Definition and Classification of Chromatography Chromatography course Aim Separation Techniques1-Biological fluids are extremely complex in composition.2-Chemical analysis would be impossible if it were necessary tocompletely isolate each substance prior to its measurement.3- An optimal method tests for a specific substance in thepresence of all others, requiring no isolation of the substanceunder analysis.4- A test is specific when none of the other substances presentinterfere. However, virtually all chemical tests are subject to atleast some interference.5-This is one of the most important problems in clinicalchemistry. Therefore some type of separation procedure isrequired.7-Separation in clinical chemistry usually is based ondifferences in the size, solubility or charge of the substancesinvolved.Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 1صفحة
Definition and Classification of Chromatography Chromatography courseINTRODUCTION The Russian botanist M. S. Tswett is discovery ofchromatography. He used a column of powdered calciumcarbonate to separate green leaf pigments into a series ofcolored bands by allowing a solvent to percolate through thecolumn bed. Since these experiments by Tswett many scientistshave made substantial contributions to the theory and practice ofchromatography. Not least among these is A. J. P. Martin whoreceived the Nobel Prize in 1952 for the invention of partitionchromatography (with R. L. K. Synge) and in the same yearwith A. T. James he introduced the technique of gas-liquidchromatography. Chromatography is now an important toolused in all branches of the chemical and life sciences.1-Definition of Chromatography Chromatography is essentially a physical method ofseparation in which the components to be separated aredistributed between two phases one of which is stationary(stationary phase) while the other (the mobile phase) throughit in a definite direction.2- Classification of chromatographic methods The common feature of all chromatographic methods istwo phases, one stationary and the other mobileA classification can be made depending upon whether thestationary phase is solid or liquid. If it is solid, the method isDr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 2صفحة
Definition and Classification of Chromatography Chromatography coursetermed adsorption chromatography; if it is liquid the methodis partition chromatography.One of the two phases is a moving phase (the mobile phase),while the other does not move (the stationary phase). Themobile phase can be either a gas or a liquid, while thestationary phase can be either a liquid or solid.3- Classification scheme One classification scheme is based on the nature of the twophases. All techniques which utilize a gas for the mobile phasecome under the heading of gas chromatography (GC). Alltechniques that utilize a liquid mobile phase come under theheading of liquid chromatography (LC). Additionally, wehave gas–liquid chromatography (GLC), gas–solidchromatography (GSC), liquid–liquid chromatography (LLC),and liquid–solid chromatography (LSC),4- Main Type of Chromatography In general, there are four main types which can beclassified as follows:4.1-Liquid-Solid chromatography Classical adsorption chromatography (Tswett column) Ion-exchange chromatography4.2. Gas-Solid chromatography4.3. Liquid-Liquid chromatography Classical partition chromatography Paper chromatographyDr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 3صفحة
Definition and Classification of Chromatography Chromatography course4.4 Gas-Liquid chromatography5-Separation techniquesTechnique Property DescriptionPrecipitation Solubility Some of the substances precipitate while the others remain dissolvedUltra-filtration or Molecular size Some of the substancesDialysis pass through a layer or sheet of porous material while the other substances are retainedExtraction Solubility Some of the substances dissolve (partition) more in water. While other substances dissolve more organic solvent in contact with the waterThin layer Solubility Some of the substancesChromatography dissolve (partition) more or in the immobile file of water on a solidColumn supporting medium (orChromatography stick more to the exposed areas of the solid supporting medium) while the other substances dissolve more in the surrounding film of flowing organic solventDr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 4صفحة
Definition and Classification of Chromatography Chromatography courseGas liquid Solubility Some of the substancesChromatography dissolve more in the immobile film of wax or oil-like material on a solid supporting medium. While the others dissolve more in surrounding stream of flowing gas.Gel filtration Molecular Size Some of the substancesChromatography diffuse into the pores in a porous, solid material while others remain outside in the surrounding stream of flowing waterIon-exchange Electrical charge Some of the substancesChromatography are bound by immobile charges on the solid supporting medium while others are not boundElectrophoresis Electrical charge The substances withChromatography more charge move faster and, therefore, further. Substances with opposite charges move in opposite directions.Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 5صفحة
Definition and Classification of Chromatography Chromatography course6-Adsorption chromatography Adsorption column chromatography is the oldest form ofchromatography. Whether two or more substances of amixture can be separated by adsorption chromatographydepends on a number of factors. Most important is the strengthwith which each component of mixture is adsorbed and itssolubility in the solvent used for elution. The degree to whicha particular substance is adsorbed depends on the type of bondswhich can be formed between the solute molecules and thesurface of the adsorbent. Chromatography Adsorption Chromatography Solid stationary phase Partition Chromatography Liquid Stationary Phase Liquid Gas mobile Liquid Gas mobilemobile phase phase mobile phase phaseDr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 6صفحة
Adsorption Chromatography Chromatography course All chromatographic separations are carried out using amobile and a stationary phase, the primary classification ofchromatography is based on the physical nature of the mobilephase. The mobile phase can be a gas or a liquid which givesrise to the two basic forms of chromatography, namely, gaschromatography (GC) and liquid chromatography (LC). Thestationary phase can also take two forms, solid and liquid,which provides two subgroups of GC and LC, namely; gas–solid chromatography (GSC) and gas–liquid chromatography(GLC), together with liquid solid chromatography (LSC) andliquid chromatography (LLC). The different forms ofchromatography are summarized in Table.1Most thin layer chromatography techniques are consideredliquid-solid systems although the solute normally interacts witha liquid-like surface coating on the adsorbent or support or, insome cases an actual liquid coating.Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 7صفحة
Adsorption Chromatography Chromatography course Table 1: The Classification of Chromatography1-ADSORPTION CHROMATOGRAPHY In adsorption chromatography the compounds to beseparated are adsorbed onto the surface of a solid material. Thecompounds are desorbed from the solid adsorbent by elutingsolvent.2-Separation of the compounds depends on1-The relative balance between the affinities of the compounds for the adsorbent and their solubility in the solvent.2-The chemical nature of the substances.3-The nature of the solvent.4-The nature of the adsorbent.Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 8صفحة
Adsorption Chromatography Chromatography courseSolid adsorbents commonly used are alumina, silica gel,charcoal (active carbon), cellulose, starch, calcium phosphategels, calcium hydroxylapatite, and sucrose.Solvents commonly used are hexane, benzene, petroleumether, diethyl ether, chloroform, methylene chloride, variousalcohols (ethyl, propyl, n-buryl and t-butyl alcohols), andvarious aqueous buffers and salts, some in combination withorganic solventsAdsorption chromatography is a column that is packed with theadsorbents. The adsorbent is prepared and poured into thecolumn with an inert support at the bottom. Suitable supportsinclude plastic discs, or sheets of nylon or Teflon fabrics.The adsorbent bed must be homogeneous and free of bubbles,cracks, or spaces between the adsorbents and the walls of thecolumn.The choice of the eluting solvent, although very important,depends on the nature of the substances to be separated andthe adsorbent, and hence affords considerable latitude. Theprocess of eluting the sample components from the adsorbent bythe solvent is termed development. As illustrated in Figure 1,the compounds in the mixture that are more soluble in thesolvent and have less affinity for the adsorbent move morequickly down the column.If the substances are colored, as they were in Tswettsexperiment, they are readily visible as they separate, However,Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 9صفحة
Adsorption Chromatography Chromatography coursemany substances are not colored, and in these instances, as thedevelopment proceeds, fractions are collected at the bottom ofthe column, and the different fractions are analyzed forcompounds of the types that are being separated, For example, ifproteins are being separated,the fractions would be analyzed for protein by measurement ofthe UV absorbance at 280 nm. If carbohydrates or nucleicacids are being separated analytical measurements forcarbohydrates or nucleic acids. The collection of fractions by anautomatic fraction collector, Figure 1: Collection of fractions from a column by an automatic fractionDr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 01صفحة
Adsorption Chromatography Chromatography coursea device that accumulates from an elution column the samepredetermined volume in each of a series of tubes thatautomatically change position when the proper volume has beencollected This may be accomplished in various ways. Forexample, set volume, with a timer, or by counting drops with adrop counter. The latter is frequently used and is usually themost reliable and flexible. The fraction collector may beequipped with a detection cell that automatically measures someparameter of the solution going into the tubes and maycorrelated with fraction number and automatically recorded. Thedetection cell is frequently a small spectrophotometer that canmeasure absorbances at a fixed wavelength or at variablewavelengths. Other detecting cell use index of refraction,optical rotation, and other properties.Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 11صفحة
