Chromatography Notes PDF

CHROMATOGRAPHY DEFINITION OF TERMS -- Analyte -- the substance to be separated during chromatography Analytical chromatography -- this is used to determine the existence and possible also the concentration of analyte in a sample A bonded phase -- this is a stationary or immobilised phase that is...

CHROMATOGRAPHY DEFINITION OF TERMS -- Analyte -- the substance to be separated during chromatography Analytical chromatography -- this is used to determine the existence and possible also the concentration of analyte in a sample A bonded phase -- this is a stationary or immobilised phase that is covalently bonded to the support particles or to the inside walls of the column tubing A chromatogram -- this 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 mixtures A chromatograph this is the equipment that enables a sophisticated separation eg gas chromatographic separation or liquid chromatographic separation Chromatography -- this is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary -- stationary or bonded phase- while the other -- mobile phase moves in a definite direction. The eluate -- this is the mobile phase leaving the column The eluent -- this is the solvent that carries the analyte Eluotropic series -- this is a list of solvents ranked according to their eluting power A preparative chromatography -- this is used to purify sufficient quantities of a substance for further use rather than analysis The retention time -- this is the characteristics time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions The sample -- the sample is the matter analysed in chromatography. It may contain a single component or a mixture of components. When the sample is treated in the course of analysis the phase or phases containing the analytes of interest is or are referred to as sample where as everything out of interest separated from the sample before or in the course of the analysis is referred to as waste The solute -- this is the sample component in partition chromatography The mobile phase -- this is the phase which moves in a definite direction. It may be a liquid or a gas (gas chromatography) or a supercritical fluid (supercritical fluid chromatography). The mobile phase consisting of the sample being separated or analyte and the solvent that moves the sample through the column. In the case of HPLC the mobile phase consists of a nonpolar solvent such as hexane in normal phase or polar solvent in reverse phase chromatography and the sample being separated. The mobile phase moves through the chromatographic column where the sample interacts with the stationary phase and is separated. Chromatography is the collective term for a set 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 based on different partitioning between the mobile and stationary phases. Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for further use and is thus a form of purification. Analytical chromatography is done with smaller amount of material and is for measuring the relative proportion of analyte in a mixture. The general chromatographic technique requires that a solute undergoes distribution between two phases, one of them fixed, (stationary phase), the other moving (mobile phase). It is the mobile phase that transfers the solutes through the medium until eventually emerges separated from the other solutes that are eluted earlier or later. Generally the solute is transferred through the separating medium by means of a flowing stream of a liquid or gaseous solvent known as eluent. The stationary phase may act through adsorption as in the case of adsorbents such as activated charcoal, activated alumina, silica gel or ion exchange resins, or it may act by dissolving the solute, thus partitioning the later between the stationary phase and mobile phases. In the later process, a liquid coating held on an inert support serves as the stationary phase. Partitioning is the predominant mechanism of separation in gas liquid chromatography, paper chromatography, and forms of liquid chromatography. In practice, separation frequently result from a combination of adsorption and partitioning effects. USE OF REFERNCE SUBSTANCES IN IDENTITY TEST -- In paper and TLC the ratio of the distances (this distance being measured to the point of maximum intensity of the spot) travelled on the medium by a given compound, to the distance travelled by the front of the mobile phase from the point of application of the test substance is called the R~f~ value of the compound. The ration between the distances travelled by a given compound and a reference substance is the R~R~ value. If the substance to be identified, all the chromatograms agree in colour and R~F~ value and the mixed chromatogram yields a single spot ie R~R~ = 1.0. Then the test compound is same as the reference compound. LOCATION OF COMPONENTS -- The spots produced by paper or thin layer chromatography may be located by a) direct inspection if the compounds are visible under white or visible short wavelength or long wave length UV light; b) inspection in white or UV light after treatment with reagents that will make the spots visible. Reagents are most conveniently applied with an atomiser; c) use of a Geiger -- Muller counter or autoradiographic techniques in case of the presence of radioactive substance; d) evidence resulting from stimulation or inhibition of bacterial growth by placing of removed portion of the adsorbent and the substance on inoculate media. In open column chromatography, in pressurised liquid chromatography and in gas