Lab Technique
Introduction Organic molecules have complex behavior. Their structure is complicated and need to be clearly understood. Chemists have been developing various techniques to see inside the organic molecules. These techniques can be broadly classified as spectrometry, separation and spectroscopy. Mass Spectrometry Mass spectrometry is a technique and an analytical tool in the hand of chemists which is used for measuring the molecular mass or atomic mass. The molecular masses of biomolecules, can be measured up to an accuracy of 0.01%. For smaller organic molecules, the molecular mass can be measured within an accuracy of 5 ppm or less. This accuracy is sufficient to confirm the molecular formula of a compound Principal of Mass Spectrometry Ions are electrically charged particles and they can be deflected by magnetic fields as electrically charged particles are affected by a magnetic field. Higher the mass less is deflection and lower the mass more is the deflection. Process of Mass Spectrometry The molecules of the sample are bombarded with electron. The atoms of molecules are ionized by losing one or more electrons to give a positive ion. This is true even for particles which normally produce negative ions (chlorine, for example) or never form ions at all (argon, for example). Mass spectrometers always work with positive ion. The largest ion is the size of original molecule less one electron. It is a cation. For example, methane will produce a cation of the type CH4+. This cation is called molecular ion. The ions are accelerated through a magnetic field so that they all have the same kinetic energy. A magnetic force is experienced by the charged ion causing the deflection from their original path.
The ions are deflected by the magnetic field according to their masses. Magnetic field can be varied to allow ions of different mass to pass through the spectrometer. The lighter they are, the more they are deflected. The amount of deflection also depends on the number of positive charges on the ion. In other words, on how many electrons were knocked off in the first stage. The more the ion is charged, the more it gets deflected. The deflection is along a curved path and radius of deflected curved path depends upon mass to charge ratio of the ion. The beam of ions passing through the machine is detected electrically. A schematic diagram of mass spectrometer is shown
Most of ions are having charge of 1+ after loosing one electron. Most of the ions have positive one charge because it very difficult to knock off 2nd electron after one electron is removed. Most of the ions passing through the mass spectrometer will have a charge of 1+, so that the mass/charge ratio will be the same as the mass of the ion. The ions which have the smaller value of m/z will need less deflection to bring them on to the detector, and less deflection is possible by using a weaker magnetic field (a smaller sideways force). For heavier ion, stronger magnetic field will be needed. In other words all ions heavier or lighter can be brought to detector by controlling the magnetic field. The detector produces a current which is proportional to the number of ions arriving. The mass of each ion being detected is related to the size of the magnetic field used to bring it on to the detector. The output from the chart recorder is usually processed by a computer and is represented as a mass spectrum. This spectrum shows the relative current produced by ions of varying mass/charge ratio. Mass Spectrum Mass spectrum will usually be presented as a vertical bar graph, in which each bar represents an ion having a specific mass-to-charge ratio (m/z) and the length of the bar indicates the relative abundance of the ion. The most intense ion is assigned an abundance of 100, and it is referred to as the base peak. Most of the ions formed in a mass spectrometer have a single charge, so the m/z value is equivalent to mass itself. Modern mass spectrometers easily distinguish (resolve) ions differing by only a single atomic mass unit (amu), and thus provide completely accurate values for the molecular mass of a compound. The highest-mass ion in a spectrum is normally considered to be the molecular ion, and lower-mass ions are fragments from the molecular ion, assuming the sample is a single pure compound. The following diagram displays the mass spectra of three simple gaseous compounds, carbon dioxide, propane and cyclopropane. The molecules of these compounds are similar in size. CO2 and C3H8 both have a nominal mass of 44 amu,
and C3H6 has a mass of 42 amu. The molecular ion is the strongest in the spectra of CO2 and C3H6, and it is moderately strong in propane. The unit mass resolution is readily apparent in these spectra (note the separation of ions having m/z=39, 40, 41 and 42 in the cyclopropane spectrum). Even though these compounds are very similar in size, it is a simple matter to identify them from their individual mass spectra.