Adsorption Chromatography Chromatography courseFigure 2: Adsorption chrornatographyA = adsorbent, S=Sample, ES = eluting solvent(A) Application of sample to the of the column.(B) Adsorption of sample onto adsorbent.(C)Addition of elution solvent.(D) and (E) Partial fraction of sample components.(F) Complete fractionation of sample.(G) and (H) Separation of all three components at various stages on the adsorbents.(I) Elution of the first component from the column.Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 21صفحة
Adsorption Chromatography Chromatography courseThe substances adsorbed on the column support can beeluted in three ways (a) in the simplest method, a single solvent continuously flows through the column until the compounds have been separated and eluted from the column (b) Stepwise elution, in which two or more different solvents of fixed volume are added in sequence to elute the desired compounds. (c) Gradient elution, in which the composition of the solvent is continuously changing. The latter method is used to effect separations that are difficult because of a tendency of component to be eluted in broad. Trailing bands when a single solvent is used. Gradient elution frequently provides a means of sharpening the bands, a simple linear gradient has two solvents, A and B, in which A is the starting solvent and B is the final solvent. Solvent B is allowed to flow into solvent A as solvent A flows into the column. The composition of solvent A is, thus, constantly changing as solvent B is constantly being added to A (Fig. 3).Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 31صفحة
Adsorption Chromatography Chromatography course Figure 3: Gradient elution. Flow of solvent B into solvent A With mixing, continuously changing the composition of solvent A as it flows into column Figure 4: Elution of chromatography column with a gradient of increasing salt concentration.Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 41صفحة
Adsorption Chromatography Chromatography course Gradients other than linear gradients (e.g., exponential, concave. or convex) may be obtained by introducing a third vessel and varying the composition of the solvents in the vessels. These eluting methods are also used with other column chromatographic methods.3-Activation of adsorbent Many adsorbents such as alumina, silica gel, and activecarbon and Mg silicate can obtain commercially, but theyrequire activation before use. Activation is achieved by heatingand there is usually an optimum temperature for activation, fore.g. alumina is about 400oC. For reduced activity by thecontrolled addition of water, and the subsequent activity isrelated to the amount of water added. Brookman and Schodderestablished five grades of alumina Grade I is the most active 0and the is simply alumina heated at about 350 C for severalhours. Grade II has about 2-3% water, Grade III 5-7%, GradeIV 9-11 %, Grade V film. (Least active) about 15%.4-RetentionThe retention is a measure of the speed at which a substancemoves in a chromatographic system. In continuous developmentsystems like HPLC or GC, where the compounds are eluted withthe eluent, the retention is usually measured as the retentiontime Rt or tR, the time between injection and detection. Ininterrupted development systems like TLC the retention ismeasured as the retention factor Rf, the run length of theDr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 51صفحة
Adsorption Chromatography Chromatography coursecompound divided by the run length of the eluentfront:The retention of a compound often differs considerably betweenexperiments and laboratories due to variations of the eluent, thestationary phase, temperature, and the setup. It is thereforeimportant to compare the retention of the test compound to thatof one or more standard compounds under absolutely identicalConditions.5-Plate theory The plate theory of chromatography was developed byMartin and Synge. The plate theory describes thechromatography system, the mobile and stationary phases, asbeing in equilibrium. The partition coefficient K is based onthis equilibrium, and is defined by the following equation:K is assumed to be independent of concentration, and canchange if experimental conditions are changed, for exampletemperature is increased or decreased. As K increases, it takeslonger for solutes to separate. For a column of fixed length andflow, the retention time (tR) and retention volume (Vr) can bemeasured and used to calculate KDr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 61صفحة
Adsorption Chromatography Chromatography course6- Column chromatography1. Small plug of wool (or cotton)2. Sand to cover "dead volume"3. Silica gel, length = 5.5 - 6 inch (Note 1inch=2.54cm).4. Tap column on bech (carefully) to remove air bubbles inside the column5. Add solvent system6. Add sand on top of silica7. The top of the silica gel should not be allowed to run dry.8. Sample is diluted (20-25% solution)9. The sample is applied by pipette10. Solvent used to pack the column is reused11. Walls of column are washed with a few milliliters of eluant12. Column is filled with eluant13. Flow controller is secured to column and adjusted 2.0 in /min.Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 71صفحة
Adsorption Chromatography Chromatography course Column as that illustrated in Fig.5 may be used: Typical chromatographic column. Mixture sorbed on top of column. Partial separation Complete separation Table 2: Common adsorbents and the type of compounds Solid Suitable for separation ofAlumina Steriods, vitamins, ester, and alkaloidsSilica gel Steriods, amino acids, alkaloidsCarbon Peptides, carbohydrates, amino acidMagnesium PorphyrinscarbonateMagnesium Steriods, ester, glycerides, alkaloidssilicateMagnesia Similar to alumina.Ca(OH)2 Carotenoids.CaCO3 Carotenoids and xanthophylls.Ca Phosphate Enzymes, protein, and polynucleotideStarch Enzymes.Sugar Chlorophyll.Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 91صفحة
Thin layer chromatography Chromatography course Thin layer chromatography This technique is particularly useful for the separation of verysmall amounts of material. The general principle involved is similar tothat involved in column chromatography, i.e. it is primarily adsorptionchromatography, although other partition effects may also be involved. Aglass sheet is covered by a uniform thin layer of an adsorbent.Adsorbents used in TLC, differ from column adsorbents. It contain abinding agent such as calcium sulphate, which facilitates the adsorbentsticking to the glass plate. The plates are prepared by spreading slurry ofadsorbent in water over them, starting at one end, and movingprogressively to the other and then drying them in an oven at 100-120°C. Drying serves to remove the water and to leave a coating ofadsorbent on the plate. Equipment is available which will ensure theproduction of an even coating of adsorbent over a series of glass plates.The normal thickness of slurry layer used is 0.25 mm for qualitativeanalysis, but layers up to 5-10 mm thick may be made for preparativework.Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 02صفحة
Thin layer chromatography Chromatography courseThe sample is applied to the plate by micropipette or syringes, as spot2.5 cm from one end and at least an equal distance from the edge. Thesolvent is removed from the sample by the use of an air blower. All spotsshould be placed on equal distance from the end of the plate.Separation takes place in glass tank which contains the developingsolvent (mobile phase) to a depth of 1.5 cm , this is allowed to stand forat least 1 hour with a glass plate over the top of the tank to ensure thatthe atmosphere within the tank becomes saturated with solvent vapor.Then, the thin layer plate is placed vertically in the tank so that, it standsin the solvent with the end bearing the sample in the solvent.The cover plate is replaced and separation of the compounds then occursas the solvent travels up the plate. After the solvent had reached thewanted level, the run is stopped. The chromatographic separation iscompleted the spots of the component substances can be detected bydifferent methods:1-Many commercially available TLC adsorbents contain a fluorescentdye, the plate is examined under UV light, the separated componentsshow up as blue, green, black area.2. Spraying the plate with 50% sulphuric acid and heating so,the compounds become charred and show spotsDr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 12صفحة
Thin layer chromatography Chromatography course3. Spraying the plates with specific color reagents will stain up certaincompounds e.g. ninhydrin for amino acid (aa) , aniline for aldoses.SolventsUniversal TLC System:petroleum ether - ethyl acetateVery polar solvent additives:methanol > ethanol > isopropanolModerately polar additives:acetonitrile > ethyl acetate > chloroform, dichloromethane >diethyl ether > tolueneNon-polar solvents:cyclohexane, petroleum ether, hexane, pentaneTLC Visualization (Detecting the spots)Non-destructive techniques:1. Ultraviolet lamp. Shows any UV-active spots2. Plate can be stained with iodine.Bottle containing silica and a few crystals of iodine(especially good for unsaturated compounds)Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 22صفحة
Thin layer chromatography Chromatography courseDestructive techniquesStaining Solutions immerse the plate as completely as possible in thestain and remove it quickly. Heat carefully with a heating Stains Use/CommentsAnisaldehyde Good general reagent, gives a range of colorsPMA Good general reagent, gives blue/green spotsVanillin Good general reagent, gives a range of colorsCeric sulfate Fairly general reagent, gives a range of colorsDNP Mainly for aldehydes and ketones, gives orange spotsPermangante Mainly for unsaturated compounds and alcohols, gives yellow spotsThin-Layer Chromatography of Amino acidsAmino acids may be separated by two-dimensional TLC using eithersilica gel or cellulose as the separating medium. Two different solventsare used for each type of TLC plate and a different type of separation isachieved for each type. The amino acids are visualized with two types ofninhydrin spray for the silica gel and the cellulose gel media.Ninhydrin Sprays for amino acid detectionFor silica gel TLC: The plate is sprayed with a solution of 300 mg ofninhydrin + 3 ml of glacial acetic acid + 100 ml of butyl alcohol andheated for 10 minutes at 110°C.Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 32صفحة
Thin layer chromatography Chromatography courseFor cellulose TLC: The plate is sprayed with a solution of 500 mg of ninhydrin + 350ml of absolute ethanol + 100 ml of glacial acetic acid + 15 ml of 2,4,6-trimethylpyridine and heated for 10 minutes at 110°C.Two-dimensional TLC separation of amino acids.On silica gel G withsolvent I, chlorolorm-17% methanol (v/v)-ammonia (2:2:1, v/v/v) andsolvent II, phenol-water (75:25, v/v).on cellulose MN 300 withsolvent III, 1-butanol-acetone-diethylamine-water (10:10:2:5,v/v/v/v,pH 12.0) andsolvent IV, 2-propanol-formic acid (99%)-water (40:2:10, v/v/v, pH 2.5)Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 42صفحة