chromatography, the retention time, t, defined as the time elapsed between the sample injection and appearance of the peak concentration of the eluted sample zone may be used as a parameter of identification. The ratio of the retention of the test substance, the reference compound and a mixture of these, to the retention time of an internal standard is called the relative retention time R~R~ and is also used frequently as a parameter for identification. TYPES OF CHROMATOGRAPHIC TECHNIQUES 1. Technique by chromatographic bed eg paper chromatography, thin layer chromatography, column chromatography 2. Displacement chromatography -- gas chromatography, liquid chromatography 3. Affinity chromatography -- supercritical fluid chromatography 4. Technique by separation mechanism -- ion exchange chromatography, size exclusion chromatography 5. Special techniques -- reversed phase chromatography, two dimensional chromatography, simulated moving bed chromatography, pyrolysis gas chromatography, fast protein liquid chromatography Choice of solvent - Solvents for chromatography are selected based on their polarity and the nature of the compounds to be separated.\ \ In chromatography, the choice of solvent, also known as the mobile phase, is crucial as it directly influences the separation of components in a mixture. The main factor to consider when selecting a solvent is its polarity, which should match the polarity of the compounds to be separated. This is because like dissolves like; polar compounds will dissolve in polar solvents and non-polar compounds in non-polar solvents.\ \ For instance, if you are trying to separate a mixture of polar compounds, you would choose a polar solvent such as water or methanol. On the other hand, if you are dealing with non-polar compounds, a non-polar solvent like hexane would be more suitable. Sometimes, a mixture of solvents may be used to achieve the desired polarity.\ Another factor to consider is the solvent\'s ability to dissolve the sample. The solvent should be able to dissolve a sufficient amount of the sample to allow for effective separation. Additionally, the solvent should not react with the sample or the stationary phase (the material on which the sample is placed).\ The boiling point of the solvent is also important. A solvent with a low boiling point is preferred as it will evaporate quickly, speeding up the chromatography process. However, if the solvent\'s boiling point is too low, it may evaporate before the separation is complete, which would not be ideal.\ Lastly, the solvent should be safe to use. It should not be toxic or flammable, and it should be easy to dispose of after use. Safety should always be a priority in any laboratory procedure, including chromatography. In summary, the selection of solvents in chromatography is a careful balance of several factors, including polarity, solubility, boiling point, and safety. GAS CHROMATOGRAPHY The distinguishing features of gas chromatography are a gaseous mobile phase and a solid or immobilised liquid stationary phase. Gas chromatography also 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 in capillary. Gas chromatography is based on a partition equilibrium of analyte between a solid stationary phase(often a liquid silicone based material) and a mobile phase(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 temperature used in GC make it unsuitable for high molecular weight biopolymers or proteins frequently encountered in biochemistry. It is well suited for use in petrochemical industries, environmental monitoring and industrial chemical fields. It is also used extensively in chemistry research. Liquid stationary phases are available in packed or capillary columns. In the packed column, the liquid phase is deposited on a divided inert solid support such as diatomaceous earth, porous polymers or graphitised carbon which is packed into a column that is typically 2-4mm in internal diameter and 1 -- 3M in length. In capillary column which contain no packing , the liquid phase is on the inner surface of the column and may be chemically bonded to it. In gas chromatography, the solid phase is an active adsorbent such as alumina, silica gel or carbon packed into a column. Polyaromatic porous resin, which are sometimes used in packed columns, are not coated with a liquid phase. When a vaporized compound is introduced into the carrier gas and is carried into the column, it is partitioned between the gas and the stationary phase by a dynamic counter current distribution process. The compound is carried down the column by the carrier gas, retarded to a greater or lesser extent by sorption and desorption on the stationary phase. LIQUID CHROMATOGRAPHY Liquid chromatography 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 utilises very small packing particles and a relatively high pressure is called high performance liquid chromatography or high pressured liquid chromatography -- HPLC. In this technique, the sample is forced through a column that is packed with irregular 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 eg toluene as the mobile phase and silica as the stationary phase, is called normal phase liquid chromatography. And the opposite eg water methanol mixture as the mobile phase and octadecylsilyl as the stationary phase is called reverse phase liquid chromatography. The normal phase has fewer application and reverse phase is used considerably more. Separation by HPLC are achieved by partition, adsorption or ion exchange processes depending upon the type of stationary phase used. HPLC