate ion under alkaline conditions. This anion can bind to the 1,2‐ cis – diol group and the interaction is enhanced in the presence of Mg2+ ions and inhibited by amine containing buffers. • HEPES and Morpholine are the basic buffers used for absorption while Tris and Sorbitol are used as desorption ( eluting l ti ) Buffers. B ff • Lectin Affinity Chromatographyl‐ Lectins are non‐ immune system proteins that have the ability to recognize and bind certain types of carbohydrate residues

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Applications of Affinity Chromatography • Lectin Affinity Chromatography – Concanavalin A, which binds to ‐D‐mannose and ‐D‐ glucose residues, and wheat germ agglutinin, which binds to D‐N‐acetylglucosamine – isolation of many carbohydrate‐containing compounds, such as polysaccharides, glycoproteins, and glycolipids – has been in the separation and analysis of isoenzymes like immobilized wheat germ agglutinin was used to distinguish between the liver liver‐ and bone bone‐derived derived isoenzymes of alkaline phosphatase in human serum – Concanavalin A have been used to separate apolipoprotein A‐ and apolipoprotein B‐containing lipoproteins in human plasma

Applications of Affinity Chromatography • Protein A/G Affinity Chromatography – ligands that have been used in direct analyte detection by affinity chromatography are antibody‐binding proteins such as protein A and protein G – protein A and protein G are bacterial cell wall proteins produced by Staphylococcus aureus and group G streptococci, respectively – Protein A and protein G bind most strongly to immunoglobulins at or near neutral pH, but readily dissociate from these solutes when placed in a buffer with a lower pH. – The ability of protein A and protein G to bind to antibodies make these good ligands for the analysis of immunoglobulins, especially IgG‐class antibodies, in humans.

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Applications of Affinity Chromatography • Immunoaffinity Chromatography

– The term "immunoaffinity chromatography" (IAC) is used for an affinity chromatographic method in which the stationaryy p phase consists of an antibodyy or antibody‐related y reagent – 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 and transferrin – determination of fibrinogen in human plasma • the amount of fibrinogen in the retained peak was determined by the 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 p and analysis y of gglutathione S‐transferase separation isoenzymes in human lung and liver samples. • "Immobilized metal ion affinity chromatography (MIAC)", also known as "metal chelate affinity chromatography", is another method that has been widely used in purification processes – The affinity ligand is a metal ion that is complexed with an i immobilized bili d chelating h l i agent. Iminodiacetic I i di i acid id is i the h most common chelating agent used, but carboxymethylaspartic acid,tris‐carboxymethylethylenediamine,tris(2‐aminoethyl) amine, or dipicolylamine sometimes are also used. The metal ions 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 as histidine, tryptophan, or cysteine) and the metal ions within the immobilized metal chelate and has been used commercial i l purification ifi i off severall peptides, id proteins, i and d amino acids have been purified commercially since its discovery • The demand for pharmaceutical grade plasmid DNA (pDNA) is expected to grow in the future, placing pressure on industry to produce the required volumes of pDNA • Affinity ff chromatography h h offers ff a solution l to the h problem bl of purifying pDNA from E.coli for gene therapy and vaccine applications without co‐ purification of undesirbale 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 the synthesized h d polymer. l • It is also used for characterization of food polysaccharides as they are typically polydisperse compounds with wide distribution 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 seed sources. In foods and beverages, polysaccharides may find use as thickening agents, emulsifiers, emulsion stabilizers, or to add structure to solids.

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Applications of Gel permeation Chromatography • They are also important for their ability to modify fat and water‐holding water holding properties, properties and to control aroma and/or flavor release • GPC has also been employed for detection of Pesticide contamination in lanolin ( a base material for cosmetics & pharmaceutical application) – GLC application. application

IEC & Applications • Ion‐exchange chromatography is a process that allows the separation p of ions and p polar molecules based on the charge properties of the molecules. • Used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids .

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IEC PRINCIPLE ƒ Ion exchange chromatography retains analyte molecules based on coulombic (ionic) interactions. ƒ Stationary S i phase h surface f di l displays i i functional ionic f i l groups that interact with analyte ions of opposite charge TYPES ƒ Cation exchange : positively charged cations because the stationary phase displays a negatively charged functional group such as a phosphonic acid ( NET NEGATIVE CHARGE) ƒ Anion exchange : negatively charged anions using positively charged functional group such as a quaternary ammonium cation ( NET POSITIVE CHARGE)

Column Chromatography ƒ Net Charge on the Ion exchanger‐measure to retain opposite charge ion clearly depends upon the pH of the mobile phase with which it is contact. ƒ Net charge is also a function of pKa of the ion group involved. ƒ (R+ X‐) + P‐

( R+P‐) + X‐ ( Absorption)

ƒ ( R+P‐) + S‐

(R+S‐) + P ( Desorption)

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

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Ion Exchange Chromatography ƒ Wide range of materials have been used for forming ion exchangers‐ cross linked polystyrene; cross linked polydextrans, cellulose and silica. ƒ Silica Gel based ion exchangers are not widely used as they have poor stability in extreme pH conditions. ƒ Branch of Ion exchange chromatography involving separation of simple inorganic cations and anions is known as Ion chromatography.