Thin layer chromatography Chromatography courseThin-Layer Chromatography of Carbohydrates Carbohydrates may be separated on commercial silica gel platesusing a variety of solvents to achieve specific separations. The results ofthe separation depend on the particular plate used. Whatman K5 silica geland Merck silica gel 60 plates give good results.Solvent for TLC separations of carbohydratesSolvent: Acetonitrile-water (35:15, v/v) with four ascents (45 minuteseach for a 20-cm plate) will separate mono-, di , and trisaccharidesThe visualization of carbohydrates on thin layer silica gel plates isobtained by spraying with sulfuric acid-methanol (1: 3, v/v) followed byheating for 10 minutes at 110-120°C. Most carbohydrates give black tobrown spots on a white background.Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 52صفحة
Thin layer chromatography Chromatography courseExamples of some TLC separation systemsCompounds Adsorbent Solvent system (v/v)Amino acids Silica Gel G 96% Ethanol/water (70/30) Butan-1-ol/acetic acids/ water (80/20/20)Mono and di Kieselguhr G (sodium Ethyl acetate/propan-1-olsaccharides acetate) (65/35). Butan-1-ol / Kieselguhr G acetone/phosphate buffer (sodium phosphate pH5) pH5 (40/50/10)Neutral lipids Silica Gel G Petroleum ether/diethyl ether/acetone (90/10/1)Cholesterol Silica Gel G Carbon tetrachloride/Esters chloroform (95/5)Carotenoids Kieselguhr G Petroleum ether/propan-1- ol (99/1)Phospholipids Silica Gel G Chloroform/methanol/water (65/25/4)Advantages of TLC. The speed at which separation is achieved. With a volatile solventas the mobile phase the time involved may be as low as 30 minutes, buteven with non-volatile solvents the time involved is rarely longer than90 minutes.Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 62صفحة
PAPER CHROMATOGRAPHY Paper chromatography is a type of liquid-liquid partitionchromatography that may be performed by ascending or descendingsolvent flow. Each mode has its advantages and disadvantages.Ascending chromatography involves relatively simple and inexpensiveequipment compared with descending chromatography and usually givesmore uniform migration with less diffusion of the sample "spots."Descending chromatography, on the other hand is usually faster becausegravity aids the solvent flow and with substances of relatively lowmobility. The solvent can run off the paper. Giving a longer path formigration. To resolve compounds with low mobility. Ascendingchromatography may be performed more than once utilizing a multiple-ascent technique. For descending chromatography, papers 22 cm wide and 56 cmlong can be used. To facilitate the flow of solvent from the paper, thebottom of the paper is serrated with a pair of pinking shears. Three pencillines are drawn 25 mm apart at the top of the sheet, and small aliquot ofthe sample (10-50 ml) is placed at a marked spot on the third line. Thespot is kept as small as possible by adding the aliquot in smallincrements. With drying in between. This may be expedited with a hairdryer. Several samples, including standards, are placed 15-25 mm apart.
The paper is then folded along the other two lines and placed in thesolvent trough of the descending tank (Fig. 1). Which has beenequilibrated with solvent beforehand to ensure a saturated atmosphere.The paper is irrigated with solvent until the solvent reaches the bottom orfor a longer period, allowing the solvent to flow off the end of the paper,if necessary. The chromatogram is then removed dried and developed toreveal the locations of the compounds. (Part II gives methods of locatingcarbohydrates, amino acids. proteins. nucleotides and nucleic acids andlipids.) In ascending chromatography, a paper approximately 25 cm x 25cm is used. A pencil line 20-25 mm from the bottom is drawn across thepaper Fig. 1 Steps in descending paper chromatography
and aliquots (10-50l) of the samples and standards are spottedapproximately 15-25 mm apart along the line. The spots are dried and thepaper is rolled into a cylinder and stapled so that the ends of the paper arenot touching (Fig. 2). Solvent is poured into the bottom of achromatographic chamber, and the cylinder is placed inside. The chamberis closed and solvent is allowed to flow up Fig. 2 Steps in ascending paper chromatographythe paper by capillary action. The chamber may be a simple wide-mouth,screw top, gallon jar or a cylinder with a ground-glass edge and a glassplate top. As with descending chromatography, the chamber should beequilibrated with solvent beforehand. Contrary to a popularmisconception, if the chamber has been sealed and is airtight, the paperdocs not have to be removed as soon as the solvent reaches the top. When
multiple ascents are performed, the paper is removed, thoroughly dried,and returned to the chamber for another ascent of solvent.The resolved compounds on a paper chromatogram may be detected bytheir color if they are colored, by their fluorescence if they arefluorescent, by a color that is produced from a chemical reaction on thepaper after spraying or dipping the chromatogram with various reagents,or by autoradiography if the compounds are radioactive. Identification ofcompounds on a chromatogram is usually based on a comparison withauthentic compounds (standards). A quantitative comparison may bemade by measuring the Rf , which is the ratio of the distance thecompound migrates to the distance the solvent migrates. A bettercomparison is the ratio of the distance a particular compound migrates tothe distance a particular standard migrates. For example, in the separationof carbohydrates, the standard might be glucose and the ratio would beRGlc or for amino acids, the standard might be glycine and the ratio wouldbe RGly A useful modification is two-dimensional paper chromatography,in which the sample is spotted in the lower left-hand corner and irrigatedin the first dimension with solvent A. The chromatogram is removed fromthe solvent dried, turned 90, and irrigated in the second dimension withsolvent B, giving a two-
Fig. 3 Two-dimensional paper or thin-layer chromatographydimensional separation (Fig. 3). An application of this procedure hasbeen developed for the study of enzyme specificity in which a solution ofthe enzyme is sprayed onto the chromatogram between the first irrigationand the second to see what products are formed by the action of theenzyme on the compounds separated in the first dimension. Paper chromatography has been used to establish the structuralhomology of a series of oligomers obtained by enzymic synthesis, by acidor enzymic hydrolysis, or by isolation from a natural source. The R F ofeach separated homologue is determined and a French-Wild plot is madeby plotting log [RF / (1-RF)]against the number of monomers in the oligomer. If the isolatedcompounds fall on a straight line of this plot, they belong to ahomologous series, differing from each other by one monomer residue
(Fig. 4). Compounds separated by paper chromatography may bequantitatively determined. Aliquots (50-200,l) of the solutioncontaining the substances to be separated and quantitatively determinedare streaked along the separation line. Aliquots of the solution (5-10,l)are also spotted on the two outside edges of the streak and are used aslocation standards. The chromatogram is irrigated in the usual way, andvertical sections of the location standards are cut out and developed toreveal the positions of the compounds. After drying, these standards areplaced alongside the streaked sections and the undeveloped compoundsare located; horizontal strips containing the individual compounds are cutout and
Fig. 4. French-Wild plots (log RF / 1-RF), versus number of monomer units permolecule) correlating paper chromatographic mobility with the number ofhomologous monomer residues in oligosaccharide molecules.Fig. 5. Elution of compounds from paper chromatograms for preparativechromatography or quantitative determination
eluted with water. To accomplish the elution, tabs of chromatographicpaper are stapled to the narrow ends of each strip. As shown in Figure 5,one end is fitted with two pieces of glass (cut microscope slides), whicharc held together with rubber bands, and the bottom end is cut tapered,like a pipet tip. This assembly is played so that one end lies in achromatographic trough containing water, and the elution of the stripoccurs by capillary flow of the water down the paper strip into a baker. Usually less than 1 mL of water is sufficient to effect quantitativeelution, the samples are quantitatively diluted to a specific volume, and achemical analysis is performed for the specific compound separated. Thistechnique also may be used as a preparative procedure to obtain smallquantities of pure compound from a mixture of compounds.In an alternate quantitative procedure, the compounds in the sample areradioactively labeled and separated in the usual way, and anautoradiogram is prepared. The labeled compounds are located on thechromatogram by comparing their positions on the autoradiogram. Theradioactive compounds are cut out and placed into a liquid scintillationcocktail, and the radioactivity is determined by heterogeneous liquidscintillation counting
Paper Chromatography What is Chromatography? Chromatography is a technique for separating mixtures into theircomponents in order to analyze, identify, purify, and/or quantify themixture or components. • Analyze Separate • Identify • Purify • Quantify Mixture Components Uses for ChromatographyChromatography is used by scientists to: Analyze – examine a mixture, its components, and their relations to one another Identify – determine the identity of a mixture or components based on known components Purify – separate components in order to isolate one of interest for further study Quantify – determine the amount of the a mixture and/or the components present in the sample
Real-life examples of uses for chromatography: • Pharmaceutical Company – determine amount of each chemical found in new product • Hospital – detect blood or alcohol levels in a patient’s blood stream • Law Enforcement – to compare a sample found at a crime scene to samples from suspects • Environmental Agency – determine the level of pollutants in the water supply • Manufacturing Plant – to purify a chemical needed to make a product Definition of ChromatographyDetailed Definition: Chromatography is a laboratory technique that separatescomponents within a mixture by using the differential affinities of thecomponents for a mobile medium and for a stationary adsorbing mediumthrough which they pass. Terminology: • Differential – showing a difference, distinctive • Affinity – natural attraction or force between things • Mobile Medium – gas or liquid that carries the components (mobile phase) • Stationary Medium – the part of the apparatus that does not move with the sample (stationary phase)Simplified Definition: Chromatography separates the components of a mixture bytheir distinctive attraction to the mobile phase and the stationary phase.
