Chromatography

Chromatography

History Mikhail Tswett, Russian, 18721919 Botanist In 1906 Tswett used to chromatography to separate plant pigments He called the new technique chromatography because the result of the analysis was 'written in color' along the length of the adsorbent column Chroma means “color” and graphein means to ”“write

Importance Chromatography has application in every branch of the physical and biological sciences Nobel prizes were awarded between 1937 and 1972 alone 12 for work in which chromatography played a vital role

Chromatography is a physical method of separation in which the components to be separated are distributed between two phases one of which is stationary (stationary phase) while the other (the mobile phase) moves .through it in a definite direction The chromatographic process occurs due to differences in the distribution constant of .the individual sample components

Chromatography Is a technique used to separate and identify the components of a mixture. Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium. Molecules that spend most of their time in the mobile phase are carried along faster.

Classification of chromatography according :to mobile phase Liquid chromatography: mobile phase is a -1 .liquid. (LLC, LSC) Gas chromatography : mobile phase is a -2 .gas. (GSC, GLC)

Classification according to the packing of the :stationary phase Thin layer chromatography (TLC): the stationary -1 phase is a thin layer supported on glass, plastic or .aluminium plates Paper chromatography (PC): the stationary phase -2 is a thin film of liquid supported on an inert .support Column chromatography (CC): stationary phase -3 .is packed in a glass column

Plastic TLC Sheets Aluminum TLC Sheets

Glass TLC

PC

CC

Classification according to the force of :separation .Adsorption chromatography -1 .Partition chromatography -2 .Ion exchange chromatography -3 .Gel filtration chromatography -4 .Affinity chromatography -5

Thin layer chromatography (TLC) is a method for identifying substances and .testing the purity of compounds TLC is a useful technique because it is relatively quick and requires small .quantities of material

Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and a .mobile phase :The stationary phase is a thin layer of adsorbent (usually silica gel or alumina) .coated on a plate :The mobile phase is a developing liquid which travels up the stationary .phase, carrying the samples with it Components of the samples will separate on the stationary phase according to how much they adsorb on the stationary phase versus .how much they dissolve in the mobile phase

Thin Layer Chromatography (TLC)

TLC

Preparing the Chamber To a jar with a tight-fitting lid add enough of the appropriate developing liquid so that it .is 0.5 to 1 cm deep in the bottom of the jar Close the jar tightly, and let it stand for about 30 minutes so that the atmosphere in the jar .becomes saturated with solvent

Preparing the Plates for Development With a pencil, etch two small notches into the adsorbent .about 2 cm from the bottom of the plate The notches should be on the edges of the plate, and each notch should be the same distance up from the bottom of .the plate The notches must be farther from the bottom of the plate .than the depth of the solvent in the jar Using a drawn-out capillary tube, spot the samples on the .plate so that they line up with the notches you etched

Question: What is wrong with the plate shown ?below

Developing the Plates After preparing the development chamber and spotting .the samples, the plates are ready for development Be careful to handle the plates only by their edges, and try to leave the development chamber uncovered for as .little time as possible When the plates are removed from the chamber, quickly trace the solvent front (the highest solvent level on the .plate) with a pencil

Identifying the Spots (visualization) If the spots can be seen, outline .them with a pencil If no spots are obvious, the most common visualization technique is to hold the plate under a UV .lamp Many organic compounds can be seen using this technique, and many commercially made plates often contain a substance which aids in the visualization of .compounds

Visualizing Agents Alkaloids: Dragendorff’s reagent Cardiac glycosides: Antimony trichloride Sugar: Aniline phthalate Amino acids: Ninhydrin

Interpreting the Data The Rf (retention factor) value for each spot .should be calculated It is characteristic for any given compound on the same stationary phase using the same .mobile phase for development of the plates Hence, known Rf values can be compared to those of unknown substances to aid in their .identifications

Note: Rf values often depend on the temperature and( .the solvent used in the TLC experiment the most effective way to identify a compound is to spot known substances – authentic - next to unknown ).substances on the same plate In addition, the purity of a sample may be estimated .from the chromatogram An impure sample will often develop as two or more pure sample will show only one spot spots, while a

