Chromatography Dr Qhobosheane 16/03/2023 – It is an analytical technique commonly used for separating a mixture of chemical substances into its individual components so that the components may be analysed thoroughly. – Separation is based on differences in rates of migration when the sample components are transported by a mobile phase through a stationary phase – The mobile phase can be a gas, a liquid or a supercritical fluid, and the stationary phase may be a solid, a liquid or a gel. – Supercritical fluid: fluid that cannot be characterised as a liquid or gas General principles of chromatography – Two phases are involved in chromatography – Mobile phase: a phase that moves – Stationary phase – The mobile phase is made up of one or many solvents – The stationary phase is made up of a solvent immobilised on a solid support, and is immiscible with the mobile phase solvent. – – The tube with the stationary phase is called a column – The sample solution is delivered onto the stationary phase – Then more mobile is phase is applied to wash the solute through the stationary phase by applied force or capillary action. – Consider a sample made up of two components; A and B. – If A is more soluble in the mobile phase than the stationary phase, its concentration will be higher in the mobile phase than the stationary phase – A separation is effected by moving the mobile phase past the stationary phase. – When the mobile phase moves past the stationary phase, more molecules of A will be in the mobile phase than the stationary phase. – However, a small fraction will be retained in the stationary phase – If B is more soluble in the stationary phase than the mobile phase, it will be retained longer in the stationary phase, and its transport will be retarded i.e. it will migrate through the column at a lower speed. Example Chromatographic separation of components A and B. – (a) Stationary phase. – (b) At time= 0 min, where the sample is applied on the stationary phase. – (c) At time > 0 min, the sample starts to separate into components under the influence of a mobile phase. – Differential migration of individual components through the column depends on the equilibrium distribution of each component between the stationary phase and the mobile phase. – Differential migration is determined by experimental variables that affect this distribution, such as composition of the mobile phase, composition of the stationary phase and the temperature – One of these variables must be changed in order to alter migration and improve separation The sample components partitioning between the two phases is represented by equilibrium equation: πΆπ πΎ= πΆπ – Where CS is the concentration of component in the stationary phase, and Cm is the concentration of component in the mobile phase – The distribution constant K, is also called the partition coefficient – In summary; if KA< KB, A moves faster with the mobile phase than B because it interacts more with the mobile phase – The speed by which each component migrates through the column is determined by the number of molecules of that component which is in the mobile phase at any instant – Components with a large distribution constant have a large portion of its molecules in the stationary phase, and they are strongly retained in the column – Components with a small distribution constant have a small portion of its molecules in the stationary phase and they are less retained – A chromatographic system generally consists of the mobile phase reservoir, injection system, column and detection system. – The sample solution is introduced onto the column and is subjected to a flow of mobile phase. – The mobile phase carries the sample components along the column at different rates from the point of introduction to the end of the column at which point a detector detects the components. – When the sample migrates and separates into components under the mobile phase influence, the sample is said to be eluted. – – The detector response is measured and recorded as a function of time. – A plot of detector response versus time is called a chromatogram. – The response is usually a Gaussian peak whose corresponding time is measured at the middle to give identification information. – The height or area of the response is measured for quantitation – The time from sample injection to maximum detector response of a component is called the retention time, tR. – The retention time of a substance in a given chromatographic system is a characteristic of its physicochemical properties and is used for identification. – The time it takes for the mobile phase, which is less retained by the column, is called the void / dead time, tM. – Although substances of a sample solution are eluted from the column as Gaussian peaks with different retention times, it is not certain that they are completely separated – For substances to be completely separated, the detector response must reach a baseline between the chromatographic peaks. – Draw overlapping and completely separated peaks as an example Chromatographic Parameters – An ideal chromatogram must exhibit completely separated peaks for it to be practically useful. – For peaks to be completely separated, sufficiently different retention times between components are necessary. – Retention times are easily affected by chromatographic variables such as mobile phase speed and column length and vary a lot. – A constant retention or capacity factor (k) is used instead. – It can be determined from a chromatogram. – Retention factor is a term used to describe the migration rate of an analyte on a column and is