IntroductionChromatography involves a sample (or sample extract) being dissolved in a mobile phase (which may be a gas, a liquid or a supercritical fluid). The mobile phase is then forced through an immobile, immiscible stationary phase. The phases are chosen such that components of the sample have differing solubilities in each phase. A component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase. Chromatography (from Greek :chroma, colour and :"grafein" to write) is the collective term for a family of laboratory techniques for the separation of mixtures. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture and allows it to be isolated. Chromatography may be preparative or analytical. Preparative chromatography seeks to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography normally operates with smaller amounts of material and seeks to measure the relative proportions of analytes in a mixture. The two are not mutually exclusive. Chromatography, although primarily a separation technique, is mostly employed in chemical analysis. Nevertheless, to a limited extent, it is also used for preparative purposes, particularly for the isolation of relatively small amounts of materials that have comparatively high intrinsic value. Chromatography is probably the most powerful and versatile technique available to the modern analyst. In a single step process it can separate a mixture into its individual components and simultaneously provide an quantitative estimate of each constituent. Samples may be gaseous, liquid or solid in nature and can range in complexity from a simple blend of two entantiomers to a multi component mixture containing widely differing chemical species. Furthermore, the analysis can be carried out, at one extreme, on a very costly and complex instrument, and at the other, on a simple, inexpensive thin layer plate.

Chromatography has been defined as follows, Chromatography is a separation process that is achieved by distributing the components of a mixture between two phases, a stationary phase and a mobile phase. Those components held preferentially in the stationary phase are retained longer in the system than those that are distributed selectively in the mobile phase. As a consequence, solutes are eluted from the system as local concentrations in the mobile phase in the order of their increasing distribution coefficients with respect to the stationary phase; ipso facto a separation is achieved.

What is Chromatography?Chromatography is a powerful technique for separating mixtures. There are different types of chromatography, such as paper, thin layer, or column chromatography (amongst others), each with its own strengths and weaknesses. Chromatography systems have a stationary phase (which can be solid or liquid) and a mobile phase (usually liquid or gas). In column chromatography both phases are placed in a column.

Advantages of chromatographyhigh selective process due to the large number of stationary and mobile phase combinations can separate very complex mixtures: sugars, proteins, enantiomers, drugs, fine chemicals, flavorings, foods, ... separated components can be collected individually only small sample sizes needed (for analyses) analyses can be highly accurate and precise easy upscaling simple and rapid

separation step for the purification of high value products with highest purityThe basis of all forms of chromatography is the partition or distribution coefficient. (Kd), which describes the way in which a compound distributes itself between two immiscible phases. For the most part, all chromatography systems consist of the stationary phase which for most of biochemical purposes are solids or gels. The mobile phase is liquid or gaseous. The mobile phase flows over and around the stationary phase. In practice separating molecules requires that the various molecules will have a different partition between the two phases. Examples include: Adsorption Equilibrium - hydrophobic chromatography is an example. Proteins with a high concentration of hydrophobic amino acids or modified with lipids will often distribute themselves more with the mobile phase Ion Exchange Chromatography A flexible technique used mainly for the separation of ionic or easily ionizable species. The stationary phase is characterized by the presence of charged centers bearing exchangable counterions. Ion Exchange chromatography is one of the most used and well defined chromatographic methods. Gel Filtration - For this type of chromatography the stationary phase is the liquid trapped inside of the matrix. Affinity Chromatography - is an equilibrium between a stationary immobilized ligand and a mobile liquid phase. Factors which influence chromatography Theoretical Plates the number of equilibrations that a compound makes with the stationary phase

DESCRIPTION OF DIFFERENT TYPES OF CHROMATOGRAPHY I. PARTITION CHROMATOGRAPHY Differential solubility of solutes in mobile and stationary phases - paper - thin layer - gas liquid PAPER AND THIN LAYER Stationary phase Liquid which is physically or co-valently attached to inert support matrix Mobile Phase: Paper, TLC Solvent less polar than stationary phase ?less polar solutes partition preferentially into mobile phase and migrate more quickly

Rf value Distance migrated by solute Distance of solvent front Advantages of TLC over paper chromatography - Better defined, uniform support matrix - better resolution of solutes - less spreading of solutes (zone spreading), ?sharper spots - shorter times required for separations - can be adapted to variety of separation procedures Disadvantages of TLC - smaller sample size - more sensitive to presence of salts, interfering substances (streaking) GAS LIQUID CHROMATOGRAPY Run under conditions of high temperature (to maintain solutes being separated in gaseous phase) and high pressure (to maintain flow rate) Mobile Phase: Inert gas N2, He, Ar Stationary Phase - liquid (organic) bonded to surface of an inert support, (eg.: glass beads, teflon) - columns generally 0.5 cm in diameter and between 1m and 20 m in length. Columns are frequently coiled to accommodate length. - high boiling temperature; remains liquid at high temperature - Capillary chromatography: liquid phase bound to surface of capillary tube, inside diameter 0.25mm, length 10 to 100m. - samples may have to be derivatized to make them volatile under conditions of chromatography Only significant limitation: requires heat stability and volatility of samples. II. ADSORPTION CHROMATOGRAPHY Some types of adsorbents used in adsorption chromatography

Alumina silica gel Fluorisil hydroxyapatite

small organic molecules, lipids amino acids, lipids, carbohydrates neutral lipids proteins, neutrallipids

separation based on differential strength of adsorption of solutes to surface of finely powdered adsorbent.

