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Dr.Ehab

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    2 biochemistry practical topic 2 biochemistry practical topic Document Transcript

    • Practical ChromatographyWhat is it?Some materials appear homogenous, but are actually a combinationof substances. For example, green plants contain a mixture ofdifferent pigments. In addition, the black ink in the pens that areused in this experiment is a mixture of different colored materials. Inmany instances, we can separate these materials by dissolving themin an appropriate liquid and allowing them to move through anabsorbent matrix, like paper.Chromatography is a method used by scientists for separatingorganic and inorganic compounds so that they can be analyzed andstudied. By analyzing a compound, a scientist can figure out whatmakes up that compound. Chromatography is a great physicalmethod for observing mixtures and solvents.The word chromatography means "color writing" which is a way thata chemist can test liquid mixtures. While studying the coloringmaterials in plant life, a Russian botanist inventedchromatography in 1903. His name was M.S. Tswett.Chromatography is such an important technique that two Nobel prizeshave been awarded to chromatographers. Over 60% of chemicalanalysis worldwide is currently done with chromatography or avariation there on. Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 1
    • Chromatography is used in many different ways. Some people usechromatography to find out what is in a solid or a liquid. It is alsoused to determine what unknown substances are. The Police andother detectives use chromatography when trying to solve a crime. Itis also used to determine the presence of cocaine in urine, alcohol inblood, PCBs in fish, and lead in water.Chromatography is used by many different people in many differentways.Chromatography is based on differential migration. The solutes in amobile phase go through a stationary phase. Solutes with a greateraffinity for the mobile phase will spend more time in this phase thanthe solutes that prefer the stationary phase. As the solutes movethrough the stationary phase they separate. This is calledchromatographic development.How it worksIn all chromatography there is a mobile phase and a stationary phase.The stationary phase is the phase that doesnt move and the mobilephase is the phase that does move. The mobile phase moves throughthe stationary phase picking up the compounds to be tested. As themobile phase continues to travel through the stationary phase it takesthe compounds with it. At different points in the stationary phase thedifferent components of the compound are going to be absorbed and Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 2
    • are going to stop moving with the mobile phase. This is how theresults of any chromatography are gotten, from the point at which thedifferent components of the compound stop moving and separatefrom the other components.In paper and thin-layer chromatography the mobile phase is thesolvent. The stationary phase in paper chromatography is thestrip or piece of paper that is placed in the solvent. In thin-layerchromatography the stationary phase is the thin-layer cell. Both thesekinds of chromatography use capillary action to move the solventthrough the stationary phase.What is the Retention Factor, RF?The retention factor, Rf, is a quantitative indication of how far aparticular compound travels in a particular solvent. The Rf value isa good indicator of whether an unknown compound and a knowncompound are similar, if not identical. If the Rf value for the unknowncompound is close or the same as the Rf value for the knowncompound then the two compounds are most likely similar oridentical.The retention factor, Rf, is defined as Rf = distance the solute (D1)moves divided by the distance traveled by the solvent front (D2)Rf = D1 / D2 where Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 3
    • D1 = distance that color traveled, measured from center of the bandof color to the point where the food color was appliedD2 = total distance that solvent traveledThe Different Types of ChromatographyThere are four main types of chromatography. These are LiquidChromatography, Gas Chromatography, Thin-Layer Chromatographyand Paper Chromatography.Liquid Chromatography is used in the world to test water samplesto look for pollution in lakes and rivers. It is used to analyze metalions and organic compounds in solutions. Liquid chromatographyuses liquids which may incorporate hydrophilic, insoluble molecules.Gas Chromatography is used in airports to detect bombs and isused is forensics in many different ways. It is used to analyze fiberson a persons body and also analyze blood found at a crime scene. Ingas chromatography helium is used to move a gaseous mixturethrough a column of absorbent material.Thin-layer Chromatography uses an absorbent material on flatglass or plastic plates. This is a simple and rapid method to check thepurity of an organic compound. It is used to detect pesticide orinsecticide residues in food. Thin-layer chromatography is also usedin forensics to analyze the dye composition of fibers. Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 4
    • Paper Chromatography is one of the most common types ofchromatography. It uses a strip of paper as the stationary phase.Capillary action is used to pull the solvents up through the paper andseparate the solutes.Therefore, Chromatography basically involves the separation ofmixtures due to differences in the equilibrium distribution of samplecomponents between two different phases. One of these phases is amobile phase and the other is a stationary phase. Concentration of component A in stationary phaseDistribution Coefficient = -------------------------------------------------- Concentration of component A in mobile phaseDifferent affinity of these two components to stationary phase causesthe separation.Kinds of Chromatography1. Liquid Column Chromatography2. Gas Liquid Chromatography3. Thin-layer Chromatography Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 5
