Instrumental Methods of Analysis B Pharmacy PDF

Page 1 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a BP701T Instrumental Method of Analysis Unit IIlI:...

Page 1 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a BP701T Instrumental Method of Analysis Unit IIlI: rm (Dr. S. K. Patro, Asst. Professor, IPT Salipur) A. Chromatography 1. Introduction to Chromatography ha 2. Column Chromatography 3. Thin layer Chromatography 4. Paper Chromatography B. Electrophoresis 1. Paper Electrophoresis lP 2. Gel electrophoresis 3. Capillary electrophoresis l we re Ca www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 2 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a A. Chromatography Introduction: rm Chromatography was invented by the Russian botanist Mikhail Tswet in the year 1903. He employed the technique to separate various plant pigments (i.e. Chlorophylls and Xanthophylls) by passing solutions of these substances (in petroleum-ether extract) through a glass column packed with finely powdered CaCO3. The separated species appeared as separate bands having colored bands; the various pigments migrating through the column at different rates (because of differences in ha their distribution ratios). The separated species appearedasseparate bands having colored bands: the various pigments migrating through the column at different rates (because of differences in their distribution of ratios). The various solutes were isolated by cutting and sectioning of the chalk packing. Twswett choose to designate the name of such a process of separation as chromatography (Chroma-color, lP graphein-writing). Tswett's original experiments remained unnoticed in the literature for several decades. It was not until 1931 when kuhan and Lederer investigated polyene pigments that interest in such a technique was renewed. But later on a diversify group of techniques which allow the separation of closely related components of the complex mixtures. In this technique, the sample is l moved in a mobile phase, may be a gas, a liquid or a supercritical fluid. Such a we mobile phase is then allowed to flow through an immiscible stationary phase. Chromatography is a physical method of separation in which the components to be separated or distributed between two phases, one of which is stationary (stationary phase), while the other, the mobile phase moves in a definite direction. re Ca 2 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 3 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Classification of Chromatographic methods: rm Type of General Type of Stationary Mobile equilibration Name of the Technique Classification method Phase Phase Process Paper Chromatography (PC) Liquid- Liquid Partition Thin layer Liquid ha Liquid Supported between the Chromatography Chromatography Liquid Or on a solid immiscible (TLC) (LC) Partition surface liquids High Performance thin layer Chromatography (HPTLC) lP Liquid-Solid, Adsorption Column Or Solid Liquid Adsorption Chromatography (ACC) adsorption Adsorption (using very Very finely Liquid-Solid divided much higher l pressures for the HighPerformance liquid Or solid Liquid flow of mobile Chromatography (HPLC) we adsorption packed in a phase) column re Ca 3 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 4 of 37 Carewell Pharma - A Family of Learning Type of General Types of Subscribe Stationary Us onMobile YouTube equilibration Name of the Classification method Phase Phase Technique Process Solid (ion lon-exchange lon- a exchange Liquid Partition/Sieving chromatography Exchange resign) (IEC) Group rm Specific liquid Affinity usually uses bonded to a solid surface (an Partition between enzymesS Liquid Affinity antibody, surface liquia chromatograpny. Or (immobilized) immobilized on Antigen- a stationary and mobile phase Antibody phase by ha highly covalentldy specific bindingtoit interactions an affinity ligand) Gas-solid lP Gas - Solid chromatography Adsorption (or Gas (GSC) Solid adsorption) Gas Gas-Liquid Chromatography |Liquid adsorbed Partition between Gas -liquid Chromatography on a solid Gas and liquid or Gas (GLC) l Partition) we Super-critical fluid Supercritical Bio specific Organic Species Partition between reritica Supercritical chromatography fluid adsorption or bonded to a Super-critical fluid | fluid (SFC) or chromatography bio affinity solid surface and bonded species Bioaffinity (SFC) re chromatography (BC) Ca www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 5 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Column Chromatography When a column of stationary phase is used, the technique is called as column chromatography. Based on the nature of the stationary phase i.e. whether it is solid or liquid, it is called as column rm adsorption chromatography or. Column partition chromatography is not widely used. Principle:. This technique is based on the principle of differential adsorption where different molecules in a mixture have different affinities with the adsorbent present in the stationary phase. 2. The molecules having higher affinity remain adsorbed for a longer time decreasing their speed of movement through the column. ha 3. However, the molecules with lower affinity move with a faster movement, thus allowing the molecules to be separated in different fractions. 4. Here, the stationary phase in the column chronmatography also termed the adsorbent is a solid (mostly silica) and the mobile phase is a liquid that allows the molecules to move through the column smoothly. The type of interaction between the stationary phase lP (adsorbent) & the solute is reversible in nature. The rate of movement of a component (R) is given as follows R Rate of movement of component = Rate of movement of mobile phase The equation can be simplified as follows: Distance moved by the solute l R= Distance moved by the solvent we When a liquid mobile phase is used, the equation is written as Am Am+ a As Where a is the Partition coefficient =. Conc.in stationary phase Conc in mobile phase Am is the average cross section of mobile phase re As is the average cross section of stationary phase Practical Requirement 1. Stationary Phase 2. Mobile Phase 3. Column characteristics Ca 4. Preparation of the column 5. Introduction of sample 6. Development technique (elution) 7. Detection of components 8. Recovery of components 5 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 6 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Stationary Phase Adsorbents are used in this technique may be organic and inorganic classes of compounds. The ideal requirements of adsorbent are: rm It should produce only adsorption of the analyte over it ii. The particles should have uniform size distribution and have spherical shape. Particle size: 60-200u. ii. It should have high mechanical stability iv. It should be inert & should not react with the solute or other components. V. Insoluble in the solvents or mobile phases used. Vi. It should be colorless to facilitate observations of zones and recovery of components. ha lP mobile phase loaded sample sample separation stronger interactions stationary phase -resolved- weaker bands interactions l we fractions eluted collection molecules re Figure: 1 Column chromatography. Table: 1 Adsorbents and Solvents Adsorbents Solvent Ca Sucrose Petroleum ether Starch Carbon tetrachloride Weak Inulin Cyclohexane talc Carbon di-sulphide Sodium carbonate Ether (ethanol free) 6 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 7 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Calcium carbonate Acetone Medium Calcium phosphate Benzene rm Magnesium carbonate Toluene Magnesium oxide Esters Calcium hydroxide Chloroform Activated magnesium silicate Acetonitrile Activated alumina Alcohols Activated charcoal Water Strong ha Activated magnesia Pyridine Activated silica Organic acids Mixtures of acids or bases Fuller's earth with ethanol or pyridine lP The most commonly used adsorbent is Silica gel of 80-100 mesh or 100 - 200 mesh size which has a particle size of 60-200u. Selection of Stationary Phase The selection of stationary phase in column chromatography depends on the following: 1. Removal of impurities: When a small quantity of impurity is present and there is difference in affinity when compared to the major component, a weak adsorbent is l sufficient. 