Chromatography Techniques PDF

Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Chromatography Chromatography is a physical method of separation in which the components to be separated are distributed between two phases one of which is stationary (stationary phase) while the other (...

Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Chromatography Chromatography is a physical method of separation in which the components to be separated are distributed between two phases one of which is stationary (stationary phase) while the other (the mobile phase) moves through it in a definite direction. The chromatographic process occurs due to differences in the distribution constant of the individual sample components. Is a technique used to separate and identify the components of a mixture. Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium. Molecules that spend most of their time in the mobile phase are carried along faster. Classification of chromatography according to mobile phase 1.)Liquid chromatography: mobile phase is a liquid. (LLC, LSC) 2.)Gas chromatography : mobile phase is a gas. (GSC, GLC ) Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Classification according to the packing of the stationary phase 1-Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminium plates 2-Paper chromatography (PC): the stationary phase is a thin film of liquid supported on an inert support 3-Column chromatography (CC): stationary phase is packed in a glass column. Classification according to the force of separation 1.Adsorption chromatography 2.Partition chromatography 3.Ion exchange chromatography 4.Gel filtration chromatography 5. Affinity chromatography All chromatography methods mainly aim at separating two or more substances. The method usually begins when two items are separated when one of them holds a more static phase than the other phase, which in turn tends to move with the mobile phase. Chromatography Applications 1- Used to diagnose and identify vehicles by using the offset value from start to finish (Rf rate) compared to the standard material. 2- Used to purify compounds especially when storing for occasional damage. 3- Used for separation and isolation operations and for precise chemical compounds usually. 4- Determine unknown molecular weights compared to standard molecular weight known compounds. Paper Chromatography Paper chromatography is one of the simplest forms of chromatography and the oldest. Paper chromatography is generally used to separate water-loving compounds such as amino acids, peptides and sugars. A method of partition chromatography using filter paper strips as carrier or inert support. The factor governing separation of mixtures of solutes on filter paper is the partition between two immiscible phases. Partition occurs between the mobile phase and the stationary aqueous phase bound by the cellulose. Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D In this technique, the normal filtration paper consisting of cellulose fiber (99-98% alpha-cellulose) is used to give a clear and good differentiation during the separation. Based on the poetic properties, the solvent dissolved in the solvent is the mobile phase and the container on the mixture to be separated will move upward or (When using different solvents or using a different solvent system) and the paper is the constant phase. The distance traveled by the solvent on the paper divided by the distance traveled by the solvent gives the value of Rf, which is called the velocity of the flow. For example, each amino acid has a fixed Rf value when using a particular solvent. In this way, the amino acid can be separated and analyzed in the mixture Rf = distance traveled by the solute / distance traveled by the solvent Thin layer chromatography (TLC) is a method for identifying substances and testing the purity of compounds. TLC is a useful technique because it is relatively quick and requires small quantities of material.Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and a mobile phase. The stationary phase : is a thin layer of adsorbent (usually silica gel or alumina) or non-organic salts mixed with calcium sulfate. It is sprayed in the form of a thin layer on a glass plate. After separation, the compound can be scraped. Easily distinguishable from other materials. The mobile phase : is a developing liquid which travels up the stationary phase, carrying the samples with it. Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Components of the samples will separate on the stationary phase according to how much they adsorb on the stationary phase versus how much they dissolve in the mobile phase A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). A small amount of the mixture to be analyzed is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid. This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary action. As the solvent moves past the spot that was applied, an equilibrium is established for each component of the mixture between the molecules of that component which are adsorbed on the solid and the molecules which are in solution. In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others. When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and the separated components of the mixture are visualized. Usually the compounds are not colored, so a UV lamp is used to visualize the plates. If the compounds are colored, visualization is straightforward. Many organic compounds can be seen using this technique, and many commercially made plates often contain a substance which aids in the visualization of compounds Visualizing Agents Alkaloids: Dragendorff’s reagent Cardiac glycosides: Antimony trichloride Sugar: Aniline phthalate Amino acids: Ninhydrin Interpreting the Data The Rf (retention factor) value for each spot should be calculated. It is characteristic for any given compound on the same stationary phase using the same mobile phase for development of the plates. Hence, known Rf values can be compared to those of unknown substances to aid in their identifications. Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Note: Rf values often depend on the temperature and the solvent used in the TLC experiment the most effective way to identify a compound is to spot known substances – authentic - next to unknown substances on the same plate. In addition, the purity of a sample may be estimated from the chromatogram. An impure sample will often develop as two or more spots, while a pure sample will show only one spot. Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D One of the advantages of this method compared to paper chromatography 1.Increased separation efficiency. 2.Chapter speed. 3.Freedom to choose a fixed stage. 4.The static phase does not affect the acid or the bases used in the diagnosis of some compounds. In the case of paper chromatography, the nature of fibrous cellulose helps to spread the compounds at the starting point. This is called capilary diffusion, whereas this is not in the fixed phase used in thin layer chromatography, as is the increase in the surface area of the phase Fixed in this technique. Columnar Chromatography (CC) This includes chromatographic methods in which the stationary phase is packed into a column. The mobile phase is a moving liquid or gas. According to the mechanism of separation of solutes, five major types of CC are distinguished. Usually, one mechanism predominates but does not exclude the others Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Affinity chromatography A special or modified method for adsorption chromatography. This method depends on the existence of a tendency or affinity between the stationary phase and the Compound to be separated in the mobile phase. For example, when Antigen is added to the column and the Antibody passes a moving phase on the column, Of the column using a solution with a specific pH. In this type of separated, there is a special type of attraction is not between the charges, but the overlap or affinity between the compounds. This method is an important method for the purification of enzymes in which a qualitative correlation occurs between the reaction material or the competitive inhibitor. As a result, a covalent bond occurs with the stationary phase (steel) of the carrier (eg, Agarose). When the mixture passes through the phase systems containing the stationary phase, The enzyme that is associated with a change in pH or ionic strength in the solution can be extracted. In this way, the enzyme bond in the columns can be weakened. This can be illustrated in Figure Figure: Affinity Chromatography technique, and notes how the glucose units are correlated through their overlap or Affinity with the separation units in the column Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Using this method, enzymes can be purified in one step from the crude extract. There are many enzymes that can be purified in this way, such as the enzyme Nuclease of Staphylococcus bacteria. The purification of enzymes in the way of Chromatography of Affinity faces many difficulties 1-It may be difficult to complete the link with the appropriate reaction material, since interactions related to the bond can not be fully described 2-Bonding with the stationary phase can cause interference with the enzyme's side groups and lead to loss of activity when the association occurs 3-The strength of the bonding of the stationary phase and the enzyme must be in the correct range 4-Specific problems related to the enzyme are more important than the reaction substance alone. Therefore, it is expected that all enzymes dehydrogenases depend on NAD + and therefore you should know that the link leads to the formation of NAD + matrix bound and the difference between dehydrogenase is in the side groups that have a secondary link with the reaction material and should be noted during the occurrence of the eluent in the column Despite the difficulties, this technique contributes to the purification of enzymes and other molecules such as antibodies. It can be used on the aggregates are inverted such as Antigen - Antibody Inhibitor or substance - the enzyme Associated protein - Hormone Nucleic acid - complementary sequence Ion – exchange chromatography Principle : Process by which ions of an electrolyte solution are brought into contact with an ion exchange resin. The ion exchange resin is an insoluble polymer consisting of a "matrix" (Lattice or framework) that carries fixed charges (not exchangeable) and mobile active ions "counter ions" which are loosely attached to the matrix Modulation method for adsorption chromatography, which is determined based on the tendency of ions or molecules to non-moving materials (the stationary phase), which have distinct charges. The ions or molecules that carry one or more positive charges exchange with negative charges in the resin(the stationary phase). This process is called cation exchanger. The resin that carries the positive charge, called Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D the anion exchanger, is attracted to the ions that carry The negative charge or in other words is called negative or positive resin based on the charge that is exchanged rather than the charge it possesses. Figure :The ion exchange material is usually made up of transformed derivatives Figure: Ion exchange chromatography, noting how compounds (eg proteins) are separated by positive or negative charge Examples of ion exchangers include A. Polystyrene Which brings copolymerization and has several types such as: Dowex - 50, Dowex - 1, Dowex - 3, IR – 45. Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D B. Polysaccharides They are used to separate proteins and nucleic acids and prepare for some chemical alterations of certain polysaccharides. Examples are: DEAE Cellulose, DEAE - Sephadex, Carboxy methyl - cellulose, Phospho – cellulose. Figure: Two types of ionic exchangers bearing a positive charge such as A) DEAE Cellulose B) Carboxy methyl - cellulose Types of Exchangers. Cation Exchangers Active ions (counter ions) are cations The polar groups attached to the matrix are acidic (sulphonic acids, carboxylic acids, phenols, phosphoric acids) e.g. a cation exchanger in the free carboxylic acid form Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D - + X-COO H X = Frame work (matrix) - -COO = Fixed charge (anionic), Non-exchangeable + H = Counter ion (cation), Exchangeable + - They are usually (but not always) supplied in the Na form: X-COO Na+ Anion Exchangers Active ions (counter ions) are anions. The polar groups attached to the matrix are tertiary or quaternary ammonium groups (basic). e.g. Anion exchanger in the quaternary ammonium form: + – X. NR3 OH X = Framework (matrix) + -NR3 = Fixed charge (cationic) Non exchangeable – -OH = counter ion (anion), Exchangeable They are supplied as the chloride rather than the hydroxide as the chloride form is + - a more stable. Represented as: X. NR3 Cl The process of purification using ion exchange chromatography is mainly based on the attraction between the different charges. This effect can be influenced by gradually increasing the pH or increasing the ionic strength of the solution or both to increase separation efficiency and reduce time. Ion exchange can be used with high efficiency on a small scale or wide scale, and large-scale application with selective elution is applied electronically in batches. For example, the enzyme is adsorbed by adding ion exchange material to the solution and then placed in columns to control the absorption or separation of the gradual increase in Ionic Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D power of solution or pH or both. Applications of Ion Exchange Chromatography 1- Water softening: Removal of Ca2+, Mg2+ & other multivalent ions causing hardness of water by filtration through a layer of strong cation resin. 2-Water demineralization: Removal of cations & anions dissolved in water. Usually carried by the two step technique in which two columns of strongly acid cation exchanger in [H+] form & strongly basic anion exchanger in [OH-] form are used in sequence. 3- Neutralization: Cationic exchanger in [H+] can be used to neutralize alkali hydroxide & anionic exchanger in [OH+] form to neutralize the acidity. 4- Separation of electrolytes from non-electrolytes. 5- Separation of carbohydrates & their derivatives: Uronic acids separated on anion exchanger. Sugars converted into ionized form by using borate& separated on strong anion exchanger.  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Hexosamines separated on strong cation exchanger. Gel Filtration chromatography Size exclusion chromatography (SEC), also known as gel permeation chromatography (GPC) or gel filtration chromatography (GFC) Separation of molecules on the basis of size (and molecular weight) Gel filtration (chromatography), is also known as molecular sieve chromatography. Gel filtration chromatography separates molecules according to their size and molecular weights. The stationary phase consists of beads containing pores that span a relatively narrow size range. Smaller molecules spend more time inside the beads than larger molecules and therefore elute later (after a larger volume of mobile phase has passed through the column). In this way, a mixture of peptides or proteins can be separated based on the difference in size and molecular weight. In addition, the gel filtration granules act as a molecular filter by separating proteins from salts such as ammonium sulphate by successive periods. Gel filtration is one of the most important methods used separation of proteins from Desalting salts.  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D The gel filtration technique shows that a mixture of small proteins passes through the granules of the gel while the large proteins can not pass from the inside so they will pass from the grain environment and pass faster than the small proteins (so that the order of descent during the size of Rogan in Start small then medium then large.  