BASICS OF CHROMATOGRAPHY AND THEIR FORENSIC APPLICATION.pptx

THIS PRESENTATION IS A BRIEF OF BASICS OF CHROMATOGRAPHY AND THEIR FORENSIC APPLICATION

1. TOPIC : BASICS OF CHROMATOGRAPHY AND THEIR FORENSIC APPLICATION PRESENTED BY: PALLAVI KUMARI ID NO: 19MSFS001 M . Sc 1 st SEMESTER 2019-21

2. CONTENTS: INTRODUCTION HISTORY BASIC TERMS RELATED TO CHROMATOGRAPHY TYPES AND THEIR APPLICATIONS CONCLUSION

3. INTRODUCTION Chromatography comes from Greek Word chroma : colour graphein : writing What is chromatography? Chromatography is a laboratory technique by which a mixture is separated by distributing its components between two immiscible phase.

4. HISTORY Chromatography was first invented by a Russian Botanist Mikhail Tswett in 1903. He separate plant pigments such as chlorophyll,xanthophyll and carotenoids by using calcium carbonate as stationary phase and hydrocarbon as mobile phase.

5. STATIONARY PHASE MOBILE PHASE ELUTION ELUTION VOLUME ELUTION TIME VOID VOLUME RETENTION FACTOR CHROMATOGRAM RESOLUTION BASIC TERMS RELATED TO CHROMATOGRAPHY

6. TYPES OF CHROMATOGRAPHY ADSORPTION CHROMATOGRAPHY PARTITION CHROMATOGRAPHY ION EXCHANGE CHROMATOGRAPHY SIZE/MOLECULAR EXCLUSION CHROMATOGRAPHY AFFINITY CHROMATOGRAPHY

7. 1.ADSORPTION CHROMATOGRAPHY DEFINITION: It is atype of chromatography which based on the principle of adsorption. Here the separation is based on the interaction of the absorbate with the adsorbent. Principle: It involves the analytical separation of a chemical mixture based on the interaction of the absorbate with the adsorbent that adsorbs different compounds at different rate Adsorbent : silica gel, alumina ,etc. Mobile phase : either liquid or gas

8. TYPES OF ADSORPTION CHROMATOGRAPHY Adsorption column chromatography Principle: Main principle of this technique is adsorption. Mixture of components dissolved in the mobile phase is introduced in to the column and components move depending upon their relative affinities. The compounds which has more affinity towards stationary phase travels slow and the compound which has lesser affinity towards stationary phase travels faster. Stationary phase: The stationary phase or adsorbent in this type is a solid. The most commonly used is silica gel and alumina. Mobile phase: The mobile phase or eluent is a solvent or a mixture of solvents. History: Russian botanist Mikhail Tsvet invented column chromatography in 1906 as a means of studying plant pigments.

9. Instrumentation: Applications: Purification of tincture of alkaloids from pigments before determination of their alkaloidal content. Drug analysis, urine or other body samples ( high pH urine will elute first). Isolation and purification of vitamins and hormones.

10. B. Thin layer chromatography (TLC) PRINCIPLE: Similar to other chromatographic method, TLC is also based on the principle of separation. The separation depends on the relative affinity of compounds towards stationary phase and mobile phase. High affinity travels slower and other travels faster. HISTORY: TLC is discovered by A.A. Izmailov and M.S. Shraiber in 1938. INSTRUMENTATION: TLC plate ii. TLC chamber Mobile phase iv. A Filter paper

11. Applications of TLC: To check the purity of given samples. Identification of compounds like acids,alcohols,proteins,alkaloids,amines , antibiotics and more. Used for identification of drugs, dyes ,inks, etc. Comparison of drugs, dyes and inks. Analysis of urine and blood.

12. C. Gas solid chromatography PRINCIPLE: The basic principle of gas chromatography is that the resolution of the constituents by partitioning them between the mobile phase and the stationary phase inside the gas chromatography column. HISTORY: The father of modern gas chromatography is John Porter Martin who also developed the first liquid chromatograph in 1950. INSTRUMENTATION: Carrier gas Flow controller Sample injector Column Oven Detector

13. Applications:   Gas chromatography is used to determined if a deceased person has taken any alcohols or drugs prior to death as well as determining if they had been poisoned. Separation and identification of volatile materials , plastics, natural, and synthetic polymers, paints, and microbiological samples. Analysis of drug products like antibiotics(penicillin), antiviral( amantidine ), general anesthetics ( chloroform,ether ), sedatives or hypnotics(barbiturates), etc. In determining the levels of metabolites in body fluids like plasma, serum, urine, etc.

