Chromatography and its classifications along with principles and applications and chromatogram

CHROMATOGRAPHY BRIEF DESCRIPTION IS GIVEN.

1. Presentation on Chromatography Submitted by Sukanta Debnath M.Pharm 1 st Semester Submitted to Mr. Rajat Gosh Tripura University Modern Pharmaceutical Analytical Techniques

2. Contents Introductions Principle Classification TLC Gas Chromatography HPTLC Column Chromatography Ion Exchange Chromatography Gel Filtration Chromatography High performance Liquid Chromatography Affinity Chromatography References

3. Introduction Chromatography is a physical process where the components of sample mixture are separated as a results of there differential distribution between stationary phase and mobile phase.

4. Principle Chromatography is usually based on principle of partition of solute between two phases. It usually consists of a Mobile Phase and a Stationary Phase. The Mobile Phase usually refers to the mixture of the substances to be separated 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.

5. Classification Thin Layer Chromatography(TLC) High Performance Thin Layer Chromatography(HPTLC) Gel Chromatography C olumn Chromatography Ion Exchange Chromatography Gel Filtration Chromatography High performance Liquid Chromatography Affinity Chromatography

6. Thin Layer Chromatography Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar to paper chromatography However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel , alumina , or cellulose Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents.

7. Cover plates Solvent mixture Thin Plate

8. Application It is widely used in separating multicomponent pharmaceutical formulations. It is used to purify of any sample and direct comparison is done between the sample and the authentic sample. It is used in the food industry, to separate and identify colors,  sweetening agent , and preservatives It is used in the cosmetic industry.

9. Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas.lt is the method of choice for the separation of volatile substances or the volatile derivatives of certain non-volatile substances Stationary phase is an inert solid material impregnated with a non-volatile liquid Gas Chromatography

10. The principle of separation in GC is "partition." The mixture of component to be separated is converted to vapour and mixed with gaseous mobile phase The component which is more soluble in stationary phase travel slower and eluted later. The component which is less soluble in stationary phase travels faster and eluted out first No two components has same partition coefficient conditions. So the components are separated according to their partition coefficient. Partition coefficient is "the ratio of solubility of a substance distributed between two immiscible liquids at a constant temperature.’’ Principle

11. He, N, Hydrogen

12. The cylinder/ gas tank is fitted with a pressure controller to control the pressure of gas, a pressure gauge that indicates the pressure, a molecular sieve to transfer filtered dry gas and a flow regulator to ensure a constant rate of flow of mobile phase to the column. It should meet the following criteria…. -- Should be chemically inert -- Should be cheap and readily available -- Should be of high quality and not cause any fire accidents --Hydrogen has low density and batter thermal conductivity. Nitrogen is inexpensive and gives reduced sensitivity.

13. Retention Of GC For example- Take a compound mixture of X,Y,Z And calculate and determined the major product . Formula :- Area of peak Total area X 100

14. Applications GC-MS is used in research and development, production, and quality control. It is used in identification of impurities in active pharmaceutical ingredients. In medicinal chemistry, GC-MS is used in the synthesis and characterization of compounds and in pharmaceutical biotechnology.

15. High Performance Thin Layer Chromatography HPTLC (High performance thin layer chromatography) is the automated, sophisticated form and improved method of TLC. It is a powerful analytical method equally suitable for qualitative and quantitative analytical tasks. It is also known as planer or flat bed chromatography.

16. Principle Same theoretical principle of TLC (Adsorption chromatography )i.e. the principle of separation is adsorption. Mobile phase flow by capillary action effect. And component move according to their affinities towards the adsorbent. The component with higher affinity toward adsorbent travels slowly. And the component with lesser affinity towards the stationary phase travels faster. Thus the components are separated on a chromatographic plate according to their affinity and separation also based on their solubility in mobile phase.

17. Sample preparation Application of Sample Chromatography Development Detection of Spots Screening and Documentation Selection Of Chromatographic Layer Pre washing Pre conditioning Steps Involve in HPTLC

18. HPTLC is suitable for both the qualitative and quantitative analysis of  herbal  extracts and formulations. Due to integrated software, all the analysis parameters and results can be stored digitally. It enables rapid analysis of herbal extracts with a minimum mobile phase. In HPTLC, the application of sample, scanning, and data analysis can be performed individually . Applications of HPTLC

19. Column chromatography separates substances based on differential adsorption of compounds to the adsorbent as the compounds move through the column at different rates which allow them to get separated in fractions. This technique can be used on a small scale as well as large scale to purify materials that can be used in future experiments.  Column Chromatography

20. Principle When the mobile phase along with the mixture that needs to be separated is introduced from the top of the column, the movement of the individual components of the mixture is at different rates. The components with lower adsorption and affinity to stationary phase travel faster when compared to the greater adsorption and affinity with the stationary phase. The components that move fast are removed first whereas the components that move slowly are eluted out last. The adsorption of solute molecules to the column occurs in a reversible manner. The rate of the movement of the components is expressed as: R f  = the distance travelled by solute/ the distance travelled by the solvent R f  is the retardation factor.