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Sample components are eluted isocratically (single buffer, no gradient). Separation can be performed within a broad pH 3–12 , ionic strength, and temperature range, and the medium accepts a variety of additives: co-factor, protein stabilizers, detergents (1% SDS), denaturing agents (8 M urea or 6 M guanidine hydrochloride) Gel Filtration can be used for protein , DNA purification, buffer exchange, desalting, or for group separation in which the sample is separated in two major groups. Gel Filtration is an easy to use method for separation of molecules with different molecular sizes, using mild conditions. Gel Filtration uses the size of molecules in solution to determine separation. SpinColumns have short media packing so the samples are separated by size, the large molecules travel out of the column with the void volume the smaller molecules, salts, etc. remain in the column. Desalting and buffer exchange: a group separation where small molecules such as salt or free labels are separated from a group of larger molecules such as proteins. Types of gels used 1-The gels used as molecular sieves are cross linked polymers. 2-They are uncharged and inert i.e. don’t bind or react with the materials being analyzed. Three types of gels are used: 1- Dextran: is a homopolysaccharide of glucose residues. it’s prepared with various degrees of cross-linking to control pore size. It’s bought as dry beads, the beads swell when water is added. The trade name is sephadex.  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D It’s mainly used for separation of small peptides and globular proteins with small to average molecular mass. 2- Polyacrylamide: these gels are prepared by cross linking acrylamide with N,N-methylene bis acrylamide. The pore size is determined by the degree of cross-linking. The separation properties of polyacrylamide gels are mainly the same as those of dextrans. They are sold as bio-gel P. They are available in wide range of pore sizes. 3- Agarose: linear polymers of D-galactose and 3,6 anhydro-1-galactose. It forms a gel that’s held together with H bonds. It’s dissolved in boiling water and forms a gel when it’s cold. The concentration of the material in the gel determines the pore size. The pores of agarose gel are much larger than those of sephadex or bio-gel p. It’s useful for analysis or separation of large globular proteins or long linear molecules such as DNA. Table :shows the types of gel used in this technique and the molecular weight that can be separated  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Table: Types of gel used in gel filtration chromatography technology. types of gel Exclusion limit * 1. Sephadex G – 10** 700 G - 15 1500 G - 25 5000 G - 50 30000 G - 75 70000 G - 10 150000 G - 150 300000 G - 200 600000 2. Acrylamide S – 200 5000 - 250000 S – 300 10000 - 1500000 S – 400 10000 - 2000000 3. Agarose 2B 70000 - 40000000 4B 60000 - 20000000 6B 10000 - 4000000 * Exclusion limit: Known as the molecular weight of a compound that does not enter the holes on the surface of the gel granules. G - 10 ** The numbers after the letter G (10, 15.....) represent the amount of water absorbed when the dry gel granules are placed in the water and measured in the unit (mL / g) and G - 10 has the highest amount of cross linking of dextran.  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Ex. The gel filtration material that will be called Sephadex G-75 and it will separate molecules with molecular weights from 3,000 to 70,000. Molecules with molecular weights larger than 70,000 will be excluded from the beads. Notes on use of gel filtration chromatography 1- The choice of matrix depends on the range of size of molecules to be separated and the goal of the separation. Different bead types have pores of different sizes. 2- The matrix beads normally come in dry form and must be swollen before use. It is important not to use a magnetic stirrer when preparing the beads, or the beads can be fragmented. It takes several days to swell beads like the Sephadex that you will use today. One short cut, however, is to autoclave the solution. This causes the beads to swell more rapidly without damaging them. 3- Never allow a gel filtration column to dry out. If it dries out, the column must be re-poured. It is crucial for good separation that the column be consistent from top to bottom (without any bubbles). Advantages of Gel filtration It’s the best method for separation of molecules differing in molecular weight because: 1- It doesn’t depend on temperature, pH, ionic strength and buffer composition. So separation can be carried out under any conditions. 2-There is very little adsorption 3-There is less zonal spreading than in other techniques. 4-The elution volume is related to the molecular weight Uses of gel filtration chromatography 1.Separation of compounds of different sizes and molecular weights. 2.Purification of compounds ) enzymes and other proteins (. 3.Isolate compounds and quantify compounds in the mix. 4.Estimation of molecular weight mainly for globular proteins. Estimation of molecular weight: To do this, several proteins with known molecular weights are run on the column and their elution volumes determined. If the elution volumes are then plotted against the log molecular weight of the corresponding proteins, a straight line is obtained for the separation range of the gel being used. If the elution volume of a  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D protein of unknown molecular weight is then found, it can be compared to the calibration curve and the molecular weight determined. Elution Profile Idealised Elution Profile 2.5 2 Amount 1.5 1 0.5 0 1 3 5 7 9 13 15 17 19 21 23 25 11 0. Fraction number Ve = Elution volume (volume of solvent between injection and elution). Dictated by proportion of porous matrix available to molecules (Kd).  