analytical techniques in biochemistry and biophysics for macro molecules

A

SEMINAR ON

BY-Dr. ARUNIMA KARKUN

ASST. PROFESSOR

G.D. RUNGTA COLLEGE OF SCIENCE AND TECHNOLOGY KOHKA- KURUD ROAD, BHILAI.

ANALYTICAL TECHNIQUES IN BIOCHEMISTRY AND BIOPHYSICS FOR MACRO MOLECULES

INTRODUCTION VARIOUS ANALYTICAL TECHNIQUESDIALYSISPAPER CHROMATOGRAPHYION-EXCHANGE CHROMATOGRAPHY GEL FILTRATION CHROMATOGRAPHY AFFINITY CHROMATOGRAPHY HPLC PAPER ELECTROPHORESIS GEL ELECTROPHORESIS ISOELECTRIC FOCCUSINGUV-VISIBLE SPECTROSCOPY MASS SPECTROSCOPY CONCLUSION SUMMARY REFERENCES

SYNOPSIS

ANALTICAL TECHNIQUES IN BIOCHEMISTRY AND BIOPHYSICS FOR MACRO MOLECULES

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Biochemistry is not pure, but an applied discipline & is both analytical & quantitative to explain the cause & effect of relationship in molecular terms.

Analysis means getting to bottom of things.

In biochemical studies , experimental models are first subjected to qualitative analysis i.e., the complex system is broken down & constituents are separated & identified.

Definition : An analytical technique is a method that is used to determine the concentration of a chemical compound.

INTRODUCTION


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ANALYTICAL

TECHNIQUES

There are variety of analytical techniques availabel for the quantification of macromolecules such as : Dialysis

Paper chromatography

Ion exchange chromatography

Affinity chromatography

Paper electrophoresis

Isoelectric focusing

Gel electrophoresis

UV-Visible spectroscopy

Mass spectroscopy 3

INTRODUCTION:

In this process small solutes diffuse from a high concentration solution to a low concentration solution across a semi permeable membrane until equilibrium is reached.

PRINCIPLE:

The porous membrane selectively allows small solute to pass while retaining larger species.

It is a separation process based on size selection.

DIALYSIS


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PROCEDURE:

This method is specifically used for separation of polysaccharides & lipoproteins.

In dialysis a porous membrane is present which allows only the selective molecules to pass through it & macromolecules are left behind.

The dialysis membrane consists of a spongy matrix of cross linked polymers, the pore rating referred to as indirect measure of the retention performance.

Since a solute’s permeability us also dependent upon molecular shape, degree of hydration , ionic charge & polarity, it is recommended to select a MWCO that is half the size of the molecular weight of the species to be retained or twice the size of the molecular weight of the species intended to pass through


DIALYSIS

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INTRODUCTION :

This technique is commonly used for the separation of amino acids, sugars, sugar derivatives & peptides.

PRINCIPLE :

The filter paper are used to support a stationary water phase while mobile organic phase moves down the suspended paper strip in a cylinder.

Separation is based on a liquid-liquid partition of the compounds.

Thus, this is essentially a form of partition chromatography between 2 liquid phases, although adsorption to the paper may also take place.


PAPER CHROMATOGRAPHY 6


PAPER CHROMATOGRAPHY

PROCEDURE :

In paper chromatography a few drops of solution containing a mixture of the compounds to be separated is applied (spotted) at one end, usually ~ 2 cm above a strip of filter paper.

The paper is dried & dipped into a solvent mixture ( for amino acid a mixture of butanol, acetic acid & water in 4:1:5 ratio commonly called BAW).

The aqueous component of the solvent system binds to the paper & forms a stationary phase.

The organic component that migrates on the paper is the mobile phase.

When the migration of the solvent is upwards, it is referred to as ascending chromatography.

In descending chromatography, the solvent moves downwards.