- Binding involves weak - Non-ionic forces: e.g. H-bonding, van der Waal interactions - OH and aromatic groups generally tend to enhance binding - stationary phase and uses: - silica amino acids, lipids, carbohydrates - alumina - lipids hydroxyapatite : proteins, nucleic acids (separation of single stranded DNA from double stranded DNA) Other media include charcoal and cellulose - Mobile phase - polarity similar to molecules being separated III. ION EXCHANGE CHROMATOGRAPHY - Anionic exchange R-(+) Cl- + HX - Cationic exchange R-(-) Na+ + HX R-(-) H+ + NaX R-(+) X- + HCl

STEPS IN EXCHANGE REACTION 1) Diffusion to resin FAST 2) Diffusion to exchange site SLOW 3) Exchange VERY FAST 4) Diffusion of exchanged ion back to surface of resin SLOW 5) Diffusion of exchanged ion away from resin FAST Note: Small ions equilibrium rx with functional groups on resin - large polyvalent ions (e.g. proteins) binding usually all or none PROPERTIES OF RESIN 1 Composition of resin (matrix) 2 Nature of functional (exchange) group 3 Identity of counter ion 4 Pore size 5 Particle (bead) size

Composition of resin A) Synthetic resins, often cross linked polystyrene (Dowex Type) Separate molecules < 5000 Mr Styrene For structures see your notes

Divinyl benzene Polystyrene i) % Cross Linking - higher the x-linking the more rigid the bead and the lower the porosity x-2, x -4 x - 8 x -12 x -16 - high porosity, use for molecules with Mr > 300 400 - medium porosity - use for inorganic ions - low porosity, rigid

ii) Bead size Mesh Number The larger the mesh number the smaller the bead. eg. Mesh # Bead diameter 20 50 0.88 1.19 mm 400 < 0.04 mm Mesh size 20-50 50-100 100-200 200-400 uses and flow rate crude preparative work, very high preparative work, high analytical separations, medium high resolution analytical work, low

iii) Nature of Exchange Group Anionic exchangers e.g. strong base R-CH3N+(CH3)3 Cl-

Weak base R-NH3+ClCationic exchanger e.g. weak acid R- COO-H+ strong acid R-SO3-H+ iv) Nature of Counter ion -can change counter ion by washing resin with appropriate acid, base or salt solution N.B. Electrical neutrality maintained charges on counter ions = charges on resin v) Capacity # of exchange sites/gm dry wt. /wet volume theoritical - number of functional groups per unit resin Nature of solute: Charge and size of solute determine binding. i) In general the greater charge the stronger the binding. e.g. trivalent > divalent > monovalent e.g. At pH 8.4 glu will bind more strongly to anionic exchanger than His

ii) Size of hydrated ion may influence binding. For e.g. among monovalent cations the binding of K+ > Na+ > Li+ The radius of the hydrated ion is largest for and it binds with lowest affinity. Elution procedures: Potential for exchange reaction determined by 1) strength of ion exchanger weak or strong acid or base 2) strength of exchanging ions in buffer are they weakly or strongly ionized - strong ion exchangers form stable salts with strongly ionized counter ions ( e.g. Na+, K+, Cl-, SO4-) and weaker associations with weak counter ions (e.g. NH4+, COO-) e.g. acetate will elute before glu-6-P from Dowex-1 (a strong basic exchanger) because

COO- is a weaker acid than PO4=. - a strong counter ion readily displaces a weak counter ion from a strong ion exchanger (eg Cl- will displace formate-) - with weak acid or base exchangers intermediately stable associations are formed with strong counter ions and unstable associations are formed with weak counter ions. AFFINITY CHROMATOGRAPHY (most powerful) - Stationary phase - Inert matrix with co-valently attached ligand - ligand: molecule with specific affinity for desired molecule - substrate analogue - competitive inhibitor - linker to avoid steric interference with inert support - Elution: high salt, pH change, competing molecule - Immuno affinity chromatography - bound ligand antibody which recognizes and binds to desired protein - Lectin affinity chromatography - lectins proteins which recognize and bind to specific types of carbohydrates. - bind to glycoproteins on basis of oligosaccharide associated with the protein - used for isolation of glycoproteins HYDROPHOBIC (Reverse Phase) CHROMATOGRAPHY - alkyl chains are attached to inert support - hydrobicity determines strength of interaction with stationary phase - most hydrophobic associates most strongly Elute by increasing hydrophobicity of eluting solvent - hydrophobic molecules eluted as non-polarity of eluting solvent increases THEORITICAL ASPECTS PLATE THEORY Counter current distribution Partition coefficient: Clower phase / Cupper phase Effective partition coefficient: Ke = (Cl x Vl) / (Cu x Vu)