2. No. of components to be separated: When few components are to be separated, weak we adsorbent is used. When more components are to be separated, a strong adsorbent is used. 3. Affinity differences between different components: When components have similar affinities, a strong adsorbent will be effective. When there is more differences in affinities, a weak adsorbent is selected. 4. Length of the column used: When a shorter column is used, strong adsorbent has to be re used. When a longer column is used, a weak adsorbent can be used. 5. Quantity of adsorbent used: 20 or 30 times the weight of the adsorbent is used for effective separation. Adsorbate: Adsorbent = 1: 20 or 1:30. Mobile Phase:Mobile Phase is the very important and they are several function: Mobile is acting Ca as solvent, developer, and as eluent. The functions of a mobile phase are: To introduce the mixture into the column As solvent - To develop the zones for separation As developing agent - To remove pure component out of the column As eluent - 7 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 8 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Different mobile phases used: It is used in increasing order of polarity or elution strength. The solvents are given in the above Table 1. These solvents can be used in either pure form or as a mixture of solvents of varying compositions rm ColumnCharacteristies Column is mostly best quality of neutral glass since it should not be affected by solvents, acids or alkalies. An ordinary burette can be used as column for separation. Length/diameter ratio is 10-15:1. For more efficiency, the length/diameter ratio is 100:1. Column length ha a. Multi-component system long column b. Components with similar affinities for adsorbent long column C. Components with different affinities for adsorbent short column Preparation of the Column The column mostly consists of a glass tube packed with a suitable stationary phase. lP Glass wool/cotton wool or an asbestos pad is placed at the botton of the column before packing the stationary phase. After packing, a paper disc kept on the top, so that the stationary layer is not disturbed during the introduction of sample or mobile phase. There are two types of preparing the column, they are: 1. Dry packing/ dry filling l In this the required quantity of adsorbent is poured as fine dry powder in the column and the solvent is allowed to flow through the column till equilibrium is reached. we 2. Wet packing/ wet filling In this, the slurry of adsorbent with the mobile phase is prepared and is poured into the column. It is considered as the ideal technique for packing. Before using column, it should be washed properly and dried. Introduction of the Sample re The sample which is usually a mixture of components is dissolved in minimum quantity of the mobile phase or a solvent of minimum polarity The entire sample is introduced into the column at once and gets adsorbed on the top portion of the column. From this zone, individual sample can be separated by a process of elution. Ca C. Elution (Development technique By elution technique, the individual components are separated out from the column. It can be achieved by two techniques: Isoeratie elution technique: Same solvent composition or solvent of same polarity is used throughout the process of separation. 8 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 9 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Eg. Use of chloroform alone or Pet.ether: Benzene = 1:l only, etc. Gradient elution technique: Solvents of gradually 1 (increasing) polarity or t (increasing) elution strength are used during the process of separation. rm E.g. initially benzene, then chloroform, then ethyl acetate then chloroform Other techniques like Frontal analysis and Displacement analysis where a graph of concentration of eluate Vs. volume of eluate will give an idea of how compounds are eluted out from the column. D. Detection of Components 1. If the compounds separated in a column chromatography procedure are colored, the progress of the separation can simply be monitored visually. ha 2. If the compounds to be isolated from column chromatography are colorless. Then the technique depends upon the properties of the components. Different properties which can be used are 3. Absorption of light (UV/Vis) Using UV-Visible Spectropotometer - 4. Flourescence or light emission characteristics Using fluorescence detector -. lP By using flame ionization flame detector. Refractive index detector- based on the refractive index difference between the mobile phase and mobile phase + component 7. Evaporation of the solvent and weighing the residue 8. Small fractions of the eluent are collected sequentially in labeled tubes and the composition of each fraction is analyzed by TLC (thin layer chromatography). l Recovery of components: Earlier, recoveries of the components were done by cutting the column into several distinct zones. Later, extrusions of the column into zones were done by using plunger. we The best technique is to recover the components by a process called aselution. The components are called as eluate, the solvent called as eluent and the process of removing the components from the column is called as elution. The different elution techniques like isocratic elution technique and gradient elution technique.Recovery is done by collecting different fractions of mobile phase of equal volume like 1Oml, 20ml etc or unequal volume. They can also be collected time wise i.e. a fraction every 10 or 20 minutes etc. The recovered fractions are detected by using the techniques re discussed earlier. Similar fractions are mixed so that the bulk of the compound of each type is obtained in a pure form. If a fraction still contains several components, it can be resolved by using another column. Applications: 1. Separation of mixture of compounds: Separation of glycosides, amino acids, plant extracts Ca 2. Removal of impurities Isolation of the active constituents from the plant extract or from formulations 3. Isolation of metabolites from the biological fluids: 17-ketosteroids from urine, cortisol 4. Estimation of drugs in formulations or crude extracts i. Determination of % w/w of strychnine in syrup of ferrous phosphate with quinine and strychnine www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 10 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a ii. Separation of diastereomers. iii. Separation of tautomers and racemates Factor affecting Column efficiency rm 1. Dimensions of the column 2. Particle size of the adsorbent 3. Nature of the solvent 4. Temperature of the column 5. Pressure ha Advantages: 1. Any type of mixture can be separated by column chromatography. 2. Any quantity of the mixture can also be separated (pg to mg of substance). 