As the solvent flows, it takes along with it the unknown substances. 7


PAPER CHROMATOGRAPHY

Fig.2:-Technique of paper chromatography

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The rate of migration of the molecules depends on the relative solubility in the stationary phase (aqueous) & mobile phase (organic).

The paper (chromatography) is then removed, dried & developed for the identification of the specific spots.

Identification of the compound :- The distance travelled by a particular component of a mixture (or solute)is used to identify it.Resolution Factor(RF) = Distance from origin run by the solute Distance from origin run by the solvent

In some cases , a second run is carried out by a different solvent system, in a direction perpendicular to the first run. This is referred to as two- dimensional chromatography.


PAPER CHROMATOGRAPHY 9

Introduction:- Ion exchange chromatography is used for separation of amino acids, proteins, nucleotides.

Principle :- Ion exchange chromatography involves the separation of molecules on the basis of their electric charges. Ion exchange resins- cat ion exchanges & anion exchanges are used for this purpose. Procedure :-

An anion exchanges (R A ) exchanges its anion (A ) with another anion (B ) in ⁺ ⁻ ⁻ ⁻ solution. R A + B = R B + A⁺ ⁻ ⁺ ⁺ ⁻ ⁻ A cation exchanges ( H R ) exchanges its cat ion (H ) with another cat ion ( C ) ⁺ ⁻ ⁺ ⁺in solution. H R + C = C R + H⁺ ⁻ ⁺ ⁺ ⁻ ⁺ Thus, in ion exchange chromatography, ions in solutions are reversibly replaced by ion-exchange resins. The binding abilities of ions bearing positive or negative charges are highly pH dependent, since the net charge varies with pH.


ION

EXCHANGE CHROMATE 1

0


ION

EXCHANGE CHROMATE

Procedure :-

An anion exchanges (R A ) exchanges its anion (A ) with another anion ⁺ ⁻ ⁻(B ) in solution.⁻ R A + B = R B + A⁺ ⁻ ⁺ ⁺ ⁻ ⁻

A cat ion exchanges ( H R ) exchanges its cat ion (H ) with another cat ⁺ ⁻ ⁺ion ( C ) in solution.⁺ H R + C = C R + H⁺ ⁻ ⁺ ⁺ ⁻ ⁺

Thus, in ion exchange chromatography, ions in solutions are reversibly replaced by ion-exchange resins.

The binding abilities of ions bearing positive or negative charges are highly pH dependent, since the net charge varies with pH.

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ION

EXCHANGE CHROMATE

FIG.3:- Ion exchange chromatography12


ION

EXCHANGE CHROMATE

For protein purification two common cellulose derivatives, carboxymethyl cellulose (CMC,cationic derivative) & diethyl aminoethyl cellulose (DEAE, anionic derivative) are used.

An even more versatile substances for the separation of protein is a crosslinked dextrin called sephadex.

When packed in a column & equilibrated with buffer, the polysaccharide acts as a molecular serve for protein molecules. In these proteins are given negative charge treating with SDS.

Thus, electrostatic attraction of oppositely charged ions on a polyectrolyte surface forms the basis of exchange chromatography.

The protein mixture is passed through a column of the modified cellulose contained in a glass tube & allowed to become to the column material.

Increasing salt concentration & buffer of varying pH values are then passed through columns.

Protein will be washed off the column at different times depending on their molecular structure, net charge & side group.

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Introduction :-

It is used for the separation of both smaller & bigger molecules.

Its apparatus consists of a column packed with sponge like gel beads (usually crosslinked polysaccharides ) containing pores.

This technique is also referred to as molecular sieves or molecular exclusion chromatography.

Principle :-

In this technique the separation of molecules is based on the size, shape & molecular weight.

Procedure :-

The gel serves as molecular sieves for the separation of smaller & bigger molecules.

The solution mixture containing molecules of different size (say protein) is applied to column & eluted with a buffer.


GEL

FILTRATION

CHROMAT

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GEL

FILTRATION

CHROMAT

The larger molecules cannot pass through the pores of gel & therefore move faster.