    • LIQUID COLUMN CHROMATOGRAPHYA sample mixture is passed through a column packed with solidparticles which may or may not be coated with another liquid. Withthe proper solvents, packing conditions, some components in thesample will travel the column more slowly than others resulting in thedesired separation.DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHYFOUR BASIC LIQUID CHROMATOGRAPHYThe 4 basic liquid chromatography modes are named according tothe mechanism involved: Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 6
    • 1. Liquid/Solid Chromatography (adsorption chromatography)A. Normal Phase LSCB. Reverse Phase LSC2. Liquid/Liquid Chromatography (partition chromatography)A. Normal Phase LLCB. Reverse Phase LLC3. Ion Exchange Chromatography4. Gel Permeation Chromatography (exclusion chromatography)LIQUID SOLID CHROMATOGRAPHYThe separation mechanism in LSC is based on the competition of thecomponents of the mixture sample for the active sites on anabsorbent such as Silica Gel.Example: Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 7
    • WATER-SOLUBLE VITAMINS1.Niacinamide2.Pyridoxine Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 8
    • 3. Riboflavin4. Thiamin Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 9
    • LIQUID-LIQUID CHROMATOGRAPHYIn Liquid-Liquid Chromatography the stationary solid surface iscoated with a 2nd liquid (the Stationary Phase) which is immiscible inthe solvent (Mobile) phase. Partitioning of the sample between 2phases delays or retains some components more than others toeffect separation. Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 11
    • ION-EXCHANGE CHROMATOGRAPHY Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 11
    • Separation in Ion-exchange Chromatography is based on thecompetition of different ionic compounds of the sample for the activesites on the ion-exchange resin (column packing). MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 12
    • GEL-PERMEATION CHROMATOGRAPHYPractical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 13
    • Gel-Permeation Chromatography is a mechanical sorting ofmolecules based on the size of the molecules in solution. Smallmolecules are able to permeate more pores and are, therefore,retained longer than large molecules.SOLVENTSPolar SolventsWater > Methanol > Acetonitrile > EthanolNon-polar SolventsN-Decane > N-Hexane > N-Pentane > CyclohexaneRetention TimeTime required for the sample to travel from the injection port throughthe column to the detector. Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 14
    • SELECTIVITY ()Ratio of Net Retention Time of 2 components.(Equilibrium Distribution Coefficient) Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 15
    • RESOLUTION EQUATION Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 16
    • HEIGHT EQUIVALENT TO A THEORETICAL PLATELength of a column necessary for the attainment of compounddistribution equilibrium (measure the efficiency of the column). Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 17
    • EXAMPLES OF THEORETICAL PLATE, SELECTIVITY AND HEIGHT EQUIVALENT TO A THEORETICAL PLATEPractical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 18
    • Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 19
    • GENERAL FACTORS INCREASING RESOLUTION1. Increase column length2. Decrease column diameter3. Decrease flow-rate4. Pack column uniformly5. Use uniform stationary phase (packing material)6. Decrease sample size7. Select proper stationary phase8. Select proper mobile phase9. Use proper pressure10. Use gradient elution Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 21
    • Practical Theory of Paper chromatographyWhen you look at a leaf, the green pigment chlorophyll is usually theonly pigment that appears to be present.Actually, chlorophyll is only one of many types of pigments present inthe leaf and one of several that are involved in the process ofphotosynthesis. Once removed from the leaf, the photosyntheticpigments can be separated from one another and identified using aprocess called chromatography.Theory of paper chromatographyA small sample of a mixture is placed on porous paper which is incontact with a solvent. The solvent moves through the paper due tocapillary action and dissolves the mixture spot. The components ofthe sample start to move along the paper at the same rate as thesolvent.Components of the mixture with a stronger attraction to the paper(stationary phase) than to the solvent will move more slowly that thecomponents with a strong attraction to the solvent (mobile phase).The difference in the rates with which the components travel alongthe paper, over time, leads to their separation.Particular mixtures will have chromatographic patterns that areconsistent and reproducible as long as the paper, solvent, and time Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 21
    • are constant. This makes paper chromatography a qualitative methodfor identifying some of the components in a mixture.Objectiveso Prepare a leaf pigment solution.o Prepare a paper chromatogram.o Separate pigments of spinach leaves by paper chromatographyo Calculate the Rf values for various photosynthetic pigmentsMaterials1. Chromatography Jar2. Mortar & Pestle3. Leaf4. Chromatography paper5. Chromatography solvent (90% Isopropyl Alcohol)6. Ruler7. Capillary tube8. CalculatorSolution Preparation:1. Place a large piece of spinach into your pestle and addapproximately 5ml of 90% isopropyl alcohol.2. Thoroughly macerate the spinach/alcohol mixture to develop athick liquid, Chromatogram Preparation: Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 22
