1 Chromatography  is the collective term for a set of laboratory techniques for separate and identify the components of a mixture. Mikhail Tswett, Russian, 1872-1919 Botanist In 1906 Tswett used 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” 2 Classification of chromatography according to mobile phase: 1. Liquid chromatography: mobile phase is a liquid. (LLC, LSC). 2. Gas chromatography : mobile phase is a gas. (GSC, GLC). Classification according to the packing of the stationary phase: 1. Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2. Paper chromatography (PC): the stationary phase is a thin film of liquid supported on an inert support. 3. Column chromatography (CC): stationary phase is packed in a glass column. 3 General Principles:  Separation of molecules :by distribution between a stationary phase and a mobile phase.  A stationary phase can be solid, gel, or liquid. Also called matrix, resin, or beads.  The mobile phase is the solvent and it is usually a liquid, but may also be a gas.  The compounds to be separated are considered solutes.  The separation of materials is based on differential partitioning [retardation]between the mobile and stationary phases. 4 1-Paper chromatography [PC]:  method for testing the purity of compounds and identifying substances.  useful technique because it is relatively quick and requires small quantities of material.  substances are distributed between a stationary phase and a mobile phase.  [solutes are separated according to their solubility in mobile phase, or their attraction to the stationary phase]. The mobile phase: is a developing solution that travels up the stationary phase, carrying the samples with it. [shallow layer of solvent] The stationary phase: is usually a piece of high quality filter paper. [cellulose ] polar substance 5 6 Separation is depend on: Components of the sample will separate on the stationary phase according to how strongly they adsorb to the stationary phase versus how much they dissolve in the mobile phase. This paper is made of cellulose [a polar substance] and the compounds within the mixture travel farther if they are non-polar. So, More polar substances bond with So: the cellulose paper more quickly, and therefore do not travel as far. Different compounds in the sample mixture travel at different rates due to  differences in solubility in the solvent.  differences in their attraction to the fibers in the paper. 7 Paper Chromatography Descending:  In this method, the solvent is kept in a trough at the top of the chamber and is allowed to flow down the paper.  The liquid moves down by capillary action as well as by the gravitational force.  In this case, the flow is more rapid as compared to the ascending method. Ascending:  In this method, the solvent moves upward against gravitational force.  The only force that cause the motion[of solvent and the compounds] is capillary force. So the speed of the process is slow. Substances that cannot be separated by ascending method, can be separated by the above descending method. 8 Descending Paper Chromatography 9  2. Thin Layer Chromatography (TLC):  The method is rapid and separations can be completed in less than one hour.  TLC is a widely used. The stationary phase[Stationary phase: adsorbent]:  is a thin layer (0.25 – 0.5 mm) of adsorbent like silica gel - a polar substance- , [aluminium oxide or magnesium silicate] spread uniformly over the surface of a flat, inert surface of the glass plastic plate.  [The stationary phase + support medium should be inert].  Mobile phase: solvent. 10 separation depend on:  Partition of a solute between a moving solvent phase and a stationary aqueous phase. The solute therefore moves in the direction of a solvent flow at a rate determined by the solubility of the solute in the moving phase. Thus a compound with high mobility is more attracted to the moving solvent [mobile phase]than to the stationary phase. Traveling of solvent via capillary action 11 Factors effect the separation [resolution effects] : 1. Ion exchange effect: Any ionized impurities in the support medium will tend to bind or attract oppositely charged ions and will therefore reduce the mobility of these solutes. [resulting in bad resolution] 2. Temperature: since temperature can affect the solubility of a solute in a given solvent temperature is also an important factor and often a chromatography laboratory has a fixed temperature for optimum results. [increased temperature  increase solubility] 12 Factors effect the separation [resolution effects] : cont’d----3. The molecular weight of a solute affects the solubility and hence chromatographic performance. [increased M wt less solubility]. 4. Adsorption of compound (solute) onto support medium: Although the support medium is inert, this is not always the case. If a solute tends to bind to the support medium this will slow down its mobility in the solvent system. 5. Composition of the solvent: Since some compounds are more soluble in one solvent than in the other the mixture of solvents used affect separation of the compounds. 13 Thin layer chromatography[TLC] 14 15 Samples Analysis Amino acids  Specific color reagents are sprayed onto the plate or the paper [ninhydrin]. sugars  spraying the plate or the paper with [aniline-diphenylamine].  The paper or plate remaining after the experiment is known as the Chromatogram. A visualization method can be: Ultraviolet light Iodine vapors to stain spots Reagents that selectively stain spots while leaving others unaffected. 16 Samples Analysis [Expression of the results]: -Relative flow [Rf]:  Used to express the performance of a solute in a given solvent system/stationary medium.  Rf value may be defined as the ratio of the distance moved by a compound to that moved by the solvent.  