3. Wider choice of mobile phase. lP 4. In preparative type, the sample can be separated and reused. 5. Automation is possible. Limitation or Disadvantages of Column chromatography. Time consuming method 2. More amounts of solvents are required which may be expensive. 3. Automation makes the technique more complicated and costly. l we re Ca 10 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 11 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Thin layer Chromatography (TLC) Introduction:The history of thin layer chromatography dates back to 1938 when Izmailov and rm Shraiber separated plant extracts using 2mm thick and firm layer of alumina set on glass plate. In 1944, Consden, Goden and Martin used filter papers for separating amino acids. In 1950, Kirchner identified terpens on filter paper and later glass fibre paper coated with alumina. Only in 1958, Stahl developed standard equipment for analyzing by Thin layer chromatography. Principle: Thin Layer Chromatography can be defined as a method of separation or identification of a mixture of components into individual components by using finely divided adsorbent coated or spread over ha a chromatographic plate. The mobile phase solvent flows through because of capillary action (against gravitational force). The components move according to their affinities towards the adsorbent. The component with more affinity towards the stationary phase travels slower. The component with lesser affinity towards the stationary phase travels faster. Thus the components are separated on a thin layer chromatographic plate based on the affinity of the components towards the lP stationary phase. OH QH OH OH OH l Fig 2: Silica extended structure and surface. we Silica (Si02) is a solid with an extended structure of tetrahedral silica atoms bridged together by bent oxygen atoms. On the surface of the silica particles, the solid terminates in very polar silanol (Si-0-H) groups. The silica is the stationary phase because it remains adhered to the glass plate and does not move during the chromatography process. The Silica extended structure and surface is shown in the Fig 2. The developed TLC plate is shown in the Fig 3. re SOLVENT FRONT.-CENTER OF SPOT Ca STARTING POINT OF SPOT Fig 3: Developed TLC Plate 11 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 12 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Advantages of TLC 1. It is a simple process with a short development time. 2. It helps with the visualization of separated compound spots easily. rm 3. It helps in isolating of most of the compounds. 4. The separation process is faster and the selectivity for compounds is higher (even small differences in chemistry is enough for clear separation). 5. The purity standards of the given sample can be assessed easily. 6. It is a cheaper chromatographic technique. 7. TLC offers a faster and more efficient separation than paper chromatography and the majority of paper chromatographic separations have now been superseded by the TLC ha Procedures. Practical Requirement: 1. Stationary Phase: There a several adsorbents which can be used as stationary phases. Some of the stationary phases, their composition and the ratio in which they have to be mixed with water or other solvents to form a slurry for preparing thin layer chromatographic plates are lP given in the below Table 2: Name Composition Adsorbent: Water ratio SilicagelH Silicagel without binder 1:1.5 Silicagel GF | Silicagel + Binder + Fluorescent indicator 1:2 Silicagel G Silicagel +CaSOa(gypsum) 1:2 l Alumina 1:1.1 Neutral Al2O3 without binder we Basic Acidic AlLO G AlO3 +binder 1:2 Cellulose Cellulose without binder 1:5 powder re Cellulose Cellulose with binder 1:6 powder Kieselguhr Diatomaceous earth + binder 1:2 G Polyamide Polyamide 1:9 (CHCI3 : CH3OH= Ca powder 2:3) 12 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 13 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Fluoroscent indicator Zine silicate Silica gel and alumina are available with different specific surface areas and these grades are identified by a number, e.g., silica gel 60 (or 40 or 150) which indicates the mean pore size in rm Angstroms (10-m).The particle size of silica gel for TLC is 10-40 um (average 15 um). 2. Preparation of the Glass Plates: The sizes of the glass plates for use with commercially available spreaders are usually 20 X 20, 20 X 10 or 20 X 5 cm. Microscopic slides can also be used for some applications like monitoring the progress of chemical reaction. In general, the glass plates should be of good quality and should be withstand temperatures used for drying the plates. ha General method: Mix 30gm of the adsorbent in a mortar to a smooth consistence with the requisite amount of water or solvent specified in the manufacturer's instruction and transfer the slurry quickly to the spreader. Spread the mixture over 4 to 5 plates (20 X 20cm) or a proportionately larger number of smaller plates and allow the thin layers to set (about 4minutes when CaS04 is lP present). Transfer the plates carefully to a suitable holder and after a further 30minutes, dry at 100-120 °C for 1 hour to activate the adsorbent. Cool and store the plates in a desiccator over silica gel. The thickness of the moist thin layer should be about 0.25 mm. Special methods: a. Preparative thin layer. The layers are 0.5 - 2mm thick, prepared as described under the general method, but using a smaller quantity of water and allowing a longer time for the initial drying of the plate. l b. Mieroscopic slides are conveniently coated by a dipping technique in the following way: we prepare slurry of the adsorbent by shaking withchloroform or chloroform-methanol (2:1) and insert two microscope slides (back to back) into the slurry. Withdraw the slides; allow draining, separating the slides and drying. C. The slurry, prepared in the normal way, is sprayed onto the surface of glass plates, using a laboratory spray gun. d. Theadsorbent, mixed with an organic solvent, e.g., chloroform or ethyl acetate, is distributed evenly over a glass plate by careful tilting, and, after evaporation of solvent, re is dried in the normal way. In all the methods the plates should be tidied before use by cleaning the edges and backs (microscope slides). 3. Application of Sample: In order to get good spots, the concentration of the sample or standard solution can be 2-5ul of a %1 solution of either standard or test sample is spotting Ca using a capillary tube or micropipette. The spots can be placed at random or equidistant from each other by using a template, with markings. The spot should be kept at least 2cm above the base of the plate and the spotting area should not be immersed in the mobile phase in the development tank. 13 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 14 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a 4. Development Tank: For the purpose of development, a developing tank or chamber of different sizes to hold TLC plates of standard dimensions are used. These require more solvents for developing the chromatogram. When a new method is developed, it is better to rm develop in glass beakers or specimen jars, etc, to avoid more wastage of solvents. When developed method or standard method is used, it is better to use development tank. In the new type of development tanks have hump in the middlle, which require less solvent. The development chamber or tank should be lined inside with filter paper moistened with the mobile phase so as to saturate the atmosphere. If this