    • 1. Obtain a chromatography jar, a piece of fresh leaf, and a length ofchromatography paper (just long enough to fit from top to bottom ofthe jar.).2. Cut the tip of the paper such that it forms a point of a triangle.3. Draw a line across the paper 1 cm up from the triangle. This is your“start line”.4. Using a capillary tube transfer a drop of the green pigment solutionto the center of your start line.5. Pour approx. 1 cm of chromatography solvent into thechromatography jar.6. Open chromatography jars and hang the papers into the jar so thetip of the triangle dips into the solvent. Do not submerge pigment linesbelow the solvent level. Recap the jars immediately.7. Allow the solvent to rise for about 15 minutes or until the solventline nears the top of your papers.8. When the solvent line is about 1cm from the top of your paper.Remove the papers and mark the farthest point of the solventsprogress before this line evaporates.9. Allow the filter papers to dry, and then make a sketch of thechromatogram. Some possible colors and the pigments theyrepresent are:o Faint yellow - carotenes Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 23
    • o Yellow - xanthophyllso Bright green - chlorophyll ao Yellow-green - chlorophyll bo Red - anthocyanin10. Measure the distance from the start point to the front line andeach of the pigment lines. Record these measurements in the datatable. Calculate the Rf values for each pigment according to thefollowing formula;Calculation of Rf Distance the pigment travels from the original spot of solventRf = ---------------------------------------------------------------------------------- distance to the solvent front Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 24
    • Practical Theory of Thin Layer ChromatographyThin layer chromatography (TLC) is among the most useful tools forfollowing the progress of organic chemical reactions and for assayingthe purity of organic compounds. TLC requires only a few ng (nanograms) of sample for a successful analysis and can be accomplishedin a matter of minutes. Like all chromatographic methods, TLC takesadvantage of the different affinity of the analyte with the mobile andstationary phases to achieve separation of complex mixtures oforganic molecules.Theory of ChromatographyStationary PhaseSilica gel, the most commonly used stationary phase, has theempirical formula SiO2. However, at the surface of the silica gelparticles, the dangling oxygen atoms are bound to protons. Thepresence of these hydroxyl groups renders the surface of silica gelhighly polar. Thus, polar functionality in the organic analyte interactsstrongly with the surface of the gel particle and nonpolar functionalityinteracts only weakly. Polar functionality in the analyte molecules canbind to the silica gel in two ways: through hydrogen bonds andthrough dipole-dipole interactions. The total strength of the interaction Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 25
    • is a sum of these two components. It should be noted that the shapeof the organic analyte is also a factor in predicting the strength of itsinteraction with silica gel. Thus, an analyte that displays multiple polargroups in position to interact with the surface of the stationary phasewith interact more strongly than an analyte that displays the samepolar functionality in a way that does not permit multidentate binding.Modes of Interaction of Analyte with Silica GelFor silica gel chromatography, the mobile phase is an organic solventor mixture of organic solvents. As the mobile phase moves past thesurface of the silica gel it transports the analyte past the particles ofthe stationary phase. However, the analyte molecules are only free tomove with the solvent if they are not bound to the surface of the silica Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 26
    • gel. Thus, the fraction of the time that the analyte is bound to thesurface of the silica gel relative to the time it spends in solutiondetermines the retention factor of the analyte. The ability of ananalyte to bind to the surface of the silica gel in the presence of aparticular solvent or mixture of solvents can be viewed as a the sumof two competitive interactions. First, polar groups in the solvent cancompete with the analyte for binding sites on the surface of the silicagel. Therefore, if a highly polar solvent is used, it will interact stronglywith the surface of the silica gel and will leave few sites on thestationary phase free to bind with the analyte. The analyte will,therefore, move quickly past the stationary phase. Similarly, polargroups in the solvent can interact strongly with polar functionality inthe analyte and prevent interaction of the analyte with the surface ofthe silica gel. This effect also leads to rapid movement of the analytepast the stationary phase. The polarity of a solvent to be used forchromatography can be evaluated by examining the dielectricconstant (ε) and dipole moment (δ) of the solvent. The larger thesetwo numbers, the more polar is the solvent. In addition, the hydrogenbonding ability of the solvent must also be considered. For examplemethanol is strong hydrogen bond donor and will severely inhibit theability of all but the most polar analytes to bind the surface of thesilica gel. Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 27
