chromatography and its application.ppt
- 1. 8/8/2022 1 Pharmaceutical Analysis II Chromatography By: D.A April, 2021
- 2. Introduction to chromatography History Russian botanist Tswett M. (1872–1919) ◦ Used a column packed with a stationary phase of calcium carbonate to separate colored pigments from plant extracts. ◦ The sample was placed at the top of the column and carried through the stationary phase using a mobile phase of petroleum ether. Chroma-color, graphy-writing
- 3. Cont… Chromatography ◦ Process by which complex mixtures can be resolved and separated Mixture could be organic or inorganic . The name (from the Latin khromatos) implies color ◦ No direct connection except that the first compound separated by this process were pigments. ◦ Is a separation process that is achieved by distributing the components of a mixture between two phases, a stationary phase and a mobile phase ◦ Those components held preferentially in the stationary phase are retained longer in the system than those that are distributed selectively in the mobile phase. ◦ As a consequence, solutes are eluted from the system in the order of their increasing distribution coefficients with respect to the stationary phase
- 4. Essential features of chromatographic separation Mixtures to be separated is introduced in to,Two phases Where each phase competes with each others Aiming at containing different components (analytes) ◦ As a result Each components will be distributed in the two competitive phase. According to certain distribution coefficients. These coefficients depends on physical properties of a molecules such as, Solubility Adsorption Volatility Molecular size and I onic charge in ionizable molecule.
- 5. Cont… Existence of two substance with exactly similar properties is highly improbable ◦ Different substance will exhibit different properties. ◦ Such different causes chromatographic separation. Mobile phase ◦ is a moving phase that continuously flows through the stationary phase and carries the analyte. Stationary phase ◦ The fixed or non-moving phase, immobile phase.
- 6. Cont… Separates components in mixture: Based on Polarity boiling point ionic group on ionizable molecule size There are different types of chromatography Based on the nature of stationary and mobile phase but more particularly the nature of stationary phase
- 7. Partition Chromatography Liquid SP and Liquid/gas MP Molecules will partition into the stationary phase based upon affinity/polrity for stationary phase & eventually partition into mobile phase again Thin layer of the liquid SP is coated onto inside of GC column or on small particles on LC column
- 8. Adsorption Chromatography Solid SP and Liquid/gas MP Very similar to partition chromatography Adsorption just on surface, partition into thin layer 8/8/2022 INSTRUMENTAL METHODS 8
- 9. Ion Exchange Chromatography 8/8/2022 INSTRUMENTAL METHODS 9 A solid polymeric stationary phase containing replaceable ions Ionic liquid mobile phase Separation of either cations or anions Separation based on relative strength of ionic bond Solute ions of the opposite charge in the mobile liquid phase are attached to the resin by electrostatic force. Anion exchange has cations ((e.g., – N(CH3)3+ on surface Cation exchange has anions (eg. SO3-) on the surface
- 10. Molecular Exclusion Chromatography An inert gel which acts as a molecular sieve and a liquid mobile phase is used. Separation based on size Small molecules get trapped in pores & take longer to get out 8/8/2022 INSTRUMENTAL METHODS 10
- 11. Affinity Chromatography Very selective Specific binding site is used to concentrate analyte on column Used a lot in biological applications Eg. Antibody SP might be used to separate protein mix. 11
- 12. Chromatographic techniques Planar Cr. SP-plane support ◦ Paper chromatography Liquid SP-soaked in cellulose paper ◦ Thin layer chromatography (TLC) adsorbent (Al2O3 or SiO2, usually) coating a sheet of plastic or glass Separated cpds-appear as spot 12 Column chromatography SP-in column ◦ Gas chromatography(GC) ◦ High performance liquid Cr. (HPLC) Separated cpds-appear as a peak
- 13. Cont… CC, TLC, & HPLC, ◦ May be of partition, adsorption, ion exchange, exclusive or affinity Paper chromatography ◦ Is primarily a partition process GC may be ◦ A partition process an in GLC ◦ An adsorption process as in GSC The term liquid chromatography Some times used to denote techniques in which the mobile phase is a liquid, PC, CC, TLC, HPLC.
- 14. Application of chromatography It has wide application ◦ Evaluation of drugs and pharmaceutical products ◦ Drug extraction, isolation and purification ◦ Identification of active principal in plant research Application can be classified in three groups ◦ Preparative application ◦ Qualitative application ◦ Quantitative application Qualitative application The presence or absence of certain constituents ◦ Techniques of choice are, HPLC GC TLC
- 15. Cont… Gives valuable information about complexity. ◦ Number of spots in PC and TLC ◦ Number of peaks in GC and HPLC Used to check purity and identity Used to establish finger print for, Extracts, tincture, volatile oil or pharmaceutical preparation. So the purity and identity of a sample can be judged. Quantitative application ◦ Quantitative chromatography determines, Percentage of any components in a sample ◦ Either using Absolute amount Relative to another components All chromatographic techniques can applied.