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Calculation of Ve , For a molecule that can partially enter the pores: Ve = Vo + Kd (Vs) or Ve = Vo + Kav (Vt-Vo) Despite the advantages of gel filtration, it is not without some drawbacks, such as dilution of the sample through the column during the separation process and the duration of the separation process, as well as the inability to use oxidizing agents with Sephadex due to the interference of separated parts and gels sometimes. Disadvantages are, for example, that only a limited number of bands can be accommodated because the time scale of the chromatogram is short, and, in general, there has to be a 10% difference in molecular mass to have a good resolution. Differential scaning calorimetry(DSC) It is a technique used to measure the transition temperutre (Tm) of phospholipids. The solids are usually measured by the melting point, which is the thermal degree in which the solids are converted to liquid, a solid physical characteristic of solids. Liquid substances use the boiling point, which is a fixed form of liquid. In the phospholipid compounds, in addition to measuring the degree of fusion, it is noted that it has another degree called temperature transition, which is defined as the degree that dissolves the groups of acyl fatty only in the phospholipids, which is the water-damaging part of the hydrophobic The transition temperature is affected by several factors, most importantly 1-The length of fatty acid chain in phospholipid : The higher the length of the chain in the fatty acids, the greater the interaction between the phospholipids molecules so they need higher energy to melt, which increases the value of Tm. 2-Existence of unsaturated bonds : (Double bonds) in the fatty acids of the phospholipids and the increase of the ( double bonds Tm less than the lack of energy needed to melt due to the lack of degree of backing between the groups of acyl fatty. 3- Percentage of phosphotidyl ethanol amine to phosphotidyl chlorine phosphotidyl ethanol amine and phosphotidyl Choline are one of the types of phospholipids, but their composition is different. phosphotidyl ethanol amine that has the ability to form hydrogen bonds between hydrogen in the amino group and  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D the carbonyl group of fatty acid. This leads to increased phospholipids binding and thus increases the energy to melt. Tm. 4- Cholesterol : The cholesterol molecule can increase the permeability of the phospholipids or reduce the interference depending on its distribution in the phospholipids. If combined with the acyl fatty acid groups in the phospholipids, the increases the value of Tm because it needs energy to remove the cholesterol and then dissolves the acyl group of fatty acids 5- Link sites proteins with phospholipids Both on the head and on the Acyl group of fatty acids in the Phospholipids bilayer. Applications of DSC technology 1- The value of Tm is fixed for phospholipids, which gives an indication of the quality of phospholipids and the purity of the material. 2-Measurement of the Tm value gives an indication in the case of an interaction between the protein and the phospholipids, since when the interference occurs, increase the value of Tm. Electrophoresis Positive or negative electrical charges are frequently associated with biomolecules. When placed in an electric field, charged biomolecules move towards the electrode of opposite charge due to the phenomenon of electrostatic attraction. Electrophoresis is a method used to separate charged molecules in an applied electric field. The relative mobility of individual molecules depends on several factors. The most important of which are net charge, charge/mass ratio, molecular shape and the temperature, porosity and viscosity of the matrix through which the molecule migrates. Complex mixtures can be separated to very high resolution by this process. Electrophoresis is a method used to separate charged particles from one another based on differences in their migration speed. In the electrophoresis, two electrodes (typically made of an inert metal, e.g. platinum). By using an electric power supply, electric potential (E) is generated between the two electrodes. Due to the electric potential, molecule move by a wire between the two electrodes. More specifically, molecule move from the anode to the cathode. Hence, the anode will be positively charged, while the cathode will be negatively charged. There must be two conditions for using this technique A. Provides a charge on the molecule until it travels through the electric medium  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D B. The electric field provides any negative electrode to move molecules that carry a positive charge and a positive electrode towards which molecules that carry a negative charge move All types of Electrophoresis are based on a basic principles, which are illustrated by the following equation v = E.q / f E electric field of field strength , ν The velocity of movement ,where f is the frictional coefficient and q is the net charge on the molecule. The movement of the molecule increases when an electric current passes by increasing the electrical voltage used or by increasing the total charge on the molecule. The speed of the movement is reduced by increasing the molecular friction resulting from the size and shape of the molecule. It is used during Electrophoresis, constant and equal electrical voltage on the broad sections of the supporting tapes, the generation gel tubes and the solutions used. The movement of molecules is affected when the voltage