e.g. if K = 10 and Vl = 0.1 ml Vu = 1 ml then Ke (10 x 0.1) / (1 x 1) = 1 i.e. amount of solute in upper and lower phases is equal __________________ Insert Diagram Here see notes __________________ Upper phase = Mobile phase Lower phase = Stationary phase K = Cs / Cm Ke = (Cs x Vs) / (Cm x Vm)

practical - actual # of exchange sites available for reaction

will increase the separation

of similar but unique compounds. Chromatography columns are considered to consist of a number of adjacent plates or zones where there is enough space for a compound to achieve complete equilibrium between the mobile and stationary phase. Each zone is called a theoretical plate and the length of the column the plate height. The more theoretical plates the better the resolution of protein. Consider three columns each with a different number of theoretical plates It is important to remember that the plates do not really exist; they are a figment of the imagination that helps us understand the processes at work in the column.They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column, N (the more plates the better), or by stating the plate height; the Height Equivalent to a Theoretical Plate (the smaller the better). Peak resolution there are several factors which alter the ability of a column to separate or resolve proteins. Separation of compounds on a liquid chromatography column are dependent on the nature of the solute, the type of chromatography and the method of elution. Take for example two closely related proteins whose isoelectric points are close. Using a very steep gradient these proteins will not resolve. However a

shallower gradient which will effect the equilibrium between the solid and mobile phases to be discriminated between the two proteins can often increase the separation. Another factor that will effect resolution is peak broadening. This will result in overlap of closely eluted samples. Column matrix with large void volumes leads to preak broadening. The cost of using very small matrix is increasing back pressure. That is the same kind of thing that happens when you put your finger over the garden hose. Column velocity Since the solute

molecules are carried through the column by the mobile phase, it is natural that its speed has an influence on the process in the column. A slow flow rate will increase the time a protein is in the mobile phase and diffusion occurs resulting in peak broadening. On the other hand a very high flow rate will decrease the interaction of the protein with the stationary phase and decrease the number of possible equilibration, resulting in a reduction of theoretical plates possible. A Van Deemter plot is a plot of plate height vs. average linear velocity of mobile phase. Such plots are of considerable use in determining the optimum mobile phase flow rate.

1 THEORY OF CHROMATOGRAPHYSeparation of two sample components in chromatography is based on their different distribution between two non-miscible phases. The one, the stationary phase, a liquid or solid, is fixed in the system. The other, the mobile phase, a fluid, is streaming through the chromatographic system. In gas chromatography the mobile phase is a gas, in liquid

chromatography it is a liquid. Mobile Phase, m v

AStationary Phase, s Figure 1 Schematic presentation of a chromatographic system with partition of analyte A between the phases; v mobile phase velocity. The molecules of the analytes are distributed between the mobile and the stationary phase. When present in the stationary phase, they are retained, and are not moving through the system. In contrast, they migrate with the velocity, v, of the mobile phase when being there. Due to the different distribution of the particular analytes the mean residence time in the stationary phase differs, too, resulting in a different net migration velocity (see Figure 2). This is the principle of chromatographic separation. Principle of chromatographic separation: Different distribution of the analytes between mobile and stationary phase results in different migration velocities The position of the distribution equilibrium determines the migration velocity. It reflects the intermolecular interactions of the analyte with the stationary and the mobile phase. If only this process is considered, separation of the analytes as schematically shown in Figure 2 would be the result.tainer.Ernst Kenndler: Introduction to Chromatography

4 partitioning steps occur: thus it increases with increasing migration distance. The result is the formation of broadened analyte peaks, leading to a chromatogram as schematically depicted in Figure. 3. An appropriate theory of chromatography must be able to describe quantitatively the two counteracting phenomena: (i) the different, specific migration velocity and (ii) peak dispersion. It should finally enable to express the extent of peak separation by a characteristic number, the chromatographic resolution. From the methods used in practise (see Table about the systematic), the following discussion will concentrate on 8 1.1.3 Retention time, tRi The retention time is the time in which halve of the quantity of a solute, i, is eluted from the

chromatographic system. With other words, it is identical with the position of the peak maximum in case of a Gaussian elution profile. It is determined by the length of the column, L, and the migration velocity of the solute v Lk u tLii Ri

(1 + , ) = = (11) with tR0 , the residence time of a non-retained component (12) the dead time, which is the time the mobile phase needs to stream through the capillary column with an average linear velocity v measured over entire length, L. The retention time is given by (1 , ) Ri Ro i t = t + k (13) We can rewrite this equation as,

t = t + t k (14) and can interpret it as following: the total retention time consists of two contributions Ri m s t = t + t The one is tm, which is identical with tR0, the time the molecules are in mobile phase. This is equal for all components (and therefore non-specific). In the mobile phase the molecules exhibit the same velocity. The second part is the solute-specific contribution on the total retention time, that part which determines the separation selectivity. It is the k-fold dead-time (tR0.k). This contribution depends on Ki, and consequently on the chemical interactions between analyte and stationary liquid. Separation selectivity can thus be established due to the selection of the phase system, when differences in the partition constants are achieved. Transformation of eq. 13 leads to an expression that enables the experimental determination of the capacity factor:Ri R0 R0 i 0 ,,0 R RiR

t tt k = (15) ki can be calculated from the retention time of the analyte, and the residence time of a nonretained compound.i

R0

t

v L=

ChromatographyTerm applied to a wide variety of separation techniques based upon the the sample partitioning between a moving phase and a stationary phase. Earliest reference Exodus XV 23-25. Moses in the wilderness ofShurcame acrossMarahwhere the waters were bitter. On the advice of an expert he through a tree into the water. ion-exchange?