    • The TLC ExperimentThe first step in conduction a TLC experiment is to select the elutionsolvent. For most organic molecules, a good starting point is 2 partsethyl acetate to 3 parts hexanes. Place about 10 mL of the elutionsolvent in a 100 mL beaker covered with a watch glass. To ensurethat the atmosphere in the elution chamber is saturated with solventvapor, place a piece of filter paper, torn into a square, along theinside wall of the beaker. Be sure that the bottom of the filter papertouches the solvent. Using a pencil, draw a line on the TLC plateabout 5 mm from the bottom. Cross the line in three places with shortpencil lines. These three intersections are the locations onto whichyou will place the sample. Prepare a solution of you sample in theleast polar solvent in which it is soluble. About 1 mg (a speck) ofsample dissolved in two to three drops of solvent is all that isrequired. The sample is introduced onto the TLC plate using a microcapillary. Dip the end of the micro capillary into the sample solution. Asmall volume of the solution will flow into the micro capillary. Now youcan spot the capillary onto the pencil lines on your TLC plate. Be surethat the spots on the TLC plate are no more than 3 mm in diameter.Let the spotting solvent evaporate for a few seconds and then placethe TLC plate in the elution chamber with the sample spots at the Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 28
    • bottom. Note that the sample spots should be above the level of theelution solvent. If this is notthe case, use a pipette to remove a small amount of the elutionsolvent from the chamber. Now let the sample elute to a point wherethe solvent front is about 5 mm from the top of the TLC plate. Tovisualize the spots on you TLC plate you will use UV light, iodine or aseries of chemical stains. You may need to adjust the polarity of thesolvent if the retention factor (RF) of you analyte is too large or toosmall. The Rf is calculated by dividing the distance traveled by theanalyte by the distance traveled by the solvent. The ideal solventgives the analyte an Rf of 0.3. Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 29
    • Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 31
    • Analysis of Proteins by Thin-Layer ChromatographyPractical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 31
    • 1. Equipment and SuppliesThe following equipment is needed for a single development,conventionalTLC analysis:1) Amber glass storage bottles (250 ml)2) Capillary pipettes (1 .0 and 0.2 l size)3) Conventional TLC chamber with a lid4) Glass vials with caps (1 and 4 ml)5) Graduated cylinder (100 ml)6) Oven7) Reagent sprayer8) Ruler (inch and metric)9) Saturation pad (20 x 20 cm)10) Spray box11) Spray stand2. Chemicals and Materials1) 0.1 N hydrochloric acid2) Eluent components  Butanol  Acetic acid  Water3) Ethanol Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 32
    • 4) Cellulose plate, 20 x 20 cm5) Methanol6) Ninhydrin (Caution: toxic reagent, handle with care)7) Amino acid standard solutions (1 mg/ml)  Glutamic acid Tyrosine  Hydroxyproline Proline  Lysine Threonine  Serine8) Binding media reference materials (hydrolyzed)  Whole egg Egg white  Egg yolk Casein3. SamplesSamples may be taken from facsimile paintings or unknowns. Thesample should be approximately 500 mg in weight and contain onlythe paint layer or material of interest. The paint layer or material beinginvestigated should be separated from all other layers, such as theground, varnish layers, or support. Samples are hydrolyzed beforeanalysis,Protocol KAmino acid standard solutions are made with glutamic acid,hydroxyproline, lysine, proline, serine, threonine, and tyrosine.Each standard solution is made in a concentration of 1 mg/ml by Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 33
    • weighing 2 mg of an amino acid into a 4-ml glass vial and adding 2ml of 0.1 N HCI. These solutions can be used for 3-4 weeks afterpreparation. Reference solutions of binding media are made fromwhole egg, egg white, egg yolk, casein. These solutions areprepared by hydrolysis following the same procedure as for thesamples (Protocol K).The reference materials should be prepared in a concentration of 2.0-2.5 g/l in 0.1N HCI.4. Preparation ProceduresPreparation for TLC analysis includes prewashing the TLC plate,making fresh eluent systems and detection reagents, and saturatingthe TLC chamber.The following preparation procedures are started 24 hours prior toanalysis:1) Prepare cellulose TLC platesThe cellulose plate must be washed in methanol before analysis. Thisprocedure takes approximately 4 hours. Place 30-60 ml of methanolin a clean conventional TLC chamber. Allow the chamber toequilibrate with methanol for approximately 30 minutes. The celluloseTLC plate is inserted vertically into the methanol, and the chamber iscovered with the lid. Allow the methanol to rise to the top of thecellulose TLC plate. Remove the plate from the chamber and dry it in Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 34
    • a fume hood. Store the cleaned cellulose TLC plate in a desiccatorcontaining silica gel.2) Prepare eluentMix butanol, acetic acid, and water in an 80:20:20 volume ratio.Seal the solution in an amber bottle to maintain freshness before use.Prepare 60 ml of the eluent fresh daily for an analysis.3) Prepare TLC chamberPresaturate chamber with solvent system at least 4 hours beforeanalysis.(Note: It is useful to presaturate the chamber overnight.) To do this,place 30-60 ml of the eluent inside a clean, dry conventional TLCchamber. Insert a saturation pad into the solvent system. Cover thechamber with a lid.4) Prepare ninhydrin detection reagent Weight 0. 158 g ofninhydrin into a 250-ml amber bottle. Add 100 ml of ethanol. Mixthoroughly. The reagent can be stored in a refrigerator for 4-5 weeks.5. TLC Analysis ProceduresTo analyze protein hydrolysates by TLC, the samples are spotted inindividual lanes at the baseline of a prewashed cellulose plate. Theplate is placed in a saturated conventional TLC chamber containing asaturation pad and the eluent (butanol: acetic acid : water, 80:20:20). The development of the plate is complete when the eluent front Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 35