kind of saturation of the atmosphere is not done, "edge effect" occurs where the solvent front in the middle of the TLC plate moves faster than that of the edge. The development tank examples are shown in the below Fig 3. ha Old type New type Glass TLC plates plates lP Mobile phase Hump solvent EES Mobile Fig 3: Development tank 5. Mobile Phase:Selection of the mobile phase depends upon the below factors. Nature of the substances to be separated l 11. Nature of the stationary phase used we 111. Mode of chromatography (Normal phase or reverse phase) 1V. Separation to be achieved Analytical or preparative - Pure solvents or mixture of solvents are used. The following gives a list of solvents (of increasing polarity). Petroleum ether, Carbon tetrachloride, Cyclohexane, Carbon di-sulphide, Ether, Acetone, Benzene, Toluene, Ethyl acetate, Chloroform, Alcohols like methanol or ethanol, Water, re pyridine. The solvent composition is done by trial and error method only but with a review of literature and other logical considerations like solubility of the substance, polar or non-polar character of the samples, etec. 6. Development technique: Different development techniques are used for efficient separations. They are Ca 1. Vertical development (One dimensional) 11. Two dimensional development ii. Horizontal development 1V. Multiple development 14 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 15 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a i. Vertical development (One dimensional): In this technique, the plates are kept vertical and the solvent flows against gravity, because of capillary action. rm Two dimensional techniques: For complex mixtures this technique is used. First, the plates are developed in one axis and the plates after drying are developed in the other axis. When large number compounds cannot be separated by using one dimensional technique. 7. Detecting or Visualizing Agents After the development of TLC plates, the spots should be visualized. Detecting colored spots can be done visually. But for detecting colorless spots, any one of the following ha techniques can be used. a. Specific methods: In this method particular detecting agents are used to find out the nature of compounds or for identification purposes. Examples are Ferric chloride- for phenolic compounds and tannins. 1. Ninhydrin in acetone- for amino acids ii. Dragendroff's reagent- for alkaloids lP iv. 2,4- Dinitrophenyl hydrazine - for aldehydes and ketones b. Nonspecific methods: Where the number of spots can be detected, but not the exact nature or type of compound. Examples 1. lodine chamber method: Where brown or amber spots are observed when the TLC plates are kept in a tank with few iodine crystals at the bottom. l 1. Sulphuric acid spray reagent: 70-80% v/v of sulphuric acid with few mg of either potassium dichromate or potassium permanganate or few ml of nitric acid as we oxidizing agent is used. This reagent after spraying on TLC plates is heated in an oven. Black spots are seen due to charring of compounds. 11i. Using fluorescent stationary phase: When the compound s are not flourscent, a fluorescent stationary phase is used. When the plates are viewed under UV chamber, dark spots are seen on a fluorescent background. Examples of such stationary phase is Silica gel GF re The detecting techniques can be categorized as i. Destructive technique: Specific spray reagents, Sulphuric acid spray reagent, etc where the samples are destroyed for detection. ii. Non-Destructive technique: like UV chamber method, lodine chamber method, densitometric method, ete where the sample is not destroyed even after detection. These Ca detecting techniques are used in TLC method development and in preparative TLC. In densitometric method, Densitometer is used which measures quantitatively the density of the spots. When the optical density of the spots for the standards and test solution are measured, the quantity of the substance can be calculated. The plates are neither destroyed nor eluted with the solvents to get the compounds. This method is also called as in-situmethod. 15 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 16 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a 8. Qualitative analysis The Rf value is calculated for identifying the spots in qualitative analysis. Rf value is the ratio of distance travelled by the solute to the distance travelled by the solvent front. rm Distance travelled by solute Rf = Distance travelled by the solvent front The Rf value ranges from 0 to 1. The ideal value is 0.3 to 0.8. The Rf value is constant for every compound in a particular combination of stationary and mobile phase. When the Rf value of a sample and reference compound is same, the compound is identified by its standard. When the Rf value differs, the compound may be different from its reference standard. ha Rx value is the ratio of distance travelled by the sample and the distance travelled by the standard. Rx value is always closer to 1. Rm value used to find out whether the compounds belong to a homologous series. If they i belong to a homologous series, the ARm values are constant. The ARm values for a pair of adjacent member of a homologous series are determined by using the formula: lP = Rm log 9. Quantitative Analysis Indirect method: Quantitative analysis can be done after eluting the individual spotswith solvent and filtering off the stationary phase. The solution can be concentrated and the exact quantities of the conmpound determined by the methods like UV-Visible spectrophotometry, fluorescence method, flame photometric method, electrochemical methods of analysis. l Direct method: It can be done after eluting the individual spots with solvent and filtering off the stationary phase. The solution can be the concentrated and the exact amount of the we compound deterimined by the various methods like UV-visible spectrophotometry, flourscence method, flame photometric method, electrochemical methods of analysis etc. 10. Application of TLC 1. Separation of mixtures of drugs of chemicals or biological origin, plant extracts etc ii. Separation of carbohydrate, vitamins, antibiotics, proteins, alkaloids. glycosides etc re ii. Identification of drugs Drug Stationary Phase Mobile Phase Detecting agent Amoxycillin trihydrate Silica Gel G.F-254 6:| NaOH+ Buffer pH acetone (4:1) Starch+glacial acetic acid+lodinein Ca potassium iodide Ampicillin for oral Cellulose M.N-300 Citric acid Butyl Starch iodide reagent : suspension alcohol(5:) 16 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 17 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Test for impurities, decomposition & related substances in pharmaceutical products (as per British Pharmacopoeia substances and preparations) Substance Tested for Mobile Phase Detection rm Chlorpropamide p-Chlorobenzene Chloroform Sodium sulphonamide and methanol:cyclohexane:13.5M hypochlorite | NN'-diropylurea ammonia followed by (0.33%) (100:50:30:11.5) potassium iodide in starch mucilage ha Nitrazepam Tablets | Decomposition and Nitromethane ethyl 254nm radiation related substances acetate(85:15) 0.5% Desipramine Iminodibenzyl Toluene: ethyl acetate: Potassium Hydrochloride (0.2%) ethanol: diethylamine dichromate lP (20:20:4:1) (0.5%) in sulphuric acid:water(4:4) l we re Ca 17 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 18 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Paper Chromatography Paper chromatography (PC) is a type of a planar chromatography whereby chromatography procedures are run on