given by: – where k is the retention factor, tR and tM are the retention and dead (hold up) times respectively. – (tR- tM) is the time the compound stays in the stationary phase and tM the time it stays in the mobile phase. – Ideal values of the retention factor range from one to five. – Retention factors are manipulated by varying the mobile and stationary phase compositions. – Another parameter that determines adjacent peaks separation is the selectivity factor (α). – Selectivity expresses the retention of a component relative to another closely eluting component. – Substituting the k terms for A and B the equation transforms and allows the selectivity factor to be determined from a chromatogram: – If species A elutes faster than species B, the selectivity factor is always greater than one. – If retention times are not sufficiently different, peaks have to be sufficiently narrow and symmetrical to avoid overlap. – Ideally narrow, symmetrical peaks are desired for a good chromatographic separation. – The width is measured at half height, W1/2. – Chromatographic separations are described using two theoretical models; the plate theory and the rate theory. – Read about the plate theory model and the rate theory of chromatography Chromatographic techniques – Chromatographic techniques are classified based on mobile phases – There is Liquid Chromatography (LC) for liquid mobile phases. – Gas Chromatography (GC) is for gaseous mobile phases. Liquid chromatography – In LC, the stationary phase is typically porous particles with large surface area. – The solute migrates under the competing influences between the interaction with the stationary phase active surfaces and the mobile phase solubility. – LC can further be classified by the stationary phase into Planar and Column Liquid Chromatography. – Planar Chromatography includes Thin Layer Chromatography (TLC) and Paper Chromatography (PC). – The most widely used column chromatography is High Performance Liquid Chromatography (HPLC) Thin layer chromatography – TLC is a liquid chromatography separation technique where the stationary phase is dispersed as a thin layer placed on the surface of a plate made of glass or alumina foil. Chromatographic development Mobile phase – the mobile phase is prepared by mixing various solvents depending on the polarity desired and is referred to as the eluent. – The polarity is measured using polarity indices (Table 1 at the end). – The mobile phase is placed in a developing tank, the tank covered to saturate with a vapour of the mobile phase. – Saturation helps to achieve faster elution. Stationary phase – a very dilute solution (1mg/ml) of the analyte sample is prepared. – In the meantime, the samples are prepared in a volatile solvent – A line (baseline) is drawn at the bottom of the plate, 1cm from the bottom using a very light pencil. – The line is marked with points to show where the spots are to be applied. – Using a spotter that was dipped into the sample solution, the plate is touched very lightly for a very short period of time. – After every application, the spot is let to dry before application of the next one. – The spot diameter must not exceed 2 mm otherwise the plate will be overloaded. – Overloading leads to tailing and overlap of the separated spots. – The sample solvents are then evaporated and the plate inserted in a mobile phase tank – The analytes in a spot migrate up the plate depending on their distribution between the mobile phase and stationary phase – Those that are strongly adsorbed will lag behind, while those that are weakly adsorbed will move with the eluent. – When the mobile phase has moved a certain distance towards the top of the plate (1 cm from the top), the TLC plate is removed from the tank of mobile phase and the solvent front is marked with a pencil Locating the analyte – For coloured analytes, visual inspection is done to locate the separated components of the analyte. – Circles are drawn with a pencil around the components. – For non-coloured analytes several methods for location are used. – For aromatics or conjugated substances, a UV light is shined over the plate and the regions where the separated analyte spots are located will become dark blue against a fluorescent background. – Under UV light a circle is drawn around the spots. – Alternatively the plate is sprayed with colour developing chemicals. – Eg. Amino acids/amines display a blue-violet colour with Ninhydrin. Phosphomolybdic acid/cerium sulphate solution is ideal for most functional groups at very low concentration. – Most organic compounds develop a colour with iodine and sulphuric acid. – Some silica gel stationary phases are incorporated with a fluorescing material. – The separated spots quench the fluorescence and where they are located, no fluorescence develops while the rest of the plate glows and fluoresces. – Note: 1. UV damages the eyes, never look directly into the UV lamp. 2. Soon as the spots are visualised, mark them with a pencil as most spots are not permanent. – When the development is done, the distances moved by the solvent front and the various separated spots and the reference if available are measured. – These distances are used to calculate the Rf (retention factor) value using the following equation: π π = π·ππ π‘ππππ π‘πππ£πππππ ππ¦ π‘βπ π π’ππ π‘ππππ π·π π·ππ π‘ππππ π‘πππ£πππππ ππ¦ π‘βπ π πππ£πππ‘ πππππ‘ π·π π – If the Rf value is below 0.5 then the separation is good – Rf values are dependent on ambient conditions, adsorbent, solvent and analyte. – Therefore no Rf from different experiments will be the same and be used to determine