Analytical Techniques

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IR spectroscopy

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Chemical compounds absorb light energy depending on their structure; this absorption can be used to detect and quantify the presence of a compound. The chemical bonds in molecules are constantly vibrating; the interaction between radiation in the infrared region and these vibrations forms the basis of IR spectroscopy.

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Electromagnetic spectrum

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Electromagnetic spectrum is divided into three regions the far-infrared = 300–10 cm−1 (1000–30 μm), has low energy and may be used for rotational spectroscopy.

The mid-infrared = 4000–300 cm−1 (30–2.5 µm) may be used to study the fundamental vibrations and associated rotational-vibrational structure.

The higher energy near-IR, approximately 14000–4000 cm−1 (2.5–0.8 µm) can excite overtone.

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UV

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One of the most common pieces of apparatus in chemical laboratories is a UV-visible spectrometer which measures the absorption of light in the ultraviolet and visible region of the electromagnetic spectrum by a sample. Absorption is due to electronic transitions within atoms and molecules.

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Column chromatography

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In this technique, the solid x particles are packed into a column, and the solvent flows down through the particles by gravity.

The mixture is introduced at the top of the column and, as the continually flowing solvent (mobile phase) flows through the column, the different components move down at different rates. Each component flows out of the other end of the column at a different time in the mobile phase, as they are separated by the chromatographic process. By collecting the solvent in portions called fractions, each component of the mixture can be isolated as it leaves the column.

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Gas chromatography

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GC is column chromatography in which the mobile phase is a gas. The stationary phase can be in either a packed column or a capillary column. Packed column are shorter and have a wide bore than capillary column.

A packed column will contain material that can itself be used as the stationary phase or be coated with a liquid layer as the stationary phase. The stationary phase is a liquid, it is gas–liquid chromatography (GLC), and this is the most common technique because of its speed and efficiency. In GLC different separations are achieved through changing the nature of the liquid stationary phase; the gas mobile phase is invariably an inert gas such as helium. Gas–solid chromatography is also used, in which the stationary phase is a solid, but this is less common.

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Thin- layer chromatography

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The stationary phase (adsorbent) consists of a very thin layer of small particles attached to a flat plate, which can be made of glass, plastic or metal foil; this is called a TLC plate. The adsorbent in TLC is commonly silica gel, a highly absorbent silica powder. Paper chromatography is essentially TLC using a sheet of absorbent blotting paper.

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Chromatography techniques

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Chromatography is used in two ways:

1. to identify components of a mixture and/or determine how much there is of each component - this is called analytical chromatography;

2.to separate and purify a complex mixture to give a pure or purer compoundwithout, or with fewer contaminants: this is called preparative chromatography

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Fluorescence microscopy

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is the property of a substance that, when subjected to radiation of a specific wavelength, will absorb that radiation (excitation) and re-emit it at a different, but also specific wavelength. Stokes noticed that the emission wavelength is always longer than the wavelength of the excitation light.

Fluorescence occurs when a substance absorbs light of a certain wavelength and consequently emits light having a longer wavelength.

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Dark - field microscopy

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This is a microscopy method that excludes unscattered beams of light from the image viewed through the objective. (DFM) can be used on a normal microscope with some modifications. A normal, light microscope works by light from a source being concentrated by a condenser lens under the stage to produce a cone shape which then is focused on the specimen. The specimen is visualised according to its refractive index and how opaque it is, and the optical effects induced when light passes through it.

However, specimens with similar refractive indices to their surroundings, or which are very thin, are very hard to see. In these cases, either a type of differential staining should be used, or another technique such as DFM.

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Electron microscopy

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A beam of electrons produced by an electron gun passes through a series of magnets, which focus it into a thin beam - rather like a conventional glass lens does with light.This focused beam may be steered to any spot on the sample, again by the action of the magnetic lenses, and scanning coils move the focused beam back and forth over the surface around this point. During this process the beam dislodges electrons from atoms within the sample. The electrons generated are detected and amplified, and a final image built up from the number of electrons emitted from each spot on the sample.

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PCR

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Polymerase chain reaction (PCR) is widely used throughout analytical science and has made possible the detection and identification of minute amounts of DNA. The technique is used to copy sections of DNA from animals, plants and microorganisms, and to give enough material to analyse that region by a variety of techniques. The term ‘amplification’ is used for the process of increasing the amount of DNA present and the equipment in which the PCR reaction takes place is called a thermocycler

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HPLC

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HPLC is very widely used for the analysis of many classes of compounds from ions through to proteins. It is a powerful technique as it can fairly rapidly achieve excellent separations of complex mixtures.

Separation is achieved by using columns packed with small particles through which the mobile phase is pumped from a reservoir under high pressure. In fact HPLC was originally called high-pressure liquid chromatography because of this.

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Mass spectrometry

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The basic components of a mass spectrometer consist of an ion source, a mass analyser and a detector. Accelerator mass spectrometry ( AMS) is similar, except that it involves the acceleration of the ions beyond the energies used in many other mass spectrometers. Whilst ions are accelerated to thousands of electronvolts (measured using keV – kilo electronvolts) for SEM; in AMS, they are accelerated to millions of electronvolts (measured in MeV – mega electronvolts) .

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Titration

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A titration is a classical analytical procedure used to determine the unknown concentration of a solution of a known solute.

Start with a solution you know the concentration of to a high degree of accuracy, this is known as a standard solution

The substance that is weighed out accurately and used to make up the standard solution is known as a primary standard. A primary standard must be: soluble in whatever solvent you’re using, pure, chemically stable either as a solid or in solution and it should not react with air or atmospheric moisture.