Chromatography terms

The analyte is the substance which is to be separated during chromatography. Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in a sample. A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing. A chromatogram is the visual output of the chromatograph. in the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.

Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example obtained by a spectrophotometer, mass spectrometer or a variety of other detectors) corresponding to the response created by the analytes exiting the system. In the case of an optimal system the signal is proportional to the concentration of the specific analyte separated.

A chromatograph is equipment that enables a sophisticated separation e.g. gas chromatographic or liquid chromatographic separation. 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 in a definite direction. The effluent is the mobile phase leaving the column. An immobilized phase is a stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing. The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC and CEC), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). A better definition: The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column. In one case of HPLC the solvent consist of a carbonate/bicarbonate solution and the

sample is the anions being separated. The mobile phase moves through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated. Preparative chromatography is used to nondestructively purify sufficient quantities of a substance for further use, rather than analysis. The retention time is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. The sample is the matter analysed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste. The solute refers to the sample components in partition chromatography. The solvent refers to any substance capable of solubilizing other substance, especially the liquid mobile phase in LC. The stationary phase is the substance which is fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography.

Chromatography theoryChromatography is method of separating mixtures and identifying their components i.e. it's a separation method that exploits the differences in partitioning behavior of analytes between a mobile phase and a stationary phase to separate components in a mixture. The interaction of the components of a mixture with the two phases is influenced by several different intermolecular forces, includiding ionic, dipolar, nonpolar, and specific affinity and solubility effects.

The Rate Theory of Chromatography

A more realistic description of the processes at work inside a column takes account of the time taken for the solute to equilibrate between the stationary and mobile phase (unlike the plate model, which assumes that equilibration is infinitely fast). The resulting band shape of a chromatographic peak is therefore affected by the rate of elution. It is also affected by the different paths available to solute molecules as they travel between particles of stationary phase. If we consider the various mechanisms which contribute to band broadening, we arrive at the Van Deemter equation for plate height; HETP = A + B / u + C u where u is the average velocity of the mobile phase. A, B, and C are factors which contribute to band broadening.

A - Eddy diffusionThe mobile phase moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths.

B - Longitudinal diffusionThe concentration of analyte is less at the edges of the band than at the center. Analyte diffuses out from the center to the edges. This causes band broadening. If the velocity of the mobile phase is high then the analyte spends less time on the column, which decreases the effects of longitudinal diffusion.

C - Resistance to mass transferThe analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes.

Van Deemter plotsA plot of plate height vs. average linear velocity of mobile phase.

Such plots are of considerable use in determining the optimum mobile phase flow rate.

RetentionThe retention is a measure of the speed at which a substance moves in a chromatographic system. In continuous development systems like HPLC or GC, where the compounds are eluted with the eluent, the retention is usually measured as the retention time Rt or tR, the time between injection and detection. In interrupted development systems like TLC the retention is measured as the retention factor Rf, the run length of the compound divided by the run length of the eluent front:

The retention of a compound often differs considerably between experiments due to variations of the eluent, the stationary phase, temperature, sample matrix and the setup. It is therefore important to compare the retention of the

test compound to that of several standard compounds under absolutely identical conditions. During the chromatographic process the analyte experiences zone broadening as a result of diffusion. Two analytes with different retention times yet with large broadening do not resolve and this is why in any chromatographic system broadening needs to be minimized. This is done by selecting the proper stationary and mobile phase, the eluent velocity, the track length and temperature. The Van Deemter's equation gives an ideal eluent velocity taking into account several physical parameters.

Plate theoryThe plate theory of chromatography was developed by Archer John Porter Martin and Richard Laurence Millington Synge. The plate theory describes the chromatography system, the mobile and stationary phases, as being in equilibrium. The partition coefficient K is based on this equilibrium, and is defined by the following equation:

K is assumed to be independent of concentration, and can change if experimental conditions are changed, for example temperature is increased or decreased. As K increases, it takes longer for solutes to separate. For a column of fixed length and flow, the retention time (tR) and retention volume (Vr) can be measured and used to calculate K.

Distribution of analytes between phasesThe distribution of analytes between phases can often be described quite simply. An analyte is in equilibrium between the two phases; Amobile Astationary The equilibrium constant, K, is termed the partition coefficient; defined as the molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase. The time between sample injection and an analyte peak reaching a detector at the end of the column is termed the retention time (tR ). Each analyte in a sample will have a different retention time. The time taken for the mobile phase to pass through the column is called tM.