On the other hand, the smaller molecules enter the gel beads & are left behind which come out slowly.

By selecting the gel beads of different porosity, the molecules can be separated.

The commercially available gel include sephadex (G-10, G-25, G-100), bio gel (P-10, P-30, P-100) & sepharose (6B, 4B, 2B).

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GEL

FILTRATION

CHROMAT

Fig:4:-Gel Filtration Chromatography

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AFFINITY

CHROMATOGRAPHY

Introduction :- Affinity chromatography is useful for purification of enzymes, vitamins, nucleic acids, drugs, hormones, receptors, antibodies, etc.

Principle :- The principle of affinity chromatography is based on the property of specific & noncovalent binding of proteins to other molecules, referred to as ligands. For instance enzymes bind specifically to ligands such as substrates or cofactors.

Procedure :-

This technique involves the use of ligands covalently attached to an inert & porus in a column.

The immobilized ligands acts as molecular fish hooks to selectively pick up the desired protein while the remaining proteins pass through the column.

The desired protein captured by the ligands, can be eluted by using free ligand molecules.

Alternatively, some reagents that can break protein- ligand interaction can also be employed for the separation. 17


AFFINITY

CHROMATOGRAPHY

Fig:5:-Affinity chromatography

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HPLC

INTRODUCTION:

High Performance Liquid Chromatography (HPLC) is used to separate components of a mixture by using a variety of chemical interactions between the substances being analyzed & the chromatography column.

PRINCIPLE:

It may employ the principles of adsorption, partition, ion-exchange, exclusion & affinity chromatography. This makes it an extremely versatile technique.

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HPLC

FIG.6:-outline of HPLC20

PROCEDURE:

The sample to be analyzed is introduced in a small volume to the stream of mobile phase.

It is retorted to specific chemical or physical interactions with the stationary phase. It travels the length of the column.

The amount of retardation depends on the nature of the analyte, stationary phase & mobile phase composition.

At the time at which a specific analyte elutes is called the retention time.

The use of pressure increases the linear velocity giving the components less time to diffuse with in the column.


HPLC

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PAPER

ELECTROPHORESIS

Introduction :-

It is used for the separation of serum proteins.

Principle :-

Separation of charged particles is determined by differences in their migration rate which varies with electrical charge, size of particles & shape of particles.

Procedure :-

In this the sample is applied on a strip of filter paper wetted with dried buffer solution.

The end of the strip are dipped into the buffer reservoirs in which electrodes are placed. Then electric current is supplied allowing the molecules to migrate for sufficient time.

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PAPER

ELECTROPHORESIS

The paper is removed, dried & stained with a dye that specifically colors the substance to be detected which can be identified by comparing with a set of standards run simultaneously.

For the separation of serum proteins whatman No.1 filter paper, veronal or tris buffer at pH 8.6 & the stains amido black or bromophenol blue are employed.

FIG.7;- process of paper electrophoresis

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GEL

ELECTROPHORESIS

Introduction :- It is used for the separation of proteins & nucleic acid.

Principle :-This technique involves the separation of molecules based on their size, in addition to electric charges.

Procedure :- In this technique gel is used as a stabilizing media.

Gel contains wells made with the help of comb.

Buffer is added in the apparatus.

In the well the sample is loaded.

The electric current is switched on. 24


GEL

ELECTROPHORESIS

The sample components get separated on the basis of their size & electric charge.

The separated components can be identifie either by labeling with a radio isotope or by UV- visible spectrophotometer.

FIG.8:- GEL GLECTROPHORESIS

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ISO

ELECTRIC

FOCUSING

Introduction :- It is used for purification of proteins.

Principle :- This technique is primarily based on the immobilization of the molecules at isoelectric pH during electrophoresis.

Procedure :- Stable pH gradients are set up (usually in gel) converting the pH range to include the isoelectric points of the components in a mixture.

As the electrophoresis occurs, the molecules migrate to positions corresponding to their isoelectric points, gets immobilized & form sharp stationary bonds.