    • reaches a distance of 17 cm from the baseline. The plate is removedand dried in a fume hood before spraying with the ninhydrin reagent.This reagent reacts with the amino acid components to producecolors that aid in the visualization of the separation zones or spots.After 24 hours the plate can be documentedThe following nine steps describe the procedure for analysis:1) Draw the base lineUsing a ruler and pencil, lightly draw a line 1 cm from the bottomedge of the plate. Very lightly mark the lanes with short tick marks atintervals of 1 cm along this baseline, for a total of 19 lanes. In theupper left corner, number the plate with a reference number, used torelate the TLC to information in the research notes. Beside thenumber, place the date and the analysts initials. Place a mark 17 cmfrom the baseline as a reference to help determine the completion ofthe development.2) Apply the standard and reference solutions to the plateAll solutions are applied following the spotting procedure noted inProtocol H.Apply 1.0 I of the reference or standard solution to a tick mark onthe origin of a lane using a capillary pipette. The total volume may beapplied in a series of smaller volumes to minimize the diameter of thespot. An air gun may be used to rapidly evaporate the carrier solvent Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 36
    • between applications. Take care not to get the air gun too close to thepipette, as the sample will evaporate.3) Apply unknown sample solutionsIf possible, apply each unknown sample in two different volumes. Forexample, in one lane apply 1.0 I of the unknown sample, and in asecond lane apply 0.2 I of the same solution. (The unknown samplemay or may not be very concentrated, and this procedure minimizesthe possibility of overloading the plate.)4) Develop the TLC plateOnce the plate is spotted, either develop immediately or store in adesiccator.To develop the plate, quickly insert the spotted cellulose TLC plateinto the saturated chamber, with the baseline oriented toward thebottom of the chamber and the front facing away from the saturationpad. Replace the lid of the chamber. Do not leave the chamber openfor any length of time, as the vapor phase equilibrium will be lost. Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 37
    • 5) Completion of developmentDevelop the plate until the solvent front travels a distance of 17 cm.Development usually takes about 4 hours.6) Dry the plateRemove the plate from the chamber, hang it vertically, and let it dryfor about 30 minutes at room temperature in the fume hood. Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 38
    • 7) Prepare to spray plateSpraying of a ninhydrin reagent should always be performed under awell ventilated fume hood or some other device to ensure effectiveremoval of the reagent cloud and solvent vapors, which are toxic.Protective glasses, laboratory gloves, and a respirator should alwaysbe worn during spraying. Set the plate on a clean, dry spray standinside a spray box. Fill the reagent sprayer with 15-20 ml of ninhydrindetection reagent.8) Spray plate with ninhydrinHold the reagent sprayer 8-10 cm from the surface of the TLC plateand spray the plate slowly back and forth, then up and down, until theplate is evenly covered (generally until the cellulose layer just beginsto turn transparent). Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 39
    • 9) Heat plateDry the plate for 15-20 minutes in the fume hood, and then place itfor 10 minutes in a preheated oven at 100 °C.6. Data Analysis ProceduresAfter the separation zones are visualized with the detection reagentEvaluation of the plate can include qualitative or semi quantitativetechniques. The migration distances, color, and intensity of theseparation spots are noted. The Rf value for each spot is calculated Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 41
    • Practical 1-Separation of Amino Acid by TLCA-Reagent 1- Mobile Phase [ butanol: formic Acid: water] ratio [70:1:29] 2- Silica gel 3- Standard Amino acids (1%) 4- Ninhydrine spray [0.2 g dissolve in 100ml acetone]B-Procedure 1- Weight 1g of silica gel, then dissolved in 3 ml distilled water and mix for 5 min. [note: wash glass plate by alcohol and accurate is clean before add silica gel on it ] 2- Pour silica gel solution on slide from glass, dry in oven for 1h 3- Load of standard amino acid and unknown sample spots 4- Put the slide with sample in mobile phase container, allow to run 5- After finished the reaction, dry slide in air 6- Visualize spots by spraying with ninhydrin 7- Calculate Rf for each spotC-calculationCalculate Rf for each amino acid sample spot from law Rf=x/y Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 41
    • Practical 2-Separation of Amino Acid by paper chromatographyA-Reagent 5- Mobile Phase [butanol: glacial acetic Acid: water] ratio [12:3:5] 6- Whattman filter paper number 1 7- Standard Amino acids (1%) 8- Ninhydrine spray [0.2 g dissolve in 100ml acetone]B-Procedure 1- Load of standard amino acid and unknown sample spots 2- After finished the reaction, dry slide in air 3- Put the sample in mobile phase container ,allow to run 4- Dry the paper 5- Visualize spots by spraying with ninhydrin 6- Calculate Rf for each amino acid spotC-calculationCalculate Rf for each amino acid sample spot from law Rf=x/y Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 42
    • Practical 3-Identification of sugars by paper chromatography and TLCA-Reagent 1-Mobile Phase [Methanol: glacial acetic Acid: water] ratio [60:10:30] 2-Whattman filter paper number 1 1. Standard sugars (1%) [Glucose, fructose, Maltose etc] 2. Diphenylamine spray [0.5 g dissolve in 50 ml acetone until dissolve completely then add the following to it + [0.5ml aniline+10ml H3PO4+ 50 ml acetone]B-Procedure 3. Load of standard glucose, fructose, maltose and unknown sample spots 4. After finished the reaction, dry slide in air 5. Put the sample in mobile phase container ,allow to run 6. Leave spot to dry in air ( paper or TLC) 7. Visualize spots by spraying with Diphenylamine reagent mixture 8. Dry in oven until the spots are appear 9. Calculate Rf for each sugar spotC-calculationCalculate Rf for each sugar sample spot from law Rf=x/y Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 43