- 16. Theoretical Concepts In a typical chromatographic system ◦ Different compounds move through the system With different rates of movements In the form of band or zones Different rates of movements (differential migration) takes into consideration. ◦ The equilibrium distributions of a compounds between Stationary phase and Mobile phase The speed with which each compounds move through a system ◦ Determines by the number of molecules of that compound in mobile phase at any instant . Sample molecules in the stationary phase do not move through the system.
- 17. Cont… There fore, compound that spends most of their time in the stationary phase Move through the system rather slowly Compound whose molecules are found in the mobile phase most of the time Move through the system more rapidly. Molecules of solvent or mobile phase move through the system At faster possible rate Except in gel chromatography Partition chromatography Some times called liquid to liquid chromatography (LLC)
- 18. Cont… Sample solution is introduced into two phase Namely liquid-liquid system One of the phase kept as a stationary phase On a suitable support Filter paper, cellulose powder etc Have the ability to absorb and fix the stationary phase The other moves past through the stationary liquid and support material. The two phases should be immiscible Adsorption chromatography Some times liquid-solid chromatography (LSC) Stationary phase is solid Such as alumina or silica gel
- 19. Cont… The mobile phase is liquid Separation occurs when components get, Adsorbed on the solid phase with different strength In theory, adsorption chromatography, More complex than partition chromatography
- 20. Thin layer Chromatography (TLC) The term “thin-layer chromatography”, introduced by N.A Izamailov and M.S Shraiber in 1938, means a chromatographic separation process in which the stationary phase consists of a thin layer applied to a solid substrate or “support”. In TLC, the sample solution is applied as a spot or band on the origin of the layer spread on a support (plate). After evaporation of the sample solvent, the plate is placed in a sealed chamber containing a solvent chosen as a mobile phase.
- 21. Cont… Development occurs as the mobile phase moves through the layer, and the components of the sample move at different rates to create the separation. Environmental, pharmaceutical, biomedical and food samples are among the sample types commonly analyzed by TLC
- 22. Cont… . Advantages of TLC include Rapid analysis time because many samples can be analyzed simultaneously, Low solvent usage, A high degree of accuracy and precision for instrumental TLC, and Sensitivity in the nanogram or pictogram range. Coupling of TLC with other analytical methods such as high performance liquid chromatography (HPLC), mass spectrometry (MS), and Fourier transform infrared spectrometry (FTIR) provides enhanced opportunities for sample analysis.
- 23. Cont… Data Analysis The position of a substance zone (spot) in a thin-layer chromatogram can be described with the aid of the retardation factor Rf. This is defined as the quotient obtained by dividing the distance between the substance zone and the starting line by the distance between the solvent front and the starting line
- 24. Cont… This value is calculated as follows: The Rf value is characteristic for the compound under those particular chromatographic conditions. The response of a compound to a particular detection reagent (e.g. spray) is also characteristic of the substance. Figure : (a) A TLC plate before elution (spot 1 contains one application, spot 2, two applications and spot 3,three applications); (b) Running a TLC chromatogram; (c) Example after elution. 82 . 0 8 . 6 6 . 5 ) ( b a pink Rf 41 . 0 8 . 6 8 . 2 ) ( a c blue Rf
- 25. Cont… Application of TLC analysis Application of TLC include analysis of the following types: starting raw ,material (plant extracts ,extracts of animal origin, fermentation mixtures); intermediates( crude products, reaction mixtures, mother liquors, and secondary products); Pharmaceutical raw material ( identification, purity testing , assay, separation of closely related compounds, stability testing); formulated products ( identification, purity testing, assay, separation of closely related compounds, stability testing under storage and stress, content uniformity test, dissolution test); and Analysis of drugs and their metabolites in biological media such as urine, plasma, or gastric fluid(pharmacological , toxicological, pharmacokinetic, metabolic, bioequivalence, forensic and compliance and pharmacodynamic studies).
- 26. Qualitative Analysis TLC is often used by BP monographs as part of a number of identity tests performed on pure drug substances. Table 1 lists a few compounds which have their identity checked by TLC and a variety of location reagents and mobile phases to illustrate the fact that there is much less uniformity about TLC methodology than there is in the case of HPLC and GLC methodology.
- 27. Stationary phase Mobile phase Visualizatio n reagents Comments Framycein sulphate Methyl prednisolone Aprotinin Levamisole Pentagastrin Silica gel + carbomer binder Silica gel GF254 Silica gel Silica gel with fluorescent indicator Silica gel G 10%w/v KH2PO4 Ether/toluene /butanol-1-ol saturated with water (85:10:50 Acetate buffer Toluene/aceto ne/13.5M ammonia (60:40:1) Analyte is examined by TLC in three different mobile phases Naphthalen ediol / H2SO4 UV light 254nm then ethanolic sulfuric acid (20%),+ heat to 1200C Ninhydrin spray UV light 254 nm 4- dimethylam ino- benzaldhyd e in methanol/H Cl Rf and color of the sample are compared with a pure standard. The resolution of the analyte from streptomycin is checked Rf and color of the sample are compared with a pure standard. Also Rf of an oxidation product is used as an additional check Rf and color of the spot obtained is matched to that of a standard Rf and the size of the spot obtained is matched to that of a standard The Rf of the analyte in three different mobile phases is determined and the color of its spot is matched to that of the standard