The gel blocks can be stained & identified.

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ISOELECTRIC

FOCUSING

Fig.9:- Isoelectric focusing

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INTRODUCTION:

Ultra-violet visible spectroscopy refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectra.

Principle:

It uses the visible & adjacent ranges. The absorption or reflectance in the visible range directly affects the perceived colour of the chemicals involved.

In this region of electromagnetic spectrum , molecules undergo electronic transitions.

The absorption measures transition from the ground state to the excited state.


UV

VISIBLE

SPECTROSCOPY

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Fig:10:UV-VIS Spectroscopy


UV

VISIBLE

SPECTROSCOPY

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PROCEDURE:

It is used in analytical chemistry for the quantitative determination of different analytes such as transition metal ion, organic compounds & biological macromolecules.

Determination is usually carried out in solution. Organic solvents may have significant UV absorption.

The Beer-Lambert law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution & the path length.

It is necessary to know how quickly the absorbance changes with concentration. This can be taken from references or more accurately , determined from a calibration curve.


UV

VISIBLE

SPECTROSCOPY

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MASS

SPECTROSCOPY

INTRODUCTION:

A mass spectrometer is an instrument that can measure the mass/charge ratio(m/z) of ions in a vaccum.

From these data , molecular masses can be determined with a high degree of accuracy, allowing the molecular composition of a given sample or analyte to be determined .

Principle:-

Mass spectrometers have three principle components:

a) A source of ions : Converst the analyte into gas phase ions in a vaccum.

b) A mass analyser : Seperates ions according to their mass/charge ratios.

c) Anion detector : Record the impart of individual ions.

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PROCEDURE: The analyte is dissolved & forced

through a narrow needle held at a high voltage.

A fine spray of charged droplets emerges from needle & is directed into the vaccum chamber of the mass spectrometer through a small orifice.

As they enter the mass spectrometry , the droplets are dried using a stream of inert gas, resulting in gas phase ions that are accelerated through the analyzer towards the detector.

Because electrospray ionization(ESI) produces gas-phase ions from solutions , it is readily integrated with upstream protein seperation by liquid chromatography(LC).


MASS

SPECTROSCOPY

Fig:11:Mass Spectroscopy

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CONCLUSION

Analytical technique is very important .

It can be used both for separation & identification of macromolecules & micro moleculses.

In biochemistry, the term macromolecule is applied to the four conventional biopolymers ( nucleic acids, proteins , carbohydrates & lipids ) as well as non polymeric molecules with large molecular mass.

The constituent molecules from which macromolecules are assembled are called monomers, dimers or tetramers.

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An analytical technique is a method that is used to determine the concentration of a chemical compound. A variety of analytical techniques are availabel for the quantification of macromolecules. Paper chromatography is commonly used for the separation of amino acids, sugars, sugar derivatives & peptides. Ion exchange chromatography is used for separation of amino acids, proteins, nucleotides. Gel filtration chromatography is used for the separation of both smaller & bigger molecules. Affinity chromatography is useful for purification of enzymes, vitamins, nucleic acids, drugs, hormones, receptors, antibodies, etc. Paper electrophoresis is used for the separation of serum proteins. Gel electrophoresis is used for the separation of proteins & nucleic acid. Isoelectric focusing is used for purification of proteins.It is used in analytical chemistry for the quantitative determination of different analytes such as transition metal ion, organic compounds & biological macromolecules. A mass spectrometer is an instrument that can measure the mass/charge ratio(m/z) of ions in a vaccum. Analytical technique is a very important.


SUMMARY

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UPADHYAY,UPADHYAY & NATH 2009 BIOPHYSICAL CHEMISTRY PRINCIPLES & TECHNIQUES

J.L JAIN 2007 FUNDAMENTALS OF BIOCHEMISTRY

U.SATYANARAYAN & U.CHAKRAPANI 2008 BIOCHEMISTRY


REFERENCES

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analytical techniques in biochemistry and biophysics for macro molecules

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