Explanation: • Compound is placed on stationary phase • Mobile phase passes through the stationary phase • Mobile phase solubilizes the components • Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases Illustration of Chromatography Stationary Phase Separation Mobile Phase Mixture ComponentsComponents Affinity to Stationary Phase Affinity to Mobile PhaseBlue ---------------- Insoluble in Mobile PhaseBlack Red Yellow
Principles of Paper Chromatography Capillary Action – the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity. Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents. Separation of components depends on both their solubility inthe mobile phase and their differential affinity to the mobile phaseand the stationary phase. Paper Chromatography Experiment What Color is that Sharpie?
Overview of the ExperimentPurpose: To introduce students to the principles and terminology ofchromatography and demonstrate separation of the dyes in Sharpie Penswith paper chromatography.Time Required: Prep. time: 10 minutes Experiment time: 45 minutesCosts: Less than $10 Materials List • 6 beakers or jars • 6 covers or lids • Distilled H2O • Isopropanol • Graduated cylinder • 6 strips of filter paper • Different colors of Sharpie pens • Pencil • Ruler • Scissors • Tape
Preparing the Isopropanol SolutionsPrepare 15 ml of the following isopropanol solutions in appropriatelylabeled beakers: - 0%, 5%, 10%, 20%, 50%, and 100% Preparing the Chromatography Strips Cut 6 strips of filter paper Draw a line 1 cm above the bottom edge of the strip with the pencil Label each strip with its corresponding solution Place a spot from each pen on your starting line
Developing the Chromatograms Place the strips in the beakers Make sure the solution does not come above your start line Keep the beakers covered Let strips develop until the ascending solution front is about 2 cm from the top of the strip Remove the strips and let them dry Developing the Chromatograms
Protein purification Chromatography1. Ammonium Sulfate Fraction of Protein Mixtures Increasing the salt concentration to a very high level willcause proteins to precipitate from solution without denaturation ifdone in a gentle manner. First, we want to understand why theprotein precipitates. A protein in a buffer solution is very highlyhydrated, in other words, the ionic groups on the surface of theprotein attract and bind many water molecules very tightly: This graphic illustrates how proteins in solution are hydratedby water molecules. When a lot of salt, such as ammonium sulfate,is added to the protein solution, the salt ions attract the watermolecules away from the protein. This is partly since the salt ionshave a much greater charge density than the proteins. So as the saltis added and these small ions bind water molecules, the proteinmolecules are forced to interact with themselves and begin toDr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 1صفحة
Protein purification Chromatographyaggregate: So when enough salt has been added, the proteins will bebegin to precipitate. If this is carried out at a cold temperature likein ice, the proteins will precipitate without denaturation. Thus, theproteins can be collected by centrifugation and then redissolved insolution using a buffer with low salt content.This process is called "Salting Out" and works best with divalentanions like sulfate, especially ammonium sulfate which is highlysoluble at ice temperatures.Salting out or ammonium sulfate precipitation is useful forconcentrating dilute solutions of proteins. It is also useful forfractionating a mixture of proteins. Since large proteins tend toprecipitate first, smaller ones will stay in solution. Thus, byanalyzing various salt fractions, one can find conditions where theDr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 2صفحة
Protein purification Chromatographyprotein you are studying precipitates and many of the otherproteins in the mixture stay in solution. The end result is that youare also able to increase the purity of your protein of interest whileyou concentrate it using an ammonium sulfate fractionationprocedure.2. Dialysis of Proteins After a protein has been ammonium sulfate precipitate andtaken back up in buffer at a much greater protein concentrationthan before precipitation, the solution will contain a lot of residualammonium sulfate which was bound to the protein. One way toremove this excess salt is to dialyze the protein against a bufferlow in salt concentration. This graphic illustrates the dialysis process. First, theDr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 3صفحة
Protein purification Chromatographyconcentrated protein solution is placed in dialysis bag with smallholes which allow water and salt to pass out of the bag whileprotein is retained. Next the dialysis bag is placed in a largevolume of buffer and stirred for many hours (16 to 24 hours),which allow the solution inside the bag to equilibrate with thesolution outside the bag with respect to salt concentration. Whenthis process of equilibration is repeated several times (replacing theexternal solution with low salt solution each time), the proteinsolution in the bag will reach a low salt concentration: The graphic illustrates the complete dialysis process, exceptfor it suggests you do this with distilled water. Really you want todo this process with buffer to prevent the protein from denaturingdue to the fact that distilled or deionized water is too low in saltand may have an undesirable pH for your protein, which mayDr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 4صفحة
Protein purification Chromatographycause it to denature.In fact, dialysis is a good way to exchange the buffer the protein isin at the same time you get rid of excess salt. For example, theGOT after ammonium sulfate precipitation contains a mixture ofbuffers as well as excess salt. So we use the buffer we want for thenext step in the purification, which is ion-exchangechromatography, as the external solution during dialysis. After thedialysis process, the protein solution is dialyzed against the startingbuffer for the ion-exchange chromatography step, not only will thesalt be removed but the protein will now be in the buffer neededfor the next step and ready to go. Sometimes, proteins willprecipitate during the dialysis process and you will need tocentrifuge the solution after dialysis to remove any particles whichwould interfere with the next step – such as ion-exchangechromatography where particles would clog the column andprevent the chromatography step from working. In addition, youmay lose enzyme activity during dialysis. So it is a good idea tokeep some of your protein solution as a sample before it is put inthe dialysis bag so that it can be assayed for enzyme activitybefore and after dialysis.3. Alternative Methods for Desalting and Concentration of Proteins There are several ways to get rid of excess salt in a proteinsolution. One rapid method is to use a small gel filtration columnDr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 5صفحة
Protein purification Chromatographywhich contains a gel with very small pores which will only allowwater and salt inside the gel particles and will exclude the protein.This method works very well and can be done at 4°C so that littleor no enzyme activity is lost during processing. A small amount ofdilution of the protein solution will take place during processing,but it is possible by this method to exchange the buffer and preparethe protein solution.Another way to both concentrate a protein and exchange thebuffer, which completely avoids precipitation, is calledultrafiltration:Ultrafiltration is done a device which can withstand highpressure. First, the ultrafiltration device is fitted with an ultrafiltermembrane of the desired molecular weight cut off such that youprotein of interest will be retain in the cell. Next, the pressure cellDr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 6صفحة
Protein purification Chromatographyis filled with the protein solution and nitrogen gas at about 50 psi isapplied while the cell is stirred gently at 4°C. After about 1 hour,the solution will be decreased in volume usually without loss ofactivity. To exchange the buffer the cell is filled with the desiredbuffer and the concentration process are repeated.Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 7صفحة
Desalting Before an ion-exchange chromatographic step or after anammonium sulfate fractionation, it is usually necessary to remove the saltfrom the solution of protein. Desalting is accomplished in one of twoways: dialysis or gel filtration.Dialysis Dialysis is performed by filling a section of dialysis tubing (a semipermeable membrane) having a sufficiently small molecular weight "Cut-off", with the protein solution, and placing the filled tubing in a largevolume of buffer. The decrease in salt concentration can be calculatedeasily from the ratio of the volumes inside and outside of the bag.