21. Applications Column Chromatography is used to isolate active ingredients. It is very helpful in separating compound mixtures. It is used to determine drug estimation from drug formulations. It is used to remove impurities. Used to isolate metabolites from biological fluids.

22. Ion- exchange chromatography is based on an exchange of ions between a charged stationary surface and ions of the opposite charge in mobile phase. Depending on the conditions, solutes are either cations (positively charged) or anions (negatively charged). These are also known as ion exchangers. Ion Exchange Chromatography Principle

23. Ion -exchangers are made up of two parts- an insoluble matrix and chemically bonded charged groups within and on the surface of the matrix. It is classified as cationic or anionic based on whether it exchanges cation or anions. Cation exchanger -also known as acidic ion exchanger. Anion exchanger -also called basic ion exchanger. Each type of exchanger is also classified as strong or weak depending on the ionizing strength of the functional group.

24. Ion exchange chromatography can be applied for the separation and purification of many charged or ionizable molecules such as proteins, peptides, enzymes, nucleotides, DNA, antibiotics, vitamins and etc. from natural sources or synthetic origin. An important use of ion-exchange  chromatography  is in the routine analysis of  amino acid  mixtures Applications

25. Gel Filtration Chromatography Principle Gel filtration is also known as size-exclusion chromatography or molecular-sieve chromatography. In this process, separation is based on the differing ability (due to differing molecular size) of molecules in the sample to enter the pores of the gel-filtration medium. The stationary phase in this technique consists of beads of a hydrated, sponge-like material that has pores of molecular dimensions and with a narrow range of sizes.  When an aqueous solution, containing molecules of various sizes, is passed through a column containing such ‘molecular sieves, molecules that are larger than the pores of the filtration medium move quickly through the column. Smaller molecules enter the pores of the gel and move slowly through the column.

26. Gel filtration chromatography, also known as size exclusion chromatography, is used to separate molecules of different sizes Fractionation of molecules and complexes within a predetermined size range Size analysis and determination Removal of large proteins and complexes Buffer exchange Applications

27. HPLC Chromatography High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase). The sample is carried by a moving carrier gas stream of helium or nitrogen.

28. The purification takes place in a separation column between a stationary and a mobile phase. The stationary phase is a granular material with very small porous particles in a separation column. The mobile phase, on the other hand, is a solvent or solvent mixture which is forced at high pressure through the separation column. Via a valve with a connected sample loop, i.e. a small tube or a capillary made of stainless steel, the sample is injected into the mobile phase flow from the pump to the separation column using a syringe. Subsequently, the individual components of the sample migrate through the column at different rates because they are retained to a varying degree by interactions with the stationary phase. After leaving the column, the individual substances are detected by a suitable detector and passed on as a signal to the HPLC software on the computer. At the end of this operation/run, a chromatogram in the HPLC software on the computer is obtained The chromatogram allows the identification and quantification of the different substances.  Principle

29. HPLC Chromatogram Interpretation Interpretation of Some Standard & Pure known Compounds . Like :- Caffeine, Aspartame, Benzoic Acid, Sorbic Acid

30. Base Line Configuration

31. Analysis of drugs Analysis of synthetic polymers Analysis of pollutants in environmental analytics Determination of drugs in biological matrices Isolation of valuable products Applications

32. Affinity chromatography is a type of liquid  chromatography  for the separation, purification or specific analysis of sample components. It utilizes the reversible biological interaction or molecular recognition called affinity which refers to the attracting forced exerted in different degrees between atoms which cause them to remain in combination. Affinity Chromatography

33. The stationary phase consists of a support medium, on which the substrate (ligand) is bound covalently, in such a way that the reactive groups that are essential for binding of the target molecule are exposed. As the crude mixture of the substances is passed through the chromatography column, substances with binding site for the immobilized substrate bind to the stationary phase, while all other substances is eluted in the void volume of the column. Once the other substances are eluted, the bound target molecules can be eluted by methods such as including a competing ligand in the mobile phase or changing the pH, ionic strength or polarity conditions. Principle

34. Affinity chromatography is one of the most useful methods for the separation and purification of specific products. It is essentially a sample purification technique, used primarily for biological molecules such as proteins. Its major application includes: Separation of mixture of compounds. Removal of impurities or in purification process. In enzyme assays Detection of substrates Applications

35. References https://www.thermofisher.com/blog/ask-a-scientist https://www.khanacademy.org/science/in-in-methods-of-purification-of-organic-compounds/a/principles-of-chromatography Journal of Chromatography A  publishes research papers and critical reviews on all aspects of  fundamental and applied separation science . The scope includes  chromatography  and related techniques The Journal of Chromatography and Separation Techniques is a peer-reviewed (refereed) The journal of Mixed solvents in gas—liquid chromatography : Activity coefficients

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