or current used by the friction factors and the total charge on the molecule is determined as in the previous equation. The molecule that carries a larger charge is attracted to the electric and runs faster than the other molecules. The friction or slippage depends on the shape and size, as well as on the molecular weight (the molecule with the high molecular weight is less than the low molecular weight. Gel types In general the macromolecules solution is electrophoresed through some kind of matrix. The matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size. The kind of supporting matrix used depends on the type of molecules to be separated and on the desired basis for separation: charge, molecular weight or both. The most commonly used materials for the separation of nucleic acids and proteins are agarose and acrylamide. Table. Some media for electrophoresis Medium Conditions Principal Uses Starch Cast in tubes or slabs Proteins Agarose gel Cast in tubes or slabs Very large proteins, nucleic No cross-linking acids, nucleoproteins etc Acrylamide gel Cast in tubes or slabs Proteins and nucleic acids  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Cross-linking Agarose gels Agarose is a linear polysaccharide (average relative molecular mass about 12 000) made up of the basic repeat unit agarobiose, which comprises alternating units of galactose and 3,6-anhydrogalactose. Agarose is one of the components of agar that is a mixture of polysaccharides isolated from certain seaweeds. The most widely used polysaccharide gel matrix nowadays is that formed with agarose. This is a polymer composed of a repeating disaccharide unit called agarobiose which consists of galactose and 3,6-anhydrogalactose. Agarose is usually used at concentrations of between 1% and 3%. Agarose gels are formed by suspending dry agarose in aqueous buffer, then boiling the mixture until a clear solution forms. This is poured and allowed to cool to room temperature to form arigid gel. Agarose gives a more uniform degree of porosity than starch and this may be varied by altering the starting concentration of the suspension (The pore size in the gel is controlled by the initial concentration of agarose; large pore sizes are formed from low concentrations and smaller pore sizes are formed from the higher concentrations ). This gel has found wide spread use especially in the separation of DNA molecules (although it may also be used in some electrophoretic procedures involving protein samples such as immunoelectrophoresis). Because of the uniform charge distribution in nucleic acids, it is possible accurately to determine DNA molecular masses based on mobility in agarose gels. Acrylamide gels: A far stronger gel suitable for electrophoretic separation of both proteins and nucleic acids may be formed by the polymerization of acrylamide. The inclusion of a small amount of acrylamide cross linked by a methylene bridge (N,N′ methylene bisacrylamide) allows formation of a cross linked gel with a highly- controlled porosity which is also mechanically strong and chemically inert. For separation of proteins, the ratio of acrylamide : N,N′ methylene bisacrylamide is usually 40:1 while for DNA separation it is 19:1. Such gels are suitable for high-resolution separation of DNA and proteins across a large mass range. Gels of 15% polyacrylamide are useful for separating proteins in the range Mr 100 000 to 10 000. It is obvious, therefore, that the choice of gel to be used depends on the size of the protein being studied. The smaller the known weight, the higher the  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D percentage that should be used.The fractionation range of different percentage acrylamide gels is shown in Table. Table. The relationship between acrylamide gel concentration and protein fractionation range Acrylamide concentration (%) Protein fractionation range (Mr x 103) 5 60–350 7.5 30- 220 10 15–200 15 10–100 Starch The gels are slightly more opaque than acrylamide or agarose. Non-denatured proteins can be separated according to charge and size. They are visualised using Napthal Black or Amido Black staining. Typical starch gel concentrations are 5% to 10%. Staining of gel One of the most important aspects of gel electrophoresis technique is staining. Once sample molecules have separated in the gel matrix it is necessary to visualize their position. This is achieved by staining with an agent appropriate for the sample. Some of the more common staining methods used in biochemistry are listed in Table. Table. Commonly used stains for biopolymers after electrophoretic separation in agarose or polyacrylamide gels. Stain Use Detection limit (a)(ng) Amido black Proteins 400 Coomassie blue Proteins 200 Ponceau red Proteins (reversible) 200 Bis-1-anilino-8-Naphthalene sulphonate Proteins 150 Nile red Proteins (reversible) 20 SYPRO orange Proteins 10 Fluorescamine (protein treated prior to electrophoresis) Proteins 1 Silver chloride Proteins/DNA 1 SYPRO red Proteins 0.5 Ethidium bromide DNA/RNA 10 a These limits of detection should be regarded as approximate since individual proteins may stain more or less intensely than average.  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Preparation and running of standard gels The equipment and supplies necessary for conducting gel electrophoresis are relatively simple and include: An electrophoresis chamber and power supply Gel casting trays, which are available in a variety of sizes and composed of UV transparent plastic. The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis. Sample combs, around which molten medium is poured to form sample wells in the gel. Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate- EDTA (TBE). Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded. Staining Fig.. Preparation, loading and running of gel in electrophoresis Applications of Electrophoresis: -To determine the purity of the material, for example, study the effect of -.storage on the stability of the protein -To know the number of compounds or units of the molecule and diagnose: - - For example, it is possible to know whether there is one protein or more and also can separate the secondary protein units (such as Isoenzymes containing more than )one unit can be separated and the diagnosis of disease or to study.  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D -Estimating the relative molecular weights of compounds by comparing them - with standard molecular weight information. -Isolation of proteins using special methods of Electrophoresis - - The human genome Project , Human DNA: ~three billion nucleotides -To Isolation and purity of the DNA, RNA and nucleotides - Species Electrophoresis 1-Paper Electrophoresis - - Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D One of the most widely used methods of separating small molecules and less - commonly used for compounds with large molecules such as proteins due to a change in the nature of proteins (denatured ) 2- Cellulose acetate and cellulose nitrate Electrophoresis Use small quantities of the sample and use tapes composed of Cellulose acetate and cellulose nitrate Structural formula of cellulose acetate. This technique can be used to alleviate heat problems generated during electrophoresis. The separation process can be performed under laboratory temperature conditions, as well as separation of blood proteins and follow-up of the distribution of Isoenzymes in this technique The cellulose acetate membranes are not strong enough. They are fragile when they are dry and weak when wet. This has led to the use of other thin layer technologies. An electric backing is placed on an inert panel such as TLC. Use  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D cellulose on glass or plastic in horizontal position with paper strips for conduction in the two electrode 3- Gel Electrophoresis In this technique, a media of support gel is used for cross-linking. This method has the following advantages A. Use a large number of samples, more than those used in cellulose acetate and cellulose nitrate B. The composition of the gel can be changed to the desired method by adding chains and multiple of molecules cross-linking There are several types of gel used in biochemistry labs such as gel Starch, Agarose and Polyacrylamide. 4- Polyacrylamide with Sodium dodecyl sulphate(SDS) Electrophoresis : SDS PAGE , PAGE=Poly Acrrylamide Gel Electrophoresis This technique is used to diagnose the number of peptide chains and to estimate their molecular weights. To explain this A -The protein is first treated with compounds containing the thiol group (- SH) , eg. Β - mercaptoethanol or Dithiothreitol to break down the two-sulphide bonds found in the protein and convert them into the reduced form as in the following equation B-SDS decomposes the protein by providing variable-size chains surrounded by SDS molecules with a negative charge (Figure). These chains move by  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D electrophoresis based on the molecular weights of the peptide chains only. Figure : shows the treatment of SDS protein, after treatment, which binds the protein to the SDS molecules carrying the negative charge C. Polyacrylamide Polyacrylamide used in the electrophoresis technique consists of polymerization of monomers of acrylamide and Biacrylamide as a cross linker with cofactor sodium persulphate or riboflavin to form free radicals and tetramethyl ethylene diamine (TEMED) which it helps in the polymerization process and the formation of a gelatinous with Cross linker , different sizes depending on the concentration of acrylamide. The process of polymerization is slow or stops with the presence of oxygen, preferred degassing when the process of polymerization( Figure ) – Figure : Molecular structure of the polyacrylamide gel. The three-dimensional molecular network comes into being by a radical polymerization of acrylamide monomers and cross-linking N,N'-methylenebisacrylamide components with cofactor Sodium persulphate (Na2S2O8). Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D The advantages of conducting an electrophoresis using Polyacrylamide are 1-The thick layer gel is able to absorb the resulting heat at the passed of the electric current and distribute it to itself, so the amount of the denatured of the protein will decrease 2- Because of the nature of the gel used, the large molecules will move toward the electrode slower than the small molecules, that is, adding another characteristic, the size, as well as the charge in the process of separation using the gel. Two types of devices are used in electrophoresis 1. Slab (THIN LAYER GEL) gel electrophoresis 2. Disc (CAPILLARY ) gel electrophoresis 1. Slab (THIN LAYER GEL) gel electrophoresis In this technique it is possible working of strips with different Measurements suitable although the 20 × 10 cm gel strips are required and the strips are usually thickness (0.1 - 0.25 mm). electrophoresis may be performed by stabilizing the gel with a vertical