Chromatography & instrumentation in Organic Chemistry What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components. Types of Chromatography TYPE STATIONARY PHASE MOBILE PHASE Paper solid (filter paper) Liquid Thin Layer Chromatography (tlc) solid (silica) Liquid Column solid (silica) Liquid High Pressure Liquid Chromatography (hplc) Gas Liquid Chromatography (glc) solid (silica) solid or liquid Liquid gas Uses for Chromatography (a) Analyse examine a mixture, its components, and their relations to one another (b) Identify determine the identity of a mixture or components based on known components (c) Purify separate components in order to isolate one of interest for further study (d) Quantify determine the amount of the a mixture and/or the components present in the sample Uses for Chromatography
(a) (b) (c) (d) (e) Pharmaceutical Company determine amount of each chemical found in new product Hospital detect blood or alcohol levels in a patient s blood stream Law Enforcement to compare a sample found at a crime scene to samples from suspects Environmental Agency determine the level of pollutants in the water supply Manufacturing Plant to purify a chemical needed to make a product Definition of Chromatography Detailed Definition: Chromatography is a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass. Terminology: Differential showing a difference, distinctive Affinity natural attraction or force between things Mobile Medium gas or liquid that carries the components (mobile phase) Stationary Medium the part of the apparatus that does not move with the sample (stationary phase) Definition of Chromatography Simplified Definition: Chromatography separates the components of a mixture by their distinctive attraction to the mobile phase and the stationary phase. Explanation: Compound is placed on stationary phase (usually paper or silica) Mobile phase (suitable solvent) passes through the stationary phase
Mobile phase solubilises the components Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases Illustration of Paper Chromatography R f value Under similar conditions, a component should always travel at the same speed. Its identity can be found by comparing the distance it moves relative to the solvent. Comparison can be a problem if components have similar R f values The unknown substance is new and there is no previous chemical to compare it with
Types of Chromatography (a) Paper Chromatography separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) (b) Thin-Layer Chromatography separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase) (c) Column Liquid Chromatography separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase) (d) Gas Chromatography (GLC & HPLC) separates vaporized samples with a carrier gas (mobile phase) and a column composed of a Liquid or of solid beads (stationary phase) Paper Chromatography Stationary phase Paper (Filter or Chromatography) Mobile phase Suitable organic solvent (water /Alcohol) Thin Layer Chromatography Here the mobile phase is a liquid Flowing past a thin layer of powder on a solid support (silica). Substances that are less attracted to the solid or are more soluble in the liquid move faster. And so move further up the plate by the time that the process has been stopped by taking the plate out of the liqiud. - larger Rf The surface of the plate consists of a very thin layer of silica on a plastic or aluminum backing. The silica is very polar. This is the stationary phase. Spot the material at the origin (bottom) of the TLC plate. Place the plate into a glass jar with a small amount of a solvent in the glass jar. This solvent acts as the moving phase. Remove the plate from the bottle when the solvent is close to the top of the plate.
Non-polar compounds will be less strongly attracted to the plate and will spend more time in the moving phase. This compound will move faster and will appear closer to the top of the plate. Polar compounds will be more strongly attracted to the plate and will spend less time in the moving phase and appear lower on the plate. Be sure to remove the TLC plate when it appears that the solvent front isn t moving! Reason: the solvent is evaporating as it moves up the plate. Results: If you don t remove the plate all of the spots will appear near the top of the plate!!!!! This isn t a pretty sight and makes it difficult to get good R f values! Visualization Method Some plates shows colored spots. Most of the time, the spots won t show unless they are visualized! Vizualization is a method that is used to render the TLC spots visible. A visualization method can be: (a) Ultraviolet light (b) Iodine vapors to stain spots (c) Colored reagents to stain spots (d) Reagents that selectively stain spots while leaving others unaffected.
Thin-Layer Chromatography: Determination of R f Values Column Chromatography Solid Phase extraction column Vary in size and polarity There are two ways to run a column: 1. Allow gravity to draw samples and buffers through the column resin. 2. Use pumps to push a sample and buffers through a column. Stationary phase silica Mobile phase suitable organic solvent Separation components interact with the stationary phase to different extents
Method (a) a chromatography column is filled with solvent and silica (b) drops of the mixture are placed on top of the silica A (c) the tap is opened to allow the solvent to flow out (d) additional solvent is added on top to replace that leaving (e) components travel through at different rates and separate B (f) batches of solvent are collected at intervals C (g) the solvent in each batch is evaporated to obtain components Gas Liquid Chromatography Here the mobile phase is an unreactive gas ( eg Nitrogen) flowing through a tube. And the stationary phase is an non-volatile liquid held on particles of a solid support. Suitable for separating volatile mixtures In practice the Column is contained in a thermostatic oven. (Why?) About 1μL of liquid is injected into one end of the column. As each component reaches the other end it is detected and registered on a chart recorder.
The Retention Time is characteristic of a particular substance. (for the same column, temperature, gas flow etc.) The area under each peak indicates the relative quantities. Uses of Gas Chromatogephy (a) Drug Testing (b) Blood Alcohol Tests (c) Used in conjunction with a Mass Spectrometer (d) GLC separates and the MS analyses each part separately High Performance Liquid Chromatography HPLC is suitable for the separation of the components of non volatile mixtures
Mobile phase is a solvent and the stationary phase is a fine silica powder contained in a slim column The solvent has to be pushed through the silica using high pressure Separation is based on the retention of the mixture components by the silica based column A better form of column chromatography. Instead of draining down through the stationary phase, the solvent is forced through under high pressure. Stationary phase silica Mobile phase suitable solvent Separation similar to column chromatography Method 1. A sample is injected 2. Solvent and sample are pushed through under pressure 3. Different compounds have different retention times 4. Output can be detected by compounds absorbing UV 5. Can be connected to a mass spectrometer
Uses Used where mixtures are less volatile and so cannot be separated using GC Used to analyse vitamins in food and growth promoters in meat Spectroscopy Spectroscopy is analysis of the interaction between electromagnetic radiation and matter. Different types of radiation interact in characteristic ways with different samples of matter The interaction is often unique and serves as a diagnostic "fingerprint" for the presence of a particular material in a sample Spectroscopy is also a sensitive quantitative technique that can determine trace concentrations of substances.
Mass Spectrometry Used to calculate the relative atomic mass of an element Separates positive ions according to their mass to charge ratio Lighter ions are deflected more by a magnetic field Uses Along with measuring relative atomic mass of elements It measures the relative molecular mass of substances Can be used to identify unknown components of substances Molecules are ionised and assessed according to their relative masses with different molecules having different mass spectra Used to assess pollutants
Ultra Violet Spectrometry Involves the absorption of Ultra violet radiation Used to identify the amount of substance in a sample (quantitative) A sample and blank is placed between a detector and a UV source and the amount of light absorbed by the solution is detected A spectra is obtained which is unique to each element The technique is quantitative and is used in drug analysis Infra Red Absorption Spectometry Each organic compound has a unique infra red absorption spectra IR is used to detect organic compounds (plastics and drugs) The detector measures light intensity following passage through a sample
Atomic Absorption Spectrometry
Colorimetry A colorimeter used to test the concentration of a solution by measuring its absorbance of a specific wavelength of light. Different solutions are made, and a control (a mix of distilled water and another solution) is filled into a cuvette and placed inside a colorimeter to calibrate the machine. After calibration you find the densities and/or concentrations of other solutions.