A term called the retention factor, k', is often used to describe the migration rate of an analyte on a column. You may also find it called the capacity factor. The retention factor for analyte A is defined as; k'A = t R - tM / tM t R and tM are easily obtained from a chromatogram. When an analytes retention factor is less than one, elution is so fast that accurate determination of the retention time is very difficult. High retention factors (greater than 20) mean that elution takes a very long time. Ideally, the retention factor for an analyte is between one and five. We define a quantity called the selectivity factor, , which describes the separation of two species (A and B) on the column; = k 'B / k 'A

When

ca

lculating the selectivity factor, species A elutes faster than species B. The selectivity factor is always greater than one.

Types of Chromatography

Adsorption ChromatographyAdsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibriation between the mobile and stationary phase accounts for the

separation solutes.

of

different

Partition ChromatographyThis form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibriates between the mobile phase and the stationary liquid.

Ion Exchange ChromatographyIn this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.Affinity Chromatography This is the most selective type of chromatography employed. It utilizes the specific interaction between one kind of solute molecule and a second molecule

that is immobilized on a stationary phase. For example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH.

The Classification of Chromatography

[edit] Techniques by chromatographic bed shape[edit] Column chromatographyFor more details on this topic, see Column chromatography.

A diagram of a standard column chromatography and a flash column chromatography setup Column chromatography is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary

phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sample.[2] In 1978, W. C. Still introduced a modified version of column chromatography called flash column chromatography (flash).[3] The technique is very similar to the traditional column chromatography, except for that the solvent is driven through the column by applying positive pressure. This allowed most separations to be performed in less than 20 minutes, with improved separations compared to the old method. Modern flash chromatography systems are sold as pre-packed plastic cartridges, and the solvent is pumped through the cartridge. Systems may also be linked with detectors and fraction collectors providing automation. The introduction of gradient pumps resulted in quicker separations and less solvent usage. In expanded bed adsorption, a fluidized bed is used, rather than a solid phase made by a packed bed. This allows omission of initial clearing steps such as centrifugation and filtration, for culture broths or slurries of broken cells.

[edit] Planar Chromatography

Thin layer chromatography is used to separate components of chlorophyll Planar chromatography is a separation technique in which the stationary phase is present as or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography).

[edit] Paper ChromatographyFor more details on this topic, see Paper chromatography. Paper chromatography is a technique that involves placing a small dot of sample solution onto a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent and sealed. As the solvent rises through the paper it meets the sample mixture which starts to travel up the paper with the solvent. Different compounds in the sample mixture travel different distances according to how strongly they interact with the paper. This allows the calculation of an Rf value and can be compared to standard compounds to aid in the identification of an unknown substance.

[edit] Thin layer chromatographyFor more details on this topic, see Thin layer chromatography. Thin layer chromatography (TLC) is a widely-employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. Different compounds in the sample mixture travel different distances according to how strongly they interact with the adsorbent. This allows the calculation of an Rf value and can be compared to standard compounds to aid in the identification of an unknown substance.

[edit] Techniques by physical state of mobile phase[edit] Gas chromatographyFor more details on this topic, see Gas chromatography. Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column, which is typically "packed" or "capillary" (see below) . Gas chromatography (GC) is based on a partition equilibrium of analyte between a solid stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium). The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical chemistry; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat will denature them), frequently encountered in biochemistry, it is well suited for use in the petrochemical, environmental monitoring, and industrial chemical fields. It is also used extensively in chemistry research.

[edit] Liquid chromatographyLiquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high performance liquid chromatography (HPLC). In the HPLC technique, the sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile phase) at high pressure. HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases. Technique in which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) is called normal phase liquid chromatography (NPLC) and the opposite (e.g. water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is called reversed phase liquid chromatography (RPLC). Ironically the "normal phase" has fewer applications and RPLC is therefore used considerably more. Specific techniques which come under this broad heading are listed below. It should also be noted that the following techniques can also be considered fast protein liquid chromatography if no pressure is used to drive the mobile phase through the stationary phase. See also Aqueous Normal Phase Chromatography.

[edit] Supercritical fluid chromatographyFor more details on this topic, see Supercritical fluid chromatography. Supercritical fluid chromatography is a separation technique in which the mobile phase is a fluid above and relatively close to its critical temperature and pressure.

[edit] Techniques by separation mechanism[edit] Ion exchange chromatographyFor more details on this topic, see Ion exchange chromatography. Ion exchange chromatography utilizes ion exchange mechanism to separate analytes. It is usually performed in columns but the mechanism can be benefited also in planar mode. Ion exchange chromatography uses a charged stationary phase to separate charged compounds including amino acids, peptides, and proteins. In conventional methods the stationary phase is an ion exchange resin that carries charged functional groups which interact with oppositely

charged groups of the compound to be retained. Ion exchange chromatography is commonly used to purify proteins using FPLC.

[edit] Size exclusion chromatographyFor more details on this topic, see Size exclusion chromatography. Size exclusion chromatography (SEC) is also known as gel permeation chromatography or gel filtration chromatography and separates particles on the basis of size. Smaller molecules enter a porous media and take longer to exit the column, whereas larger particles leave the column earlier. It is generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins, especially since it can be carried out under native solution conditions.