Rf value is constant for a particular compound, solvent system and stationary phase. Its value is always between zero and one Rf = Distance moved by solute Distance moved by solvent 17 [Rf] Value:  If Rƒ value of a solute is closer to zero, the solute has more attraction to stationary phase.  If Rƒ value of a solute is closer to 1, then the solute has more affinity for the mobile phase and travels further.  The final chromatogram can be compared with other known mixture chromatograms to identify sample mixes, using the Rf value in an experiment. 18 19 (TLC) paper chromatography. -less time required than paper chromatography. -less time required. -It has better separations than paper chromatography. -separate the sample. It has better resolution and to allow for quantitation. Low resolution but also allow for quantitation. -It is faster runs than paper, Wide choice between different adsorbents[stationary phase]. compact zonal spread[concentrated for quantitation analysis in need ]. -It is fast. cellulose Expanded zonal spread[ not concentrated for quantitation analysis in need ]. 20 - Applications[TLC, PC]:     Identifying unknown compounds present in a given substance. Qualitative and semi quantitation tests. Separation of compounds in a sample. Identify the purity of the sample. 21 COLUMN CHROMATOGRAPHY Column Chromatography was developed by the American chemist D.T Day in 1900, M.S. Tswett, the Polish botanist, in 1906 used adsorption columns in his investigations of plant pigments. 22 Different Types of chromatography according to the force of separation Mode or type Stationary phase Mobile phase Mechanism Adsorption Chromatography 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 Thin film of liquid formed on the surface of a solid inert support Liquid or gas 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 are immobilized Liquid or gas Special kind of solute molecules interact with those immobilized on the stationary phase 23 Term Definition 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. Solvent Effluent Eluent Eluate Retention volume (VR) Mobile liquid phase with no affinity to the stationary phase (i.e. inert towards it) & no effect on solutes. Any liquid that passes out of the column. Any liquid that has lesser affinity to the stationary phase than solutes but is capable to move them out of the column. Fraction of eluent containing a required specific substance. (or retardation volume): Volume of mobile phase that passes out of the column, before elution of a specific substance. 24 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 methods. In all cases avoid inclusion of air bubble 2. Sample application 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). 25 Elution techniques Technique Procedure Isocratic elution 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. 26 Factors affecting solutes separation in CC ( Factors affecting column efficiency) Factor Effect Particle size of solid stationary phase (or of support) Decrease of size improves separation (but very small 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 broadening 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. 27 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. 28 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. 29 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. 30 Selection of the solid support The support material should: adsorb & retain the 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. 31 Selection of the mobile phase The liquid stationary & mobile phases should have a considerable difference between their solvent strength parameters. 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). 32 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. Gel-Permeation Chromatography is a mechanical sorting of molecules based on the size of the molecules in solution. Small molecules are able to permeate more pores and are, therefore, retained longer than large molecules. 33 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. 34 Affinity Chromatography Mobile phase Mobile phase is a liquid as water or dilute alcohol 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) of the solute. Vo ~ 1 / M. 35 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 mono-polysaccharides & soluble RNA. Separation of myoglobin & hemoglobin. Separation of alkaloids & purification of enzymes. 36 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". 37 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 38 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 - + is the matrix) Na2SO4 solution Na2+2 SO4-2 SO4-2 + 2 Na+Cl - 39 Regeneration of the resin Ion exchange process is generally reversible example in the following: 2 Na+ + Ca++ 2Cl - → Ca++ + 2 Na+ Cl – 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 example a solution of NaCl. 40 Applications of Ion Exchange Chromatography 1- Water softening: Removal of Ca2+ , Mg2+ & other multivalent ions causing hardness of water by filtration through a layer of strong cation resin. 2-Water demineralization: Removal of cations & anions dissolved in water. Usually carried by the two step technique in which two columns of strongly acid cation exchanger in [H+] form & strongly basic anion exchanger in [OH-] form are used in sequence. 3- Neutralization: Cationic exchanger in [H+] can be used to neutralize alkali hydroxide & anionic exchanger in [OH-] form to neutralize the acidity. 41 Application cont’d---4- Separation of electrolytes from non-electrolytes. 5- Separation of carbohydrates & their derivatives: Uronic acids separated on anion exchanger. Sugars converted into ionized form by using borate& separated on strong anion exchanger. Hexosamines separated on strong cation exchanger.