Chromotography Chromotography is a technique of resolution or separation. Chromatography involves a sample being converted into a solution. The solvent is called mobile phase (which may be a gas, a liquid or a supercritical fluid). The mobile phase is then forced through a stationary phase. The phases are chosen such that components of the sample have differing solubility 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. Different types of chromatography are discussed in the following paragraphs. Solid to Liquid 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 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 (open column). Differences in rates of movement through the medium are calculated to different retention times of the sample. Each component can be subsequently be collected as it elutes with solvent and drips down the coloumn. Paper Chromotography A small concentrated spot of solution preferably colored that contains the sample is applied to a strip of chromatography paper about 2 cm away from the base of the plate. This is absorbed onto the paper and may form interactions with it. Any substance that reacts or bonds with the paper cannot be measured using this technique. The paper is then dipped in to a suitable solvent, such as ethanol or water, taking care that the spot is above the surface of the solvent, and placed in a sealed container. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the solvent molecules to the paper and to one another. As the solvent rises through the paper it meets and and dissolves the sample mixture, which will then travel up the paper with the solvent. Different compounds in the
sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the fibers in the paper. Paper chromatography takes anywhere from several minutes to several hours. Result is is a series of colored dots representing different components of the sample which can be noticed on the paper. The distance between the different dots is indication of their property of solubility and affinity to paper in comparison to solvent. The thin layer chromotogrphy is similar to paper chromotography except that a coated glass is used and the results are analysed by iodine vapor chamber. Gas to liquid. Gas chromatography - specifically gas-liquid chromatography - involves a sample being vaporized and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous (mobile phase). The column itself contains a liquid (stationary phase) which is adsorbed onto the surface of an inert solid. The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is often dependent upon the type of detector which is used. The carrier gas system also contains a molecular sieve to remove water and other impurities. Compounds in the mixture interact with liquid at different rate and elute as individual component at the exit port Distillation Distillation can be defined as a process in which a liquid or vapor mixture of two or more substances is separated into its component fractions of desired purity, by the application and removal of heat. Distillation is based on the fact that the vapor of a boiling mixture will be richer in the components that have lower boiling points. Therefore, when this vapor is cooled and condensed, the condensate will contain more volatile components. At the same time, the original mixture will contain more of the less volatile material.
Mixtures of liquids whose boiling points are similar (separated by less than 70°C) cannot be separated by a single simple distillation. In these situations, a fractional distillation is used. Vacuum distillation is distillation at a reduced pressure. Since the boiling point of a compound is lower at a lower external pressure, the compound will not have to be heated to as high a temperature in order for it to boil. Vacuum distillation is used to distill compounds that have a high boiling point or any compound which might undergo decomposition on heating at atmospheric pressure. The vacuum is provided either by a water aspirator or by a mechanical pump. Azeotropes Azeotrope is a mixture of two liquids which show positive of Raults law and mixture solution will boil at lower temperature than either of the two components. Such mixtures are called azeotropes. Azeotropes cannot be separated by distillation; e.g. a mixture of 5% water and 95% ethanol make an azeotrope. Azeotropes can also be formed when the mixture boils at higher temperature than either of the components. Crystallization Crystallization refers to the formation of solid crystals from a homogeneous solution. It is essentially a solid-liquid separation technique and a very important one. In order for crystallization to take place a solution must be "supersaturated". Supersaturation refers to a state in which the liquid (solvent) contains more dissolved solids (solute) than can ordinarily be accommodated at that temperature. It is based on the principle that pure substance crystallizes more easily than impure substance. Therefore in an impure solution pure substance crystallizes leaving behind an impure solution. Crystallization is very inefficient method of separation and it is a very difficult to arrive at a pure substance through crystallization.
Extraction Extraction is a very common laboratory procedure used when isolating or purifying a product. Organic chemistry employs solid-liquid, liquid-liquid, and acid-base extractions. The following applies to liquid-liquid extractions, which will be used in this discussion It is very common to obtain a mixture of product in any synthesis. These products are to be separated by some technique. Extraction is widely used method. A separatory funnel is used for this process and the principal of like dissolve like is followed. The organic solvent used for extracti