    • Practical4-Identification of sugars in milk by paper chromatography and TLCA-Reagent 1. Mobile Phase [Methanol: glacial acetic Acid: water] ratio [60:10:30] for paper chromatography 2. Mobile Phase [Ethyl acetate:isopropanol:pyridine:water] ratio [26:14:2:7] for TLC 3. 10% Trichloroacetic acid (TCA) [dissolve10g/100ml water] 4. Whattman filter paper number 1 5. Standard sugars (1%) [Glucose, fructose, Maltose etc] 6. Diphenylamine spray [0.5 g dissolve in 50 ml acetone until dissolve completely then add the following to it + [0.5ml aniline+10ml H3PO4+ 50 ml acetone]B-Procedure 1- 2 ml from milk + 2 ml TCA and mix well, after precipitation , make centrifuge at 3000 rpm for 5 min 2- Collect only supernatant (sample) and discard the pellet 3- Load of standard glucose, fructose, maltose and unknown sample spots 4- After finished the reaction, dry slide in air 5- Put the sample in mobile phase container ,allow to run Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 44
    • 6- Leave spot to dry in air ( paper or TLC) 7- Visualize spots by spraying with Diphenylamine reagent mixture 8- Dry in oven until the spots are appear 9- Calculate Rf for each sugar spotC-calculationCalculate Rf for each sugar sample spot from law Rf=x/y Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 45
    • Practical 5-Identification of sugars in Fruit Juice by paper chromatography and TLCA-Reagent 1- Mobile Phase [Methanol: glacial acetic Acid: water] ratio [60:10:30] for paper chromatography 2- Mobile Phase [Ethyl acetate:isopropanol:pyridine:water] ratio [26:14:2:7] for TLC 3- Absolute Ethanol 4- Whattman filter paper number 1 5- Standard sugars (1%) [Glucose, fructose, Maltose etc] 6- Diphenylamine spray [0.5 g dissolve in 50 ml acetone until dissolve completely then add the following to it + [0.5ml aniline+10ml H3PO4+ 50 ml acetone]B-Procedure 1- 2 ml from fruit juice + 3 ml Ethanol and mix well, after precipitation , make centrifuge at 3000 rpm for 5 min 2- Collect only supernatant (sample) and discard the pellet 3- Load of standard glucose, fructose, maltose and unknown sample spots 4- After finished the reaction, dry slide in air 5- Put the sample in mobile phase container, allow to run Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 46
    • 6- Leave spot to dry in air ( paper or TLC) 7- Visualize spots by spraying with Diphenylamine reagent mixture 8- Dry in oven until the spots are appear 9- Calculate Rf for each sugar spotC-calculationCalculate Rf for each sugar sample spot from law Rf=x/y Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 47
    • Practical 6-Separation of Lipid by TLCA-Reagent 1- Mobile Phase [ petroleum ether : diethyl ether: glacial acetic acid ] ratio [79:20:1] 2- Silica gel 3- Standard lipids (1%) 4- Iodine spray [0.2 g dissolve in 100ml acetone]B-Procedure 1- Weight 1g of silica gel, then dissolved in 3 ml distilled water and mix for 5 min. 2- Pour silica gel solution on slide from glass, dry in oven for 1h 3- Load of standard lipid and unknown sample spots 4- Put the slide with sample in mobile phase container, allow to run 5- After finished the reaction, dry slide in air 6- Visualize spots by spraying with ninhydrin 7- Calculate Rf for each spotC-calculationCalculate Rf for each amino acid sample spot from law Rf=x/y Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 48
    • Practical 7-ElectrophoresisPrinciple Electrophoresis means the migration of chargedparticles in a liquid medium under the influence of an electric field.When an electric field is applied to a medium containing chargedparticles or molecules (e.g. DNA or protein), the negativelycharged molecules migrate towards the positive electrode(anode) and vice versa. After separation (according to differencein charge and mass) permanent fixation of the fractions at theposition to which they migrate is done. Bands are then stained inorder to visualize them.Components1. Power supply: provide stable direct current, and has controlsfor both voltage and current output. (cathode & anode)2. Support medium: It is the heart of the system whereseparation occurs there. Its function is to provide an inert porousmedium for the electrolytes solution. Zone Electrophoresis isclassified according to the support medium type. Support media maybe Thin sheet (of paper, cellulose acetate or silica) or Gel (of starch,agarose or polyacrylamide that separate samples according to the Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 49
    • charge and size) e.g. cellulose acetate electrophoresis,polyacrylamide gel electrophoresis.3. Buffer: It serves as a multifunctional component in theelectrophoretic process as it: a) carries the applied current(electrophoresis buffer) b) establish the pH at which electrophoresis isperformed (gel buffer). c) Determine the electric charge of thesample (sample buffer). There are several considerations must bedone to select buffer: A) the buffer must be not interact with sample.B) The ionic strength and concentration of buffer must be suitable forsample. C) It must allow the sample to be charged not denaturated.4. Stains: It is used to visualize and locate the separated proteinand nucleic acid fractions e.g. Coomassie Brilliant Blue (CBB).(Tracking dye such as Bromophenol Blue (BB) is often used to seethe sample movement on gel (do not stain sample bands) thatenables us to terminate the process when the bands reach lowerbuffer reservoir. It moves faster than any macromolecules).Procedures(e.g. Poly-Acrylamide Gel Electrophoresis) SDS-PAGELaemmle (1970) Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 51