Summary A TLC plate is a sheet of glass, metal, or plastic which is coated with a .thin layer of a solid adsorbent (usually silica or alumina) A small amount of the mixture to be analyzed is spotted near the .bottom of this plate The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in .the liquid This liquid, or the eluent, is the mobile phase, and it slowly rises up the .TLC plate by capillary action As the solvent moves past the spot that was applied, an equilibrium is established for each component of the mixture between the molecules of that component which are adsorbed on the solid and .the molecules which are in solution

In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than .others When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and the .separated components of the mixture are visualized If the compounds are colored, visualization is straightforward. Usually the compounds are not colored, .so a UV lamp is used to visualize the plates

Paper Chromatography A method of partition chromatography using filter .paper strips as carrier or inert support The factor governing separation of mixtures of solutes on filter paper is the partition between two .immiscible phases One is usually water adsorbed on cellulose fibres in .the paper (stationary phase) The second is the organic solvent flows past the .sample on the paper (stationary phase)

Partition occurs between the mobile phase and the .stationary aqueous phase bound by the cellulose The isolation depends on partition coefficient of the .solute c( stationary ) K c(mobile) Where, K:Is the partition coeffcient. C (Stationary: Is the concentration of the solute in the stationary. C (Mobile): Is the concentration of the solute in the mobile.

General Procedure .Choice of paper and solvent to be used -1 .Desalting of sample -2 .Application of the sample -3 .Equilibration of paper -4 .Development -5 .Detection -6 .Identification of substances -7

Techniques of development with various flow directions Radial development Ascending development Descending development

Descending Chromatography

Multiple chromatography Multiple chromatography includes all procedures in which the development is .repeated after one development is completed A- multiple development: the chromatogram is repeatedly developed in the same direction and thus the complete resolution of two or more substances which have Rf values close .together can be obtained As the mobile phase one can use either the same .solvent system or different solvent systems

:B- two-dimensional chromatography When large numbers of substances are to be separated .on a single chromatogram Development in a direction perpendicular to the first, and with a solvent system different from that used .initially is often necessary The sample is applied on one corner of a square piece of paper and after development with the first solvent, the paper is dried , rotated 90o and developed in the .second direction Usually, different types of solvents systems are used in each direction. It is essential that the first solvent be .completely volatile

Column Chromatography (CC) This includes chromatographic methods in :which .The stationary phase is packed into a column .The mobile phase is a moving liquid or gas According to the mechanism of separation of solutes, five major types of CC are distinguished. Usually, one mechanism predominates but does not exclude the others

Different Types of chromatography Mode or type Stationary phase Mobile phase Mechanism Adsorption Chromatography (CC) Solid that attracts the solutes Liquid or gas Solutes move at different rates according to the forces of attraction .to the stationary phase Partition Chromatography (Paper & TLC) Thin film of liquid Liquid or gas formed on the surface of a solid inert support Solutes equilibrate between the 2 phases according to their partition coefficients Ion Exchange Chromatography Solid resin that carries fixed ions & mobile couterions of opposite charge attached by covalent bonds Liquid containing electrolytes Solute ions of charge opposite to the fixed ions are attracted to the resin by electrostatic forces & replace the .mobile counterions Molecular Exclusion Chromatography Porous gel with no attractive action on solute molecules Liquid Molecules separate according to :their size 1.Smaller molecules enter the pores of the gel, and need a larger volume of eluent. 2.Larger molecules pass through the column at a faster rate. Affinity Chromatography Solid on which specific molecules Liquid or gas Special kind of solute molecules interact with those immobilized on

Column Chromatography Column chromatography Stationary phase is held in a narrow tube through which the mobile phase is forced under pressure or under the effect of gravity

Term Definition Solvent Mobile liquid phase with no affinity to the stationary phase .(i.e. inert towards it) & no effect on solutes Developer Any liquid with more affinity to the stationary phase than the solvent but less than solutes and just capable to move .them through the column Effluent .Any liquid that passes out of the column Eluent Any liquid that has lesser affinity to the stationary phase .than solutes but is capable to move them out of the column Eluate .Fraction of eluent containing a required specific substance Retention volume (VR) Volume of mobile phase that :)or retardation volume( passes out of the column, before elution of a specific .substance

Open Column Chromatography (Traditional column chromatography) Traditional column chromatography is characterized by addition of mobile phase under atmospheric pressure and the stationary phase is packed in a glass .column

Packing & operating the column 1- Packing The selection of the method of packing depends mainly on the density of the solid.Techniques .used are the wet, dry & slurry methods In all cases avoid inclusion of air bubbles

