Analytical techniques ppt

Analytical techniques in biochemistry

1. A SEMINAR ON ANALYTICAL TECHNIQUES IN BIOCHEMISTRY PROF. J.P. SHARMA (DIRECTOR) DR. R.K. RAO (PRINCIPAL) GUIDED BY - HUMA NAZZ SIDDIQUI PRESENTED BY.. NIKITA DEWANGAN M.Sc.1 st SEM BIOTECHNOLOGY G.D. RUNGTA COLLEGE OF SCIENCE & TECHNOLOGY KOHKA-KURUD,BHILAI DURG (C.G.)

2. INTRODUCTION VARIOUS ANALYTICAL TECHNIQUES PAPER CHROMATOGRAPHY ION-EXCHANGE CHROMATOGRAPHY GEL FILTRATION CHROMATOGRAPHY AFFINITY CHROMATOGRAPHY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY PAPER ELECTROPHORESIS GEL ELECTROPHORESIS ISOELECTRIC FOCCUSING SPECTROPHOTOMETER CENTRIFUGE ENZYME LINKED IMMUNO SORBANT ASSAY CONCLUSION SUMMARY REFERENCES S Y N O P S I S 1 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

3. Introduction In analytical biochemistry uses instruments and used to separate, identify and qualify biomolecules . Definition An analytical techniques is a method that is used to determine the concentration of a chemical compound. I N T R ODU C T I ON ANALYTICAL TECHNIQUES IN BIOCHEMISTRY 2

4. ANA L Y T I C A L T E CHN I QUE S Chromatography Electrophoresis Spectrophotometer Centrifugation Enzyme Linked Immuno Sorbant Assay ANALYTICAL TECHNIQUES IN BIOCHEMISTRY 3

5. INTRODUCTION Chromatography is an analytical technique dealing with the separation of closely related compound from a mixture. These include protein, peptides, amino acid, lipids, carbohydrates, vitamins and drugs . PRINCIPLE Chromatography (Greek ; chroma – colour , graphein – to write) usually consists of a mobile phase and a stationary phase. The mobile phase refers to the mixture of substances( to be separate), dissolved in a liquid or a gas. The stationary phase is a porous solid matrix through which the sample contained in the mobile phase percolates. The interaction between the mobile and stationary phases results in the separation of the compounds from the mixture. TYPES OF CHROMATOGRAPHY - PAPER CHROMATOGRAPHY, ION EXCHANGE CHROMATOGRAPHY, AFFINITY CHROMATOGRAPHY. CHROMA TOGRA PHY ANALYTICAL TECHNIQUES IN BIOCHEMISTRY 4

6. INTRODUCTION Paper chromatography is useful for separating the mixture of amino acid, sugar, chemicals, lipid , urea and some drugs. 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 5 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

7. PROCEDURE – The sample mixture is applied to a piece of filter paper the edge of the paper is immersed in a solvent. 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. 6 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY PAPER CHROMATOGRAPHY Fig. 1; paper chromatography

8. 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 7 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY PAPER CHROMAT OGRAPHY

9. INTRODUCTION Ion exchange chromatography is used for separation of amino acids, proteins, nucleotides and charged molecule. 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 . I ON E X CHAN G E ANALYTICAL TECHNIQUES IN BIOCHEMISTRY 8

10. FIG.:- 2 Ion exchange chromatography ANALYTICAL TECHNIQUES IN BIOCHEMISTRY 9 I ON E X CHANG E CHROMA T E

11. INTRODUCTION Affinity chromatography  is a method of separating   biochemical   mixtures based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, Or  receptor  and  ligand. 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. ANALYTICAL TECHNIQUES IN BIOCHEMISTRY 10 AF F I N I TY CHROMATOGRAPHY

12. AF F I N I TY CHROMATOGRAPHY Fig:-3 Affinity chromatography ANALYTICAL TECHNIQUES IN BIOCHEMISTRY 11

13. HP L C 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. ANALYTICAL TECHNIQUES IN BIOCHEMISTRY 12 Fig; 4. HPLC

14. HPLC PRINCIPLE It may employ the principles of adsorption, partition, ion-exchange, exclusion & affinity chromatography. This makes it an extremely versatile technique. 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. ANALYTICAL TECHNIQUES IN BIOCHEMISTRY 13

15. E L ECTROPHORE S I S INTRODUCTION The movement of charged particles(ions) in an electric field resulting in their migration towards the oppositely charged electrode is known as electrophoresis. TYPES OF ELECTROPHORESIS – PAPER ELECTROPHORESIS, GEL ELECTROPHORESIS, ISOELECTRIC FOCUSSING. PAPER ELECTROPHORESIS - 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. ANALYTICAL TECHNIQUES IN BIOCHEMISTRY 14