[edit] Affinity chromatographyFor more details on this topic, see Affinity chromatography. Affinity chromatography is based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the purification of proteins bound to tags. These fusion proteins are labelled with compounds such as His-tags, biotin or antigens, which bind to the stationary phase specifically. After purification, some of these tags are usually removed and the pure protein is obtained.

[edit] Special techniques[edit] Reversed-phase chromatographyFor more details on this topic, see Reversed-phase chromatography. Reversed-phase chromatography is an elution procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase.

[edit] Two-dimensional chromatographyBy using an additional physico-chemical (Chemical classification) criterion for separation of the mixture of analytes (sample), the resolution and quality of chromatographic separation can be increased. As a result, higher specifity concerning the separational capability of the chromatographic technique is obtained, allowing separation and preparation or analysis of compounds indistinguishable by Onedimensional chromatography.

In Gas-Phase Chromatography, two dimensional separation is achieved by coupling a second, short column to the first long column. Coupling is achieved by different techniques, for example shock-freezing the elutes in order of elution from the first column at fixed time-intervals, and then reheating them in order of elution, releasing them into the second column. The time of traversal through the second column needs to be shorter than the time remaining until the next sample is reheated to prevent compound buildup and to fully exploit the separational capability. In addition, a Time-of-Flight Mass Spectrometer (TOFMS) can be used as the second dimension of a 2-D gas chromatoghraph. TOF-Mass Spectrometers used in gas chromatography can be very short, as there is a somewhat limited range of molecules that can be separated. In this technique, one column is used to separate analytes, followed by TOFMS detection. This allows for a greater number of analytes to be separated in one experimental run.

GELS USED IN CHROMATOGRAPHY TECHNIQUES:Term gel refers to fairly soft elastic material containing water. A gel consists of three-dimensional structure. A structural material, often consisting of cross-linked polymers, gives a gel a mechanical stability. The space within the gel not occupied by structural material is filled with liquid. Important properties of gels for chromatography 1. The matrix of the gel must be inert. 2. The gels must be stable chemically. 3. Low content of ionic groups is required to avoid ion exchange effects. 4. The mechanical rigidity of the gel grains should be a high as possible.

Dextran gelsDextran is a polysaccharide, built up from glucose residues. 1. Sephadex 2. Sepahcryl is a dextran gel manufactured by cross-linking. The cross-reaction gives a gel the very high rigidity.

Polyacrylamide gelsThey are prepared by co-polymerization of acrylamide and N,Nmethylenbis-acrylamide.

Agarose gelsAgarose gels are made of mixture of linear polysaccharides composed

mainly from D-galactose and anhydro-L-galactose residues.

TECHNIQUES OF ADSORPTION CHROMATOGRAPHY:

In adsorption chromatography, the chromatographic medium is chosen to permit the specific interaction with some of the components in the mixture to be resolved. The intermolecular forces, which are thought to be primarily responsible for chromatographic adsorption, include: 3 Van der Waals forces 3 Electrostatic forces 3 Hydrogen bonds 3 Hydrophobic interactions

1. ION-EXCHANGE CHROMATOGRAPHY

The interactions between the chromatographic medium and the proteins in the mixture are based primarily on ionic charge. Ion exchangers are resins often coupled on cross-linked polysaccharides that can exchange ions with water solutions. Most common types of ion-exchangers: DEAE (diethylaminoethyl)-cellulose; an anion exchange resin used primarily for neutral and acidic proteins CM (carboxymethyl)-cellulose; a cation exchanger used primarily for the separation of neutral and basic proteins. Elution from the column: by altering the pH of the elution buffer by increasing the ionic strength of the elution buffer

3. AFFINITY CHROMATOGRAPHY Definition of a ligand:Ligand refers to a substrate, product, inhibitor, coenzyme, allosteric effector or any other molecule that interacts specifically and reversibly with the protein or other macromolecule to be purified.

Advantages of affinity chromatography:

Adsorbent is designed and constructed specifically for the protein

to be purified. The specific adsorbent permits a rapid separation of the desired protein from inhibitors and destructive contaminants such as proteolytic enzymes. The operation of the technique, in many cases as a single step procedure, leads to a high yield of purified protein. The technique is ideally suited to the isolation of proteins present in very low concentrations. The elution of specifically adsorbed proteins: After inert proteins have been washed off the column the composition, pH, ionic strength or temperature of the buffer is changed.

The Development ProcessA solute progresses through the chromatographic system, albeit through a column or along a plate, only while it is in the mobile phase. This process, whereby the substances are moved through the chromatographic system, is called chromatographic development. There are three types of chromatographic development, elution development, displacement development and frontal analysis. Elution development is now virtually the only development technique employed in both GC and LC although some displacement development is occasionally used in preparative LC.

In TLC, the development process is confused by the frontal analysis of the multicomponent solvent that occurs as the mobile phase moves through the system. In contrast, the solutes are transported across the plate by elution development. This apparent paradox will be explained in detail in due course.