Sample Application -2 Apply evenly & in a concentrated solution to the top of the column which is protected from disturbance (e.g. add glass wool or .filter paper)

Elution techniques.1 Technique Isocratic elution Procedure Addition of solvent mixture of fixed composition .during the whole process Gradient elution Continuous or linear elution: in which there is continuous change in the composition of the mobile phase over a period of time (e.g. polarity, .pH or ionic strength) Step wise or fractional elution: in which the change is not continuous i.e. a sudden change in the composition of the mobile phase is followed by a period where the mobile phase is held .constant

Detection -4 On-column detection for colored or fluorescent compounds directly after .developing the chromatogram .Monitoring of eluted fractions (PC or TLC) Using special detectors connected to the column such as refractive index, UV detectors, etc

Factors affecting solutes separation in CC ( Factors affecting column efficiency) Factor Effect Particle size of solid stationary Decrease of size improves separation (but very small phase (or of support) .particles need high pressure) Column dimensions .Efficiency increases as ratio length / width increases Uniformity of packing Non uniform packing results in irregular movement of solutes through column & less uniform zone .formation, (i.e. band broadning or tailing) Column temperature Increase in column temperature results in speed of .elution but does not improve separation (tailing) Eluting solvent Solvents should be of low viscosity (to give efficient resolution) & high volatility (to get rapid recovery of .the substances) Solvent flow rate .Uniform & low flow rate gives better resolution Continuity of flow Discontinuous flow disturbs resolution Condition of adsorbent .Deactivation of adsorbent decreases separation Concentration of solutes .Substances of high concentration move slowly

Adsorption Column Chromatography :Adsorbents The most common are Alumina & Silica gel in which the interactions with solute molecules is due to OH groups .present on their surface More polar molecules are adsorbed more strongly & thus, will elute more slowly Strength of adsorption of polar groups (solutes) on polar support :is in the following order C C- O-CH3 -COOR C O -CHO -NH2 -OH -COOH Olefins Ethers Esters Lactones Aldehydes Amines .Phenols Acids

Applications in separation of natural products Alumina: sterols, dyestuffs, vitamins, esters, .alkaloids & inorganic compounds Not used for compounds containing phenolic or carboxylic groups .Silica gel: sterols & amino acids .Carbon: peptides, carbohydrates & amino acids .Calcium carbonate: carotenoids & xanthophylls

Partition Column Chromatography In this type, the packing consists of a theoretically inert support material coated .with a film of the liquid stationary phase The division into adsorption & partition is only of theoretical significance as in partition chromatography the adsorption effects of .the support can be felt

Selection of the solid support :The support material should .adsorb & retain the mobile stationary phase expose as large surface as possible to the mobile phase .be mechanically stable .be easy to pack not retard the solvent flow :Examples of solid supports Silica gel, diatomaceous earth (as kieselguhr, celite etc.) .& cellulose

Selection of the mobile phase The liquid stationary & mobile phases should have a considerable difference between their solvent strength .parameters Pure water Methanol Ethanol Propanol Acetone Ethyl acetate Ether Chloroform Dichloromethane Benzene Toluene Carbon .tetrachloride Cyclohexane Hexane Pentane e.g. if the stationary phase is water, pentane would be .the eluent of choice

The mobile phase is usually saturated with the stationary phase to overcome "stripping" (washing of the stationary phase from the .column by the mobile phase)

Gel Permeation Chromatography (GPC) :This type is also known as Size Exclusion Chromatography (SEC) Molecular Exclusion Chromatography (MEC) Molecular Sieve Chromatography (MSC) Gel Filtration Chromatography (GFC) .Gel Chromatography

The chromatographic method in which molecules are separated based on their size is called GEL FILTARATION CHROMATOGRAPHY

Stationary phase Porous polymeric matrix: formed of spongy particles, with pores completely filled with the liquid mobile .phase (gel) The gels (polymers) consist of open, three-dimensional networks formed by cross-linking of long polymeric .chains .The pore size varies with the degree of cross-linking The diameter of the pores is critical as separation is based on that molecules above certain size are totally excluded from the pores because they can not enter .the gel The interior of the pores is reached, partially or wholly, .by smaller molecules

Affinity Chromatography

Mobile phase Mobile phase is a liquid as water or dilute acohol Separation mechanism Based on difference between the solutes .molecular weights Molecules will distribute themselves outside & .inside the pores according to their size Larger are excluded, medium sized enter half.way & smallest permeate all the way