16. P A P ER E L ECTROPHORE S I S 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 5, PROCESS OF PAPER ELECTROPHORESIS ANALYTICAL TECHNIQUES IN BIOCHEMISTRY 15

17. GEL E L ECTROPHORE S I S INTRODUCTION It is used for the separation of proteins & nucleic acid. 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. The separated components can be identify either by labeling with a radio isotope or by UV- visible spectrophotometer . ANALYTICAL TECHNIQUES IN BIOCHEMISTRY 16

18. GEL E L ECTROPHORE S I S FIG. :- 6 GEL GLECTROPHORESIS ANALYTICAL TECHNIQUES IN BIOCHEMISTRY 17

19. I SOELECTRIC FOCUS I NG ANALYTICAL TECHNIQUES IN BIOCHEMISTRY 18 INTRODUCTION It is used for purification of proteins. 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. Fig -7 Isoelectric focusing

20. SPECTROPHOTOMETER – Spectrophotometer is a method to measure how much a chemical absorbs light, by measuring the intensity of light as a beam of light passes through sample solution. PRINCIPLE OF SPECTROPHOTOMETER - The principle of spectrophotometer is based on Lambert’s Beer’s law. :- Lambert’s law (Bouguer’s law) - this law states that the amount of light absorbed is directly propotional to the length or thickness of the solution under analysis.Thus, I / I = e -kb I = the intensity of the transmitted light, I = the intensity of the incident light, b = the absorbing thickness, better known by the term path-length, k = the linear absorption coefficient of the absorbing material, the power term in the above relationship can be removed by converting to the logarithmic form, thus, In I /I = - kb or In I /I = kb, Changing the common logarithms we get 2. 2.303 Log 10 I /I = kb Beer’s law - this law states that the amount of light absorbed is directly proportional to the concentration of the solute in solution. Thus, ANALYTICAL TECHNIQUES IN BIOCHEMISTRY SPECTROPHOTOME T E R 19

21. 2.303log 10 I /I = kʹC Where kʹ = absorptivity constant , C = the concentration of the absorbing material. The beer- lambert’s law then becomes: Log 10 I / I = abC ANALYTICAL TECHNIQUES IN BIOCHEMISTRY SPECTROPHOTOME T E R 20 Fig - 8 spectrophotometer

22. CENTRIFUGE is device for separating particles from a solution according to there size, shape, density, viscosity of the medium. PRINCIPLE : - An object moving in a circle at a steady angular velocity will experience a force F directed outwards. This is the basis of centrifugation. Angular velocity ω and the radius of rotation ‘r’ in cm collectively determine the magnitude of the force F, F = ω 2 r If F is expressed in the form of gravitational force it is divided by 980. RCF = ω 2 r / 980 RCF = relative centrifugal force But ω = 2 π radian = 2 π /60 r/min RCF = 4 π 2 ( r/m) 2 r/ 3600× 980 RCF = 1.118 × 15 -5 rpm 2 r 21 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY CENT RIFUGE

23. ENZYME LINKED IMMUNOSORBANT ASSAY ELISA is based on affinity. An ELISA test can detect both current and past infections. As well as antibodies, an ELISA test can also be used to detect antigens (substances that provoke an immune response, such as bacteria or proteins) or enzymes. 22 E L I S A ANALYTICAL TECHNIQUES IN BIOCHEMISTRY Fig ; 9ELISA

24. CONCLUS ION Analytical techniques in biochemistry provides an opportunity for researchers and scientists to explore the advanced and latest research developments in the field of biochemistry . Biochemistry deals with the structures, functions and interactions of biological macromolecules such as proteins, nucleic acids, carbohydrates and lipids which provide the structure of cells and perform many functions associated with life. It can be used both for separation & identification of macromolecules & micro molecules. 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. 23 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

25. An analytical technique is a method that is used to determine the concentration of a chemical compound. CHROMATOGRAPHY is used to separation of closely related compound from a mixture. Chromatography types – PAPER CHROMATOGRAPHY, ION- EXCHANGE CHROMATOGRAPHY, AFFINITY CHROMATOGRAPHY, HPLC. ELECTROPHORESIS – The movement of charged particle an electric field resulting in their migration towards the oppositely charged electrode . Electrophoresis types – PAPER ELECTROPHORESIS, GEL ELECTROPHORESIS, ISOELECTRIC FOCUSING. Spectrophotometer  is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. CENTRIFUGE - it is a device for separating particles from a solution according to their size, shape, density and velocity of medium. ELISA - An enzyme-linked immunoassay (ELISA) is a test that detects antibodies in the blood. SUMMARY 24 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

26. U. SATYANARAYANA 2010 BIOTECHNOLOGY J.L JAIN SIXTH EDITION 2010 FUNDAMENTAL OF BIOCHEMISTRY UPADHAYAY FOURTH EDITION2007 BIOPHYSICAL AND BIOCHEMISTRY REFERENCES 25 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

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