Displacement DevelopmentDisplacement development is only effective with a solid stationary phase where the solutes are adsorbed on its surface. The sample mixture is placed on the front of the distribution system, and the individual solutes compete for the immediately available adsorption sites. Initially, all the nearby adsorbent sites will be saturated with the most strongly held component. As the sample band moves through the system the next available adsorption sites will become saturated with the next most strongly adsorbed component. Thus, the components array themselves along the distribution system in order of their decreasing adsorption strength. The sample components are usually held on the stationary phase so strongly that they are eluted very slowly or even not at all. Consequently the solute must be displaced by a substance more strongly held than any of

the solutes (called the displacer). The displacer, contained at a low concentration in the mobile phase, first displaces the most strongly held component. In turn this component will displace the one next to it. Thus, the displacer forces the adsorbed components progressively through the distribution system, each component displacing the one in front until they are all pass through the system. The solutes will be characterized by the order in which they elute and the amount of each solute present will be proportional to the length of each band, not the height. In displacement development the solutes are never actually separated from one another. The solutes leave the system sequentially and in contact, each somewhat mixed with its neighbor. This type of development is not used in analytical chromatography and only very rarely in preparative LC. However, displacement effects can occur in overloaded distribution systems and in the development of thin layer plates with multicomponent solvents.

Elution DevelopmentElution development is best described as a series of absorption-extraction processes which are continuous from the time the sample is injected into the distribution system until the time the solutes exit from it. The elution process is depicted in Figure 1. The concentration profiles of the solute in both the mobile and stationary phases are depicted as Gaussian in form. Equilibrium occurs between the two phases when the probability of a solute molecule striking the boundary and entering one phase is the same as the probability of a solute molecule randomly acquiring sufficient kinetic energy to leave the stationary phase and enter the other phase. The distribution system is continuously thermodynamically driven toward equilibrium. However, the moving phase will continuously displace the concentration profile of the solute in the mobile phase forward, relative to that in the stationary phase which, in a grossly exaggerated form, is depicted in Figure 1. This displacement causes the concentration of solute in the mobile phase at the front of the peak to exceed the equilibrium concentration with respect to that in the stationary phase. As a consequence, a net quantity of solute in the front part of the peak is continually entering the stationary phase from the mobile phase in an attempt to reestablish equilibrium. At the rear of the peak, the reverse occurs. As the concentration profile moves forward, the concentration of solute in the stationary phase at the rear of the peak is now in excess of the equilibrium concentration.

Figure 1. The Elution of a Solute Through a Chromatographic System A net amount of solute must now leave the stationary phase and enters the mobile phase to re-establish equilibrium. Thus, the solute moves through the chromatographic system as a result of solute entering the mobile phase at the rear of the peak and returning to the stationary phase at the front of the peak. However, that solute is always transferring between the two phases over the whole of the peak in an attempt to attain or maintain thermodynamic equilibrium. Nevertheless, the solute band progresses through the system as a result of a net transfer of solute from the mobile phase to the stationary phase in the front half of the peak. This net transfer of solute is compensated by solute passing from the stationary phase to the mobile phase at the rear half of the peak. Equilibrium processes between two phases is complicated, but a simplified explanation is as follows. The distribution of kinetic energy of the solute molecules contained in the stationary phase and mobile phase is depicted in Figure 2A and 2B. Solute molecules leave the stationary phase when their kinetic energy is equal to or greater than the potential energy of their interaction with the stationary phase. The distribution of kinetic energy between the molecules dissolved in the stationary phase at any specific temperature T, can be considered to take the form of a Gaussian curve as shown in Figure 2A.

Classification according to the shape of the chromatographic bedPlanar chromatography: A separation technique in which the stationary phase is present as or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography, PC) or a layer of solid particles spread on a support, e.g. a glass plate (thin layer chromatography, TLC). Column chromatography: A separation technique in which the stationary bed is within a tube. The particles

of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open-tubular column).

Classification according to the physical state ofGas-liquid chromatography - GLC Gas solid chromatography - GSC Liquid-liquid chromatography - LLC Liquid-solid chromatography LS both phases Classification according to the physical state ofGas chromatography (GC): pro: high efficiency, universal detection contra: only for high volatile and thermally stable substances Liquid chromatography (HPLC): pro: also for low volatile and thermally unstable substances contra: lower efficiency than GC, no universal detection Supercritical fluid chromatography (SFC): pro: also for low volatile and thermally unstable substances, universal detection, higher efficiency than HPLC

contra: lower efficiency than GC the mobile phase

Classification according to the mechanism of separation 1Adsorption chromatography: based on differences between the adsorption affinities of the sample components for the surface of an active solid. Partition chromatography: based on differences between the solubilities of the sample components in the mobile and stationary phases.

Adsorption chromatography Partition chromatography

Solute adsorbed onSolute dissolved in liquid phase bonded to the surface of column surface of stationary phase Ion-exchange chromatography: based on differences in the ion exchange affinities of the sample components. Exclusion chromatography/Size exclusion chromatography (Gel filtration/Gel permeation chromatography): based on exclusion effects as differences in molecular size and/or

shape or in charge. Affinity chromatography: unique biological specificity of the sample and the ligand interaction is utilized for the separation.