    • A) Gel preparation:Fist: prepare the following solutions as follow:Second: prepare the separating and stacking gel as follow:  Separating gel: (3.5ml from 1) + (2.5ml from 2) + (0.1ml from 4) + (0.05ml from 5) + (10μl from 6)  Stacking gel: (0.6ml from 1) + (1.25ml from3) + (0.05ml from 4) + (0.05ml from 5) + (5μl from 6)Note: These gels are polymer. We can control their pores though theconcentration of their constituents .high concentration decrease poressize, and become suitable for passing low molecular weight proteinsand vice versa. B) Pouring the gel in electrophoresis unit: 1. The separating gel is transferred to the gel glass sandwich. Wait till polymerization (25min) (take care with air bubbles) 2. The stacking gel is then transferred over separating gel. The comb is inserted into the top then removed after polymerization Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 51
    • C) Sample application: 1. The sample is homogenized by sample buffer (Tris + SDS+ 2- mercapto ethanol + BB + sucrose) then centrifuged. 2. 25μl of supernatant is applied side by side in the wells inside the gel. 3. 15μl of protein marker is applied in the last well for comparisons with unknown bands. 4. The upper and lower buffer reservoir is filled with electrophoresis buffer (Tris + glycine + SDS) D) Running of samples: The power is switched on. Wait till the bands reach at lower end of gel (stopping gel) then switch off. E) Detection and quantification: 1. The plate is removed, dried then stained to see the bands. 2. Compare the separating unknown bands with the known marker bands. (The process can carry out without SDS in certain samples: Native-PAGE)Applications 1. Gel electrophoresis is used in quantitative analysis in molecular biology and genetics. 2. Nucleic acids carry negative charge on their suger-phosphte backbone so they migrate into gel with similar rates and Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 52
    • separation is done due to different molecular size. Agarose gel electrophoresis is suitable for DNA and RNA analysis. 3. Proteins have different charges, so they charged negatively (denaturated) by SDS in order to migrate into the gel with similar rates and separation is done due to different molecular size. Polyacrylamide gel electrophoresis is suitable for protein analysis 4. Other different types of electrophoresis have many applications in many fields.Factors affecting electrophoresis 1. The sample: a) Charge: migration increase with charge increase b) Size: migration decrease with size increase 2. The support media: a) If adsorption, migration decrease b) If molecular sieving, migration increase 3. The buffer: a) Composition, bad buffer decrease migration b) Concentration increase, migration decrease c) pH affect ionization Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 53
    • 4. The electric field: a) Voltage: migration increase with its increase b) Current: migration increase with its increase c) Resistance: migration decrease with its increase 5. The heat: migration increase with its increase due to fall in resistance.Types of electrophoresisElectrophoresis has two mains types according to the support media: (I) Thin sheet electrophoresis: it separates samples according to the charge. Support medium is thin sheet. 1. Paper electrophoresis: used in past to separate charged samples (support medium is thin sheet of paper) 2. Cellulose acetate electrophoresis: it suitable for separation of radio-labeled substances especially for clinical investigations (support medium is cellulose acetate that is prepared by treating cellulose with acetic anhydride) 3. Thin layer electrophoresis (TLE): as in TLC but the plate is placed in electrophoresis unit (support medium is silica) (II) Gel electrophoresis: it separates samples according to the charge and molecular size. Support medium is gel. 1. Continuous gel electrophoresis: Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 54
    • a) Starch gel electrophoresis: it is prepared heating and cooling starch in suitable buffer. b) Agar/Agarose gel electrophoresis: Agar composed of agaropectin and agarose. Separation based on charge only c) Polyacrylamide gel electrophoresis (PAGE):it is made from acrylamide monomers copolymerized with thecross linker N,N`methylenebisacrylamide in presence ofammonium persulphate and TEMED as catalyst. Separation basedon molecular size (molecular sieving). It has two types:  Native-PAGE: under non-denaturating conditions.  SDS-PAGE: under denaturating conditions. 2. Discontinuous gel electrophoresis: 3. Two dimensional gel electrophoresis Gradient gel electrophoresis: a) Isoelectric focusing b) Pulse-field gel electrophoresis c) Capillary electrophoresis Native PAGE SDS- PAGE1. used to determine total 1-used to determine fragments of proteins protein2. detergent not used to avoid 2-detergents are used to do deformation of proteins fragmentation of proteins Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 55
    • 1D 2D1-separation according to molecular weight 1-separation according to2-depends on gradient Acrylamide isoelectric point of protein 2-depends on gradient pH Material FunctionAcrylamide and Bisacrylamide To form a net structure through which, proteins will sieve according to their sizeSeparating gel Provide an inert porous medium for separationStacking gel To press all sample bands in one line in order to run with each otherSodium dodecyl sulfate (SDS) To provide a negative charge to proteinsAmm. Per sulfate (APS) Initiate the reaction between Acrylamide and BisacrylamideTEMED Increase or catalyst the reactionAmpholyte To establish a pH gradient on electrophoresis unit before running proteins. When running proteins, they move on till reaching a pH corresponding to its isoelectric point Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 56
    • Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 57
    • Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 58