a specialized paper.It is considered to be the simplest and most widely used rm of the chromatographic techniques because of its applicability to isolation, identification and quantitative determination of organic and inorganic compounds.It was first introduced by German scientist Christian Friedrich Schonbein (1865). Types of Paper chromatography: ) Paper Adsorption Chromatography: Paper impregnated with silica or alumina acts as adsorbent (stationary phase) and solvent as mobile phase. ha (ii) Paper Partition Chromatography: Moisture/ Water present in the pores of cellulose fibers present in filter paper acts as stationary phase & another mobile phase is used as solvent In general paper chromatography mostly refers to paper partition chromatography. Principle of Separation The principle of separation is mainly partition rather than adsorption. Substances are distributed between a stationary phase and mobile phase. Cellulose layers in filter paper contain moisture lP which acts as stationary phase. Organic solvents/buffers are used as mobile phase. The developing solution travels up the stationary phase carrying the sample with it. Components of the sample will separate readily according to how strongly they adsorb onto the stationary phase versus how readily they dissolve in the mobile phase. Instrumentation of Paper chromatography 2. Stationary phase & papers used l 3. Mobile phase 4. Application of sample we 5. Developing Chamber 6. Detecting or Visualizing agents 1.STATIONARY PHASE AND PAPERS: Whatmann filter papers of different grades like No.1, No.2, No.3, No.4, No.17, No.20 ete are used. In general the paper contains 98-99% of a-cellulose, 0.3 1% B - -cellulose. These papers differ in sizes, shapes, porosities and thickness. re Other modified papers like Acid or base washed filter paper, glass fiber type paper. Hydrophilic Papers Papers modified with methanol, formamide, glycol, glycerol etc. - Hydrophobie papers acetylation of OH groups leads to hydrophobic nature, hence can be used - for reverse phase chromatography. Silicon pretreatment and organic non-polar polymers can also be Ca impregnated to give reverse phase chromatographic mode. Impregnation of silica, alumna, or ion exchange resins can also be made. Size of the paper used: Paper of any size can be used. Paper should be kept in a chamber of suitable size. 18 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 19 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a 2.Application of sample: The sample to be applied is dissolved in the mobile phase and applied using capillary tube or using micropipette. Very low concentration is used to avoid larger zone 3. PAPER CHROMATOGRAPHY MOBILE PHASE rm Pure solvents, buffer solutions or mixture of solvents can be used. Some of the Examples of Hydrophilic mobile phases Isopropanol: ammonia:water 9:1:2 Methanol: water 4:1 or 3:1 n-Butanol: glacial acetic acid: water 4:1:5 Hydrophobic mobile phases ha kerosene: 70% isopropanol Dimethyl ether: cyclohexane The commonly employed solvents are the polar solvents, but the choice depends on the nature of the substance to be separated. If pure solvents do not give satisfactory separation, a mixture of solvents of suitable polarity may be lP applied. 4.CHROMATOGRAPHIC CHAMBER: The chromatographic chambers are made up of many materials like glass, plastic or stainless steel. Glass tanks are preferred most. They are available in various dimensional sizes depending upon paper length and development type. The chamber atmosphere should be saturated with solvent vapor. Development technique: l Sample loaded filter paper is dipped carefully into the solvent not more than a height of I cm and waited until the solvent front reaches near the edge of the paper. we Different types of development techniques can be used: a. ASCENDING DEVELOPMENT Like conventional type, the solvent flows against gravity. The spots are kept at the bottom portion of paper and kept in a chamber with mobile phase solvent at the bottom. (Same as in TLC) (Fig 4) Cotton Gas jar cover thread re Hangers Solvent front Paper Spacers Paper stnp Original spot wi bes of mixture Origin line Ca solvent lxo Solvent tray (i) single strip jar od bo 0) Muttisheet frame Fig4 19 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 20 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a b. DESCENDING TYPE: This is carried out in a special chamber where the solvent holder is at the top.The spot is kept at the top and the solvent flows down the paper.In this method solvent moves from top to bottom so it is called descending rm chromatography (Fig: 5). Through for solvent s Anti-siphon rod NOZzle Wires fixed to sides 9 Rubber of tank on which bulb trough can restmie Jno bat ha Paper Glass tank 8Side Visualisation- Serrated edge reagent lP Fig 5: Descending technique and sprayer to spray the visualizing agent c. ASCENDING DESCENDING DEVELOPMENT: A hybrid of above two - techniques is called ascending-descending chromatography.Only length of separation increased, first ascending takes place followed by descending. d. CIRCULAR/ RADIAL DEVELOPMENT Radial mode Cirular Paper l we Solvent flow Sample - Wick Solvent front. re Fig 6: Circular/Development technique Spot is kept at the centre of a circular paper.The solvent flows through a wick at the centre& Ca spreads in all directions uniformly. Hence the individual spots after development look like concentric circles. By making perforations radially, number of quadrants can be created allowing more number of samples to be spotted (Fig 6). e. Two dimensional developments: This technique is very similar to 2-Dimensional TLC. Here the chromatogram development occurs in two directions at right angles (Fig 7). In this mode, the samples are spotted to one corner of rectangular paper and 20 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 21 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a allowed for first development. Then the paper is again immersed in the mobile phase at a right angle to the previous development for the second chromatogram. In the second direction, either the same solvent system or different solvent system can be rm used for development. 1st Development 2nd Development Original Spot ha D Fig 7: Two dimensional techniques lP f. Drying of Chromatogram: After the development, the solvent front is marked and the left to dry in a dry cabinet or oven. g. Detection: After the development of chromatogram, the spots should be visualized. Detecting colored spots can be done visually. But for detecting colorless spots, any one of the following technique can be used. l a. Nonspecific methods: where brown or amber of spots can be detected, but not the exact nature or type of the compound. we Examples (i) lodine chamber method where brown or amber spots are observed when the developed papers are kept in atank with few iodine crystals at the bottom. ii) UV chamber for fluorescent compounds: When compounds are viewed under UV chamber, at 254nm (short ) or at 365nm (long ), fluorescent re compounds can be detected. Bright spots can are seen against a dark background. b. Specific methods: Specific spray reagents or detecting or visualizing agents are used to find out the nature of compounds or identification purposes. a. Ferric chloride- For phenolic compounds and tannins Ca b. Ninhydrin in acetone- for amino acids c. Dragendroff's reagent- for alkaloids d. 3,5-Dinitro benzoic acid- for cardiac glycosides e. 2,4-Di-nitrophenyl hydrazine- for aldehydes and ketones 21 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 