has distinct advantages over gas chromatography for the analysis of organic compounds. Compounds to be analysed are dissolved in a suitable solvent and most separation take place at room temperature thus most drugs being volatile or thermally unstable compounds can be chromatographed without decomposition. Most pharmaceutical analysis are based on partition chromatography and are completed within 30mins. Size-exclusion chromatography Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a [chromatographic](https://en.m.wikipedia.org/wiki/Chromatography) method in which molecules in [solution](https://en.m.wikipedia.org/wiki/Solution_(chemistry)) are separated by their size, and in some cases [molecular weight](https://en.m.wikipedia.org/wiki/Molecular_weight). It is usually applied to large [molecules](https://en.m.wikipedia.org/wiki/Molecule) or macromolecular complexes such as proteins and industrial polymers. Typically, when an [aqueous solution](https://en.m.wikipedia.org/wiki/Aqueous_solution) is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name [gel permeation chromatography](https://en.m.wikipedia.org/wiki/Gel_permeation_chromatography), which is used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are composed of dextran polymers (Sephadex), agarose (Sepharose), or polyacrylamide (Sephacryl or BioGel P). The pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a widely used [polymer characterization](https://en.m.wikipedia.org/wiki/Polymer_characterization) method because of its ability to provide good [molar mass distribution](https://en.m.wikipedia.org/wiki/Molar_mass_distribution) (Mw) results for polymers. [~...~](https://en.m.wikipedia.org/w/index.php?title=Size-exclusion_chromatography&action=edit&section=1) Application - The main application of gel-filtration chromatography is the [fractionation](https://en.m.wikipedia.org/wiki/Fractionation) of proteins and other water-soluble polymers, while gel permeation chromatography is used to analyze the molecular weight distribution of organic-soluble polymers. Either technique should not be confused with [gel electrophoresis,](https://en.m.wikipedia.org/wiki/Gel_electrophoresis) where an electric field is used to \"pull\" or \"push\" molecules through the gel depending on their electrical charges. The amount of time a solute remains within a pore is dependent on the size of the pore. Larger solutes will have access to a smaller volume and vice versa. Therefore, a smaller solute will remain within the pore for a longer period of time compared to a larger solute. The advantages of this method include good separation of large molecules from the small molecules with a minimal volume of eluate, and that various solutions can be applied without interfering with the filtration process, all while preserving the biological activity of the particles to separate. The technique is generally combined with others that further separate molecules by other characteristics, such as acidity, basicity, charge, and affinity for certain compounds. With size exclusion chromatography, there are short and well-defined separation times and narrow bands, which lead to good sensitivity. There is also no sample loss because solutes do not interact with the stationary phase. The other advantage to this experimental method is that in certain cases, it is feasible to determine the approximate molecular weight of a compound. The shape and size of the compound (eluent) determine how the compound interacts with the gel (stationary phase). [~...~](https://en.m.wikipedia.org/w/index.php?title=Size-exclusion_chromatography&action=edit&section=4) Method - SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping smaller molecules in the pores of the [adsorbent](https://en.m.wikipedia.org/wiki/Adsorbent) (\"stationary phase\"). This process is usually performed within a column, which typically consists of a hollow tube tightly packed with micron-scale polymer beads containing pores of different sizes. These pores may be depressions on the surface or channels through the bead. As the solution travels down the column some particles enter into the pores. Larger particles cannot enter into as many pores. The larger the particles, the faster the elution. The larger molecules simply pass by the pores because those molecules are too large to enter the pores. Larger molecules therefore flow through the column more quickly than smaller molecules, that is, the smaller the molecule, the longer the retention time. One requirement for SEC is that the analyte does not interact with the surface of the stationary phases, with differences in elution time between analytes ideally being based solely on the solute volume the analytes can enter, rather than chemical or electrostatic interactions with the stationary phases. Thus, a small molecule that can penetrate every region of the stationary phase pore system can enter a total volume equal to the sum of the entire pore volume and the interparticle volume. This small molecule elutes late (after the molecule has penetrated all of the pore- and interparticle volume---approximately 80% of the column volume). At the other extreme, a very large molecule that cannot penetrate any the smaller pores can enter only the interparticle volume (\~35% of the column volume) and elutes earlier when this volume of mobile phase has passed through the column. The underlying principle of SEC is that particles of different sizes [elute](https://en.m.wikipedia.org/wiki/Elution) (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size. Provided