Ion Exchange Chromatography FIVE BASIC STEPS OF ION EXCHANGE CHROMATOGRAPHY Starting Conditions

+ ++ ++

Adsorption Of Sample

+ ++ ++

Staring Buffer Counter ions Substances to be separated

Start of Desorption

End of Desorption

+ ++ ++

+ ++ ++

regeneration

+ ++ ++

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 pH and ionic strength, which allows the binding of the desired solute molecules. • The exchanger groups are associated at this time with exchangeable counter‐ions (usually simple anions or cations, such as chloride or sodium). • The second stage is sample application and adsorption, in which solute molecules carrying the appropriate charge displace counter counter‐ions ions and bind reversibly to the gel. • Unbound substances can be washed out from the exchanger bed using starting buffer.

Ion exchange chromatography • Third stage: substances are removed from the column by changing to elution conditions unfavourable for ionic bonding of the solute molecules. This normally involves increasing the ionic strength of the eluting buffer or changing its pH. • The fourth and fifth stages are the removal from the column of substances not eluted under the previous experimental conditions and re‐equilibration at the starting 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 exchanger due to differences in their charges, charge densities and distribution of charge on their surfaces. surfaces • These interactions can be controlled by varying conditions such as ionic strength and pH. • In ion exchange chromatography one can choose whether to bind the substances of interest and allow the contaminants to pass through the column, column or to bind the contaminants and allow the substance of interest to pass through.

Ion exchange chromatography MATRIX OF ION EXCHANGE COLUMN CHROMATOGRAPH

• Ion exchanger consists of an insoluble matrix to which charged groups have been covalently bound. The charged groups are associated with mobile counter ions. ions • These counter‐ions can be reversibly exchanged with other ions of the same charge without altering the matrix. • Positively charged exchangers have negatively charged counter‐ions (anions) available for exchange and are called anion exchangers. • Negatively charged exchangers have positively charged counter‐ions (cations) and are termed cation exchangers.

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Ion exchange chromatography MATRIX OF ION EXCHANGE COLUMN CHROMATOGRAPH

• The matrix may be based on inorganic compounds, synthetic resins or polysaccharides. • The Th characteristics h t i ti off the th matrix t i determine d t i it its chromatographic properties such as efficiency, capacity and recovery as well as its chemical stability, mechanical strength and flow properties. The nature of the matrix will also affect its behavior towards biological substances and the maintenance of biological activity. i i • The first ion exchangers designed for use with biological substances were the cellulose ion exchangers developed by Peterson and Sober

Ion exchange chromatography MATRIX OF ION EXCHANGE COLUMN CHROMATOGRAPH

• Many cellulose ion exchangers had low capacities (otherwise the cellulose became soluble in water) and h d poor flow had fl properties due d to their h irregular l shape. h • Ion exchangers based on dextran (Sephadex), followed by those based on agarose (Sepharose CL‐6B) and cross‐linked cellulose (DEAE Sephacel) were the first ion exchange matrices to combine a spherical form with high porosity, leading to improved flow properties and high capacities for macromolecules.

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Ion exchange chromatography MATRIX OF ION EXCHANGE COLUMN CHROMATOGRAPH

Function Group Used on Ion Exchangers

Ion exchange chromatography APPLICATIONS • Purification of Biologically Active proteins‐ ENZYMES like Creatine Kinase from Chicken Breast Muscle is recovered by 90%. • Purification of Immunoglobulins like IgG from Cell culture preparations ( Eli Lilly, USA) • Nucleic acid separations – Purification of Plasmid HB101 (pBR322) has been separated using anion exchange chromatography on Q Sepharose High performance. Time required for preparation through IEC – 1 hr. and traditional CsCl density gradient centrifugation – 8 hours.

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Ion exchange chromatography APPLICATIONS • Separation of peptides fragments from Collagen.

from

Cyanogen

bromide

• Peptide Mapping • Separation of Oligonucleotides • Cation exchange chromatography used for fermentative production 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 chromatography steps using DEAE adsorbent.

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