22 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a The detecting techniques can also be categorized as a.Destructive technique: Specific spray reagents ete where the samples are destroyed before detection e.g. Ninhydrin reagent. rm b. Non-destructive technique: UV chamber method, lodine chamber method, densitometric method, e.t.c, where the sample is not destroyed even after detection. For radioactive materials, detection is by using autoradiographyor Geiger muller counter. For antibiotics, the chromatogram is layed on nutrient agar inoculated with appropriate strain and the zone of inhibition is compared. ha Quantitative Analysis Direct technique: Densitometer is an instrument whichmeasures quantitatively the density of the spots. When the optical densities of the spots for the standard and test solution are determined, the quantity of the substance can be calculated. The papers are neither destroyed nor eluted with solvents to get the compounds. The method is also known as in-situ method. lP Indirect techniques: In this technique, the spots are cut into portions and eluted with solvents. The solution can be analyzedby any conventional techniques of analysis like spectrophotometry, electrochemical methods, etc. Qualitative Analysis: a. Rf value Distance travelled by solute Rf Distance traveled by solvent front l The Rf value ranges from 0 to But the ideal values are from 0.3 to 0.8. 1. b. Rx value: It is always closure to 1. we Distance travelled by solute Rx Distance traveled by standard c. Rm value: It is mainly used to find out whether the compounds belong to a homologous series. If they belong to a homologous series, the ARm values are constant. The ARm values for a pair of adjacent member of a homologous series are determined by using the below formula: re Rm = log(p1) Application: ) To check the control of purity of pharmaceuticals, ii) For detection of impurities Ca Drug Mobile phase Detecting agent | Hydroxocobalamin Butyl alcohol: acetic Elution and measurement of acid potassium cyanide absorbance at 361nm. 22 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 23 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a (ii) Detect the contaminants in foods and drinks, (iv) For the detection of drugs Drug Mobile phase Detecting agent rm Gentamycin Chloroform: Methanol:| Ninhydrin in pyridine- Ammonia: Water acetone mixture (10:5:3:2) Vancomycin t-Amylalcohol: Nutrient agar containing Acetone: water (2:1:2) Bacilus subtilis ha (v) In analysis of cosmetics (vi) Analysis of the reaction mixtures in biochemical labs. (vii) Identification of decomposition products (vii) Analysis of metabolites of drugs in blood, urine etc. (ix) In the study of ripening and fermentation lP Advantages of Paper Chromatography: 1. Simple and Rapid 2. Paper Chromatography requires very less quantitative material. 3. Paper Chromatography is cheaper compared to other chromatography methods. 4. Both unknown inorganic as well as organic compounds can be identified by paper chromatography method. l 5. Paper chromatography does not occupy much space compared to other analytical methods or equipment's. we Limitations of Paper Chromatography 1. Large quantity of sample cannot be applied on paper chromatography. 2. In quantitative analysis paper chromatography is not effective. 3. Complex mixture cannot be separated by paper chromatography. 4. Less Accurate compared to HPLC or HPTLC re Ca 23 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 24 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a B. ELECTROPHORESIS Electrophoresis is a physical method of analysis based on themigration of electrically charged rm proteins, colloids, moleculesor other particles dissolved or dispersed in an electrolytesolution in the direction of the electrode bearing the oppositepolarity when an electric current is passed through it. The electrophoretic mobility is the rate of movement in metre per second of the charged particles under the action of an electric field of I volt per metre and is expressed in square metres per volt second. For practical reasons it is given in square centimetres per volt second, cmV "S". The mobility is specific for a given electrolyte under precisely determined operational conditions. ha 6Ttrn Where = Electrophoretic mobility Q-Net charge on the ion r- lonic radius of the solute n-viscosity of the medium lP The electrophoretic mobility is directly proportional to net charge and inversely proportional to molecular size and viscosity of the electrophoresis medium. The pH of the solution affects the mobility of the ion by Depending on the method used, the electrophoretic mobility is either measured directly or compared with that of a reference substance. Based upon the type of apparatus used, electrophoretic methods may be divided into two categories, l one called free or moving boundary and the other called zone electrophoresis (using a supporting medium). Zone electrophoresis include paper, gel such as agar, starch or poly acrylamide. we re Ca 24 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 25 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Paper electrophoresis: It is separation technique, where ions of different charges are separated on a medium of paper rm (moistened with a buffer), by the application of a voltage between two electrodes, which are in contact with the paper. Principle of Separation: A mixture of ions or ionisable substances is applied on the centre of a paper, previously immersed in a buffer of known ionic strength. This paper is placed across two trays, filled with buffer, into which two electrodes are immersed. When a voltage is applied across these electrodes, the ions or ionisable substances migrate towards anode or cathode, based on their charge and other factors. Neutral or non-ionisable substances do not migrate. Anionic substances ha move towards anode and cations move towards cathode. Ultinmately, there is separation of anionic, cationic and non-ionie / zwitterionic substances. The spots/bands which migrate can be detected bu using appropriate spray reagents or visualizing agents as in paper chromatofraphy and can be quantified by using densitometer. Both qualitative and quantitative analysis can be performed in paper electrophoresis. Components of paper electrophoresis lP a. Normally Whatman® filter paper (Grade 3 MM or No. 1) of suitable dimension (2.5cm to Scm) with a length so that ends of the strip of paper touch the buffer solution, kept in the electrode vessels. The paper to be used is washed with double distilled water followed by 0.1 M HCI or 0.01 M EDTA to remove impurities. b. Electrodes and voltage to be applied The electrode in the form of a thin wire is made up of carbon or platinum. A DC voltage of l about 8-15 V/cm length of paper is normally applied. In low voltage electrophoresis, the voltage across two electrodes is about 100-300V, with a current of 0.4mAmp per cm width we or 1.5mAmp/strip. In high voltage electrophoresis, a potential of about 50-215V/cm(Total 10,000V/strip) is applied across the electrodes. C. Buffers used: The pH of buffer to be used depends upon the types of compounds to be separated. The following are some of the buffers used: re 1. Barbitone buffer (Veronal buffer) (0.07mole/litre, pH 8.6). lonic Strength -0.05. 