or horizontal position, and a typical installation of the vertical position can be observed in Figure  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D Figure : The technique of electrophoresis in slab and vertical position 2. Disc gel electrophoresis CAPILLARY METHODS This method is used in glass tubes with a diameter of 7 mm and a length of 10 - 20 cm. advantageous of , It is easy to add the sample and experiment and get the material quantitatively but it is disadvantageous that it uses one sample per tube and it is difficult to compare clearly between models and standard molecules. Isoelectric focusing (IEF) This technique can be used to separate proteins that have similar molecular weights but vary in charge by focusing on the Isoelectric point pI of each protein molecule. PI is defined as the pH where positive and negative charges are equal to the molecule or protein molecule known as the point of charge and the total charge on the vessel is equal to zero. The process is carried out in a column containing substances with a gradient in the pH called Ampholytes, which are multiple amino acids with different properties in their charge and then a difference in the electric point pI , Add to it low-concentration acrylamide gel as well as sugar (no shipments) as well as protein to be analyzed A mixture of amphoteric materials gives a pH gradient based on its type, and is composed of various activiting groups such as carboxyl, amidazole, amines, etc. It is associated with diluted acid and a base electrode. The electrical current causes the transmission of ions based on the charges it carries. And that have points equal to the charge is low (Such as the carboxylic group) move towards the base pole, giving a gradient in the pH of the ampholytes. The large molecules (such as proteins) required to decompose are moved based on the charges they carry. Their movement depends on negative and positive aggregates and regions The  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D pH produces a separate special package that can be clearly observed in Figure. For example, the proteins that stop at the beginning are acidic, then neutral and then base, and thus can be overwhelmed by the nature of the protein. It is possible to separate different brands with a small difference (0.1 pH unit) at the electrolyte point. This method is therefore of great importance in the purification and separation of enzymes. Figure : Proteins move in a charge-like focus technology based on the value of pI with the pH values between 3 and 9 at a constant current Two-dimensional (2D) electrophoresis The most effective separation can be achieved if the combined consecutive separation steps rely on absolutely independent physicochemical properties. A good example of this is the very high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) that combines two already discussed electrophoresis methods, isoelectric focusing and SDS-PAGE (Figure 7.6). As the first step of 2D gel electrophoresis, isoelectric focusing is performed to separate proteins based on their pI values. Only a single sample is loaded on a gel strip in this step. the separation is based on a single well-defined property, the pI value After the first separation step has been completed in the first dimension, the gel strip is soaked in an SDS solution and is fitted tightly to one side of a “classical” SDS polyacrylamide gel  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D The second separation step is traditional SDS-PAGE, which separates proteins based on their molecular mass. This second step represents a second dimension and the property utilised in the second step (molecular mass) is completely )independent of the one utilised in the first step (pI Figure 7.6. Two-dimensional (2D) electrophoresis. 2D electrophoresis is the combination of isoelectric focusing and SDS-PAGE. Proteins are first separated based on their pI values and then based on their molecular mass. Automated amino acid analyzer The process of analysis of amino acids is done after obtaining the mixture of the amino acid compounds of the protein resulting from the cracking of proteins to polypeptide using several methods (such as chromatography) and the analysis of peptides acidic analysis or basalysis or enzymatic decomposition After the composition of amino acid is transferred to the Automated amino acid analyzer, Which includes a column containing a positive ion exchange resin (such as the number of salfunat alsatarin)  Prof.Dr.Thikra A.Allwsh Biotechniques M.Sc and Ph.D The acid solution (pH = 3) of the amino acid mixture is placed on the upper surface of the resin and then an buffer solution is passed to filter through the column. Amino acids are positive at pH 3 as their positive charges vary in their ionization range. Thus, high-positive amino acids (basal amino acids such as lysine, arginine and histidine) will be strongly associated with the resin. The lowest amino acids charge will first be removed and mixed with the ninhydrine solution. The resulting color is then measured by means of a photometer. The results are then recorded by means of the recorder graphically showing the absorption versus the retention time of the jitter part. Since the time taken to remove the specific amino acid is specific to that of the amino. acid, and the amount of absorption is directly related to the amount of amino acid, so the diagram of the recording machine directly indicates the type and quantity of amino acids present in the sample.