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Solisa, Daryll Mc Jay V. MT11310 Analytical Methods 1.

Spectometry Spectrophotometry

June 26, 2013 Clinical Chemistry

A spectrophotometer consists of two instruments, namely a spectrometer for producing light of any selected color (wavelength), and a photometer for measuring the intensity of light. The instruments are arranged so that liquid in a cuvette can be placed between the spectrometer beam and the photometer. The amount of light passing through the tube is measured by the photometer. The photometer delivers a voltage signal to a display device, normally a galvanometer. The signal changes as the amount of light absorbed by the liquid changes. Principle: Spectrophotometric techniques are used to measure the concentration of solutes in solution by measuring the amount of light that is absorbed by the solution in a cuvette placed in the spectrophotometer. Spectrophotometry takes advantage of the dual nature of light. Light has a particle nature which gives rise to the photoelectric effect and a wave nature which gives rise to the visible spectrum of light. Atomic Absorption The technique makes use of absorption spectrometry to assess the concentration of an analyte in a sample. It requires standards with known analyte content to establish the relation between the measured absorbance and the analyte concentration and relies therefore on the BeerLambert Law. In short, the electrons of the atoms in the atomizer can be promoted to higher orbitals (excited state) for a short period of time (nanoseconds) by absorbing a defined quantity of energy (radiation of a given wavelength). This amount of energy, i.e., wavelength, is specific to a particular electron transition in a particular element. In general, each wavelength corresponds to only one element, and the width of an absorption line is only of the order of a few picometers (pm), which gives the technique its elemental selectivity. The radiation flux without a sample and with a sample in the atomizer is measured using a detector, and the ratio between the two values (the absorbance) is converted to analyte concentration or mass using the BeerLambert Law. Mass Spectrometry Mass spectrometry is a powerful analytical technique used to quantify known materials, to identify unknown compounds within a sample, and to elucidate the structure and chemical properties of different molecules. The complete process involves the conversion of the sample

into gaseous ions, with or without fragmentation, which are then characterized by their mass to charge ratios (m/z) and relative abundances. This technique basically studies the effect of ionizing energy on molecules. It depends upon chemical reactions in the gas phase in which sample molecules are consumed during the formation of ionic and neutral species. Principle: A mass spectrometer generates multiple ions from the sample under investigation, it then separates them according to their specific mass-to-charge ratio (m/z), and then records the relative abundance of each ion type. The first step in the mass spectrometric analysis of compounds is the production of gas phase ions of the compound, basically by electron ionization. This molecular ion undergoes fragmentation. Each primary product ion derived from the molecular ion, in turn, undergoes fragmentation, and so on. The ions are separated in the mass spectrometer according to their mass-to-charge ratio, and are detected in proportion to their abundance. A mass spectrum of the molecule is thus produced. It displays the result in the form of a plot of ion abundance versus mass-to-charge ratio. Ions provide information concerning the nature and the structure of their precursor molecule. In the spectrum of a pure compound, the molecular ion, if present, appears at the highest value of m/z (followed by ions containing heavier isotopes) and gives the molecular mass of the compound. The instrument consists of three major components: 1. Ion Source: For producing gaseous ions from the substance being studied. 2. Analyzer: For resolving the ions into their characteristics mass components according to their mass-to-charge ratio. 3. Detector System: For detecting the ions and recording the relative abundance of each of the resolved ionic species. In addition, a sample introduction system is necessary to admit the samples to be studied to the ion source while maintaining the high vacuum requirements (~10-6 to 10-8 mm of mercury) of the technique; and a computer is required to control the instrument, acquire and manipulate data, and compare spectra to reference libraries.


2. Luminescence Luminescence is emission of light by a substance not resulting from heat; it is thus a form of cold body radiation. It can be caused by chemical reactions, electrical energy, subatomic motions, or stress on a crystal. This distinguishes luminescence from incandescence, which is light emitted by a substance as a result of heating. Types: Chemiluminescence, a result of a chemical reaction Bioluminescence, emission as a result of biochemical reaction by a living organism Electrochemiluminescence, a result of an electrochemical reaction Crystalloluminescence, produced during crystallization Electroluminescence, a result of an electric current passed through a substance Cathodoluminescence, a result of a luminescent material being struck by the electrons Mechanoluminescence, a result of a mechanical action on a solid Triboluminescence, generated when bonds in a material are broken when that material is scratched, crushed, or rubbed Fractoluminescence, generated when bonds in certain crystals are broken by fractures Piezoluminescence, produced by the action of pressure on certain solids[3] Sonoluminescence, a result of imploding bubbles in a liquid when excited by sound Photoluminescence, a result of absorption of photons Fluorescence, photoluminescence as a result of singletsinglet electronic relaxation (typical lifetime: nanoseconds) Phosphorescence, photoluminescence as a result of tripletsinglet electronic relaxation (typical lifetime: milliseconds to hours) Radioluminescence, a result of bombardment by ionizing radiation Thermoluminescence, the re-emission of absorbed light when a substance is heated