Dialysis requires a few hours, after which the bag may betransferred to fresh buffer if the reduction in salt concentration effectedby one cycle is deemed to be insufficient. In dialysis, all small molecules,including salt ions, metal ions and cofactors, pass through the membrane,which retains only macromolecules. Neither tightly bound metal ions andcofactors, nor counterions to the macromolecule are effectively removed. Since the initial solution in the bag is of much greater osmoticstrength than the surrounding buffer, the bag generally increases involume. The volume of the contents of the bag must be measured afterdialysis if either total protein or total enzyme units are to be calculated.Ion Exchange Chromatography Since proteins have different net charge and charge distribution,ion exchange chromatography can be an effective purification tool. Forbench-top preparations, usually gravity-flow columns are employed, butHPLC and automated HPLC-like systems have grown in popularity. Forgravity flow or for use with low pressure peristaltic pumps, ion exchangemedia are usually carbohydrate based. Charged groups are attached tosolid supports (“inert phase”) such as Sepharose, Sephadex and cellulose.Since these carbohydrates are compressible, they are not used in higher-pressure systems, and more rigid inert phases such as TSK (a polyether-coated gel) are used. For higher pressures, reinforced Polysaccharides,
and organically coated silica (e.g., TSK) are used. The resins, especiallypoly (styrenediviny1benzene) described by HIRS for use with enzymeswere used by MOORE and STEIN in their famous amino acid analyzer.They are commonly employed for ion exchange chromatography of smallmolecules, but have given way to the ion exchange polysaccharides forpreparative applications in enzymology. The charged groups used withthe solid supports depend to some extent on the chemistry of the supportmaterial itself, but are remarkably similar. Groups containing chargednitrogen atoms are almost universally used for anion exchange media.These include, from strong to weak, quaternary amino methyl or ethyl(QAE), tertiary amino (diethylaminoethyl, DEAE, or diethylaminomethyl)and secondary plus tertiary nitrogens (polyethylenimine, PEI). Thequaternary amino compounds are positively charged at any pH, but theothers must be used at a pH below the pK, of the protonated form (- 10,for DEAE). The conjugate base of the strongly acidic sulfonic acid (i.e.,alkyl or aryl sulfonate) and the weakly acidic carboxylic acids (e.g.,carboxymethyl, CM) are the most common charged groups employed incation exchangers. The carboxymethyl packing must be used at a pHabove their pK4. Methods for determining the optimal pH for separationof proteins depends, of course, on the proteins. Since most proteins areacidic, they are negatively charged at pH 7-8. They therefore adsorb to apositively charged stationary phase to which they act as counterions,
providing that other anions are not available to play the role ofcounterion. The cationic stationary phase is known as an anionexchanger because it functions by exchanging one anionic counterion foranother. Anionic proteins may bind more tightly to anion- exchangestationary phases than simple salts because they possess more negativecharges than a simple anion. However, it is not the total charge on aprotein, but the charge density that determines the affinity. Moreprecisely, it is the charge distribution. Since a protein may interact with astationary phase on one side at a time, proteins with densely chargedpatches may be bound more tightly. At pH values below the isoelectricpoint of a protein, the net charge is positive, so negatively chargedstationary phases (cation exchange phases) are used. If a protein has anisoelectric point near neutrality, either a cation exchange or an anionexchange system can be used, depending on the pH employed. Theimportant considerations in choosing an optimal pH for separation ofenzymes by ion exchange chromatography have been reviewed. Proteinsolutions are generally desalted, then loaded onto a column packed with astationary phase having the appropriate charge. Loading can often bedone as rapidly as the columns will flow without undue pressure; proteinsthat adsorb are retained at the top of the column. As long as the capacityof the column is not exceeded, liters of a (desalted, buffered) crudeextract can be loaded onto a column of modest size, so that a pre-
chromatography concentration step is not needed. After loading, thecolumn is washed with the loading buffer to remove unabsorbed andweakly adsorbed proteins. The adsorbed proteins are then eluted bywashing the column with buffers of increasing salt concentration (e.g.,NaCl), which corresponds to increasing solvent strength. This method ofelution using a series of isocratic (constant strength) elutions ofprogressively increasing strength is known as batch elution. The ionhaving a charge of the same sign as the protein can act as a displacing ionby competing for charged sites on the stationary phase. At someconcentration, the eluting ion competes effectively with the protein,which accordingly, spends a larger fraction of its time in the mobilephase, leading to elution. This concentration would be ideal to purify theprotein of interest providing that more loosely bound proteins wereremoved first, because it affords the maximum discrimination amongthe charge densities of the proteins still on the column. However, theprotein might elute as a broad, dilute band. A simple and commonsolution to elution is to employ a linear concentration gradient of salt,Such a gradient can cover a range from 0 to 1 M NaCl over the volume ofa few hundred ml to a few liters, depending on the dimensions of thecolumn and the steepness of the gradient desired.
A major advantage of gradient elution is that proteins having awide range of affinities for the column can be eluted in a single run. Theinformation obtained from a gradient elution may be used to determine anoptimum salt concentration to be used in isocratic elution, but theprocedure is not straightforward. The theory of gradient elution is messy,even in the simplest case. One egregious misstatement appears innumerous papers on enzyme purification “the enzyme elutes at such andsuch a concentration of sodium chloride”. Because the gradient travelsmuch more rapidly in the column than the protein (the protein is retainedto some extent), the concentration of sodium chloride in which theenzyme actually appears at the bottom of the column is much higher thanthe concentration at which it began to elute appreciably. Thus, theconcentration in which it appears to elute (concentration of sodiumchloride in the fraction in which the activity appears) is much too strongfor use as an isocratic eluent. In addition, the concentration in which theenzyme appears varies with the dimensions of the column; longercolumns cause the enzyme to appear to elute in a higher saltconcentration, simply because the gradient progresses as the enzymemoves down the column. To exercise maximum control over the system,it is useful to separate the effects of pH from those of ionic strengthduring ion exchange chromatography. One of the ions involved in thebuffering system bears the same charge as the protein and can therefore
act as a displacing ion. The concentration of this ion should not changewith pH, so it should not be the one involved in the equilibrium withsolvent protons. Buffering ions selected for use in ion exchangechromatography should have the same charge as the column, i.e., cationsfor an anion exchange column, anions for cation exchange. Hence,phosphate buffers are used for cation exchange chromatography, andTris (for instance) buffers are used for anion exchange. It is necessaryfor the column to be completely equilibrated with the starting solvent.Equilibration can be checked by measurement of both pH and ionicstrength (e.g., by conductivity) prior to loading the column. Elution froman ion-exchange column could also be accomplished using a change inpH. Stepwise pH changes are sometimes employed, but do not generallyproduce high resolution of complex mixtures. Reproducible continuouspH gradients are difficult to obtain because so many of the components inthe system engage in acid-base equilibria. A workable system along theselines has been devised using a proprietary mixed-bed packing and amulti-component buffer system to elute proteins at their isoelectric pH.The process is called chromatofocusing because of a loose analogy toisoelectric focusing gel electrophoresis.