that all the particles are loaded simultaneously or near-simultaneously, particles of the same size should elute together. Each size exclusion column has a range of molecular weights that can be separated. The exclusion limit defines the molecular weight at the upper end of the column \'working\' range and is where molecules are too large to get trapped in the stationary phase. The lower end of the range is defined by the permeation limit, which defines the molecular weight of a molecule that is small enough to penetrate all pores of the stationary phase. All molecules below this molecular mass are so small that they elute as a single band. The filtered solution that is collected at the end is known as the **eluate**. The **void volume** includes any particles too large to enter the medium, and the solvent volume is known as the **column volume**. Following are the materials which are commonly used for porous gel beads in size exclusion chromatography **Fractionation range (Molecular mass in Da)** ---- ---------------- ------------------------------------------------ 1 Sephadex G-10 0 to 700 2 Sephadex G-25 1000 to 5000 3 Sephadex G-50 1500 to 30000 4 Sephadex G-75 3000 to 70000 5 Sephadex G-100 4000 to 150000 6 Sephadex G-150 5000 to 300000 7 Sephadex G-200 5000 to 800000 8 Bio-gel P-2 100 to 1800 9 Bio-gel P-6 1000 to 6000 10 Bio-gel P-60 3000 to 60000 11 Bio-gel P-150 15000 to 150000 12 Bio-gel P-300 16000 to 400000 13 Sepharose 2B 2 x 10^6^ to 25 x 10^6^ 14 Sepharose 4B 3 x 10^5^ to 3 x 10^6^ 15 Sepharose 6B 10^4^ to 20 x 10^6^ [~...~](https://en.m.wikipedia.org/w/index.php?title=Size-exclusion_chromatography&action=edit&section=5) ========================================================================================================= ION EXCHANGE CHROMATOGRAPHY **ion-exchange [chromatography](https://en.m.wikipedia.org/wiki/Chromatography)** separates [ions](https://en.m.wikipedia.org/wiki/Ion) and [polar molecules](https://en.m.wikipedia.org/wiki/Polar_molecule) based on their affinity to the [ion](https://en.m.wikipedia.org/wiki/Ion) exchanger. It works on almost any kind of [charged molecule](https://en.m.wikipedia.org/wiki/Charge_(chemistry))--- including large [proteins,](https://en.m.wikipedia.org/wiki/Proteins) small [nucleotides](https://en.m.wikipedia.org/wiki/Nucleotides), and [amino acids.](https://en.m.wikipedia.org/wiki/Amino_acid) However, ion chromatography must be done in conditions that are one unit away from the [isoelectric point](https://en.m.wikipedia.org/wiki/Isoelectric_point) of a protein. The two types of ion chromatography are anion-exchange and cation-exchange. Cation exchange chromatography is used when the molecule of interest is positively charged. In this type of chromatography, the stationary phase is negatively charged and positively charged molecules are loaded to be attracted to it. Anion-exchange chromatography is when the stationary phase is positively charged and negatively charged molecules are loaded to be attracted to it. It is often used in protein purification, water analysis, and quality control. The water-soluble and charged molecules such as proteins, amino acids, and peptides bind to [moieties](https://en.m.wikipedia.org/wiki/Moiety_(chemistry)) which are oppositely charged by forming ionic bonds to the insoluble stationary phase. The equilibrated stationary phase consists of an ionizable functional group where the targeted molecules of a mixture to be separated and quantified can bind while passing through the column---a cationic stationary phase is used to separate anions and an anionic stationary phase is used to separate cations. Cation exchange chromatography is used when the desired molecules to separate are cations and anion exchange chromatography is used to separate anions. The bound molecules then can be [eluted](https://en.m.wikipedia.org/wiki/Elution) and collected using an eluant which contains anions and cations by running higher concentration of ions through the column or changing [pH](https://en.m.wikipedia.org/wiki/PH) of the column. One of the primary advantages for the use of ion exchange chromatography is only one interaction involved during the separation as opposed to other separation techniques; therefore, ion chromatography may have higher matrix tolerance. Another advantage of ion exchange is the predictability of elution patterns (based on the presence of the ionizable group). For example, when cation exchange chromatography is used, cations will elute out last. Meanwhile, the negative charged molecules will elute out first. However, there are also disadvantages involved when performing ion-exchange chromatography, such as constant evolution with the technique which leads to the inconsistency from column to column. A major limitation to this purification technique is that it is limited to ionizable group. PRINCIPLE Ion-exchange chromatography separates molecules based on their respective charged groups. Ion-exchange chromatography retains [analyte](https://en.m.wikipedia.org/wiki/Analyte) molecules on the column based ionic interactions. The ion exchange chromatography matrix consists of positively and negatively charged ions. Essentially, molecules undergo electrostatic interactions with opposite charges on the stationary phase matrix. The stationary phase consists of an immobile matrix that contains charged ionizable functional groups or ligands. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. To achieve electroneutrality, these inert charges couple with exchangeable counterions in the solution. Ionizable molecules that are to be purified compete with these exchangeable counterions for binding to the immobilized charges on the stationary phase. These ionizable molecules are retained or eluted based on their charge. Initially, molecules that do not bind or bind weakly to the stationary phase are first to wash away. Altered conditions are needed for the elution of the molecules that bind to the stationary phase. The concentration of the exchangeable counterions, which competes with the molecules for binding, can be increased or the pH can be changed. A change in pH affects the charge on the particular molecules and, therefore, alters binding. The molecules then start eluting out based on the changes in their charges from the adjustments. Further such adjustments can be used to release the protein of interest. Additionally, concentration of counterions can be gradually varied to separate ionized molecules. This type of elution is called gradient elution. On the other hand, step elution can be used in which the concentration of counterions are varied in one step. This type of [chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography. Positively charged molecules bind to cation exchange resin](https://en.m.wikipedia.org/wiki/Anion-exchange_chromatography)s while negatively charged molecules bind to anion exchange resins.^\]^ The ionic compound consisting of the cationic species M+ and the anionic species B- can be retained by the stationary phase. Cation exchange chromatography retains positively charged [cations](https://en.m.wikipedia.org/wiki/Cation) because the stationary phase displays a negatively charged functional group: Anion exchange chromatography retains anions using positively charged functional group: Note that the ion strength of either C+ or A- in the mobile phase can be adjusted to shift the equilibrium position, thus retention time. Paper Chromatography This is a technique that involves placing a small dot or line of sample solution onto a strip of chromatographic 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 start to travel up the paper with the solvent. This paper is made up of cellulose, a polar substance and the compounds within the mixture travel further if they are non polar. More polar substances bond with the cellulose more quickly and therefore do not travel thus far. The paper or adsorbent is of suitable texture and thickness. Thin layer chromatography -- TLC The adsorbent in a TLC is a relatively thin, uniform layer of dry, finely divided material applied to a glass, plastic or metal sheet or plates, that is instead of using a stationary phase of paper. It involves a stationary phase of a thin layer of silica gel, alumina or cellulose. It has an advantage of faster runs, better separations and the choice between different adsorbents. For even better resolutions, and to allow for quantification, high performance TLC can be used. The coated plate can be considered an open chromatographic column. The separation achieved may be based on adsorption, partition or combination of both effects. **Column Chromatography** Column chromatography is used for the separation and isolation of different constituents of extracts. Column chromatographic grade adsorbents of choice are used for packing the columns and various organic solvents are used as eluting solvents. A glass column of suitable size is selected depending upon the quantity of the extract and number of components present in it. The column of smaller size for laboratory scale fractionation and very large rocket columns with built-in sintered glass filter in the neck, for large scale industrial use are available for the purpose. If such a filter is not is not present it becomes necessary first to plug the neck of the column with a wad of glass or cotton wool. Silica gel is either made into slurry with the solvents or as such introduced from the top of the column. The next step is to add the sample solution to the top of the column in such a way that a narrow band is formed, for further elution. To this end the sample is dissolved in minimum volumes of solvents. The solvent used should be one of those selected for later elution and preferably the one with the lowest polarity. The extract is sometimes converted to dry slurry by using little quantity of silica gel and introduced at the top of silica gel column and cotton plug is placed on it. In order to fractionate the components of varying solubility, the gradient elution technique is followed. The column is subsequently eluted with various proportions of solvents in the order of increasing polarity. The fractions collected from the bottom are distilled to recover the solvents, while the concentrated residue of the fraction thus obtained is stored in a clean and dry glass vials and monitored by TLC studies. The mixture of component obtained by such fractionation can be subjected to re-fractionation so as to get the pure components. Column chromatography therefore becomes a purification process. Most of the extracts obtained from the crude drugs are multicomponent mixtures of several constituents. The potent component may be present in major or minor amount or even in traces. Chromatographic fractionation if skilfully used guarantees the purification of diverse types of natural products, biochemical vitamins, hormones etc Vaccume liquid chromatography -- VLC -- is same as column chromatography described above. The only difference is that a vacume or pump is input at the bottom of the column to add pressure to the column and greatly reduce time spent.