Classification according to the mechanism of separation 2

Mechanisms of chromatography 2

Ion-exchange chromatography Exclusion chromatography

Here: cation-exchange resin; only cations can be attracted to it; mobile cations held near anions that are covalently attached to stationary phase Small molecules penetrate pores of particles; large molecules are excluded

Mechanisms of chromatography 3Affinity chromatography One kind of molecule in complex mixture becomes attached to

molecule that is covalently bound to stationary phase; all other molecules simply wash through.

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Terms of chromatography

ResolutionAlthough the selectivity factor, , describes the separation of band centres, it does not take into account peak widths. Another measure of how well species have been separated is provided by measurement of the resolution. The resolution of two species, A and B, is defined as

Baseline resolution is achieved when R = 1.5 It is useful to relate the resolution to the number of plates in the column, the selectivity factor and the retention factors of the two solutes;

To obtain high resolution, the three terms must be maximised. An increase in N, the number of theoretical plates, by lengthening the column leads to an increase in retention time and increased band broadening - which may not be desirable. Instead, to increase the number of plates, the height equivalent to a theoretical plate can be reduced by reducing the size of the stationary phase particles. It is often found that by controlling the capacity factor, k', separations can be greatly improved. This can be achieved by changing the

temperature (in Gas Chromatography) or the composition of the mobile phase (in Liquid Chromatography). The selectivity factor, , can also be manipulated to improve separations. When is close to unity, optimising k' and increasing N is not sufficient to give good separation in a reasonable time. In these cases, k' is optimised first, and then is increased by one of the following procedures:

Fundamental equation of Chromatography:

Vr= Vm+ KVsK = partition coefficient of solute between stationary and mobile

phase Vm= void volume of column; volume of mobile phase in columnand volume that solute would come out in after injection even if it did not interact with stationary phase! Vr= retention volume; = volumetric flow rate x retention time (tr) Rely on differences in K values for two different solutes in order to separate them--so they have different Vr and trvalues!

The basis of all forms of chromatography is the partition or distribution coefficient. (Kd), which describes the way in which a compound distributes itself between two immiscible phases. For the most part, all chromatography systems consist of the stationary phase which for most of biochemical purposes are solids or gels. The mobile phase is liquid or gaseous. The mobile phase flows over and around the stationary phase. In practice separating molecules requires that the various molecules will have a different partition between the two phases.

Examples include:

Adsorption Equilibrium - hydrophobic chromatography is an example. Proteins with a high concentration of hydrophobic amino acids or modified with lipids will often distribute themselves more with the mobile phase Ion Exchange Chromatography A flexible technique used mainly for the separation of ionic or easily ionizable species. The stationary phase is characterized by the presence of charged centers bearing exchangable counterions. Ion Exchange chromatography is one of the most used and well defined chromatographic methods.Gel Filtration - For this type of chromatography the stationary phase is the liquid trapped inside of the matrix. Affinity Chromatography - is an equilibrium between a stationary immobilized ligand and a mobile liquid phase. Factors which influence chromatography Theoretical Plates the number of equilibrations that a compound makes with the stationary phase will increase the separation of similar but unique compounds. Chromatography columns are considered to consist of a number of adjacent plates or zones where there is enough space for a

compound to achieve

complete equilibrium between the mobile and stationary phase. Each zone is called a theoretical plate and the length of the column the plate height. The more theoretical plates the better the resolution of N=1000 N=100 N=10 Relative distribution on the columnMSUM Biotech -Chromatography

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protein. Consider three columns each with a different number of theoretical plates It is important to remember that the plates do not really exist; they are a figment of the imagination that helps us understand the processes at work in the column.They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column, N (the more plates the better), or by stating the plate height; the Height Equivalent to a Theoretical Plate (the smaller the better). Peak resolution there are several factors which alter the ability of a column to separate or resolve proteins. Separation of compounds on a liquid chromatography column are dependent on the nature of the solute, the type of chromatography and the method of elution. Take for example two closely related proteins whose isoelectric points are close. Using a very steep gradient these proteins will not resolve. However a shallower gradient which will effect the equilibrium between the solid and mobile phases to be discriminated between the two proteins can often increase the separation. Another factor that will effect resolution is peak broadening. This will result in overlap of closely eluted samples. Column matrix with large void volumes leads to preak broadening. The cost of using very small matrix is

increasing back pressure. That is the same kind of thing that happens when you put your finger over the garden hose. Column velocity Since the solute molecules are carried through the column by the mobile phase, it is natural that its speed has an influence on the process in the column. A slow flow rate will increase the time a protein is in the mobile phase and diffusion occurs resulting in peak broadening. On the other hand a very high flow rate will decrease the interaction of the protein with the stationary phase and decrease the number of possible equilibration, resulting in a reduction of theoretical plates possible. A Van Deemter plot is a plot of plate height vs. average linear velocity of mobile phase. Such plots are of considerable use in determining the optimum mobile phase flow rate.