Chemiluminescence is the generation of electromagnetic radiation as light by the release of energy from a chemical reaction. While the light can, in principle, be emitted in the ultraviolet,

visible or infrared region, those emitting visible light are the most common. They are also the most interesting and useful. Chemiluminescent reactions can be grouped into three types: 1. Chemical reactions using synthetic compounds and usually involving a highly oxidized species such as a peroxide are commonly termed chemiluminescent reactions. 2. Light-emitting reactions arising from a living organism, such as the firefly or jellyfish, are commonly termed bioluminescent reactions. 3. Light-emitting reactions which take place by the use of electrical current are designated electrochemiluminescent reactions. 3. Electrophoresis Electrophoresis is an analytical method frequently used in molecular biology and medicine. It is applied for the separation and characterization of proteins, nucleic acids and subcellular-sized particles like viruses and small organelles. Its principle is that the charged particles of a sample migrate in an applied electrical field. If conducted in solution, samples are separated according to their surface net charge density. The most frequent applications, however, use gels (polyacrylamide, agarose) as a support medium. The presence of such a matrix adds a sieving effect so that particles can be characterized by both charge and size. Protein electrophoresis is often performed in the presence of a charged detergent like sodium dodecyl sulfate (SDS) which usually equalizes the surface charge and, therefore, allows for the determination of protein sizes on a single gel. Additives are not necessary for nucleic acids which have a similar surface charge irrespective of their size. Like centrifugation, the molecules feel a force pushing them in one direction. However, in this case, the force involved is due to the electric field acting on the charge of the molecule and is given by F = EQ where F is the force, E is the electric field and Q is the charge. Obviously the greater the charge on the molecule, the greater the force. Thus, for two molecules of the same size, the one with the larger charge will move faster in the electric field. Now, it is less obvious that molecules with a larger mass will move more slowly. This actually comes about because the frictional forces that slow a molecule traveling through solution down depend on the molecules size. The speed at which molecules go through a solution is determined by the point at which the forces driving it forward are just balanced by the frictional forces generated by the motion. The greater the mass of the molecule, the greater the size, in general, and therefore the friction the molecule will generate when traveling through the solution. It turns out that in fact the electrophoretic mobility of a molecule depends on its charge to mass ratio. Two different sized molecules with the same charge to mass ratio should run with the same mobility in a uniform electric field and a perfect world. Types: SDS-PAGE. One of the most common means of analyzing proteins by electrophoresis is by using Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis. SDS is a detergent which denatures proteins by binding to the hydrophobic regions and essentially coating the linear

protein sequence with a set of SDS molecules. The SDS is negatively charged and thus becomes the dominant charge of the complex. The number of SDS molecules that bind is simply proportional to the size of the protein. Therefore the charge to mass ratio should not change with size. In solution (water), in principle all different sized proteins covered with SDS would run at about the same mobility. However, the proteins are not run through water. Instead they are run through an inert polymer, polyacrylamide. The density and pore size of this polymer can be varied by just how you make it (concentration of monomer and of cross-linking agent). Thus, the size of molecules that can pass through the matrix can be varied. This determines in what molecular weight range the gel will have the highest resolving power. Native Gels. It is also possible to run protein gels without the SDS. These are called native gels in that one does not purposely denature the protein. Here, the native charge on the protein (divided by its mass) determines how fast the protein will travel and in what direction. Electrofocusing Gels. Another variation of gel electrophoresis is to pour a gel that purposely has a pH gradient from one end to the other. As the protein travels through this pH gradient, its various ionizable groups with either pick up or lose protons. Eventually, it will find a pH where its charge is zero and it will get stuck (focused) at that point. DNA Agarose Gels. A simple way of separating fairly large fragments of DNA from one another by size is to use an agarose gel. Agarose is another type of matrix used for many purposes (such as the support for the growth of bacteria on plates). DNA does not need a detergent, since it already has a large under of negative phosphate groups evenly spaced. Thus, as with SDSPAGE, the charge to mass ratio is constant. Also like SDS-PAGE, the separation results from the matrix itself. The range of size sensitivity can be varied by changing the density of the agarose. DNA denaturing polyacrylamide gels (often called sequencing gels). To look at smaller DNA molecules with much higher resolution, people generally denature the DNA via heat and run it through a thin polyacrylamide gel that is also kept near the denaturing temperature. These gels usually contain additional denaturing compounds such as Urea. Two pieces of DNA that differ in size by 1 base can be distinguished from each other this way. Capillary electrophoresis. It has become popular to separate molecules electrophoretically by running them into and through a capillary tube. This is fast and accurate, but does not allow much sample to be loaded on the gel at once. Source: ml

4. Chromatography Principle of Chromatography Chromatography is a technique by which a mixture sample is separated into components. Although originally intended to separate and recover (isolate and purify) the components of a sample, today, complete chromatography systems are often used to both separate and quantify sample components. The term, chromatography" was coined by the Russian botanist, Tswett, who demonstrated that, when a plant extract was carried by petroleum ether through a column consisting of a glass tube packed with calcium carbonate powder, a number of dyes were separated, as shown in Figure 1. He named this analysis method "Chromatographie" after "chroma" and "graphos", which are Greek words meaning "color" and to draw," respectively.

Types of chromatography Mobile Phase : Gas Stationary Phase: Solid/Liquid Analysis: Gas chromatography (GC) Sample Types: Samples that are gaseous at ordinary temperatures and samples that vaporize when heated. Odorous samples such as petrochemicals, perfumes, and thinner are easier to analyze by GC. High molecular weight compounds are measured after pyrolysis.

Mobile Phase : Liquid Stationary Phase: Solid/Liquid Analysis : Liquid Chromatography Sample Types: Liquid samples and solvent-soluble solid samples. Compared to GC, LC has a wide range of measurement subjects. High molecular weight compounds can be analyzed, if soluble in solvent.