    • SEPARATION OF AMINO ACIDS BY THIN LAYER CHROMATOGRAPHYMATERIALS NEEDED• silica gel plate• mobile phase: 1-butanol, glacial acetic acid and water (4:1:1)• known solutions of amino acids• unknown solutions of amino acids• micropipette• developing tank• 2% ninhydrin solution• heat gun• pencil• glovesPURPOSE: To understand the concepts of chromatography and toidentify unknown amino acids.BACKGROUNDThe discovery of chromatography in 1944 revolutionized the separation anddetection of amino acids and dipeptides. The separation is based on the liquid-liquid partition of the compounds between two immiscible phases. Initially theseparations were primarily conducted on filter paper and were called paperchromatography. In paper chromatography the hydrated cellulose fibers of thepaper act as the stationary phase. A polar solvent ascends in the vertically heldpaper by capillary action and is the mobile phase. In thin layer chromatography(TLC) a thin uniform layer of silica gel acts as the stationary phase. TLC isreplacing paper chromatography because the plates are easier to use than thepaper, they give a sharper separation and the amino acids or dipeptides caneasily be collected from the plate.Many microscopic distributions of the compounds occur between the mobile andthe stationary phases. In time equilibrium is established between the two phasesand the more soluble compounds move farther along the plate; differentcompounds move 2 Amino Acids different distances from the origin. The plate isdried, sprayed with a ninhydrin solution and heated in order to locate the aminoacids. The ninhydrin reacts with the amino acids to form colored products. Theratio of the distance moved by the amino acids to the distance moved by the Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 59
    • solvent front from the original spot on the paper is defined as the Rf value and ischaracteristic of the compound. Rf compound = distance traveled by compound /distance traveled by solventRf values depend on several factors: type of silica gel plus binder used,water content of the thin layer, concentration of solute, temperature, manner ofdevelopment and distance of the starting point from the solvent. Knowncompounds are usually run on the same plate as the unknowns to assist inidentification of the unknowns rather than relying solely on published Rf values.PROCEDURE1. Put gloves on. If your developing tank isnt prepared, add enough solvent to adepth of approximately 1 cm or less.2. Get your silica gel plate. Carefully hold the plate by the sides to preventdisturbing the silica gel layer. Draw a pencil line about 1.5 cm from the bottomplate.3. Mark one point on the line for each one of your known and unknown solutions.(If you have four known solutions and 2 unknowns, mark six points.) Leavemargins of at least 1.5 cm on both sides. Number each point.4. At point number 1 apply a very small drop of one of your known 1 2 3 4 5 6 7solutions. Do not wet • • • • • • • 3 Amino Acids the silica beyond a diameter of 2-3mm. Locating the center of large spots will be difficult later when the spot hasmoved along the paper.5. After the liquid has evaporated (only a few seconds), add a second drop to thesame spot. Record the name of the amino acid and the number of the spot.6. Repeat this procedure for the remaining solution. Remember to record thename of the amino acid or unknown number and the number of the spot.7. Allow all the spots to dry completely.8. Place your TLC plate in the developing tank with the mobile phase with thespots toward the bottom.9. Allow the solvent to ascend the silica gel to at least ¾ of its height, which will torequire 1 hour or less. (The farther the solvent ascends, the greater theseparation. Immediately remove the plate, if the solvent reaches the top.)10. Remove the plate and quickly mark the farthest advance of solvent front witha pencil, unless it reached the top of the paper. Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 61
    • 11. Dry the plate with a heat gun. Be careful to move the heat gun around andnot heat one point continuously. Do this procedure in the hood.12. Spray the plate with the 2% ninhydrin solution in the hood.13. Do not allow the ninhydrin solution to stream down the plate, because thismay move some of the compounds.14. Dry the plate again with the heat gun. Do not over heat the plate. Longheating times may cause browning of the plate over the entire surface.15. Circle each colored spot with a pencil. The ninhydrin spots fade gradually, socircle at once.16. Measure the distance from the origin to the center of each colored spot andcalculate the Rf values for all spots. 4 Amino Acids17. Record the Rf values and the color of each ninhydrin spot.18. Identify the unknown amino acids.QUESTIONS1. Why does touching the silica gel with your hands potentially contaminateyour plate?2. Why can an Rf value never be greater than 1?3. What would happen if so much solvent was used (mobile phase) that theoriginal spots were covered with solvent?4. What would happen if you made the line and points with an ink pen rather thana pencil?5. You dropped and mixed up your samples. You know that one containsonly valine, one contains valine and glycylvaline, one contains valine and alanine,one contains only glycine and valine, and one contains glycine and glycylleucine.How would you determine what your samples are using TLC and the data below?Can you figure out what they all are?Rf Values for Amino Acids and Dipeptides Compound Rf ColorGlycine 0.26-0.29 purpleAlanine 0.39-0.42 purpleGlycylvaline 0.62-0.66 grayValine 0.62-0.64 purpleGlycylleucine 0.76-0.80 light brownRf values taken two days after solvents were mixed and with solvent advance100 mm in 43 minutes at a temperature of 31C. Calculating Rf Values Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 61
    • ResultsNames of amino acids found in the mixture. Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta 62