2. Tris-acetate buffer (0.07 mole/litre, pH 7.6) 3. Citrate buffer (0.07 mole/litr, pH 3.0 or pH 6.8) Other buffers of different pH and ionic strength can also be for separation, based on the type of compounds. Usually ionic strengths (IS) of 0.05-0.5 is used in most separations. Ca Types of paper electrophoresis (PE) A. Horizontal/vertical/Continuous Electrophoresis There are different types of electrophoresis instruments based on the design of the instrument. The diagrams of these 3 types of instruments are shown in Fig I to 3. 25 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 26 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Horizontal and vertical modes are used in analytical scale; whereas continuous electrophoresis is used on a preparative scale (i.e. large amount of sample mixture is used). The principles involved in all the modes are same, but the design of each rm instrument varies. In Horizontal type / Vertical (Fig 1 and 2), buffer solution of known pH and ionic strength is filled into two troughs. Appropriate grade of Whatmann filter paper and suitable width and length of filter paper are immersed in buffer solution. 10-20ul of sample solution is applied at the centre of the paper and fixed in position. The transparent lid is closed for safety as well as to prevent evaporation of buffer/solvent. A suitable potential (100-300V) is applied across two electrodes dipped ha in buffer solution. Fig 1: Horizontal Paper electrophoresis Sample application Paper strip Transparent insulating Lid -LED for safety lP Plastic shoulder pieces for grip Wick for capillary flow of buffer Buffer Electrodes Buffer Transparent insulating Lid l (for safely) we Sample application Supporting rod or string Paper Electrodes- re BuferBuffersolution- Ca Fig: 2: Vertical Paper Electrophoresis When such potential is applied across the electrodes, migration of cations and anions take place towards cathode and anode respectively. Non-ionisable / neutral substances do not move. Hence the separation of compound from the mixture takes place. 26 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 27 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a In this vertical mode, the migrations of ions are assisted by gravity and hence a typical separation takes place in about 6-8 hours. After sufficient migration, the paper is taken out and dried, to fix the spots bands. Then the compounds / bands /spots can be rm visualized by using the visualizing agent. The quantitation of spots can be done by densitometer. The horizontal mode is similar to the vertical mode, in principle. However, the paper is placed on a flat bed, as shown in Fig 2. The procedure to be followed is same as that of vertical type. In horizontal mode, it takes about 12-14 hours for separation. Continuous electrophoresis (Fig. 3) is meant for preparative samples, where a predetermined sample volume through a valve device is applied continuously on the ha centre of paper. The application of voltage causes migration of samples and hence compounds are separated as bands. Thus each band is made to fall down and pure compounds are collected in separate containers. The solvent is evaporated and pure fractions are reused. Various factors like charge of ions, size of the ions, viscosity of the medium, applied voltage, pH of buffer and ionic strength affect the migration of ions in paper lP electrophoresis aU WIl DC Supply in Fig Sample solution or separation l Valve we Buffer solution Buffer solution re Paper Ca Compound(s) or Band Collection vials 27 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 28 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Fig 3: Continuous paper electrophoresis rm B. There are two types of paper electrophoresis based on the voltage applied, i.e. Low voltage or Hligh voltage Paper electrophoresis Voltage/em Votage/strip Low voltage PE 8-15 V/ cm 100-300 Highvoltage PE 50-215V/ cm 10,000v ha High voltage paper Electrophoresis has the following advantages: 1. Separation is faster, hence less time is required for separation 2. Sharp bands are obtained, since there is less diffusion of bands. 3. As sharp bands are obtained, separation of closely related compounds can be achieved. lP 4. More number of samples can be analysed simultaneously. Disadvantages of High Voltage Paper Electrophoresis 1. It is dangerous to the operator, since high voltage is applied. 2. More heating effects are seen because of high voltage and the paper becomes dry So in most of the laboratories, low voltage paper electrophoresis is used. l Advantages of Paper Electrophoresis: 1. The technique is easy to follow we 2. Less expenditure 3. Number of samples can be separated on a sample paper, at a time. 4. Wide variety of ionisable substances such as amino acids, proteins and peptides, antibiotics, alkaloids etc., can be separated. Disadvantages 1. The time required for separation is more, i.e. 6-8 hours in vertical mode and 12- re 14hours in horizontal mode. 2. Use of high voltage may be dangerous, unless precautions are taken. Application of Paper Electrophoresis Paper electrophoresis is used mainly for the separation of ionizable substances, by using buffers of different pH and ionic strength. The following are some of the pharmaceutical Ca applications of paper electrophoresis. 1. Separation of amino acids into acidic or basic or zwitterionic type 2. Separation of proteins in serum (into albumin, al, a2, B and gamma globulins). The type of protein and the percentage of each component can be estimated using densitometer. 28 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 29 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a 3. Separation of lipoproteins in serum (in case of hyperlipidemia) 4. Separations of enzymes in blood. 5. Separation of alkaloids and antibiotics in different samples can be carried out. rm GEL ELECTROPHORESIS It is a separation technique. Gel is used as medium. The gel may be agar or agarose gel or polyacrylamide gel. The device consists essentially of a glass plate over the whole surface ofwhich is deposited a firmly adhering layer of gel of uniform thickness. The connection between the gel and the ha conducting solution is effected in various ways according to the type of apparatus used. Precautions are to be takento avoid condensation of moisture or drying of the solid layer. Vertical Gel Instrument- The schematic diagram of a vertical gel electrophoresis apparatus is given in Figure 4. It has two buffer chambers, upper chamber and a lower chamber. Both chambers are fitted with the platinum electrodes connected to the external power supply from a power pack which supplies a direct current or DC voltage. The upper and lower tank filled with the running lP buffer is connected by the electrophoresis gel casted in between two glass plates (rectangular and notched). 