14. 2. PARTITION CHROMATOGRAPHY PRINCIPLE: The separation of the components from the sample mixture is carried out by the process of partition of the components between two phases (both are in liquid phase). The liquid surface is immobilised by stationary phase which results in making it stationary phase. The mobile phase moves from the stationary phase and components get separated. The separation depends on different co-efficient. HISTORY: This separation theory was introduced by Richard Laurence Millington and Archer Martin in 1940s. APPARATUS REQUIRED: i . Chromatography jar ii. Liquid impregnated paper (stationary phase) iii. Capillary tube (to apply sample mixture) iv. Mobile phase (chloroform, methanol, acetone, ethanol)

15. SOME MAIN TYPES OF PARTITION CHROMATOGRAPHY A. Liquid liquid chromatography This is a chromatographic technique where a sheet of blotting paper is used instead of adsorption column. The components are separated based on their differential migration velocities. B.Gas liquid chromatography This is a type of chromatography technique in which the separation of the mixture is done by an inert gas along a tube. The tube is filled with with finely divided inert solid. The solid is coated with non-volatile oil. The migration of each component occurs at a rate determined by its solubility in oil as well as its vapour pressure. C. Paper chromatography PRINCIPLE: Differential partitioned co-efficient of the components between water on the solid stationary phase and the mobile phase. Solid support: Paper Stationary phase: Whatman’s paper no.1 & 3, CM-cellulose, DEAE( diethylaminoethane ) Mobile phase: solvent system (ex: chloroform, benzene , butanol , cyclohexane ,etc.)

16. INSTRUMENTATION/COMPONENTS REQUIRED Solid support Mobile phase Stationary phase Chromatographic chamber APPLICATIONS: Separation between polar and non-polar compound. To determine organic compounds , biochemicals in urine. For determination of hormones and drugs. Identification of inks.  

17. 3) ION EXCHANGE CHROMATOGRAPHY PRINCIPLE: Ion exchange chromatography separates molecules based on their respective charged groups. It retains analyte molecules on the column based on ionic interactions. HISTORY: It was first reported by Peterson and Sober in 1956. In modern form it was invented by Small, Stevens and Bauman in 1975. TYPES: 1). Cation exchange chromatography 2). Anion exchange chromatography

18. INSTRUMENTATION: i . Pump, ii. Injector, iii. Column, iv. Suppressor, v. Detector, vi. Recorder or data system. APPLICATIONS: Determination and identification of chlorate, nitrite and nitrate in cases of explosives and explosive residues were investigated. Analysis of amino acid mixtures. In treatment of water. In the analysis of products of hydrolysis of nucleic acids.

19. 4) AFFINITY CHROMATOGRAPHY PRINCIPLE: Separation in chromatography depends upon the reversal adsorption of biomolecules through biospecific interactions on the ligands. HISTORY : Discovered by Pedro and Wilcheck in 1968. COMOPONENTS: Matrix ii. Spacer arm iii. Ligand

20. To identify the biological compound binding to a specific sites. It is essentially a sample purification technique, used primarily for biological molecules such as proteins. Removal of impurities or in purification process. Nucleic acid purification. Investigation of binding sites of enzymes. In in vitro antigen-antibody reactions APPLICATIONS:

21. 5) SIZE/MOLECULAR EXCLUSION CHROMATOGRAPHY PRINCIPLE: The principle of size exclusion chromatography is that particles of different sizes or different molecular weight elute from the stationary phase at different rates. HISTORY: In 1979, Yau and Kirkland published the first text on size exclusion chromatography. TYPES: Gel permeation chromatography(GPC): In this type, a hydrophobic column packing material is used and a non-aqueous mobile phase (organic solvent) to measure the molecular weight distribution of synthetic polymers. B. Gel filtration chromatography(GFC): In this type, a hydrophilic packing material and an aqueous mobile phase is used to separate, fractionate, or measure the molecular weight distribution of molecules soluble in water, such as polysaccharides and proteins.

22. INSTRUMENTATION: INJECTION VALVE PUMP COLUMN DETECTOR RECORDER

23. APPLICATIONS: Purification of biological macromolecules like viruses, protein, enzymes, nucleic acids, antibodies and polypeptides. separation of low molecular weight components from mixture. Separation of biological molecules in food specimens.

24. CONCLUSIONS Chromatography is important because it is a versatile and small quantities of a material can be separated with ease, it is fast and accurate if the hardware is maintained. Chromatography is one of the most common techniques in analytical technology and needs only a few micrograms of material in order to work.

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