identities of substances. – In order to determine an unknown in a sample analyte, run the sample side by side with the known pure reference substance in the same chromatographic tank with all conditions identical. – TLC is used as an analytical and separation technique. – In chemical synthesis, it can be used to monitor the progress of the reaction. – A small portion of a reaction mixture is spotted on the plate which is then developed. – If say reactant A and B were supposed to have formed C, one spot from the developed plate is expected. – So at specified intervals the reaction mixture is spotted on the plate and visualised to check if one or several spots are seen. Preparative TLC – This technique uses TLC plates with thick stationary phase – Once the elution is deemed to have completely taken place and the spot visualised, the area where the spot developed is scraped off the plate using a razor. – The silica is placed in a fritted funnel and flushed with a polar solvent (like Ethyl Acetate). – The pure product can be isolated from the filtrate. Paper chromatography (PC) – Separation is achieved in the same manner as in TLC. – The only difference is that the stationary phase is a piece of adsorbent paper made up of high purity cellulose with carefully controlled porosity and thickness. High Performance Liquid Chromatography (HPLC) – HPLC is one mode of chromatography which uses a liquid mobile phase to separate the components of a mixture. – It is the most commonly used to determine drugs in pharmaceutical preparations and in biological material – The stationary phase can be a liquid or a solid phase packed in a column. – The mobile phase is a liquid forced through a column packed with a material that retards the analytes introduced into the system – The analytes are first dissolved in a solvent and then forced to flow through a chromatographic column under pressure exerted by a pump. – The mixture separates into its components and the degree to which they separate is referred to as resolution. – Resolution is dependent on the extent of the interaction between the solute components and the stationary and mobile phases. – The interaction of the solute with the mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. – As a result, HPLC has a high degree of versatility not found in other chromatographic modes and is applicable in the separation of a wide variety of chemicals. – A variety of detection systems is available for HPLC. – The most commonly used are the UV-vis detectors which can be tuned to different wavelengths. – Another common type is the electrochemical detector. – HPLC has different modes and it can be classified based on the nature of the stationary phase and the separation process. The types include: Partition/Liquid-liquid chromatography (PC) – The solute is partitioned between the liquid mobile phase and the liquid stationary phase supported by physical adsorption onto solid support particles. – Separation is achieved by differential preference of the solute of one liquid phase over the other. – Used in pharmaceuticals, biochemicals and other chemicals. Adsorption/ Liquid-solid chromatography (AC) – stationary phase is made up of an adsorbent, eg. Alumina, Silica gel or any silica based packing. – Separation is based on repeated adsorption-desorption steps. – Used in: resolution of isomeric mixtures of relatively non-polar, water insoluble organic compounds Mr< 5000. Ion- exchange chromatography (IEC) – stationary phase has an ionic charged surface. – It is used to separate ionic/ionisable samples. – The mobile phase is an aqueous buffer that regulates both pH and ionic strength that in turn control elution time. – IEC is applied for cation and anion analysis. Size exclusion chromatography (SEC) – stationary phase is made up of material having precisely controlled pore sizes. – Separation is achieved through a differential inclusion and exclusion of components into the pores based on solvated molecular size. – SEC is used for substances with Mr> 1000. – Adsorption and ion exchange chromatography can be further classified as Normal Phase and Reverse Phase Chromatography. – Normal phase (NP): stationary phase is polar (eg. Silica gel) while the mobile phase is nonpolar (eg.n-hexane). – Reverse phase (RP): stationary phase is non polar while the mobile phase is polar (mixture of water and methanol/acetonitrile). – Polarity is a major role player in PC, AC, and IEC while solvated molecular size dominates in SEC separations. Applications of HPLC – Due to its wide versatility, HPLC can be applied for various analyses. – It is used for pharmaceutical compounds which are thermo labile and is thus carried out at room temperature for: – Quantitative analysis of pharmaceutical compounds. – Monitoring the stability of API in raw materials and formulations. – Monitoring drugs and associated metabolites in biological fluids. – Determination of API physical constants like partition coefficients, drug-protein binding constants, stability constants and pKa’s. Instrumentation – Pressure was thought to be the principal criterion that lent HPLC its versatility; hence it was initially referred to as High Pressure Liquid Chromatography (HPLC). – This was however not accurate because although it has pump to apply pressure, the pressure from the pump is not so high. – Pressure develops as a result of many factors in the instrument: small packed particles, small pores, low volume samples and of course good pumping systems. – A pump is simply required to apply pressure that permits development of the desired mobile phase flow rates.