'There are additional accessories needed for casting the polyacrylamide gel such as comb (to prepare different well), spacer, gel caster etc. l we Power supply Gel cassette Electrode chamber re Comb Tank Ca Fig 4: Different components of the vertical gel electrophoresis apparatus 29 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 30 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Casting of the gel: The acrylamide solution (a mixture of monomeric acrylamide anda bi- functional eross linker bis-acrylamide) is mixed with the TEMED and APS and poured in between the glass plate fitted into the gel caster. What is the mechanism of acrylamide polymerization? rm Ammoinum persulfate in the presence of TEMED forms oxygen free radicals and induces the polymerization of acrylamide monomer to form a linear polymer. These linear monomers are interconnected by the cross linking with bis-acrylamide monomer to form a 3-D mesh with pores. The size of pore is controlled by the concentration of acrylamide and amount of bis-acrylamidein the gel. n a vertical gel electrophoresis system, we cast two types of gels, stacking gel and resolving gel. First the resolving gel solution is prepared and poured into the gel cassette for polymerization. A thin layer of organie solvent (such as butanol or isopropanol) is layered to stop ha the entry of oxygen (oxygen neutralizes the free radical and slow down the polymerization) and make the top layer smooth. After polymerization of the resolving gel, a stacking gel is poured and comb is fitted intothe gel for construction of different lanes for the samples. Different steps involves the vertical gel electrophoresis is shown in the below Fig 5. lP cathode buffer Anode buffer Gel cassette l we Electrode chamber Hamilton Power source syringe sample re Tank Separate protein bands Ca Fig 5: Different steps in performance of vertical gel electrophoresis to resolve sample Running of the gel: The sample is prepared in the loading dye containing SDS, B-mercaptoethanol in glycerol to denature the sample and presence of glycerol facilitates the loading of sample in the well. As the samples are filled vertically there is a distance drift between the molecules at the top 30 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 31 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Vs at the bottom in a lane. This problem is taken care once the samples run through the stacking gel. The pH of the stacking gel is 6.8 and at this pH, glycine is moving slowly in the front whereas Tris-HCl is moving fast. As a result, the sample gets sandwiched between glycine-Tris and get rm stacked in the form of thin band. As the sample enters into the resolving gel with a pH 8.8, the glycine is now charged, it moves fast and now sample runs as per their molecular weight (dueto SDS they have equal negative charge). After tracking dye reaches to the bottom of the gel, gel is taken out from the glass plate with the help of a spatula. Gel is stained with coomassie brilliant blue R250 dye. The dye stains protein present on the gel. A typical SDS-PAGE pattern is given in the Fig. 6. ha Potentials of discontinuous PAGE: 1. Number of disulfide bonds: Comparison of reducing and non-reducing denaturing gels can be used to provide information related to the number of disulfide bonds present in the protein. 2. Seperating Proteins based on size alone: In the presence of SDS and reducing environment, PAGE gel resolves two proteins of on the basis of molecular masses and the concentration of gel concentration. lP In SDS-PAGE, the relative mobility and the log molecular weight as given by v=Vo4logM Molecular weight of a protein can be determined by plotting relative migration Rf with the log molecular weight of standard protein. migration of protein from the lane Rf= migration of tracking dye l we Marke Sample Graph botwoen Rf and log Moleaular weight 1.2.8 re DIstance avelled y dye U. 02 0.3 04 0s 0.8 O8 leg moleular weight(kD) Ca d, d2, d,d, & d, mean distance travelled by marker protein Mean distance travelled by protein Distance travelled by dye Fig 6: Determination of molecular weight using SDS-PAGE and Determination of Rf 31 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 32 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Calculation of molecular weight of the unknown protein sample is a 5 step process: 1. Resolve the protein sample on the SDS-PAGE along with the molecular weight markers. rm 2. Calculate the relative mobility (Rf) using the following formula: Rf-migrationofproteinfromthelane/migrationoftrackingdye 3. Plotlog molecular mass (y-axis) versus relative mobility (x-axis) of the standards. 4. Perform a linear regression using a calculator or using regression software such as Microsoft Excel. ha 5. Use the linear regression equation (Y = mx + c) to estimate the mass of the unknown protein. Log Molecular Weight = (slope) (mobility of the unknown) + Y intercept Buffer and reagent for electrophoresis- The different buffer and reagents with their purpose for lP vertical gel electrophoresis is as follows- 1. N, N, N', N'-tetramethylethylenediamine (TEMED)-it catalyzes the acrylamide polymerization. 2. Ammonium Persulfate (APS)-it is an initiator for the acrylamide polymerization. 3. Tris-HCl- it is the component of running and gel casting buffer. 4. Glycine-it is the component of running buffer l 5. Bromophenol blue- it is the tracking dye to monitor the progress of gel electrophoresis. 6. Coomassie brilliant blue R250-it is used to stain the polyacrylamide gel. we 7. Sodium dodecyl sulphate-it is used to denature and providenegative charge to the protein. 8. Acrylamide- monomeric unit used to prepare the gel. 9. Bis-acrylamide- eross linker for polymerization of acrylamide monomer to form gel. re Ca 32 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 33 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a Capillary Electrophoresis Capillary electrophoresis employing a narrow bore fused quartz silica capillary tube usually rm 50-75cm long with an i.d. of 25-100um (and an o.d. of 400 um) containing an appropriate electrolyte using a direct current (DC) high voltage source, capable of producing a current of 250 uA at voltage ranging from 1000 to 30,000volts and on-line detector that similar to those HPLC are involved (high voltage electrophoresis). A cross -sectional view of such a capillary is shown in the Fig 7. The capillary is protected with an outer layer of a polyimide (polymer of imide monomer) ha O -Capillary opening lP Polyimide Coating Fused silica Fig 7: A cross - sectional view of such a capillary is shown in the Integrator or l Capillary Computer we Detector Anode- Cathode re Buffer Buffer Source Vial Sample Vial Destination Vial High Voltage Power Supply Fig 8: Capillary electrophoresis system A basic schematic of a capillary electrophoresis system is shown in fig. 8. The system's main Ca components are a sample vial, source and destination vials, a capillary, electrodes, a high voltage power supply, a detector, and a data output and handling device. The source vial, destination vial and capillary are filled with an electrolyte such as an aqueous buffer solution. To introduce the sample, the capillary inlet is placed into a vial containing the sample. Sample is introduced into the capillary via capillary action, pressure, siphoning, or electrokinetically, and the capillary is then 33 www.carewellpharma.in (free notes) This pdf is not owned by Carewell Pharma, Source - IPT Salipur  Page 34 of 37 Carewell Pharma - A Family of Learning Subscribe Us on YouTube a returned to the source vial. The migration of the analytes is initiated by an electric field that is applied between the source and destination vials and is supplied to the electrodes by the high- voltage power supply. In the most common mode of CE, all ions, positive or negative, are pulled rm through the capillary in the same direction by electroosmotic flow (EOF) (Fig 9). The analytes separate as they migrate due to their electrophoretie mobility, and are detected near the outlet end of the capillary. The output of the detector is sent to a data output and handling device such as an integrator or computer. The data is then displayed as an electropherogram, which reports detector response as a function of time. Separated chemical compounds appear as peaks with different retention times in an electropherogram. ha Electrophoretic migration Bulk EOF -Cathode +Anode solution O lP Diffuse layer Double layer