Gel filtrationBiomolecules are purified using chromatography techniques that separatethem according to differences in their specific properties, as shown inFigure 1. and Table 1. Property TechniqueSize Gel filtration (GF), also called size exclusionCharge Ion exchange chromatography (IEX)Hydrophobicity Hydrophobic interaction chromatography (HIC) Reversed phase chromatography (RPC)Biorecognition (ligand specificity) Affinity chromatography (AC)Table 1. Fig. 1. Separation principles in chromatography purification.Gel filtration has played a key role in the purification of enzymes,polysaccharides, nucleic acids, proteins and other biologicalmacromolecules. Gel filtration is the simplest and mildest of all the
chromatography techniques and separates molecules on the basis ofdifferences in size. The technique can be applied in two distinct ways:1. Group separations: The components of a sample are separated into two major groupsaccording to size range. A group separation can be used to remove highor low molecular weight contaminants (such as phenol red from culturefluids) or to desalt and exchange buffers.2. High resolution fractionation of biomolecules: The components of a sample are separated according to differencesin their molecular size. High resolution fractionation can be used toisolate one or more components, to separate monomers from aggregates,to determine molecular weight or to perform a molecular weightdistribution analysis.Gel filtration can also be used to facilitate the refolding of denaturedproteins by careful control of changing buffer conditions.Gel filtration is a robust technique that is well suited to handlingbiomolecules that are sensitive to changes in pH, concentration of metalions or co-factors and harsh environmental conditions. Separations canbe performed in the presence of essential ions or cofactors, detergents,urea, guanidine hydrochloride, at high or low ionic strength, at 37 °Cor in the cold room according to the requirements of the experiment
Gel filtration in practice Gel filtration separates molecules according to differences in sizeas they pass through a gel filtration medium packed in a column. Unlikeion exchange or affinity chromatography, molecules do not bind to thechromatography medium so buffer composition does not directly affectresolution (the degree of separation between peaks).Separation by gel filtrationGel filtration medium is packed into a column to form a packed bed. Themedium is a porous matrix in the form of spherical particles that havebeen chosen for their chemical and physical stability, and inertness (lackof reactivity and adsorptive properties). The packed bed is equilibratedwith buffer which fills the pores of the matrix and the space in betweenthe particles. The liquid inside the pores is sometimes referred to as thestationary phase and this liquid is in equilibrium with the liquid outsidethe particles, referred to as the mobile phase as shown in Figure 2. Gel filtration is used in group separation mode to remove smallmolecules from a group of larger molecules and as a fast, simple solutionfor buffer exchange. Small molecules such as excess salt (desalting) orfree labels are easily separated. Samples can be prepared for storage orfor other chromatography techniques and assays. Gel filtration in groupseparation mode
is often used in protein purification schemes for desalting andbuffer exchange . Fig. 2. Common terms in gel filtration Sephadex G-10, G-25 and G-50 are used for group separations.Large sample volumes up to 30% of the total column volume (packedbed) can be applied at high flow rates using broad, short columns. Figure3 shows the elution profile (chromatogram) of a typical group separation.Large molecules are eluted in or just after the void volume, Vo as theypass through the column at the same speed as the flow of buffer. For awell packed column the void volume is equivalent to approximately 30%of the total column volume. Small molecules such as salts that have full
access to the pores move down the column, but do not separate from eachother. These molecules usually elute just before one total column volume,Vt, of buffer has passed through the column. In this case the proteins aredetected by monitoring their UV absorbance, usually at A280 nm, andthe salts are detected by monitoring the conductivity of the buffer. Sample: (His)6 protein eluted from HiTrap™ Chelating HP with sodium phosphate 20 mM, sodium chloride 0.5 M, imidazole 0.5 M, pH 7. Column: HiTrap Desalting 5 ml Buffer: Sodium phosphate 20 mM, Sodium chloride 0.15 M, pH 7.0 Void volume :Vo, Total column volume :VtFig. 3. Typical chromatogram of a group separation. The UV (protein)and conductivity (salt) traces enable pooling of the desalted fractions andfacilitate optimization of the separation. The theoretical elution profile (chromatogram) of a highresolution fractionation. Molecules that do not enter the matrix are elutedin the void volume, Vo as they pass directly through the column at thesame speed as the flow of buffer. For a well packed column the voidvolume is equivalent to approximately 30% of the total column volume(packed bed). Molecules with partial access to the pores of the matrixelute from
the column in order of decreasing size. Small molecules such as salts thathave full access to the pores move down the column, but do not separatefrom each other. These molecules usually elute just before one totalcolumn volume, Vt, of buffer has passed through the column, Fig. 4.Fig. 4.Theoretical chromatogram of a high resolution fractionation (UVabsorbance).Separation examplesFig. 5. Cytochrome C, Aprotinin, Gastrin I, Substance P, (Gly)6, (Gly)3 and Gly
Comparison of the selectivity of Superdex 75 prep grade and Superdex200 prep grade for model proteins Figure.6Superdex 75 prep grade (a)gives excellent resolution of the three proteins in the Mr range 17 000 to67 000 while the two largest proteins elute together in the void volume.Superdex 200 prep grade (b)resolves the two largest proteins completely. The three smaller proteinsare not resolved quite as well as the larger ones or as in (a). The voidvolume (Vo) peak at 28 minutes in (b) is caused by protein aggregates. Fig. 6. Columns : a) HiLoad 16/60 Superdex 75 prep grade b) HiLoad 16/60 Superdex 200 prep grade Sample : 1. Myoglobin 1.5 mg/ml, Mr 17 000 2. Ovalbumin 4 mg/ml, Mr 43 000 3. Albumin 5 mg/ml, Mr 67 000 4. IgG 0.2 mg/ml, Mr 158 000 5. Ferritin 0.24 mg/ml, Mr 440 000 Sample volume : 0.5 ml Buffer : 0.05 M phosphate buffer, 0.15 M NaCl, 0.01% sodium azide, pH 7.0 Flow : 1.5 ml/min (45 cm/h)
Media Selection Chromatography media for gel filtration are made from porousmatrices chosen for their inertness and chemical and physical stability.The size of the pores within a particle and the particle size distributionare carefully controlled to produce a variety of media with differentselectivities. Todays gel filtration media cover a molecular weightrange from 100 to 80 000 000, from peptides to very large proteins andprotein complexes. Figure.7Superdex is the first choice for high resolution, short run times and high recovery.Sephacryl is suitable for fast, high recovery separations at laboratory and industrialscaleSuperose offers a broad fractionation range, but is not suitable for large scale orindustrial scale separations.Sephadex is ideal for rapid group separations such as desalting and buffer exchange.Sephadex is used at laboratory and production scale, before, between or after other chromatography purification steps.
The selectivity of a gel filtration medium depends solely on its poresize distribution and is described by a selectivity curve. Gel filtrationmedia are supplied with information on their selectivity, as shown forSuperdex in Figure 8. The curve has been obtained by plotting apartition coefficient Kav against the log of the molecular weight for aset of standard proteins Fig. 8. Selectivity curves for Superdex Fig. 9. Defining fractionation range and exclusion limit from a selectivity curve.
Selectivity curves are usually quite linear over the range Kav = 0.1to Kav = 0.7 and it is this part of the curve that is used to determine thefractionation range of a gel filtration medium Figure 9.Determination molecular weight Ve – V0Kav =-------------- Vt – V0where Ve = elution volume for the protein Vo = column void volume Vt = total bed volumeOn semilogarithmic graph paper, plot the Kav value for each proteinstandard (on the linear scale) against the corresponding molecularweight (on the logarithmic scale). Draw the straight line which best fitsthe points on the graph. Then, Calculate the corresponding Kav for thecomponent of interest and determine its molecular weight from thecalibration curve.Sephadex: Rapid group separation of high and low molecular weightsubstances, such as desalting, buffer exchange and sample clean upSephadex is prepared by cross-linking dextran with epichlorohydrin.Variations in the degree of cross linking create the different Sephadex