The retention volume Vo of a substance is inversely proportional to the molecular weight .(M. Wt) of the solute Vo 1 / M.wt Vo retention volume M.wt Molecular Weight

Applications of GPC to natural products .Determination of M. wt. of peptides, proteins & polysaccharides .Desalting of colloids e.g. desalting of albumin prepared with 2% (NH4)2SO4 .Separation of mixture of mono- & polysaccharides .Separation of amino acids from peptides & proteins .Separation of proteins of different molecular weights .Separation of mucopolysaccharides & soluble RNA .Separation of myoglobin & haemoglobin .Separation of alkaloids & purification of enzymes

Ion Exchange Chromatography Principle Process by which ions of an electrolyte solution are brought into .contact with an ion exchange resin The ion exchange resin is an insoluble polymer consisting of a "matrix" (Lattice or framework) that carries fixed charges (not exchangeable) and mobile active ions "counter ions" which are .loosely attached to the matrix In water, the counter-ions move more or less freely in the framework & can be replaced by ions of the same sign present in .the surrounding solution The "matrix" (framework) of a "cation exchanger" is considered as a crystalline non-ionized "polyanion" & the matrix of an "anion ."exchanger" as a non-ionized "polycation

Cation Exchangers .Active ions (counter ions) are cations The polar groups attached to the matrix are acidic (sulphonic acids, carboxylic acids, phenols, phosphoric acids) e.g. a cation exchanger in the free :carboxylic acid form X-COO- H X Frame work (matrix) ,COO- Fixed charge (anionic)Non-exchangeable H Counter ion (cation), Exchangeable

They are usually (but not always) supplied in the Na form: X-COO-Na or Na , Where Matrix e.g. exchange with CaCl2 aqueous solution Na Ca Cl2 2 Ca 2 Na Cl-

Anion Exchangers .Active ions (counter ions) are anions The polar groups attached to the matrix are .tertiary or quaternary ammonium groups (basic) e.g. Anion exchanger in the quaternary :ammonium form – X. NR3 OH X Framework (matrix) NR3 Fixed charge (cationic)Non exchangeable OH– counter ion (anion), Exchangeable-

They are supplied as the chloride rather than the hydroxide as the chloride form is a more - stable. Represented as : X. NR3 Cl or Cl - (where, e.g. exchange with 2 Cl - Na2 2 is the matrix) Na2SO4 solution -2 SO4 -2 SO4 2 Na Cl -

Regeneration of the resin Ion exchange process is generally reversible e.g :in the following – Na Ca Cl 2- Ca 2 Na Cl 2 The cation exchanger could be exhausted after exchanging all Na for Ca , the exchanger could be regenerated (loaded again with Na ) by contacting it with excess Na ions e.g. a .solution of NaCl

Types of Exchangers Ion Exchangers These are either cation or anion exchangers of either .organic or inorganic nature A- Inorganic ion exchangers Common clays, soils, minerals e.g. zeolites used for .""softening water .Disadvantage: low ion-exchange capacity :Advantages .Great resistance to high temperatures .High volume capacity .Great selectivity towards simple inorganic ions

B- Organic exchangers .They may be natural or synthetic : Preparation of organic synthetic ion exchangers Polycondensation of phenols, sulpho- & carboxyderivatives with formaldehyde cationic .exchangers Polycondensation of aromatic amines with .formaldehyde anionic exchangers These techniques yield products linear in structure & relatively soluble in water which are now replaced by resin materials based on styrene divinyl benzene .copolymers and polyacrylate

Applications of Ion Exchange Chromatography :Water softening -1 Removal of Ca2 , Mg2 & other multivalent ions causing hardness of water by filtration through a layer of strong .cation resin :Water demineralization-2 Removal of cations & anions dissolved in water. Usually carried by the two step technique in which two columns of strongly acid cation exchanger in [H ] form & strongly basic anion exchanger in [OH-] form are used in .sequence :Neutralization -3 Cationic exchanger in [H ] can be used to neutralize alkali hydroxide & anionic exchanger in [OH ] form to .neutralize the acidity

Separation of electrolytes from non- -4 .electrolytes Separation of carbohydrates & their -5 :derivatives .Uronic acids separated on anion exchanger Sugars converted into ionized form by using .borate& separated on strong anion exchanger Hexosamines separated on strong cation .exchanger

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