media and influence their degree of swelling and their selectivity forspecific molecular sizes (Table. 2 ). Product Fractionation pH stability Bed volume Particle size, range, Mr ml/g dry wet (globular Sephadex proteins)Sephadex G-10 <7×102 Long term: 2–13 2-3 55–165 μm Short term: 2–13Sephadex G-25 1×103–5×103 Long term: 2–13 4-6 170–520 μmCoarse Short term: 2–13Sephadex G-25 1×103–5×103 Long term: 2–13 4-6 85–260 μmMedium Short term: 2–13Sephadex G-25 1×103–5×103 Long term: 2–13 4-6 35–140 μmFine Short term: 2–13Sephadex G-25 1×103–5×103 Long term: 2–13 4-6 17–70 μmSuperfine Short term: 2–13Sephadex G-50 1×103–3×104 Long term: 2–10 9-11 40–160 μmFine Short term: 2–13• Sephadex G-10 is well suited for the separation of biomolecules suchas peptides (Mr >700) from smaller molecules (Mr <100).• Sephadex G-50 is suitable for the separation of molecules Mr >30000from molecules Mr<1500 such as labeled protein or DNA fromunconjugated dyes. The medium is often used to remove smallnucleotides from longer chain nucleic acids.• Sephadex G-25 is recommended for the majority of group separationsinvolving globular proteins. These media are excellent for removingsalt and other small contaminants away from molecules that are greaterthan Mr 5000. Using different particle sizes enables columns to be packedaccording to application requirements
Sephadex is prepared by cross-linking dextran withepichlorohydrin, illustrated in Figure 10 The different types ofSephadex vary in their degree of cross-linking and hence in their degreeof swelling and selectivity for specific molecular sizes, as shownFig. 10. Partial structure of Sephadex.Why use different techniques at each stage In order to final removal of trace contaminants. Adjustment of pH,salts or additives for storage. Then, end product of required high levelpurity Therefore, The technique chosen must discriminate between thetarget protein and any remaining contaminants
Gel Filtration (or)Gel Permeation Chromatography (or)Size Exclusion Chromatography Size exclusion chromatography(SEC), also called gel permeationChromatography (GPC) or gel filtration chromatography(GFC) is atechnique for separates molecules according to their molecular size. Gelparticles form the stationary phase of this type of chromatography; themobile phase is the solution of molecules to be separated and the elutingsolvent, which most frequently is water or a dilute buffer. The sample isapplied to the gel, if the molecules are too large for the pores; they neverenter the gel and move outside the gel bed with the eluting solvent. Thus,the very large molecules in a mixture move the fastest through the gel bedand the smaller molecules, which can enter the gel pores, are retarded andmove more slowly through the gel bed. In gel chromatography, moleculesare, therefore, eluted in order of decreasing molecular size
Fig.1 Gel permeation chromatography. Open circles represent porous gel molecules:large solid Circles represent molecules too large to enter the gel through the pores,and smaller solid circles represent molecules capable of entering the gel poresThree types of polymers are principally used-dextran,polyacrylamide, and agaroseDextran is a polysaccharide composed of (-1--->6)-linked glucoseresidues with (-1,3) branch linkages. It is synthesized from sucrose byan enzyme produced by the bacterium Leuconostoc mesenteroides B-512F. The dextran is cross-linked to various extents by reaction withepichlorohydrin to give gel beads with different pore sizes Fig.2. Cross-linked dextrans are commercially produced by Pharrnacia FineChemicals, lnc., (Uppsala, Sweden), and sold under the trade nameSephadex. Sephadex gels in the so-called G-series, where the G-numbers refer to the amount of water gained when the beads areswelled in water (Table 1) have different degrees of cross-linking, hencedifferent pore sizes. This gives gels that have capabilities of separatingdifferent ranges of molecular weights and have different molecular
exclusion limits. The exclusion limit is the molecular weight of thesmallest peptide or globular protein that will not enter the gel pore.Sephadex G-10, the highest cross-linked dextran, has a water regain ofabout 1mL/g of dry gel and Sephadex G-200, the lowest cross-linkeddextran, has a water regain of about 20 mL/g of dry gel. In the swellingprocess, the gels become filled with water. Fig.2. Structure of epichlorohydrin cross linked Dextran
Table 1: Properties of gels used in gel permeation (filtration) chromatography Water ExclusionMaximum Maximum Gel regain limit hydrostatic flow rate (mL/g) pressure cm (ml,min) H2O Sephadex G-10 1.0 700 200 100 Sephadex G-15 1.5 1500 200 100 Sephadex G-25 2.5 5000 200 50 Sephadex G-50 5.0 30000 200 25 Sephadex G-75 7.5 70000 160 6.4 Sephadex G-100 10.0 150000 96 4.2 Sephadex G-150 15.0 300000 36 1.9 Sephadex G-200 20.0 600000 16 1.0 6 Sepharose 6B NA 4 x 10 200 1.2 6Sepharose CL 6B NA 4 x 10 >200 2.5 6 Sepharose 4B NA 20 x 10 80 0.96 6Sepharose CL 4B NA 20 x 10 120 2.17 6 Sepharose 2B NA 40 x 10 40 0.83 6Sepharose CL 2B NA 40 x 10 50 1.25 Bio-Gel P-2 1.5 1800 >100 110 Bio-Gel P-4 2.4 4000 >100 95 Bio-Gel P-6 3.7 6000 >100 75 Bio-Gel P-10 4.5 20000 >100 75 Bio-Gel P-30 5.7 40000 >100 65 Bio-Gel P-60 7.2 60000 100 30 Bio-Gel P-100 7.5 100000 100 30 Bio-Gel P-150 9.2 150000 100 25 Bio-Gel P-200 14.7 200000 75 11 Bio-Gel P-300 18.0 400000 60 6 Bio-Gel A-0.5m NA 500000 >100 3 6 Bio-Gel A-1.5m NA 1.5 x 10 >100 2.5 6 Bio-Gel A-5m NA 5 x 10 >100 1.5 6 Bio-Gel A-15m NA 15 x 10 90 1.5 6 Bio-Gel A-50m NA 50 x 10 50 1.0 6Bio-Gel A-150m NA 150 x 10 30 0.5Bio-Gel is a trade name of Bio-Rad LaboratoriesSephadex and Sepharose are trade name of Pharmacia Fine Chemical
Polyacrylamide gels are long polymers of acrylamide cross-linked withN.Nmethylene-bisacrylamide (Fig. 3). Fig.3. Structure of cross-linked polyacrylamideThe gels are commercially produced by BioRad Laboratories, Richmond.California, as the Bio-Gel P series. Like the Sephadex G series. the Bio-Gels differ in degree of cross-linking and in pore size; the Bio-Gels,however. have a wider range of pore sizes than is available in theSephadex G series See Table. 1 for the exclusion limits and properties ofthe different Bio-Gels.Agarose is a gel material with pore sizes larger than cross-linked dextranor polyacrylamide. Agarose is the neutral polysaccharide fraction of agar.It is composed of a linear polymer of D-galactopyranose linked ( 1->4)3,6 anhydro-L-galactopyranose, which is linked (1-> 3) (Fig. 4).
D-galactose (-1->4) 3,6-Anhydro-L-galactoseFig.4. Structure of the repeating unit of agarose, D-galactopyranose linked (-1->4)to 3,6-anhydro-L-galactopyranose, which is linked (-1-3) to the next D-galactopyranose residueWhen the polysaccharide is dissolved in boiling water and cooled, itforms a gel by forming inter-and intramolecular hydrogen bonds. Thepore sizes are controlled by the concentration of the agarose. Highmolecular weight materials such as protein aggregates, chromosomalDNA, ribosomes, viruses, and cells have been fractionated on agarosegels. Bio-Rad markets the agarose Bio-Gel A series with differentmolecular exclusion limits, and Pharmacia markets agarose as Sepharoseand Sepharose CL. The latter is Sepharose cross-linked by reacting withalkaline 2,3-dibromopropanol to give an agarose gel with increasedthermal and chemical stability. Table 1 gives the properties of thedifferent Sephadex, Bio-Gel, and Sepharose gels.The separations that may be achieved by gel permeation chromatographyare based on differences in the molecular sizes of the molecules. Themethod is used for both preparative and analytical purposes. The latterhas been especially useful in determining the molecular weights ofproteins. The proteins are chromatographed on a gel column and the
elution volume of the protein determined. Proteins with known molecularweights are also chromatographed and the elution volumes determined.Then, from a plot of log molecular weight versus elution volume, themolecular weight of an unknown protein may be determined (Fig. 5).Fig.5. Molecular weight determination of proteins by gel permeation chromatographyusing Sephadex G-100 as the gel bed: log molecular weight is plotted versus elutionvolume.Gel chromatography provides a rapid and mild method of removing saltsand other small molecules from high molecular weight biomolecules. Thesample containing the biomolecules and the salt is passed over a gelcolumn whose exclusion limit is below the molecular weight of thebiomolecules. The biomolecules which do not enter the gel emerge in thevoid volume of the column, while the salts enter the gel and are retarded,and therefore are removed from the biomolecules.
Ion-exchange chromatography Ion-exchange chromatography is a variation of adsorptionchromatography in which the solid adsorbent has charged groupschemically linked to an inert solid. Ions are electrostatically bound to thecharged groups; these ions may be exchanged for ions in an aqueoussolution. Ion exchangers are most frequently used in columns to separatemolecules according to charge. Because charged molecules bind to ionexchangers reversibly. Molecules can be bound or eluted by changing theionic strength or pH of the eluting solvent. Two types of ion exchanger are available: those with chemicallybound negative charges are called cation exchangers and those withchemically bound positive charges are called anion exchangers. Thecharges on the exchangers are balanced by counterions such as chlorideions for the anion exchangers and metal ions for the cation exchangers.Sometimes buffer ions are the counterions. The molecules in solutionwhich are to be adsorbed on the exchangers also have net charges whichare balanced by counterions. As an example of an ion-exchange process,let us say that the molecules to he adsorbed from solution have a negativecharge (X-), which is counterbalanced by sodium ions (Na +). Suchnegatively charged molecules can be chromatographed on an anion
exchanger (A+), which has chloride ions as the counterion to give A+Cl-.When (Na+ X-) molecules in solution interact with the ion exchanger, theX- displaces the chloride ion from the exchanger and becomeselectrostatically bound to give A+X-, simultaneously releasing sodiumions. This process of ion exchange is illustrated in Figure 1. A similarbut opposite process would take place for positively charged molecules(Y+ Cl-) which would be chromatographed on cation exchangers (C-Na+).Thus the cation exchangers will bind positively charged molecules fromsolution and the anion exchangers will bind negatively charged moleculesfrom solution. One of the inert materials used in this type of chromatography iscross-linked polystyrene, to which the charged groups are chemicallybound. In the separation of biologically important macromolecules,such as enzymes and proteins. Figure 1. The process of anion-exchange chromatography Cellulose and cross-linked dextran (Sephadex) are used as thesolid supports and charged groups such as diethylaminoethyl (DEAE)
or carboxymethyl (CM) are chemically linked to them to give anionand cation and the exchangers respectively. The preparation andcommercial availability of these materials beginning in the 1960 providedthe biochemist with powerful tools for separation of proteins andnucleic acid Figure 2 presents partial structures of DEAE-cellulose andCM –celluloseFigure 2. Partial structures of diethylaminoethyl-cellulose and carboxymethyl-cellulose. The DEAE and CM groups are shown attached to the C6-hydroxyl groupof glucose. The DEAE and CM groups are also found attached to the hydroxyl groupsof C2 and C3. The total degree of substitution of the DEAE and CM groups must beless than one group per five glucose residues to maintain a water-insoluble product. Table 1. Pretreatment steps for DEAE-cellulose and CM -cellulose ion exchangers Cellulose First treatment Intermediate Second pH treatment DEAE 0.5 M HCl 4 0.5 M NaOH CM 0.5 M NaOH 8 0.5 M HCl
The dry ion-exchange celluloses are pretreated with acid and base toswell the exchangers so that they become fully accessible to the chargedmacromolecules in solution. The weighed exchanger is suspended in 15volumes (w/v) of the "first treatment," acid or alkali depending on theexchanger (Table. 1), and is allowed to stand at least 30 minutes but notmore than 2 hours. The supernatant is decanted and the exchanger iswashed until the effluent is at the "intermediate pH" The exchanger isstirred into 15 volumes of the "second treatment" and allowed to stand for
an additional 30 minutes. The second treatment is repeated and theexchanger is washed with distilled water until the effluent is close toneutral pH. The treated exchanger is placed into the acid component ofthe buffer (the pH should be less than 4.5) and degassed under vacuum10 cm Hg pressure) with stirring, until bubbling stops The exchanger isthen titrated with the basic component of the buffer to the desired pH,filtered, and suspended in fresh buffer to complete the pretreatment. Theexchanger is allowed to settle and the "fines" (fragments < 10 m indiameter) above the settled exchanger are removed by decantation.Buffer is added to the exchanger so that the final volume of the slurry isl50% of the settled wet volume of the exchanger. The column is thenpacked with the slurry of the exchanger, the sample is applied, andelution is performed as described for adsorption chromatography. Three general methods are used for eluting molecules from theexchanger:(a) Changing the pH of the buffer to a value at which binding isweakened (i.e., the pH is lowered for an anion exchanger and raised for acation exchanger),(b) Increasing the ionic strength by increasing the concentration of saltin the elution solvent, thereby weakening the electrostatic interactionsbetween the adsorbed molecule and the exchanger, and
(c) Performing affinity elution. In affinity elution the adsorbed moleculeis usually a macromolecule that is desorbed from the affinity ligand byadding a molecule that is charged and of opposite signs to the netcharge on the macromolecule and has a specific affinity for themacromolecule. Thus, the reduction of the net charge on themacromolecule weakens its electrostatic interaction with the exchangersufficiently to permit the elution of the macromolecule from the affinityligand.The stages of anion exchange chromatography.An example of the use of ions exchange resinsIs the purification of cytochrome C:Cytochrome C has an isoelectric point (pI) of 10.05; that is at pH 10.05the number of positive charges will equal the number of negativecharges. A cloumn containing a cation exchanger buffered, at pH 8.5,
is prepared. This column has a full negative charge. Cytochrome C atpH 8.5 has a full positive charge. An Impure solution of cytochromeC at pH 8.5 placed on the column, and water is passed throughthe column (the pI of proteins is usually 7.0 or less) butcytochrome C is held firmly by electrostatic attraction to the resinheads. If the eluting solvent pH is raised to about 10, thecytochrome C will now has a net zero charge and will pass rapidlythrough as a pure component.
Histadine ,Aspartic,glycine,tyrosineIn anion exchange chromatography,which seprate first and why
AFFINITY CHROMATOGRAPHY Affinity chromatography is a specialized type of adsorptionchromatography in which a specific type of molecule is covalently linkedto an inert solid support. This specific molecule called a ligand, has ahigh binding affinity for one of the compounds in a mixture ofsubstances. The process uses the unique biological property of thesubstance to bind to the ligand specifically and reversibly and provides ahigh degree of selectivity in the isolation and purification of biologicalmolecules Fig. 1. The steps of affinity chromatography
A solution containing the substance to be purified. Usually amacromolecule such as a protein (enzyme, antibody, hormone. etc.).Polysaccharide or nucleic acid is passed through a column containing aninsoluble inert polymer to which the ligand has been covalently attached.The ligand may be specific competitive inhibitors, substrate analogues,product analogues, coenzymes and so on. Molecules in the mixture nothaving affinity for the ligand pass through the column. Wide moleculesthat have specific affinity for the ligand are bound and retained on thecolumn. The specifically adsorbed molecules) can be eluted by changingthe ionic strength the pH or by the addition of a competing ligand. In onechromatographic step. The method is capable of isolating a singlesubstance in a pure form. It has thus become a powerful tool in theisolation and purification of enzymes, antibodies, antigens, nucleic acids.Polysaccharides, coenzyme or vitamin binding proteins, repressorproteins, transport proteins, drug or hormone receptor structures andother biochemical materials.The Inert Support and the Ligand The inert solid supports are the same materials discussed in thepreceding sections: cross-linked dextran cross-linked polyacrylamide,agarose and cellulose. The macromolecules to be separated should not beretarded by a gel filtration process but should be retarded only by the
specific interaction with the ligand. The ligand must be a molecule thatdisplay, special and unique affinity for the macromolecule to be purifiedit also must have a chemical group that can be modified for covalentlinkage to the solid support without destroying or seriously decreasing itsinteraction with the macromolecule to be purified. Also for successfulaffinity chromatography, the chemical groups of the ligand that arccritical for the binding of the macromolecule to be purified must besufficiently distant from the solid support to minimize steric interferencewith the binding process. This steric problem has been solved by adding along, hydrocarbon chain spacer arm to the solid support and coupling theligand to the end of the arm. Alternatively the hydrocarbon arm may beattached to the ligand and the arm attached to the solid support.Attachment of the Ligand to the Solid Support The polysaccharide solid supports-cross-linked dextran, agarose,and cellulose can be activated by reaction with alkaline cyanogenbromide. The products that arc formed upon coupling of the activatedpolysaccharides with amino compounds are derivatives of amino carbonicacid. The reactions are the following:
If the ligand contains an amino group, it can be coupled directly tothe activated polysaccharide. A spacer arm can be introduced bysequential reaction with a diaminoalkane and glutaraldehyde. The aminogroup on the ligand can then be coupled to the free aldehyde group. If the ligand contains an aldehyde group instead of an amino group,it can be coupled directly to the free amino group of the diaminoalkane.Ligands may be coupled to polyacrylamide by displacing the amide group
of the polyacrylamide by heating with a diaminoalkane (c), followed byreaction with glutaraldehyde (d). The Schiff base that results from the reaction of glutaraldehydewith an amino group may be stabilized by reduction with sodiumcyanoborohydride without affecting the aldehyde group. The ligand canthen be coupled to the aldehyde group.Another method of activating polyacrylamide is to form the hydrazidederivative by reaction with hydrazine hydrate. When an amino, aldehyde,or hydrazide group is incorporated onto the solid support, the supportbecomes activated so that ligands may be attached through amino,carboxyl, phenolic, or imidazole groups.
Gel electrophoresisThe movement of a charged presented by Equation 1.0 subjected to anelectric field: (Equation 1.0)whereE = the electric field in volts/cmq = the net charge on the moleculef = frictional coefficient, which depends on the mass and shape of the moleculeV = the velocity of the moleculeThe charged particle moves at a velocity that depends directly on theelectrical field (E) and charge (q) but inversely on a counteracting forcegenerated by the viscous drag (f ) The applied voltage represented by E inEquation 1.0 is usually held constant during electrophoresis, althoughsome experiments are run under conditions of constant current (where thevoltage changes with resistance) or constant power (the product ofvoltage and current). Under constant-voltage conditions, Equation 1.0shows that the movement of a charged molecule depends only on theratio q/f. For molecules of similar conformation (for example, acollection of linear DNA fragments or spherical proteins), varies withsize but not shape; therefore, the only remaining variables in Equation1.0 are the charge (q) and mass dependence of (f ) meaning that under